Davie et al: Nuclear matrix in cancer diagnosis and chromatin structure matrix consisting of residual nucleoli, surrounding nuclear pore-lamina complex, and internal matrix is revealed when nuclease-digested nuclei are extracted with salt (e.g., 0.25 M ammonium sulfate). The protocol that we use to isolate nuclear matrices is shown in F i g . 1. Briefly, nuclei are digested with DNAase I followed by extraction with 0.25 M ammonium sulfate, yielding NM1-IF [nuclear matrices (NM) with attached intermediate filaments (IF)] (Sun et al., 1994; Chen et al., 1996). Further extraction of the NM1-IF nuclear matrices with 2 M NaCl yields NM2-IF. The internal matrix of NM1-IF preparations has a fibrogranular appearance (Chen et al., 1996). Extraction of the NM1-IF with high salt removes proteins that decorate core filaments of the internal matrix (Penman, 1995; Nickerson et al., 1995). The core filament fiber network is also seen when nuclear DNA is removed from nuclease-digested cells by electroelution in solutions of physiological ionic strength (Jackson and Cook, 1988). Core filaments, composition of which is currently unknown, have a diameter of 10-13 nm. These filaments appear to be the underlying structure onto which other nuclear components are bound. The nuclear matrix is composed of protein and RNA. The nuclear pore-lamina consists of lamins and pore proteins. The internal matrix has a complex protein composition, with heterogeneous nuclear ribonuclear proteins (hnRNP) being major components (Mattern et al., 1996). Most nuclear RNA is associated with the nuclear matrix and contributes to the structural integrity of the nuclear matrix (Nickerson et al., 1995). The absence of nuclear RNA may weaken nuclear matrix internal structures. For example, nuclear matrices isolated from chicken mature erythrocytes lack nuclear RNA and internal structures, while nuclear matrices from immature erythrocytes of anemic adult birds have internal structures and nuclear RNA (Chen et al., 1996).
II. Nuclear matrix proteins and the diagnosis of cancer The protein composition of the nuclear matrix is both tissue and cell type specific, and undergoes changes with differentiation and transformation (Fey and Penman, 1988; Stuurman et al., 1990; Dworetzky et al., 1990; Cupo, 1991). Pathologists have long appreciated that irregular nuclear appearance is the signature of a malignant cell (Miller et al., 1992; Nickerson et al., 1995). Changes in the composition of nuclear matrix proteins in malignant cells may contribute to alterations in nuclear structure. Nuclear matrix proteins are informative markers of disease states (Khanuja et al., 1993; Keesee et al., 1994). Informative nuclear matrix proteins have been identified for bladder, breast, colon, prostate, head, and neck cancers (Getzenberg et al., 1991a;1996; Khanuja et al., 1993;
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Keesee et al., 1994; Donat et al., 1996). For example, the nuclear matrix protein PC-1 is found in the nuclear matrix proteins from prostate cancer but not in the nuclear matrix from normal prostate or benign prostatic hyperplasia (Getzenberg et al., 1991a). Recently, we reported that the nuclear matrix protein composition was radically altered in highly metastatic oncogene transformed mouse fibroblasts (Samuel et al., 1997b). Interestingly, highly metastatic ras-transformed 10T1/2 cells and highly metastatic festransformed NIH 3T3 cells had a similar set of nuclear matrix proteins that were not seen in poorly metastatic or non-tumorigenic parental mouse fibroblast cell lines. Clearly, this study shows a correlation between the nuclear matrix protein profile and the metastatic potential of the cell. Of potential importance is the demonstration that nuclear matrix proteins can be detected in the serum and urine of cancer patients, thus suggesting that the detection of specific nuclear matrix proteins may be of value in breast cancer diagnosis (Miller et al., 1992; ReplogleSchwab et al., 1996; Carpinito et al., 1996). We have identified informative breast cancer nuclear matrix proteins (Samuel et al., 1997a). Typically we prepare NM2-IF nuclear matrices from breast cancer cell lines or breast tumours. To remove IFs from these preparations we disrupt nuclear matrices and attached IFs with urea (F i g . 1). The IFs are then allowed to reassemble and are removed from the soluble nuclear matrix proteins (Fey and Penman, 1988). Over a broad protein concentration range, this process is independent of protein concentration, but it is dependent upon temperature (F i g . 2). Performing the reconstitution at room temperature is recommended. About 8-10% of the nuclear protein is recovered in the nuclear matrix protein fraction. In the search for informative breast cancer nuclear matrix proteins, we used human breast cancer cell lines T47D, MCF-7 and ZR-75 (ER+/hormone dependent), MDA MB231, and BT-20 (ER-/hormone independent), and T5-PRF (ER+/hormone independent). A non-tumorigenic, spontaneously immortalized human breast epithelial cell line known as MCF-10A1 (ER-/hormone independent) obtained from reduction mammoplasty was chosen as the closest representative of normal breast epithelia. Typically we isolate proteins from at least three nuclear matrix preparations of each cell line, and these proteins are electrophoretically separated on two dimensional gels. Comparative analysis of the two dimensional gel patterns identified nuclear matrix proteins of estrogen receptor (ER) positive breast cancer cells that were not found in ERbreast cancer cells or normal breast epithelial cells (Samuel et al., 1997a). Our criteria for designating a nuclear matrix protein as being informative in breast cancer was that the protein had to be present in each of the relevant preparations (either ER+ and/or ER- breast cancer cell nuclear matrix proteins), but not in the preparations of
Gene Therapy and Molecular Biology, Vol 1, page 511 nuclear matrix proteins from "normal" breast epithelial cells. Using the nomenclature proposed by Khanuja et al. (1993), we refer to these proteins as NMBCs (nuclear matrix proteins in breast cancer). Five NMBCs (1-5) exclusive to the ER+ cell lines and one NMBC (6) exclusive to the ER- cell lines were identified (Samuel et al., 1997a). The extracellular environment can alter the cellular morphology as well as the protein composition of the cytoskeletal and nuclear matrix compartments (Getzenberg et al., 1991b; Pienta et al., 1991; Fallaux et al., 1996). Thus, it was important to find out whether the changes in nuclear matrix proteins we observed with cancer cells grown on plastic were observed with cancer cells present in a breast tumour. In the preparation of nuclear matrices from breast tumours, we found that it was necessary to remove the adipose tissue surrounding the tumour. We found all NMBCs (1-5) exclusive to ER+ status in the human breast cancer cell lines as being present in the ER+ breast tumours, while NMBC-6 was not detectable (see F i g . 3 , tumour 12797). NMBC-6, but not NMBCs 1-5, were present in ER- tumour nuclear matrix proteins. Figure 1. Method to Isolate Nuclear Matrix and Nuclear Matrix Proteins.
F i g u r e 2 . Effect of Temperature on the Separation of Intermediate Filament (IF) Proteins from Nuclear Matrix Proteins (NMP). NM2-IF from human breast cancer cells was disrupted in urea and then made to different protein concentrations prior to removal of IF proteins as described (Samuel et al., 1997b).
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The effect of cellular transformation on nuclear matrix protein composition is illustrated in the following study. Nuclear matrix proteins were isolated from MCF10A1 breast cancer cells that were transformed with the human T-24 mutated Ha-ras oncogene (MCF10AneoT) or with wild type human ER (cell line 139B6). MCF10AneoT cells are transformed and show anchorage independent growth (Basolo et al., 1991). The cell line 139B6 expresses ER at a similar level to that of MCF7 breast cancer cells (Pilat et al., 1996). In the presence of estradiol, this cell line has a slight inhibition in growth. This is typical of results of studies in which the ER is expressed in a ER- breast epithelial cell line or ER- breast cancer cell line (Pilat et al., 1996; Lundholt et al., 1996). Estradiol activated ER failed to elevate the expression of endogenous estrogen responsive genes but did induce the transient expression of an estrogen responsive elementregulated reporter gene in the 139B6 cell line (Pilat et al., 1996). Analysis of the two dimensional gel patterns of the nuclear matrix proteins from these cell lines revealed several alterations in nuclear matrix protein composition when MCF10A1 cells were transformed with ras or expressing ER. These differences were seen against a pattern of proteins found in all cell lines, for example hnRNP K (hk in F i g . 3 ). With the MCF10AneoT (Haras transformed) cells, nuclear matrix proteins with a molecular mass of 47 kDa and pI range of 5.8-6.2 (constellation C in Fig. 3) were found to be exclusive to this cell line. Similarly, nuclear matrix proteins with molecular masses 50-57 kDa and pIs 5.5-5.7 (constellation B in F i g . 3 ) and proteins with molecular masses of 30-36
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Figure 3. Human Breast Cancer Nuclear Matrix Proteins Nuclear matrix proteins were isolated from MCF10A1 (parent, ER-, human breast epithelial cells), MCF10A-139B6 (parent transfected with human wild type ER), MCF10AneoT (parent transformed by T-24 Ha-ras), and human breast tumour 12797 (ER+). Protein (40 ug) was electrophoretically resolved on two-dimension gels. The gels were stained with silver. The position of the molecular weight standards (in thousands) is shown on the left side of each gel pattern. LA and LC are lamin A and C, respectively. The circles in MCF10A1 (parent) show the absence or decreased amount of nuclear matrix proteins highlighted in other gel patterns.
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Gene Therapy and Molecular Biology, Vol 1, page 513 around which DNA is wrapped. The four core histones of the octamer are arranged as a (H3-H4)2 tetramer and two H2A-H2B dimers positioned on both sides of the tetramer. The core histones have a similar structure with a basic N terminal unstructured domain, a globular domain organized by the histone fold, and a C terminal unstructured tail (Arents and Moudrianakis, 1995) (F i g . 4 ). Histone H1 binds to the linker DNA, which joins nucleosomes together, and to core histones (Boulikas et al., 1980; Ban猫res et al., 1994). H1 has a tripartite structure with a basic N terminal domain, a basic C terminal tail domain, and a central globular core (Ramakrishnan, 1994).
Figure 4. Sites of Post-Synthetic Modifications on the Histones. The structures of H2A-H2B dimers and (H3-H4)2 tetramers and the sites of modification are shown. Ac, acetylation; Ub, ubiquitination. The enzymes catalyzing reversible histone acetylation are shown.
kDa and pIs 4.5 (constellation A in F i g . 3) were determined to be exclusive to the ER expressing cell line MCF10A-139B6. However, within constellation B, a 48 kDa (pI 5.5) nuclear matrix protein (denoted by * in F i g . 3) was observed in the MCF10A parent cell line as well as in the ras- transformed and ER transfected cell lines. Relative to the parent cell line, the level of this protein in MCF10AneoT and MCF10A-139B6 was higher. NMBC1 present in the ER+ breast tumor nuclear matrix proteins was also detected in the ER expressing cell line (F i g . 3 ). The presence of NMBC1 in the ER transfected cell line suggests that ER expression has a role in the association of NMBC1 with the nuclear matrix. These results illustrate how nuclear matrix protein profiles reflect alterations in a cell's physiological state.
III. Nuclear matrix and organization of nuclear DNA Nuclear DNA is packaged into nucleosomes, the repeating structural units in chromatin (Van Holde, 1988). The nucleosome consists of an histone octamer core 513
In low ionic strength, chromatin fibers depleted of H1 have a "beads-on-a-string" structure, but with H1, folding of the fiber is evident (Leuba et al., 1994). At physiological ionic strength chromatin is folded into a 30 nm fiber. H1 stabilizes the folding of the chromatin fiber (Shen et al., 1995). The native 30 nm chromatin fiber has an irregular structure (an irregular three dimension zigzag) in vitro (Woodcock and Horowitz, 1995). Woodcock and colleagues show that the irregularities of the 30 nm chromatin fiber can be accurately reflected in a model that accounts for variability in linker DNA length and angle of trajectory that the linker DNA has as it enters and leaves the nucleosome. Thirty nm fibers are usually not seen inside nuclei (Woodcock and Horowitz, 1995). The chromatin is observed as matted patches. It appears that neighboring zigzags interdigitate, preventing individual chromatin fibers from being seen in nuclei. The core histone tails contribute to the condensation of the chromatin fiber (Garcia-Ramirez et al., 1995; Schwarz et al., 1996; Krajewski and Ausi贸, 1996). H3 and H4 tails are needed for fiber-fiber interactions (Schwarz et al., 1996). The chromatin fiber is organized into loop domains, with an average size of 86 kb (Jackson et al., 1990; Gerdes et al., 1994) (F i g . 5 ). Transcriptionally active genes are found in DNAase I-sensitive, presumably decondensed chromatin loops that are accessible to transcription factors and transcription machinery (Davie, 1995). Transcriptionally inactive genes are in higher order, interdigitated chromatin patches, being essentially invisible to transcription factors and the transcription machinery. At the base of the loop there are DNA sequences called MARs (matrix associated regions) that bind to nuclear matrix proteins (Bode et al., 1995). MARs tend to be AT-rich, but do not have a consensus sequence (Bode et al., 1995; Mielke et al., 1996). MAR-DNA binds to both internal matrix and nuclear pore-lamina, suggesting that proteins of the nuclear pore-lamina and internal matrix are involved in the organization of chroma-
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Figure 5. A Model for Transcriptionally Active and Repressed Chromatin Domains At the base of the loop are nuclear matrix associated regions (MARs). HET (SAF-B) is a nuclear matrix protein that binds MARs. The repressed chromatin loop has a condensed chromatin structure. Multiple dynamic attachment sites between the transcriptionally active domain and the internal nuclear matrix are presented in the box outline. Histone acetyltransferase (HAT A), histone deacetylase (HDAC), transcription machinery and transcription factors are shown associated with the internal nuclear matrix, mediating a dynamic attachment between transcriptionally active chromatin and the nuclear matrix. HAT A and HDAC are shown as multiprotein complexes.
IV. In situ crosslinking with cisplatin
tin (Zini et al., 1989). MAR-binding proteins include lamins, which are found in the nuclear pore-lamina and internal matrix (HozĂĄk et al., 1995), topoisomerase II, SATB1, HET (SAF-B), and attachment region binding protein which is an internal matrix protein or nuclear matrin (Pommier et al., 1990; Nakayasu and Berezney, 1991; von Kries et al., 1991; Luderus et al., 1992; Nakagomi et al., 1994; Buhrmester et al., 1995; Oesterreich et al., 1997).
Recent studies suggest that cisplatin (cis-diammine dichloroplatinum or cis-DDP) preferentially crosslink MARs to nuclear matrix proteins in situ. Either cells or nuclei can be incubated with cisplatin to crosslink protein to DNA. Most proteins crosslinked to DNA with cisplatin are nuclear matrix proteins, and the DNA crosslinked to protein is enriched in MAR-DNA sequences (Wedrychowski et al., 1986; 1989; Ferraro et al., 1992; 1995; Bubley et al., 1996; Olinski et al., 1987). F i g . 6 shows the protocol to isolate proteins crosslinked to DNA in situ. A comparison of two dimension gel patterns of nuclear matrix proteins and proteins crosslinked to DNA with cisplatin in ZR-75 human breast cancer cells shows that several abundant nuclear matrix proteins are crosslinked to DNA in the cells (F i g . 7 ). Lamins A and C, components of the nuclear pore-lamina, are crosslinked in situ to nuclear DNA consistent with in vitro data suggesting that these proteins are involved in the organization of nuclear DNA (Wedrychowski et al., 1986; 1989). Abundant nuclear matrix proteins found
Alterations in MAR-binding proteins have been reported in cancer cells. In Southwestern blotting experiments with a radiolabelled mouse IgH MAR sequence, Yanagisawa et al. (1996) detected a 114-kDa MAR binding protein expressed in breast carcinomas but not normal or benign breast tissue. Further, the levels of this MAR-binding protein were elevated in poorly differentiated breast ductal carcinomas. A recent study shows that mutant, but not wild type, p53 binds to MARs (MĂźller et al., 1996). Changes in nuclear matrix, MARbinding proteins could result in reorganization of nuclear DNA.
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Gene Therapy and Molecular Biology, Vol 1, page 515 crosslinked to nuclear DNA in situ with cisplatin are Factin and hnRNP K (Miller et al., 1991; Sauman and Berry, 1994) (F i g . 7). HnRNP K is a single-strand DNA-binding protein that is associated with the nuclear matrix and has an important role in regulating the expression of the c-myc gene (Michelotti et al., 1996; Mattern et al., 1996). Further, hnRNP K interacts with TATA-binding protein (Michelotti et al., 1996). This transcription factor is a prominent protein observed in both the nuclear matrix fraction and proteins crosslinked to DNA in situ with cisplatin in ZR-75 human breast cancer cells (F i g . 7 ).
F i g u r e 6 . Method to Isolate Proteins Crosslinked to DNA in Cells or Nuclei with Cisplatin
Figure 7. Analysis of Nuclear Matrix Proteins and Proteins Crosslinked to DNA ZR-75 human breast cancer nuclear matrix proteins (40 ug) and proteins crosslinked to DNA by cis-DDP in situ (40 ug) were electrophoretically resolved on two dimension gels. The gels were stained with silver. The position of the carbamylated forms of carbonic anhydrase is indicated by ca. The position of the molecular weight standards (in thousands) is shown to the left of the gel patterns. LA and LC show lamin A and C, respectively. HnRNP K is shown as hk.
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The ability of cisplatin to preferentially crosslink nuclear matrix proteins to nuclear DNA in situ has great potential in identifying nuclear matrix proteins involved in the organization and function of nuclear DNA. Several transcription factors are nuclear matrix proteins thought to interact with promoter and enhancer elements of specific genes. It has been proposed that the interaction of nuclear matrix bound transcription factors with regulatory DNA sequences has a role in attaching transcriptionally active chromatin to nuclear matrix (see below). Crosslinking with cisplatin may provide a method to find if the nuclear matrix associated transcription factor is bound to the DNA sequence of interest in situ. We are currently developing methods that will identify nuclear matrix associated transcription factors and their bound DNA sequences. Further, these methods are being used to find informative DNA-binding nuclear matrix proteins in the diagnosis of cancer.
Davie et al: Nuclear matrix in cancer diagnosis and chromatin structure
V. Transcription factors: activators of transcription Current evidence suggest that an interaction between an enhancer or locus control region and promoter is an essential step in forming the open chromatin domain (Reitman et al., 1993). The enhancer/locus control regionpromoter interaction is mediated by protein-protein associations between transcription factors bound to these cis-acting regulatory elements. This complex recruits the transcription initiation machinery and initiates the transcription cycle. The transcription cycle can be separated into at least four stages: initiation, promoter clearance, elongation, and termination. During the initiation stage, the pre-initiation complex (PIC) is formed at the promoter of a RNA polymerase II transcribed gene. The basal transcription factors TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH and RNA polymerase II are involved in the assembly of the PIC (for review see Orphanides et al., 1996; Pugh, 1996; Nikolov and Burley, 1997). In vitro studies show that there is a defined order by which the basal transcription factors are assembled into the PIC. TFIID, one of the first factors involved, binds to the TATA-box and consists of the TATA-binding protein (TBP) and several TBP-associated factors (TAFs).
Figure 8. Model for Histone H5 Chromatin in Chicken Immature Erythrocytes. The 3' enhancer is positioned next to the 5' promoter through protein-protein interactions. NF1 and multiprotein complexes (HAT As, HDACs, transcription machinery) are shown mediating the dynamic attachments of the histone H5 gene to the nuclear matrix at sites of transcription.
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However, there is evidence that the PIC comes partially preassembled (Maldonado et al., 1996). In the formation of the PIC at any given promoter, TFIID binding appears to be the rate limiting step. Transcription factors bound to enhancers and upstream promoter elements interact through their activation domains with the TAFs of TFIID or with other components of the PIC (e.g., TFIIB), increasing the rate that the PIC is formed at the promoter (for review see Tjian and Maniatis, 1994; Chiang et al., 1996; Gupta et al., 1996). TAFs are referred to as coactivators, proteins that mediate an interaction between transcription factors and PIC. Transcription factors bind to different TAFs, and these multiple contacts between the transcription factors and TAFs ensure the efficient recruitment of TFIID (Sauer et al., 1995; Chi et al., 1995). Transcription factors with multimerization domains also have key roles in juxtapositioning enhancer and promoter elements. For example, multimers of the transcription factor Sp1 bound at DNA sites separated by 1.8 kb will interact, resulting in a looping out of the intervening sequences (Pascal and Tjian, 1991). Once these cis-acting elements are positioned next to each other, there will be a high local concentration of activators in the vicinity of the promoter (F i g . 8 ).
Gene Therapy and Molecular Biology, Vol 1, page 517 nuclear matrix bound transcription apparatus as transcription proceeds (Cook, 1994; Hendzel and BazettJones, 1995; Iborra et al., 1996). The transcription machinery is a massive multiprotein complex (Aso et al., 1995; Chao et al., 1996; Maldonado et al., 1996). Thus, it is unlikely that RNA polymerase travels along the DNA as text book models often show. Further, the nascent RNA becomes associated with the nuclear matrix. A solid state process by which DNA is driven through the nuclear matrix bound machinery and the nascent RNA is processed at the nuclear matrix would be an efficient way of dealing with these nuclear activities.
VI. Regulation of transcription Within the DNAase I sensitive chromatin domains containing transcriptionally active genes are regions that are hypersensitive to DNAase I attack. The DNAase I hypersensitive (DH) regions of chromatin may lack nucleosomes and often mark chromatin for the presence of cis-acting regulatory DNA sequences and trans-acting factors. DH sites in human breast cancer c-myc chromatin and chicken erythrocyte histone H5 chromatin map with promoters and enhancers, and using in vitro assays we identified the transcription factors binding to these regulatory DNA elements (Penner and Davie, 1992; 1994; Sun et al., 1992; 1993; Miller et al., 1993; 1996; Murphy et al., 1996). However, in vitro assays can sometimes be misleading, and the most rigorous method in finding transcription factor occupancy is in situ footprinting (Becker et al., 1987; Mueller and Wold, 1989). We did in situ footprinting using a procedure called ligationmediated PCR to reveal the occupancy of factor binding sites in the promoter and enhancer of the H5 gene in chicken erythrocytes (Sun et al., 1996b). Some factor binding sites in the promoter and enhancer identified in vitro were not occupied in situ. Based upon our studies on the chromatin structure and transcription factors associated with the H5 promoter and enhancer, we put forth a model for the transcriptionally active H5 gene (F i g . 8 ).
VIII. Nuclear matrix and transcriptionally active chromatin Transcribed and nontranscribed sequences are precisely compartmentalized within the nucleus (Andreeva et al., 1992; Gerdes et al., 1994; Davie, 1995). Actively transcribed, but not inactive, chromatin regions are immobilized on the nuclear matrix by multiple dynamic attachment sites (F i g . 5 and 8). When histones are removed by high salt, loops of DNA are seen emanating from a central nuclear skeleton, forming a halo around this nuclear structure. Transcriptionally inactive genes are found in the halo, while DNA loops with transcriptionally active genes remain associated with the nuclear skeleton (Gerdes et al., 1994). The transcription machinery, specific transcription factors, and nuclear enzymes (e.g., histone acetyltransferase, histone deacetylase, see F i g . 8 ) are thought to mediate the dynamic attachments between transcribing chromatin and nuclear matrix (van Wijnen et al., 1993; Cook, 1994; 1995; Bagchi et al., 1995; Merriman et al., 1995).
VII. Nuclear matrix and processing of the genetic information The nuclear matrix is involved in the processing of the genetic information. In recent years we have come to appreciate that functional components (e.g., transcript domains, RNA processing sites, sites of replication) of the nucleus are highly organized (Hendzel and Bazett-Jones, 1995; Penman, 1995; Xing et al., 1995). Transcribed genes are found in discrete foci (Jackson et al., 1993; Iborra et al., 1996; Wansink et al., 1996). The nuclear matrix is the foundation from which this organization is built, providing a scaffold from which nuclear processes such as DNA replication and transcription occur (Berezney, 1991; Iborra et al., 1996). It is important to note that these functional centers in the nucleus are dynamic in their formation and dissociation. For example, sites of replication will assemble on the nuclear matrix at or near transcription foci in early S phase of the cell cycle. Once replication of these regions of the genome is complete, the replication machinery will disassemble from its site on the nuclear matrix and reassemble at other sites continuing replication of other regions of the genome (HozĂĄk et al., 1993; Bassim Hassan et al., 1994). The process of transcription occurs at the nuclear matrix, and it has been proposed that the chromatin fiber moves through the
The nuclear matrix is selective for which transcription factors it binds, and this selectivity varies with cell type (van Wijnen et al., 1993; Sun et al., 1994; 1996a). It has been postulated that the nuclear matrix has a role in the expression of genes by concentrating a subset of transcription factors at specific nuclear sites (Stein et al., 1991; Merriman et al., 1995). Transcription factors associated with the nuclear matrix include ER, HET, GATA-1, YY1, AML-1, Sp1, Oct1, mutant p53, and Rb (Dworetzky et al., 1992; Isomura et al., 1992; Vassetzky et al., 1993; van Wijnen et al., 1993; Sun et al., 1994; Merriman et al., 1995; Guo et al., 1995; MĂźller et al., 1996; Mancini et al., 1996; Kim et al., 1996; Oesterreich et al., 1997). HET is a transcriptional repressor. Interestingly, sequencing of HET revealed that it was identical to SAF-B, a protein isolated by its ability to bind MARs (Renz and Fackelmayer, 1996; Oesterreich et al., 1997). Thus, HET (alias SAF-B) is a nuclear matrix protein in breast cancer cells that binds to MARs and acts as a repressor.
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Davie et al: Nuclear matrix in cancer diagnosis and chromatin structure Protein domains involved in targeting transcription factors to the nuclear matrix have been identified. However, it is too early to know whether a consensus nuclear matrix localization signal will emerge from these studies. The N-terminal domains of the androgen and glucocorticoid receptor are involved in directing these receptors to the nuclear matrix (van Steensel et al., 1995). There are examples of where the association of transcription factors with the nuclear matrix is regulated by modifications. For example, the association of Rb with the nuclear matrix appears to be regulated by phosphorylation and is cell-cycle dependent. When Rb is in a hypophosphorylated state in G1-phase of the cell cycle, it is attached to the nuclear matrix. But the highly phosphorylated Rb of S-phase is not associated with the matrix (Mancini et al., 1994). The amino terminus of hypophosphorylated Rb binds to a p84 nuclear matrix protein (Durfee et al., 1994).
regulatory DNA elements of specific genes is currently lacking. For example, NF1 is a nuclear matrix associated transcription factor that binds to the enhancer of the chicken histone H5 gene (F i g . 8 ). In vitro footprinting and electrophoretic mobility shift assays show that NF1 isolated from immature erythrocyte nuclear matrices binds to the H5 enhancer (Sun et al., 1994). In situ footprinting shows that the NF1 binding site in the H5 enhancer is occupied in chicken immature erythrocytes (Sun et al., 1996b). We have proposed that NF1 recruits the H5 enhancer to the nuclear matrix (Davie, 1996). However, we have yet to show that nuclear matrix associated NF1 is the protein occupying the H5 enhancer NF1 binding site in erythroid cells. Cisplatin crosslinking may provide direct evidence to test this model (see above).
ER is associated with the nuclear matrix of estrogen responsive tissues (Metzger and Korach, 1990; Metzger et al., 1991; Thorburn and Knowland, 1993). In vitro reconstitution studies with nuclear matrices and hormone receptors (e.g., ER and androgen receptor) show that nuclear acceptor sites for the hormone receptors are associated with the nuclear matrix (Barrack, 1987; Metzger and Korach, 1990; Lauber et al., 1995). The binding of the ER to the nuclear matrix was saturable, of high affinity, target tissue specific, and receptor specific (Metzger and Korach, 1990). Acceptor proteins for ER have been identified in a variety of estrogen-responsive tissues (Lauber et al., 1995; Ruh et al., 1996).
Transcribed DNA is associated with acetylated histones (Hebbes et al., 1994; O'Neill and Turner, 1995; Mutskov et al., 1996). The core histones are reversibly modified by acetylation of lysines located in their basic N terminal domains (F i g . 4). Reversible histone acetylation is catalyzed by histone acetyltransferases (HATs) and deacetylases (HDACs), with the level of acetylation being decided by the net activities of these two enzymes. Histone acetylation alters nucleosome and higher order chromatin structure (Davie, 1995; 1997). For example, chromatin associated with highly acetylated histones does not undergo histone H1 mediated aggregation at physiological ionic strength, while chromatin with unacetylated histones aggregates when associated with H1 (Ridsdale et al., 1990; Davie, 1997). Besides modulating nucleosome and higher order chromatin packaging, the core histone tails bind to regulatory proteins (Ma et al., 1996; Edmondson et al., 1996). For example, yeast repressor protein Tup1 binds to the tails of H3 and H4. Acetylation of H3 and H4 prevents the binding of Tup1 (Edmondson et al., 1996). In mammalian cells and chicken erythrocytes, transcriptionally active chromatin regions have core histones undergoing high rates of acetylation and deacetylation, while in repressed chromatin regions the rate of reversible acetylation is slow (Davie, 1996; 1997). Thus, we expect that the interaction of regulatory proteins with the histone tails and chromatin structure of transcriptionally active regions of mammalian and chicken erythrocytes is in dynamic flux.
Transcription factors associated with the nuclear matrix can change throughout development and differentiation (Stein et al., 1994; Davie, 1995; Merriman et al., 1995; Bagchi et al., 1995; Sun et al., 1996a). For example, transcription factors associated with the chick erythrocyte nuclear matrix change throughout development (Sun et al., 1996a). Primitive red blood cells from 5-day old embryos have high levels of nuclear matrix-bound transcription factors, including GATA-1, CACCC-binding proteins, and NF1; factors that have key roles in erythroid-specific gene expression. In definitive red blood cells (11-day and 15-day embryos) the levels of these nuclear matrix bound transcription factors decline. Erythroid nuclear matrices preferred to bind CACCC-binding proteins and not Sp1. Promoters and enhancers of erythroid-specific genes have Sp1 binding sites that bind both CACCC-binding proteins and Sp1. It is possible that the selective nuclear matrix binding of CACCC-binding proteins gives the CACCCbinding proteins an advantage over Sp1 in binding to a Sp1/CACCC site. Although we know that transcription factors are associated with the nuclear matrix, evidence that nuclear matrix associated transcription factors are bound to 518
IX. Dynamic histone acetylation
The process of reversible histone acetylation is not dependent upon ongoing transcription (Ruiz-Carrillo et al., 1976). To date, the only histone modifications dependent upon ongoing transcription are ubiquitination of H2B (see F i g . 4 ) and phosphorylation of mouse H1b (Davie and Murphy, 1990; Chadee et al., 1997). However, interference of dynamic acetylation by inhibiting
Gene Therapy and Molecular Biology, Vol 1, page 519 deacetylation with histone deacetylase inhibitors (e.g., sodium butyrate, trichostatin A or trapoxin) greatly affects cell cycle progression, arresting cells in G1 or G2, and may enhance or repress the expression of genes (Yoshida et al., 1995; Johnston et al., 1992; Girardot et al., 1994; Miyashita et al., 1994; Laughlin et al., 1995).
F i g u r e 9 . CBP/p300 Cointegrates Diverse Signalling Pathways CBP and its functional/protein interaction domains are shown.
X. Histone acetyltransferase and gene activation Histone acetylation is not limited to transcriptionally active chromatin, but also has a role in DNA replication (deposition-related acetylation) and DNA repair (for review see Davie, 1995; 1997). Deposition-related acetylation of H4 is catalyzed by HAT B, a cytoplasmic enzyme (Kleff et al., 1995; Brownell and Allis, 1996). HAT A is responsible for transcriptionally active chromatinassociated acetylation. Nuclear HAT A is bound to chromatin and acetylates all core histones when free or within nucleosomes (Brownell and Allis, 1996). Dr. Allis and colleagues were the first to purify and clone a HAT A. There studies showed that Tetrahymena HAT A (p55) is homologous to yeast Gcn5, a transcriptional adaptor, that has HAT activity (Brownell et al., 1996). This important breakthrough provided a direct link between the process of transcription activation and histone acetylation.
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Tetrahymena HAT A (p55) and yeast Gcn5 are components of large multiprotein complexes, and the substrate specificity of the catalytic subunit is regulated by the proteins binding to it (Grant et al., 1997). Yeast Gcn5 and Tetrahymena p55 can acetylate free histone H3 but these HAT As are unable to acetylate histones in nucleosomes. Yeast Gcn5 is a component of two high molecular mass complexes (0.8 and 1.8 megadaltons) (Grant et al., 1997). These high molecular mass, multiprotein complexes acetylated histones in nucleosomes and free histones. Both HAT A complexes contain Ada2 and Ada3. Gcn5-Ada2-Ada3 is a putative adaptor complex that connects DNA-bound transcription factors (activators) to components of the PIC (Candau et al., 1997). The HAT domain of yeast Gcn5 has been localized. Gcn5 requires both the HAT domain of Gcn5 and interaction with Ada2 for transcriptional activation (Candau et al., 1997). Human homologues of Gcn5 and Ada2 have been identified (Candau et al., 1996). Tetrahymena HAT A and yeast Gcn5 have a bromodomain that is lacking in yeast Hat1p. The bromodomain, which is thought to be a protein-protein interaction domain, is found in the C-termini of these proteins (Haynes et al., 1992). Several other recently identified HAT As have the bromodomain, including TAFII250 (a 250 kDa protein that binds to TATA-binding protein), CBP/p300 but not P/CAF (Yang et al., 1996; Bannister and Kouzarides, 1996; Mizzen et al., 1997; Ogryzko et al., 1997). It is possible that HAT As interact with other transcription factors through the bromodomain, directing HAT A to specific regions in chromatin and in the nucleus (Brownell and Allis, 1996). Most HAT As are coactivators (e.g., Gcn5, TAFII250, CBP/p300). CBP/p300 binds to hormone receptors, AP1, c-Myb, SV40 large T antigen, and adenovirus E1a, and appears to be an integrator of multiple signalling pathways (Kamei et al., 1996; Avantaggiati et al., 1996; Oelgeschl채ger et al., 1996; Hanstein et al., 1996) (F i g . 9). Unlike Gcn5, Tetrahymena p55, TAFII250, or P/CAF, CBP acetylates all four core histones in nucleosomes (Ogryzko et al., 1997). The discovery that several transcription modulators or coactivators have HAT activity provides a mechanism by which chromatin structure is altered in the vicinity of DNA-bound transcription activators. A variety of transcription factors including hormone receptors, CREB, and fos-jun will bind directly or indirectly to CBP, recruiting a coactivator with histone acetyltransferase activity (F i g . 1 0 ). The HAT activity of CBP would then acetylate surrounding histones in nucleosomes, leading to the destabilization of nucleo-
Gene Therapy and Molecular Biology, Vol 1, page 520
F i g u r e 1 0 . Role of HAT As and HDACs in Transcriptional Activation and Repression Top panel: Fos-Jun is shown recruiting the coactivator CBP, resulting in the acetylation of nucleosomal histones. Bottom panel: Mad-Max is shown recruiting the corepressor HDAC multiprotein complex, resulting in the deacetylation of nucleosomal histones
some and higher order chromatin structure. Such a chromatin state is thought to be facilitate the binding of other transcription factors and, in general, aid the transcription process.
The high molecular mass chicken erythrocyte histone deacetylase complex deacetylates the four core histones in chromatin, but has a preference for H2B (Li et al., 1996). Dissociation of the multiprotein histone deacetylase complex resulted in a change in substrate preference. The 66-kDa enzyme could not deacetylate histones in chromatin and had a preference for free H3. The data suggest that proteins important in regulating HDAC activity were lost during enzyme purification (Li et al., 1996).
XI. Histone deacetylase and gene repression Histone deacetylases are nuclear enzymes that have been isolated from a variety of sources. Our studies have focused on the chicken erythrocyte histone deacetylase, an enzyme associated with the nuclear matrix (Hendzel et al., 1991). Chicken erythrocyte histone deacetylase is a component of a multiprotein complex that has a molecular mass in excess of 400 kDa (Li et al., 1996). The chicken histone deacetylase complex extracted from nuclei dissociates to a 66-kDa form in 1.6 M NaCl or when applied to an ion-exchange column (e.g., Q-sepharose). However, the high molecular mass histone deacetylase complex extracted from chicken erythrocyte nuclear matrices does not dissociate in 1.6 M NaCl, but this HDAC complex did dissociate to a 66-kDa form when applied to a Q-sepharose column (Li et al., 1996). These observations suggest that the solubilized nuclear matrix histone deacetylase is associated with proteins that stabilize the complex from dissociation into the 66-kDa form in a high concentration of salt.
Dr. Schreiber and colleagues were the first to clone a mammalian histone deacetylase (HDAC1, 55 kDa) (Tauton et al., 1996). They found that mammalian histone deacetylase was related to the yeast transcription regulator Rpd3p, providing a link between transcription regulation and histone deacetylation. At around the same time, Dr. Grunstein and colleagues purified two yeast histone deacetylase complexes, HDA (350 kDa) and HDB (600 kDa) (Carmen et al., 1996; Rundlett et al., 1996). The HDA complex consists of multiple peptides with molecular masses of approximately 70 kDa. Two peptides from the HDA complex have been sequenced, and yeast genes HDA1 (codes for p75) and HDA3 (codes for p71) isolated. HDA1 shares sequence similarity with Rpd3p, a yeast histone deacetylase. Gene disruptions of HDA1 or HDA3 resulted in the loss of the HDA, but not, HDB complex. Rpd3p is a component of HDB (Rundlett et al., 1996). Rpd3p binds to Sin3 which in turn associates with 520
Gene Therapy and Molecular Biology, Vol 1, page 521 Ume6, a DNA-binding protein required for the repression of several genes including those involved in meiosis (Kadosh and Struhl, 1997). These observations suggest that the yeast HDB complex consists of Rpd3p, Sin3 and Ume6. Mammalian histone deacetylase HDAC1 is related to yeast transcriptional regulator Rpd3p (Tauton et al., 1996). Although HDAC1 has a reported molecular mass of 55 kDa, we have found that it migrates on our SDS polyacrylamide gels with an apparent molecular mass of 66 kDa. The mammalian homologue of Rpd3p, named HDAC2, has been cloned (Yang et al., 1996). Chicken erythrocyte histone deacetylase has been purified, and the enzyme migrates as a 66-kDa band on SDS gels (J.-M. Sun, H. Y. Chen, J. R. Davie, unpublished observations). Thus, chicken erythrocyte histone deacetylase has a molecular mass similar to that of mammalian HDAC1.
Figure 1 1 . Regions of mSin3A Involved in Protein Interactions N-CoR, SMRT and Mad family members interact with different paired amphipathic helix (PAH) domains in mSin3A. The HID domain which binds to HDAC 1 and 2 is shown.
As with yeast and chicken histone deacetylases, mammalian histone deacetylases, HDAC1 and HDAC2, exist as high molecular mass, multiprotein complexes (Hassig et al., 1997). HDAC1 and HDAC2 bind to a variety of proteins, including RbAp48, YY1, and mammalian (m) Sin3A and mSin3B (Tauton et al., 1996; Yang et al., 1996; Laherty et al., 1997). These HDACbinding proteins may exist in different HDAC multiprotein complexes. For example, HDAC complexes with mSin3 do not contain YY1 (Zhang et al., 1997). HDAC1 was purified as a complex with RbAp48, a 50 kDa Rb-binding protein that binds to the C-terminus of unphosphorylated or hypophosphorylated Rb (Tauton et 521
al., 1996; Qian et al., 1993). RbAp48 has several partners in addition to HDAC1. RbAp48 is a component of human and Drosophila CAF-1 (chromatin assembly factor 1) (Verreault et al., 1996). A yeast protein similar to RbAp48, Hat2p, is component of yeast HAT B (Roth and Allis, 1996; Parthun et al., 1996). HDAC1 and/or HDAC2 are in large multiprotein complexes that contain mSin3, N-CoR, and SMRT, proteins that are corepressors (Nagy et al., 1997; Hassig et al., 1997; Laherty et al., 1997; Heinzel et al., 1997). Mammalian Sin3A and mSin3B have four paired amphipathic helix (PAH) domains thought to be involved in protein-protein interactions (F i g . 1 1 ). HDAC1 and HDAC2 bind to the region between PAH3 and PAH4, referred to as HID [the histone deacetylase interaction domain (HID)] (Laherty et al., 1997). The HID region is conserved in mSin3A, mSin3B and yeast Sin3. Mammalian Sin3A (150 kDa) interacts with many other proteins, including SAP18 (mS in3 associated protein), Mad family members (Mad1, Mad3, Mad4, Mxi1) and Max-binding repressor Mnt, SMRT, and N-CoR (Laherty et al., 1997; Zhang et al., 1997; Nagy et al., 1997; Heinzel et al., 1997; Alland et al., 1997). mSin3A does not have DNA binding ability; however, several of the proteins associated with mSin3A can direct it to specific DNA regulatory regions. The N-terminal region (SID, mS in3 interaction domain) of the Mad family members and Mnt binds to PAH2 of mSin3 (F i g . 11). Mad family members form a dimer with Max, a DNA-binding complex that binds to E-box related DNA sequences (Laherty et al., 1997). Max and Mad proteins are members of the basic region-helix-loop-helix-leucine zipper (bHLHZip) transcription factors. Myc forms a heterodimer with Max which binds to the same E-box-related DNA sequences as does Mad-Max heterodimers. However, MycMax activates genes, while Mad-Max represses their transcription. The repressive action of Mad-Max is mediated in part by the interaction of Mad with mSin3 which in turn is associated with HDAC1 and/or HDAC2. N-CoR and SMRT bind to unliganded retinoid and thyroid hormone receptors (Nagy et al., 1997; Heinzel et al., 1997). Thus, like the bHLH-Zip repressor proteins, unliganded hormone receptors recruit the HDAC multiprotein complex. HDAC has a principal role in transcription repression. Several studies show that tethering HDAC1 or 2 to a promoter by fusing HDAC to a DNA-binding domain (e.g., Gal4 DNA-binding domain) results in transcription inhibition (Yang et al., 1996; Zhang et al., 1997; Kadosh and Struhl, 1997; Nagy et al., 1997). These studies suggest that repressors recruit histone deacetylase which would deacetylate histones in nucleosomes, leading to the condensation of chromatin (Wolffe, 1997) (F i g . 1 0 ).
Davie et al: Nuclear matrix in cancer diagnosis and chromatin structure Although these studies show that HDAC is involved in repression, HDAC is associated with transcriptionally active chromatin. Both HAT As and HDACs are needed to catalyze dynamic acetylation of histones associated with transcribed chromatin domains. The presence of both HAT As and HDACs at transcriptionally active regions allows the rapid manipulation of nucleosome and chromatin structure (Wade and Wolffe, 1997).
XII. Histone acetylation and nuclear matrix Vertebrate histone acetyltransferase (HAT A) and histone deacetylase (HDAC) are associated with the nuclear matrix (Hendzel et al., 1991; 1992; 1994; Li et al., 1996). Nuclear skeletons from chicken immature erythrocytes retain 80% of the nuclear HAT A and HDAC activities, and these enzymes catalyze reversible acetylation using as substrate the chromatin fragments associated with the nuclear skeletons (Hendzel et al., 1994). These studies suggest that HAT A and HDAC are colocalized to specific sites on the nuclear matrix. However, there is no evidence that HAT A and HDAC are part of the same large complex. We proposed a model in which nuclear matrixbound HAT A and HDAC mediate dynamic interactions between the nuclear matrix and transcriptionally active chromatin (F i g . 8) (Davie and Hendzel, 1994; Davie, 1995). We have evidence that HDAC1 is associated with the matrix, but the identity of the nuclear matrix bound HAT A is currently unknown. Several transcription factors binding directly or indirectly with HAT A and HDAC are nuclear matrix proteins. For example, YY1 is a nuclear matrix protein that binds to HDACs (Yang et al., 1996; Guo et al., 1995). Estrogen receptors bound to the nuclear matrix could recruit CBP, an HAT A (Hanstein et al., 1996). Determining how HATs and HDACs are recruited to nuclear matrix sites engaged in transcription will be an important challenge.
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Acknowledgements This research was supported by grants (MT-9186, PG12809) from the Medical Research Council of Canada and the Cancer Research Society, Inc., and by a Medical Research Council of Canada Senior Scientist Award to J.R. Davie.
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Gene Therapy and Molecular Biology Vol 1, page 529 Gene Ther Mol Biol Vol 1, 529-542. March, 1998.
Structural organization and biological roles of the nuclear lamina 2
Amnon Harel 1, Michal Goldberg, Nirit Ulitzur and Yosef Gruenbaum Department of Genetics, The Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem 91904, Israel. __________________________________________________________________________________________________ Correspondence: Yosef Gruenbaum, Tel: +972-2-6585995, Fax: +972-2-5633066 or +972-2-6586975, E-mail: gru@vms.huji.ac.il 1. Present address: Department of Biology, University of California, San Diego, San Diego CA. 2. Present address: Department of Biochemistry, Stanford Medical School, Stanford CA.
Summary T h e n u c l ea r l a m i na i s a p ro t e i n m e s hw or k t ha t l i e s on the nucleoplasmic side o f the nuclear e n v e l o p e a n d i s a s s o c i a t e d w i t h t h e peripheral chromatin. It i s involved i n several biological activities including: the mitotic disassembly and reassembly of the nuclear envelope, determination of the size and shape of the nucleus, higher order chromatin organization, cell differentiation, and a p o p t o s i s . L a m i n s a r e t h e m a j o r p r o t e i n s o f t h e n u c l e a r l a m i n a . T h e y a r e type V intermediate filaments and, like all intermediate filaments, they form filamentous structures. Lamins can interact i n vitro with specific DNA sequences, with chromosomal proteins and with several proteins of the inner nuclear membrane, including otefin, LBR, LAP1 and LAP2. In this paper we show that Drosophila lamin Dm 0 and otefin proteins are required for the assembly o f the Drosophila nuclear envelope. We also demonstrate that the lack of lamin Dm 0 activity causes the dissociation of peripheral chromatin from the nuclear envelope, accumulation of annulate lamellae and lethality. In addition, we show that the carboxy (tail) domain of lamin Dm 0 c a n i n t e r a c t i n vitro with chromosomes and the central (rod) domain of lamin Dm 0 is essential and sufficient for t h e i n v i t r o a s s e m b l y o f l a m i n D m 0 into filamentous structures. These results are discussed in relationship to the biological roles of the nuclear lamina.
I. Introduction In eukaryotic cells, DNA replication and RNA processing occur in the nucleus, while protein synthesis occurs in the cytoplasm. These activities are physically separated by the nuclear envelope. The nuclear envelope is a complex structure composed of outer and inner lipid bilayer membranes. The two membranes are separated by a 20-40 nm perinuclear space and are connected at the nuclear pore complexes, which are passageways for transport of macromolecules between the nucleoplasm and the cytoplasm (reviewed in Davis, 1995; Gorlich and Mattaj, 1996). Underlying the inner nuclear membrane there is a proteinaceous meshwork of intermediate filaments termed the nuclear lamina (F i g . 1 A ; reviewed in Hutchison et al., 1994; Moir et al., 1995).
A. Proteins of the inner nuclear membrane and nuclear lamina
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Several components of the inner nuclear membrane and the lamina have been identified. These include the integral membrane proteins (IMPs): LBR (Worman et al., 1990), LAP1, LAP2 (Furukawa et al., 1995; Harris et al., 1994; Martin et al., 1995), p34 (Simos and Georgatos, 1994) and p18 (Simos et al., 1996), and the peripheral proteins: nuclear lamins (Fisher et al., 1986; McKeon, 1991), otefin (Harel et al., 1989; Padan et al., 1990) and YA (Lopez et al., 1994; Lopez and Wolfner, 1997). The existing experimental data suggests that lamins can interact with LBR, LAP1, LAP2, otefin and YA (Foisner and Gerace, 1993; Goldberg et al., 1997; Worman et al., 1988) . p18 and p34 are associated with LBR and p18 is distributed equally between the inner and the outer nuclear membranes (Simos and Georgatos, 1994). The data on the peripheral proteins indicates that otefin is closely associated with the inner nuclear membrane, lamin can associate with both the inner nuclear membrane and the peripheral chromatin, and YA is associated with the peripheral chromatin (F i g . 1 A ; Goldberg et al., 1997). These proteins are present in the insoluble NMPCL (nu-
Harel et al: Structural Organization and Biological Roles of the Nuclear Lamina
F i g u r e 1 . (A) Schematic view of the structural organization of nuclear envelope. OMN, outer nuclear membrane; INM, inner nuclear membrane; NPC, nuclear pore complex. (B ) Schematic view and the putative roles of different regions in lamin Dm 0 and otefin. The numbers are of amino acids positions in these proteins.
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Gene Therapy and Molecular Biology Vol 1, page 531 chromosomes at the same time that lamins begin to reassemble around them (Foisner and Gerace, 1993; Yang et al., 1997). The phosphorylation of LAP2 during mitosis inhibits its binding to both lamin B and chromosomes. (Foisner and Gerace, 1993). The mechanism for inner membrane targeting and retention of LAP2 probably involves lateral diffusion in the interconnected membranes of the endoplasmatic reticulum and nuclear envelope, and interaction with components of the nuclear lamina and chromatin (Furukawa et al., 1995). Y A (Young Arrest) is an essential Drosophila gene for the transition from meiosis to the initiation of the rapid mitotic divisions by early embryos (Judd and Young, 1973; Lin et al., 1991; Liu et al., 1995). The chromosome condensation state is abnormal in nuclei in YA-deficient eggs and embryos (Liu et al., 1995). The YA protein is present during the first two hours of zygotic development, where it is localized to the nuclear lamina (Lin et al., 1991). Ectopically expressed YA associates with polytene chromosomes in vivo (Lopez and Wolfner, 1997), and YA can associate with both chromosomes and lamin Dm0 (Goldberg et al., 1997; Lopez and Wolfner, 1997). Otefin is a 45 kDa peripheral nuclear envelope protein with no apparent homology to other known proteins (Padan et al., 1990). It includes a large hydrophilic domain, a single carboxy terminal hydrophobic sequence of 17 amino acids and a high content of serine and threonine residues (F i g . 1 B ). With the exception of sperm cells, otefin is present in the nuclear envelope of all cells examined during the different stages of Drosophila development. In eggs and young embryos, otefin is also associated with the maternal fraction of membrane vesicles (Ashery-Padan et al., 1997b). The COOH-terminal, 17-aa hydrophobic sequence of otefin is essential for the targeting of otefin to the nuclear periphery. Other sequences of otefin are required for its efficient targeting to the nuclear envelope and for further stabilizing otefin's interaction with the nuclear envelope (Ashery Padan et al., 1997a). Otefin is a phosphoprotein in vivo and a substrate for in vitro phosphorylation by cdc2 kinase and cAMPdependent protein kinase. Lamins are the major proteins of the nuclear envelope. They are classified as type V intermediate filaments and, like all intermediate filaments, they contain an ! helical rod domain flanked by amino (head) and carboxy (tail) domains (F i g . 1B). Unlike the cytoplasmic intermediate filaments that are 10 nm wide, lamins can make up to 200 nm thick fibers (Belmont et al., 1993; Paddy et al., 1990). The rod domain of lamins is 52 nm long and contains three ! helices, each composed of heptad repeats (reviewed in McKeon, 1987). These helices form coiled-coil interactions between lamin monomers. The lamin dimers associate longitudinally to form polar head-to-tail polymers. These polar head-to-tail polymers further associate laterally to form the 10 nm thick filaments (Heitlinger et al., 1991). The 10 nm filaments further associate to form the 50-200 thick
clear matrix-pore-complex-lamina) fraction, after salt and Triton X-100 extraction. LBR (lamin B receptor) was isolated by its ability to bind in a saturable and specific fashion to lamin B. Binding of lamin B to LBR is affected by its phosphorylation. LBR is a 58 kDa protein containing a nucleoplasmic amino-terminal domain of 204 amino acids followed by a hydrophobic domain with eight putative transmembrane segments (Worman et al., 1990). Its sequence shows high homology to the yeast sterol C-14 reductase (Gerace and Foisner, 1994). Both the first transmembrane domain (Smith and Blobel, 1993) and the amino-terminal domain of LBR (Soullam and Worman, 1993; Soullam and Worman, 1995) mediate the targeting of LBR to the inner nuclear membrane. The highly charged amino-terminal domain of LBR can also direct cytosolic proteins to the nucleus and type II integral membrane proteins to the inner nuclear membrane in transfected COS-7 cells (Smith and Blobel, 1993). LBR is phosphorylated in a cell cycle-dependent manner on serine residues in interphase and on serine and threonine residues in mitosis. Its phosphorylation is mediated by p34cdc2kinase and by an unidentified kinase that resides in the nuclear envelope and associates with LBR in vivo (Nikolakaki et al., 1997; Simos and Georgatos, 1992). LBR can interact with several proteins including p34 and p18 (Simos and Georgatos, 1994), lamin B (Worman et al., 1988) and with the human homologue of the Drosophila heterochromatin associated protein HP1 (Ye and Worman, 1996). LAP1A-C - (Lamina-associated polypeptides 1A-C) is a group of three related integral membrane proteins of the inner nuclear membrane that are recognized by monoclonal antibody RL13. LAP1 proteins can bind both type A and B lamins (Foisner and Gerace, 1993). Cloning of LAP1C revealed that it is a type II integral membrane protein with a single membrane-spanning region and a hydrophilic amino terminal domain that is exposed to the nucleoplasm (Martin et al., 1995). The different LAP1 isotypes are differentially expressed during development and appear to bind lamins with different affinities (Martin et al., 1995). LAP2 (Lamina-associated polypeptide 2 - also named thymopoietin) is a type II integral membrane protein of the inner nuclear membrane. The LAP2 gene is alternatively spliced to give rise to at least 5 different products (Theodor et al., 1997). The most abundant products: LAP2!, LAP2", and LAP2# (75 kDa, 51 kDa and 39 kDa, respectively) are present in most cell types. LAP2! is present diffusely throughout the nucleus, while LAP2" and LAP2# are confined to the inner nuclear membrane (Harris et al., 1995). LAP2" contains a large hydrophilic domain with several potential cdc2 kinase phosphorylation sites and a single putative membranespanning sequence close to its carboxy terminus. The amino-terminal domain of this protein is hydrophilic and is exposed to the nucleoplasm. LAP2 can bind directly to both lamin B and chromosomes and associates with
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Harel et al: Structural Organization and Biological Roles of the Nuclear Lamina nuclear lamina (this study). The head-to-tail binding sites are at the ends of the rod domain that are highly conserved among all intermediate filament proteins. Point mutants that cause defects in binding were mapped to these conserved regions (Stuurman et al., 1996; Zhao et al., 1996). Lamins are divided into types A and B. Type A lamins are mainly expressed in differentiated cells, have a neutral isoelectric point and are soluble during mitosis. Type B lamins are expressed constitutively in all somatic cells, have an acidic isoelectric point and remain associated with membrane vesicles during mitosis (reviewed in McKeon, 1991; Nigg, 1992). Different eukaryotes possess between one to six lamin genes. Mammalian lamins A and C are the result of alternative splicing of the same gene. Lamins B1-B3 and C2 are coded by separate genes (Alsheimer and Benavente, 1996). The two major lamins in chicken are lamins A and B2 (Peter et al., 1989). An additional minor species is termed lamin B1. Xenopus laevis has at least five different lamin genes (Stick, 1992; Stick, 1994). Drosophila melanogaster has two lamin genes, termed lamin Dm 0 and C (Bossie and Sanders, 1993; Gruenbaum et al., 1988). Caenorhabditis elegans probably has a single lamin gene, termed CeLam-1 (Riemer et al., 1993). Lamins undergo specific post translational modifications. All nuclear lamins except lamins C contain CaaX box at their carboxy terminus. The CaaX box undergoes proteolytic cleavage of the last three amino acids, farnesylation of the C-terminal cysteine, and carboxyl methylation. The isoprenylation is essential but not sufficient for the association of lamins with the nuclear envelope (Firmbach and Stick, 1995; FirmbachKraft and Stick, 1993; Hennekes and Nigg, 1994). Lamins are phosphorylated by several protein kinases in vivo and in vitro. These include: cdc2 kinase (Dessev et al., 1991; Heald and McKeon, 1990; Peter et al., 1990; Ward and Kirschner, 1990), Casein kinase II (Li and Roux, 1992), PKA (Lamb et al., 1991), "II PKC (Fields et al., 1988; Hennekes et al., 1993; Hocevar et al., 1993; Hocevar and Fields, 1991; Kasahara et al., 1991) and MAP kinase (Peter et al., 1992). The phosphorylation state of lamins is cell-cycle regulated (Ottaviano and Gerace, 1985). It is involved in lamin polymerization and disassembly, and in importing lamin molecules into the nucleus. The Drosophila lamin Dm0 undergoes post translational modifications to give rise to at least three distinct isoforms termed, Dm1, Dm 2 and Dm mit which differ in their phosphorylation pattern. Dm1 and Dm2 are present in most types of interphase nuclei as a random mixture of homo- and hetero-dimers (Smith et al., 1987; Stuurman et al., 1995). Dmmit is present in the maternal pool and in mitotic cells (Smith and Fisher, 1989). 3-D in vivo studies in Drosophila and in mammalian cells revealed that lamin fibers are closely associated with chromatin fibers (Belmont et al., 1993; Paddy et al., 1990). Studies in vitro have shown that lamins can specifically bind chromatin fragments and interphase chromatin (Hoger et al., 1991; Taniura et al., 1995;
Yuan et al., 1991), as well as condensed in vitro assembled chromatin (Ulitzur et al., 1992) or mitotic chromosomes (Glass et al., 1993; Glass and Gerace, 1990). Lamins can also bind to specific DNA sequences (Baricheva et al., 1996; Luderus et al., 1992; Luderus et al., 1994; Shoeman and Traub, 1990; Zhao et al., 1996) and to chromosomal proteins (Burke, 1990; Glass et al., 1993; Glass and Gerace, 1990; Hoger et al., 1991; Taniura et al., 1995; Yuan et al., 1991). Binding of lamins to chromatin is specific and depends on the integrity of the chromosomes. Lamin A binds in vitro to poly-nucleosomes with a dissociation constant of about 1x10-9 M (Yuan et al., 1991). A binding site for mammalian lamins A and B was localized at the tail domain (Taniura et al., 1995). In the latter study, the dissociation constant of the tail domain binding to interphase chromatin was estimated to be in the range of 3x10-7 M and the binding was mediated by histones. Since lamins form large polymers in vivo, the actual association between the lamin filament and chromatin may be stronger. A specific binding site to mitotic chromosomes was also found in the rod domain. However, the in vivo relevance of this binding is not yet clear since the rod domain binding occurred only under acidic, nonphysiological, conditions (Glass et al., 1993). Chicken lamin B and Drosophila lamin Dm 0 polymers also bind specifically to M/SARs fragments (Luderus et al., 1992; Luderus et al., 1994). These DNA sequences are several hundred base pairs long with several stretches of AT rich sequences and are likely to form an "open" form of chromatin. Indeed, the binding to these sequences could be competed to some extent with single strand DNA (Luderus et al., 1994). The binding of Drosophila lamin Dm 0 to M/SARs is mediated by the rod domain and requires its polymerization (Zhao et al., 1996). Lamin-DNA interactions can occur, for example, in the centromeric regions since the l20p1.4 Drosophila centromeric sequence has DNA composition similar to M/SAR and it binds specifically to polymers of Drosophila lamin Dm0 (Baricheva et al., 1996). Lamin polymers can also bind strongly to telomeric sequences (Shoeman and Traub, 1990).
B. Biological roles of the nuclear lamina Several functions have been ascribed to the nuclear lamina concerning nuclear organization and activity. These functions include: (i) regulating the size, shape and assembly of the nuclear envelope, (ii) a role in higher order chromatin organization by providing docking sites for chromatin , (iii) a role in DNA replication, (iv) a possible role in differentiation, as indicated by the change in lamina composition during development. In addition, the nuclear lamina is a major substrate for signals that control the cell cycle and lamins are specifically degraded in apoptosis (Nigg, 1992; Oberhammer et al., 1994). (i) Nuclear envelope disassembly.
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Gene Therapy and Molecular Biology Vol 1, page 533 During mitosis, the nuclear envelope breaks down in prophase and starts to reassemble at late anaphase. Nuclear lamins and lamina associated proteins are likely to play a role both in the assembly and disassembly of the nuclear envelope. Disassembly of the nuclear lamina is controlled by phosphorylation of sites outside the rod domain of lamins that prevents the head-to tail association of the lamin molecules. For example, mutations in Ser-22 and Ser-392 of human lamin A in transfected COS cells prevented phosphorylation at these sites and blocked the disassembly of the nuclear lamina during mitosis (Heald and McKeon, 1990). ( i i ) Nuclear envelope assembly depends on lamins and on lamin-associated proteins. Microinjection of lamin antibodies into cultured PtK2 cells resulted in daughter nuclei that remained arrested in a telophase-like configuration, and telophase-like chromatin that remained inactive (Benavente and Krohne, 1986). In mammalian cell-free extracts, antibodies directed against type A or B lamins blocked vesicles binding to chromatin, which is the first step of nuclear envelope assembly (Burke and Gerace, 1986). Similarly, anti-lamin Dm0 antibodies blocked the interaction between vesicles and chromatin in a Drosophila cell-free system that assembles nuclei from sperm chromatin (Ulitzur et al., 1992; Ulitzur et al., 1997). The role of lamin proteins in the association between nuclear vesicles and chromatin in Xenopus extracts has been the subject of debate; Depletion of lamin B3 from the assembly extract did not prevent the formation of nuclear envelopes consisting of membranes and nuclear pores. These lamin B3-depleted nuclei were small, fragile and failed to replicate their DNA (Jenkins et al., 1995; Meier et al., 1991; Newport et al., 1990). In contrast, Dabauvalle et al. (Dabauvalle et al., 1990) were able to block the formation of nuclear envelopes by using an antibody directed against both lamins B2 and B3. A major reason for the discrepancy between the above studies could be that Xenopus extracts contain lamins B2 and B1, in addition to lamin B3 (Lourim et al., 1996; Lourim and Krohne, 1993). In cell-free extracts of Xenopus eggs and Drosophila melanogaster it was shown that trypsinization of the membrane fraction abolished its ability to bind demembranated sperm chromatin and hence to support assembly of the nuclear envelope (Ulitzur et al., 1997; Wilson and Newport, 1988). Possible target proteins for the Trypsin treatment are IMPs. Indeed, several studies suggest a role for LBR, LAP1 and LAP2 in nuclear assembly. LAP2 associates with chromosomes at the same time as lamins, which suggests a role for LAP2 in initial events of nuclear envelope reassembly (Foisner and Gerace, 1993). A recent study (Yang et al., 1997) shows that LAP1 and LAP2 become completely dispersed throughout ER membranes during mitosis and proposes that the reassembly of the nuclear envelope at the end of mitosis involves sorting of IMPs to chromosome surfaces by binding interactions with lamins and chromatin. Pyrpasopoulou et al. (Pyrpasopoulou et al., 1996) analyzed the role of LBR in providing chromatin docking sites for nuclear vesicles by binding in vitro reconstituted 533
vesicles of nuclear envelopes to chromatin. The results of this study suggest that LBR is involved in providing chromatin anchorage site at the nuclear envelope. It was also suggested that the homologue of LBR in sea urchin targets membranes to chromatin and later anchors the membrane to the lamina (Collas et al., 1996). The essential role of otefin in the assembly of the nuclear envelope was recently demonstrated in a Drosophila cellfree system (Ashery-Padan et al., 1997b). The similar phenotype obtained when otefin or lamin Dm0 activities are inhibited (Ashery-Padan et al., 1997b) is probably due to the fact that otefin and lamin are part of the same protein complex in the vesicle fraction (Goldberg et al., 1997). In summary, the above data implies that the assembly of nuclear membranes following mitosis requires the function of protein complexes containing both peripheral and integral membrane proteins including: lamin, otefin, LAP2 and LBR. Lamin genes are not present in significant homology in the yeast Saccharomyces cerevisiae (Gruenbaum, Y., unpublished observations) and in the protozoon Amoeba proteus (Schmidt et al., 1995). In addition, the laminaassociated proteins LAP1, LAP2 and otefin are not present in significant homology in Saccharomyces cerevisiae (Gruenbaum, Y., unpublished observations), while LBR is the enzyme sterol C14 reductase (reviewed in Gerace and Foisner, 1994). One possible explanation for the appearance of lamins only in organisms with an open mitosis concerns their roles in nuclear envelope breakdown at the begining of mitosis and nuclear reassembly at the end of mitosis. These activities are not required in organisms with a closed mitosis. The involvement of the nuclear lamina in nuclear organization, development and DNA replication may have appeared later in evolution. (iii). Nuclear and chromatin organization. The nuclear lamina is a major component of the nuclear matrix. It was, therefore, suggested that a lamin filamentous meshwork is involved in nuclear and chromatin organization. An example for a direct involvement of a lamin protein in nuclear organization comes from an ectopic expression of the mouse spermspecific lamin B3 in cultured somatic cells. This ectopic expression resulted in transformation of the nuclear morphology from spherical to hook-shaped (Furukawa and Hotta, 1993). Also, depletion of soluble lamin B3 from Xenopus nuclear assembly extracts gave in vitro assembled nuclei that were small and fragile (Meier et al., 1991; Newport et al., 1990). Another evidence for the role of lamin in nuclear organization comes from the analysis of flies mutated in the Drosophila lamin Dm 0 gene. Flies homozygous for a strong mutation in the lamin Dm0 gene had an aberrant nuclear structure and died following 9-16 hours of development. The dissociation of chromatin from the nuclear membrane was one of the first phenotypes observed in these flies (Osman, 1992). A weak mutation in the lamin Dm0 gene (<20% of lamin expression) resulted in a retarded development, reduced viability,
Harel et al: Structural Organization and Biological Roles of the Nuclear Lamina sterility, and impaired locomotion. The nuclei in these mutant flies are enriched in nuclear pore complexes, in cytoplasmic annulate lamellae and contain defective nuclear envelopes (Lenz-Bohme et al., 1997). In vitro studies support the role of nuclear lamin in chromatin organization. As discussed above, the nuclear lamina interacts in vivo with chromatin, and lamin proteins can bind histones and specific DNA sequences. The Drosophila YA protein is needed to initiate embryonic cleavage divisions (Lopez et al., 1994). Ya is likely to be involved in mediating the association of chromosomes with the lamina (Goldberg et al., 1997), thus contributing to the organization of the nucleus in a developmental stage-specific manner (Lopez and Wolfner, 1997). Nuclei in YA-deficient eggs and embryos have abnormal chromosome condensation states (Liu et al., 1995), ectopically expressed YA associates with polytene chromosomes in vivo, and YA can associate with chromosomes in vitro (Lopez et al., 1994; Lopez and Wolfner, 1997). (iv). DNA replication requires nuclear lamins. Several reports demonstrated that, during interphase, lamin B molecules are present in foci in the nucleoplasm, in addition to their presence in the nuclear envelope. These foci coincide with sites of DNA replication (Goldman et al., 1992; Moir et al., 1994; Spann et al., 1997). In addition, nuclei assembled in Xenopus egg extracts that were depleted of lamin B3 were unable to initiate DNA replication. These lamin B3-depleted nuclei had continuous nuclear envelopes and nuclear pores and were able to import proteins required for DNA synthesis such as PCNA, MCM3, ORC2 and DNA polymerase ! (Goldberg et al., 1995; Meier et al., 1991; Newport et al., 1990; Spann et al., 1997). Addition of purified lamin B3 to the depleted extracts could rescue lamina assembly and DNA replication. Microinjection of a truncated human lamin, that was utilized as a dominant negative mutant to perturb lamin organization in mammalian cells, caused a dramatic reduction in DNA replication (Spann et al., 1997). Nuclear lamins are likely to be required for the elongation phase of DNA replication since the distribution of MCM3, ORC2, and DNA polymerase ! that are required for the initiation stage of DNA replication was not affected by the depletion of lamin B3 activity (Spann et al., 1997).
II. Results and discussion A. Mutations in the Drosophila lamin Dm0 gene reveal that it is an essential gene that is required for nuclear organization. During egg chamber development, large amounts of lamin Dm0 are secreted by the nurse cells into the developing Drosophila oocyte (Ashery-Padan et al., 1997b; Smith and Fisher, 1989; Ulitzur et al., 1992). The 534
amounts of lamin Dm0 RNA and protein that are maternally stored in the oocyte are sufficient for the assembly of many thousands of nuclei. In addition, lamin Dm0 is a very stable protein with an estimated half life of about 24 hr (Dr. Paul A. Fisher, personal communication). Therefore, flies mutated in their lamin Dm0 gene are expected to show a phenotype only following the consumption of the large maternal pool of lamin Dm 0. Drosophila melanogaster (canton S) males were mutagenized with ethyl methane sulphanate (ems) and offspring flies mutated in their second chromosome were crossed with flies containing the deletion Df(2L) gdh-A (Knipple et al., 1991). This deletion is between 25D726A7 bands and contains the 25F1 locus of lamin Dm 0 (Gruenbaum et al., 1988). One of the complementation groups was specific for a mutation in lamin Dm0 since it could be specifically rescued by a P-element mediated transformation with a CaspeR vector (Pirrotta, 1988) containing 1.2 kb upstream sequences of lamin Dm0 and either the complete genomic lamin Dm0 gene (EcoRIEcoRI fragment; Osman et al., 1990) or the two first exons and part of the third exon of the genomic lamin gene (EcoRI-HindIII fragment; Osman et al., 1990) ligated to the HindIII-EcoRI fragment of lamin Dm0 cDNA (Gruenbaum et al., 1988). A second mutagenesis screen utilized a P-element targeted gene mutation, using the Birm-2/Birm-2; ry/ry line (Ballinger and Benzer, 1989; Kaiser and Goodwin, 1990). Candidate lines for a mutation in lamin Dm 0 were crossed with an ems-mutated line in lamin Dm 0 (Osman, 1992). One of the isolated mutations, termed PM-15, was analyzed in more details. PM-15/PM15 or PM-15/Df(2L)gdh-A flies showed an abnormal chromatin organization following 9-16 hr of development. The variability in the time of phenotype appearance is likely to be due to differences in the amounts of the maternal pool of lamin Dm0. Flies homozygous or transheterozygous for these mutations eventually die. Therefore, lamin Dm0 is an essential gene of the fruit fly (Osman, 1992). One of the first phenotypes in embryos homozygous for a mutation in lamin Dm0 gene is the detachment of the peripheral chromatin from the nuclear envelope. This detachment occurs in many regions of affected nuclei and is followed by condensation of chromatin (F i g . 2 . compare panels C , D to panels A,B). The later phenotypes of these embryos include nuclei aggregation and formation of cytoplasmic annulate lamellae (F i g . 2 E ). A Drosophila line mutated in its lamin Dm 0 gene (Lenz-Bohme et al., 1997), in which the amounts of lamin Dm 0 protein are reduced to less than 20% of their normal levels, also revealed enrichment in annulate lamellae and in nuclear envelope clusters. These
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F i g u r e 2 . A mutation in lamin Dm 0 gene results in dissociation of chromatin from the nuclear envelope and accumulation of annulate lamellae (Osman et al., 1990). Embryos mutated in lamin Dm0 showed a visible phenotype following 9-16 hr of development. Electron microscope analysis of PM15/PM-15 cells (C , D ) revealed chromatin dissociation from the nuclear envelope as compared to normal cells (A , B ). (E) Annulate lamellae in PM-15/Df(2L)gdh-A embryos following degradation of nuclei. The bars in panels A,C,E represent 1 Âľm. The bars in panels B,D represent 5 Âľm.
flies showed reduced viability, retardation in their development, sterility, and impaired locomotion. In some cells, defective nuclear envelopes were also observed (LenzBohme et al., 1997). In summary, these studies demonstrate the essential role of lamins in nuclear and chromatin organization.
B. Filament assembly properties of lamin Dm0 and derivative proteins. The assembly properties of lamin Dm0 were investigated in vitro using bacterially expressed and purifies lamin Dm0 and derivatives (Ulitzur et al., 1992). To test for the ability of filamentous protein to polymerize we used the sedimentation test (Heitlinger et al, 1991), which is based on the separation of pelletable polymers from soluble protein, following incubation under various chemicals and pH conditions. Reduction of salt concentration from 0.5 M NaCl to 50-150 mM NaCl in
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pH range of 5-9 was sufficient to induce 35-95% polymerization of lamin Dm0 protein. Electron microscope analysis of negative stained pellets confirmed the formation of filamentous structures (F i g . 3). The observed paracrystals were characterized by a distinct stainexcluding pattern with 25 nm axial repeat unit, which is half the size of the lamin rod domain (F i g . 3 A,B). Figure 3C shows a relatively rare case which reveals that these paracrystals are composed of separate lamin filaments. These filaments are 8-10 nm wide, which is the normal size of cytoplasmic intermediate filaments. Although there is no evidence for the existence of paracrystals in vivo, it is noteworthy that the width of these paracrystals fits is in the size range of lamin fibers that were visualized in Drosophila cells in vivo (Paddy et al., 1990). The ability of the isolated rod domain of lamin Dm0 (amino acids 55-413) to polymerize was analyzed utilizing bacterially expressed protein that was purified to near ho-
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Figure 3. Supramolecular structures formed by lamin Dm0 at low ionic strength. Lamin Dm0 protein at 2 mg/ml in buffer H (30 mM Tris-HCl pH 7.5, 1 mM DTT) containing 0.5 M NaCl was diluted 5 times in buffer H and incubated for 80 min on ice. Samples were placed on electron microscope grid and negatively stained with 1% uranyl acetate. Tightly packed paracrystals exhibit ~25 nm axial repeat unit and their thickness ranged between 40-200 nm (A , B ). The thick paracrystalline arrays are composed of a large number of thin filaments (C).
mogeneity. Unlike the complete lamin Dm0 molecule, polymerization of the isolated rod domain was saltindependent. However, under acidic conditions (pH 5.5) and in the presence of 25 mM CaCl2, the isolated rod domain was organized in higher order structures, as judged by the sedimentation test (F i g . 4 A ) and by electron microscope analysis (F i g . 4B). The filamentous structure of the polymerized rod domain resembled that of the complete lamin protein, but lacked the 25 nm repeat unit. Under neutral and basic pH conditions the rod domain was organized into dimers which were 52 nm long and about 0.5 nm in diameter (not shown). In summary, these results demonstrate that the rod domain contains enough information to form the lamin filaments and that sequences outside the rod domain are required for the proper organization of the lamin filaments and for their assembly under physiological conditions.
C. Interaction between lamin Dm0 and chromatin. 536
Our previous analysis demonstrated that lamin Dm0 can interact specifically with sperm chromatin (Ulitzur et al., 1992). These experiments also showed that the addition of bacterially expressed lamin Dm0 to Drosophila embryonic extracts that can assemble nuclei from sperm chromatin resulted in increased amounts of lamin Dm0 around the peripheral chromatin (Ulitzur et al., 1992). The mitotic chromosome assay that measures the association between lamin and mitotic CHO chromosomes (Glass et al., 1993; Glass and Gerace, 1990) was used to analyze domains in lamin Dm0 protein that are capable of interaction with chromatin. When lamin Dm0 protein was incubated for 30-60 min at 22oC with isolated mitotic chromosomes, in the presence of excess amounts of either 5% BSA or 10% FCS, a strong lamin staining was observed following immunofluorescence analysis with anti-lamin antibodies. The staining was mostly peripheral to the chromosomes and included aggregates of lamin (not shown). These aggregations are probably due to the organization of lamin Dm0 into polymers since the
Gene Therapy and Molecular Biology Vol 1, page 537 aggregates were absent when mitotic chromosomes were incubated with lamin Dm0 containing the mutation R64>H (Zhao et al., 1996), which impairs the ability of lamin Dm 0 to form filaments (F i g . 5B). The tail domain of lamin Dm 0 (amino acids 425-622) contains specific binding site(s) to chromatin since it bound specifically to the mitotic chromosomes (F i g . 5 A ). The R64>H mutant protein was incubated with mitotic chromosomes in the presence of hundred fold molar excess of the isolated tail domain in order to find other possible domains in lamin that bind chromatin. As shown in F i g . 5C, staining with affinity purified polyclonal antibodies against the rod domain of lamin Dm 0 gave intensity levels that were close to background levels. In conclusion, lamin Dm0 binds specifically to chromatin and its binding site(s) are localized to its tail domain. The specific lamin sequence that binds to chromatin, the affinity of its binding and the target chromosomal proteins are currently under investigation.
F i g u r e 5 . Binding of lamin Dm0 to chromosomes. Lamin Dm0 protein mutated in Arginine 64 (R64>H) (Zhao et al., 1996), which is impaired in its ability to form head-to-tail polymers (Stuurman et al., 1996; Zhao et al., 1996), bound specifically to mitotic chromosomes (B). The tail domain of lamin Dm0 (amino acids 425-622) also bound specifically to mitotic chromosomes (A). The tail domain of lamin Dm 0 could compete for the binding of the complete lamin Dm0 molecule to mitotic chromosomes since addition of a hundred fold molar excess of the tail domain could efficiently compete for the binding of R64>H (C). DAPI staining of DNA, left panels; antibody staining, right panels. Affinity purified polyclonal antibodies against the rod domain of lamin Dm0 B,C; monoclonal antibody 611A3A6 anti-lamin Dm0 , A. This monoclonal antibody recognize an epitope in the tail domain. The bar represents 6Âľm and applies to all panels.
F i g u r e 4 . Polymerization properties of the isolated rod domain of lamin Dm 0 . (A) Isolated rod domain protein (2 mg/ml) in buffer H was diluted 5 folds in buffer H or in 50 mM sodium citrate pH 5.5, 25 mM CaCl 2 . Pellet (p) and supernatant (s) were separated by 30 min centrifugation at 15,000xg, boiled in sample loading buffer and subjected to SDS-10% PAGE stained with Comassie Brillant blue. The position of the size markers are shown on the left of the panel. The rod domain polymerized at pH 5.5 but not at pH 7.5. (B) The pellet fraction was placed on electron microscope grid and negatively stained with 0.75% uranyl acetate.
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F i g u r e 6 . Inhibition of lamin Dm 0 and otefin activity prevents the in vitro nuclear envelope assembly in Drosophila embryonic extracts. Twenty microliters of embryonic extracts were preincubated for 90 min with either 100 µg polyclonal antilamin Dm 0 antibodies (C), 100 µg polyclonal anti-otefin antibodies (D), or 100 µg of preimmune serum antibodies (IgG fraction), (A , B ). Sperm chromatin was added and the incubation proceeded for additional 90 min. Samples from the two experimental systems were viewed by standard transmission electron microscope. Decondensed chromatin was enveloped with nuclear membranes in preimmune antibodies-treated extracts (A,B) but not in anti-lamin Dm 0 (C) or anti-otefin (D) antibodies-treated extracts. The bar represents 1 µm.
D. Lamin and otefin are essential for nuclear envelope formation To analyze lamin Dm0 and otefin function in nuclear envelope formation, 0-6 hr old Drosophila embryo extracts, in which interphase-like nuclei can be assembled from sperm chromatin (Berrios and Avilion, 1990; Crevel and Cotterill,
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1991; Ulitzur and Gruenbaum, 1989), were incubated with either 100-300 µg polyclonal anti- Drosophila lamin Dm0 or anti-Drosophila otefin antibodies (IgG fractions). Incubation of the extract under the same conditions with 100-300 µg of preimmune rabbit sera (IgG fraction) or with normal rabbit IgG served as controls. No membrane assembly was observed when lamin Dm0 or otefin activities were inhibited
Gene Therapy and Molecular Biology Vol 1, page 539 (Ashery Padan et al., 1997; Ulitzur et al., 1992; Ulitzur et al., 1997). Electron microscope (F i g . 6), light, and fluorescent microscope analyses (not shown) revealed that while chromatin went through the characteristic decondensation process, membrane vesicles did not attach to its surface, and nuclear envelope did not assemble around it (F i g . 6 C , D ). Incubation of the extract with preimmune sera, (IgG fraction), or with commercially available normal rabbit IgG fraction had no effect on nuclear assembly, the presence of membranes around the chromatin was observed (F i g . 6 A , B ). Addition of 2 Âľg of interphase lamin isolated from Drosophila embryos to extracts that were preincubated with the anti-lamin Dm0 antibodies restored binding of vesicles to chromatin (Ulitzur et al., 1997).
Acknowledgment The isolation of Drosophila lines with mutation in lamin Dm0 gene and the analysis of these lines was performed in collaboration with Dr. R. Falk and was part of the Ph.D. thesis of Dr. Midhat Osman. This study was supported by the US-Israel Binational Fund (BSF), and by the Israel Academy of Sciences.
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Gene Therapy and Molecular Biology Vol 1, page 543 Gene Ther Mol Biol Vol 1, 543-549. March, 1998.
Analysis of mutant p53 for MAR-DNA binding: determining the dominant-oncogenic function of mutant p53 Katrin Will and Wolfgang Deppert Heinrich-Pette-Institut f체r Experimentelle Virologie und Immunologie an der Universit채t Hamburg, Martinistr. 52, D-20251 Hamburg, Germany. __________________________________________________________________________________________________ Correspondence to: Wolfgang Deppert, Tel: +49-40-48051261, Fax: +49-40-48051117, E-mail: deppert@hpi.uni.hamburg.de
Summary At least some mutant p53 proteins not s i m p l y have l o s t the wild-type p53 specific tumor suppressor function, but exhibit oncogenic functions on their o w n . Recently we showed that binding of mutant p53 to MAR/SAR elements is an activity specific for mutant p53 and clearly distinguishable from the previously reported DNA-binding activities o f p 5 3 . Since MAR/SAR elements are considered to be important regulatory elements for a variety of nuclear processes, the interaction of mutant p53 with MAR/SAR elements might form the molecular basis for oncogenic potential of mutant p53. By employing different binding assays (the target-bound DNA binding assay, the South-western blotting technique and an adapted liquid phase binding assay), we studied MAR/SAR binding of various p53 proteins to different MAR/SAR elements. Murine mutant p53 bound different MAR/SAR elements with an approximately 1,000-fold higher affinity than murine wild-type p 5 3 . Analysis o f MAR/SAR binding o f human wild-type and mutant p53 proteins revealed also high affinity MAR/SAR binding o f several human p53 mutant proteins (175 Arg H i s , 2 7 3 A r g P r o ) , b u t n o t o f h u m a n w i l d - t y p e p 5 3 , c o n f i r m i n g t h a t M A R b i n d i n g i s a general property of mutant p53. By antibody interference analysis using a panel of different p53specific monoclonal antibodies and by deletion mutant analysis the MAR/SAR binding domain on mutant p53 was mapped, revealing a bipartite domain consisting of the mutated core region and the C-terminal 60 amino acids. tumor suppressor functions of p53, and the expression of mutant p53 often is considered being equivalent to a p53 "null" situation.
I. Introduction Mutations in the p53 gene constitute the most frequent alteration in a single gene in human cancer (Soussi et al., 1994). Wild-type (wt) p53 is a tumor suppressor, whose main function is to preserve the integrity of the genome as a cell cycle checkpoint protein. Thereby p53 not only mediates DNA damage response, growth arrest or apoptosis by modulating cellular transcription, it also exhibits a variety of other biochemical activities, which are directly related to its function as major control element in preserving the integrity of the cells' genetic information. 90% of all mutations in the p53 gene are single missense point mutations. These mutations are localized mostly in the p53 core domain, which mediates most of the biochemical activities of wild-type p53. Consequently, these mutations serve to inactivate the
However, considering the many different activities exerted by wild-type (wt) p53, it is quite astounding that a single point mutation in the p53 molecule should totally eliminate p53 function. Furthermore, point mutations are a rather unique way for inactivating a tumor suppressor. All of the other known tumor suppressors are inactivated mostly by loss of functional gene expression, resulting either from gene truncations or deletions, or promoter inactivation ( Soussi et al., 1994). This, and the fact that there is a strong selection for the maintenance of mutant p53 expression, provoked the idea that mutant p53 not simply is an inactivated tumor suppressor, but exerts oncogenic functions on its own (Deppert et al., 1990, Dittmer et al., 1993, Michalowitz et al., 1991, Levine et 543
Gene Therapy and Molecular Biology Vol 1, page 544 To find a specific interaction of mutant p53 with DNA which differs from that of wt p53, our laboratory has analyzed in detail the DNA binding properties of murine wt and mutant p53. Using !DNA as a model substrate for a DNA which, due to its length and complexity, contains abundant sequence elements for sequence specific interactions, as well as structural elements for more complex interactions of a protein with DNA, we were able to demonstrate that highly purified mutant p53 from MethA cells, a methylcholanthrene induced mouse tumor cell line (DeLeo et al., 1977), binds to the 1,215 bp AluI fragment of !DNA using a target-bound DNA-binding assay (Figure 1).
al., 1995). This view very well corresponds to the initial characterization of p53 as an oncogene, when all of the available p53 cDNAs directed the expression of mutant p53 proteins. The notion that mutant p53 proteins can exhibit endogenous dominant- oncogenic functions of their own (Deppert et al., 1990, Dittmer et al., 1993, Michalowitz et al., 1991, Levine et al., 1995, Zambetti et al., 1993) is strongly supported by a variety of experimental observations, perhaps most remarkably by the findings that mutant p53 not only leads to full transformation of the weakly Abelson murine leukemia virus transformed L12 cells (Shaulsky et al., 1991), but also increases the metastatic capacity of cells of a p53-deficient murine bladder carcinoma cell line (Pohl et al., 1988). As another example, expression of mutant p53 in p53-negative human SAOS-2 or murine BALB/c (10/3) cells resulted in increased proliferation rates and higher tumorigenicity of these cells (Dittmer et al., 1993).
Computer analysis of this fragment then showed that it had both sequence and structure similarities with nuclear matrix attachment region/scaffold attachment regions (MAR/SAR elements). Both mutant and wild-type p53 previously were shown to interact with nuclear substructures within the nucleus, the chromatin and the nuclear matrix, with mutant p53 binding even more strongly to the nuclear matrix than wt p53. Therefore the binding of mutant p53 to a DNA fragment with homology to MAR/SAR elements raised the possibility that mutant p53 indeed might interact with such DNA, and prompted us to analyze this interactions in more detail using the target-bound DNA-binding assay specifically developed to detect this interaction.
The molecular basis for this gain of function of mutant p53 is still elusive. Mutant p53 has retained some of the biochemical activities of the wt p53 protein, like nonsequence-specific RNA or DNA binding and specific binding to RNA with extensive secondary structures (Mosner et al., 1995; Steinmeyer et al., 1988). Furthermore, mutant p53 binds to various cellular proteins which may lead to deregulation of cellular functions. Alterations in gene expression by mutant p53, e.g. upregulation of the mdr1 gene (Strauss et al., 1995), have been consistently reported; however, upregulation of a particular gene by a transfected mutant p53 was observed in one type of cell but was totally absent in another one (Deppert et al., 1996).
Its main features are that p53 is doubly immunopurified: p53 is immunoprecipitated with the p53specific monoclonal antibody PAb122 and protein ASepharose (PAS), then eluted by PAb122 epitope-specific peptide (Steinmeyer et al., 1988) and reprecipitated with another p53-specific monoclonal antibody, recognizing an N-terminal epitope of p53 (PAb248) and PAS. This second immune complex then is incubated with DNA under saturating conditions for both specific and competitor DNA, followed by rigorous washing to remove non-bound or non-specifically bound DNA. Specifically bound DNA and p53 are eluted separately and analysed by SDS-PAGE (WeiĂ&#x;ker et al., 1992).
This clearly indicates that stimulation of gene expression by mutant p53 must be due to a different mechanism than the wt p53 specific transactivator function, especially since most mutant p53 proteins have lost this ability, due to loss of sequence-specific DNA binding (Deppert et al., 1994). Interestingly, however, additional mutations in the transactivator domain of mutant p53 also abolish the ability of mutant p53 to upregulate the expression of certain reporter genes (Li et al., 1995). The most likely interpretation of these results thus seemed that mutant p53 still is able to interact with the cellular transcription machinery, but interacts with DNA in a different way than wt p53. Nevertheless this interaction must be specific, as there obviously is only a limited number of genes which are regulated by mutant p53.
These analyses revealed that binding of mutant p53 to the 1,215 bp AluI DNA fragment is a complex process, involving recognition of both structural and sequence determinants, as the fragment could not be narrowed down to any small consensus oligonucleotide. Binding was of high affinity (KD 10 -10 M), as shown by Scatchard analysis (Figure 1, WeiĂ&#x;ker et al., 1992). Further studies provided evidence that this type of complex DNA binding indeed reflected the affinity of MethA mutant p53 to MAR-DNA elements, as it could be extended to several bona fide MAR-DNA elements.
II. DNA binding properties of murine wt and mutant p53.
Despite the usefulness of the target-bound DNA binding assay for quantitatively assessing MAR-DNA
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Will and Deppert: Interaction of mutant p53 with MAR DNA
Figure 1 Scatchard analysis of the mutant p53 MAR-DNA binding using the target-bound DNA binding assay. (A) Equal amounts of the doubly immunopurified MethA p53 (1µg) bound to PAb248 were incubated in the target-bound DNA-binding assay with increasing amounts of pA1215 restricted with HindIII, BglI and EcoRI. Lane a, 0.05 µg, lane b, 0.1 µg, lane c, 0.2 µg, lane d, 0.4 µg, lane e, 0.6 µg, lane f, 0.8 µg, lane g, 1 µg, lane h, 2 µg, lane i, 4 µg, lane j, 8 µg. Lanes M, marker DNA (1215-bp fragment, 1369-bp EcoRI-BglI pUC18 fragment, 1118-bp BglI-BglI pUC18 fragment): M1 , 10 ng, M 2 , 50 ng, M 3 , 100 ng. DNA is marked with an arrow. (B) Binding curve of the Scatchard analysis of A. (C) Linear Scatchard plot of B (from Weißker et al., 1992)
binding by mutant p53, further comparative analyses of wt and mutant p53 MAR-DNA binding required the development of another assay system, as the target-bound DNA-binding assay had intrinsic limitations. For instance, this assay did not allow appropriate competition experiments, as the amount of competitor DNA required would be out of any experimentally feasible range. Thus it was difficult to discriminate in MAR-DNA binding by wt and mutant p53 between binding activities reflecting nonspecific DNA binding by these proteins, and their specific MAR-DNA binding properties. Furthermore, the targetbound DNA binding assay also did not allow a direct comparative analysis of the MAR-DNA binding activity of deletion fragments of mutant p53 due to the necessity of binding the p53 proteins to a monoclonal antibody, and, last not least, sterical interference of the affinity column material during the binding reaction could not be excluded. Therefore, we adapted an alternative binding assay, the South-western blotting technique, for the analysis of MAR-DNA binding by wt and mutant p53. After separation of the p53 proteins by SDS polyacrylamide gel electrophoresis, the proteins were transferred onto a nitrocellulose membrane, and renatured on this membrane. The membrane then was incubated with excess radioactively labeled MAR-DNA in the presence of unlabeled non-specific competitor DNA, washed
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extensively, and the DNA bound by p53 was visualized by autoradiography. These analyses, using the XbaI MAR/SAR fragment of the murine immunoglobulin heavy chain gene enhance locus (Cockerill et al., 1987), revealed that this binding was specific for mutant p53. The affinity of MethA p53 to MAR-DNA was approximately 1,000-fold higher than that of wt p53 (Müller et al., 1996). By antibody interference analysis using a panel of different p53-specific monoclonal antibodies (Figure 2) and deletion mutant binding studies (Figure 3), we mapped the MAR/SAR binding region on mutant p53 to a bipartite domain consisting of the mutated core region and the C-terminal 60 amino acids (Müller et al., 1996). Thus both the non-sequence specific DNA binding domain localized on the C-terminus of p53, as well as the core domain of p53 mediate MAR/SAR binding synergistically (Müller et al., 1996), thereby clearly discriminating this activity from sequence-specific DNAbinding by wt p53 (mediated by the core domain), and from non-sequence specific DNA binding of both wt and mutant p53 (mediated by the C-terminus). An important question regarding the relevance of the MAR-DNA binding observed with murine MethA mutant p53 was, whether this interaction would be limited to this special mutant p53 or exhibited also by other mutant p53
Gene Therapy and Molecular Biology Vol 1, page 546
F i g u r e 2 . Mapping of the MAR/SAR binding domain of mutant p53 by antibody-interference using South-western blotting. (A) Schematic representation of the epitopes of various anti-p53 monoclonal antibodies on the p53 molecule. (B) The influence of the anti-p53 monoclonal antibodies indicated below each panel upon binding of the IgE-MAR element by wild-type and mutant p53 was monitored in Southwestern analyses. 2µg of purified wild-type and MethA mutant p53, respectively, were analysed in the presence of a 3 x 104 fold excess of calf thymus genomic DNA. Antibodyincubation was performed prior to DNA-binding (From Muller et al, 1996).
proteins. Various murine mutant p53 proteins were selected, isolated from different cellular and recombinant sources and subsequently subjected to South-western binding analysis using the IgE-MAR element. In accordance with our earlier findings, all murine mutant p53 proteins in repeated experiments clearly showed high affinity binding to the IgE-MAR also in the presence of a
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Figure 3. Mapping of the MAR/SAR binding domain of mutant p53 by deletion analysis using South-western blotting. (A) Schematic representation of the wild-type and MethA mutant p53 deletion-molecules, constructed according to the tripartite structure of the p53 molecule. (B) 1µg of the purified wild-type and MethA mutant p53 deletion fragments were subjected to SDS-PAGE and stained with Coommasie blue. (C) South-western analysis of the binding of 2 µg of each wild-type and MethA mutant p53 deletion fragment to the IgE MAR element in the presence of a 3 x 10 4 fold molar excess of non-labeled calf thymus genomic DNA. (from Müller et al., 1996).
Gene Therapy and Molecular Biology Vol 1, page 547 F i g u r e 4 . Analysis of MARDNA binding using the liquid phase binding assay. Mutant p53 protein 175 (aa175 Arg"His) binds to the IgE-MAR element with higher affinity than wild-type p53. Equal amounts of wild-type p53 and mutant 175 p53 were added to binding buffer (SWB-buffer) including the radioactively labeled IgE-MAR-DNA fragments and increasing amounts of unlabeled competitor DNA and incubated for 30 min at room temperature. Subsequently, after incubation of the mixture with antibody PAb1018 and protein A-Sepharose (PAS) the proteine-DNA complexes were washed and the bound IgEMAR-DNA eluted. The eluates were lyophilized, dissolved and subjected to DNA-SDS-PAGE and visualised by autoradiography (manuscript in preparation).
refolding the human p53 proteins accounted for our difficulties to unequivocally demonstrate MAR-DNA binding for human mutant p53 proteins.
high excess of non-specific competitor DNA, whereas murine wt p53 failed to bind to this MAR element under such conditions.
A disturbing result was obtained when we subjected different human p53 proteins to MAR-DNA binding analysis in South-western experiments. In contrast to murine mutant p53, which reproducibly bound to MARDNA in repeated experiments, we obtained quite varying results when human mutant p53 proteins were used, ranging from weak to no binding at all. Rather than assuming that MAR-DNA binding is a property specific for murine mutant p53, we considered the possibility that the apparent lack of a reproducible MAR-DNA binding by human mutant p53 reflected technical problems related to structural differences between human and murine p53.
This forced us to develop an assay which did not require renaturation procedures. The liquid-phase binding assay fulfilled this criterion. In this assay, the desired MAR-DNA fragments were isolated and end-labeled using T4 polynucleotide kinase and # (-32-P) ATP by standard procedures and subjected to the binding assays including mutant p53 and unlabeled competitor DNAs. To avoid interference of the column material, p53 and the DNA were first incubated alone, and an N-terminal p53 specific monoclonal antibody (PAb248 for murine p53 and PAb1801 for human p53) and PAS were added later. Finally, the DNA-Protein-PAS complexes were washed and the bound DNA quantitatively eluted. The eluates were lyophilized, resuspended in sample buffer and separated by gel electrophoresis.
Although human and murine p53 share extensive homologies, there are sequence and conformational differences between these proteins, already reflected by the fact that there are species-specific monoclonal antibodies for human and murine p53. The most important step in the South-western binding assay is a renaturation step, which is very critical for reconstructing the capability for DNA binding. Therefore, we suspected that problems in
Application of this assay first for murine mutant p53 proteins in accordance with our earlier findings in repeated experiments clearly showed high affinity binding to the IgE-MAR also when high excess of non-specific DNA was added. Murine wt p53 again failed to bind to this MAR element under these conditions. When this assay then was applied to human wt and mutant p53, we in repeated experiments observed high affinity MAR-DNA binding
III. Studies with human p53 proteins
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Gene Therapy and Molecular Biology Vol 1, page 548
5. The DNA eluates were lyophilized and dissolved in 50 µl of gel loading buffer (water, 10% glycerol, bromophenol blue). Samples of 5 µl were subjected to SDS-PAGE and visualised by silver staining. Marker proteins of known concentration, electrophoresed on the same gel, served as standards.
also of human mutant p53 proteins (F i g u r e 4 , 175 Arg"His, not shown 273 Arg"Pro), but not of human wt p53, thereby confirming the assumption that MARDNA binding is a general property of mutant p53.
IV. Conclusions B. South-western DNA binding assay
Many questions remain to be resolved before MARDNA binding of mutant p53 can be related to its oncogenic activities, and before the molecular consequences of such interactions are understood within tumor cells. Most importantly, we must identify the structural features within MAR-DNA which mediate the specific interaction of mutant p53 with these DNA elements. Although our understanding of the oncogenic effects of mutant p53 is still at the beginning, the exciting possibility emerges that by interfering with MAR-DNA binding of mutant p53 it might be possible to abrogate its oncogenic functions in the tumor cell. Considering the strong selection for the maintenance of mutant p53 expression in tumor cells, one can hope that elimination of mutant p53 function in tumor cells is detrimental to tumor cell growth and will lead to its destruction.
1. Purified protein was subjected to SDS-PAGE and electrophoretically transferred to a nitrocellulose membrane soaked in transfer buffer (20mM Tris-acetate pH 8.3, 0.1% SDS, 20% 2-propanol) at 60V for 2 h. 2. Proteins were fixed on the filters with 50% 2propanol. 3. Filters were washed with demineralized water and incubated 2 times for 30 min in renaturation buffer I (50mM NaCl, 10mM Tris HCl pH 7, 2mM EDTA, 0.1mM DTT, 4M urea, 1% TritonX100) and renaturation buffer II (as renaturation buffer I, without TritonX 100), respectively. 4. A 30 min incubation at 30°C in renaturation buffer III (50mM NaCl, 10mM Tris HCl pH 7.0, 1mM EDTA, 6mM MgCl2 , 0.02% BSA, 0.02% Ficoll, 0.02% polyvinylpyrolidone, 1 µg/ml DnaK, 0.5mM DTT, 1mM ATP) strongly enhanced renaturation, but was optional for MAR/SAR binding.
V. Protocols A . Target-bound p 5 3 elements
binding
to
MAR/SAR
5. After renaturation the membranes were equilibrated and saturated in DNA binding buffer with genomic calf thymus DNA and bovine serum albumin (SWB: 50mM NaCl, 10mM Tris-HCl pH 7.0, 1mM EDTA, 6mM MgCl2 , 0.02% BSA, 0.02% Ficoll, 0.02% polyvinylpyrolidone, 100 µg/ml, calf thymus DNA with an average fragment length of 10 3 to 10 4 bp (Sigma)). Filters were incubated in a total volume of 5 ml binding buffer with 5x 106 cpm of the specific DNA probe (MAR/SAR DNA elements), which was radioactively labeled by primer extension.
1. Immunoprecipitation of p53 (approximately 1 µg total) from extracts of MethA cells, or from High five insect cells infected with recombinant baculoviruses expressing the respective mutant p53 protein using PAb122.
2. Elution of p53 from the immune complex with a 100-fold molar excess of a PAb122 epitope-specific peptide, followed by reprecipitation of p53 with an antibody recognizing a different epitope on p53. 3. Target-bound DNA binding assay of the doubly immunopurified p53: The immune complexes were washed with binding buffer (10 mM MOPS, pH 7, 150 mM NaCl, 1 mM DTT, 0.5 mM MgCl 2 ) and incubated with 8 µg of the respective DNA (restricted plasmid DNA containing the respective DNA fragment) in a total volume of 200 µl of binding buffer for 1 hr at 4°C. Immune complexes were washed three times with high-salt buffer ( 10 mM TrisHCl, pH 7.8, 10 mM NaCl) to separate bound and free DNA.
6. After 4 h the membrane was washed 3 times with DNA binding buffer and subjected to autoradiography. C . L iquid phas e binding as s ay
1.p53 was isolated from extracts of MethA cells, from High five insect cells infected with recombinant baculoviruses or from bacteria expressing the respective wild-type or mutant p53 protein using antibody PAb248 columns. PAS-antibody-p53 -complexes were washed with buffer A (30 mM KPi, pH 8.0, 50 mM KCl, 1 mM EDTA, 2 mM DTT) and eluted with buffer A including 1 M KCl. and subsequently with buffer B (100 mM KPi, pH 12, 1M KCl, 1 mM EDTA, 2 mM DTT), followed by immediate neutralisation with KH2 PO4 . Aliquots of the eluates were subjected to SDS-PAGE and protein concentrations were determined after Coomassie blue staining.
4. Two-step elution of bound DNA and p53 and SDSPAGE: DNA fragments bound to p53 immune complexes were quantitatively eluted with 500 µl of 100 mM ammonium hydrogen carbonate, pH 9.5 for 45 min at 35°C. Proteins were eluted with 50 µl SDS sample buffer and subjected to SDS-PAGE.
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Will and Deppert: Interaction of mutant p53 with MAR DNA
2.The desired MAR-DNA fragments were isolated by restriction digest and gel electrophoretic separation, purified from the gel and end-labeled using T4 polynucleotide kinase and #(-32 -P) ATP by standard procedures. 3. Afterwards equal amounts of each p53 preparation were added to the binding buffer (SWB-buffer, see Southwestern DNA binding assay) including the desired radioactively labeled MAR-DNA fragments and unlabeled competitor DNA and incubated for 30 min at room temperature. 4. Subsequently, antibodies PAb248 or PAb1810 and PAS were added and shaken for 30 min at room temperature. 5. The DNA-protein-antibody complexes were washed three times with SWB-buffer.
6. DNA fragments bound to p53 immune-complexes were quantitatively eluted with 500 µl of 100 mM ammonium hydrogen carbonate, pH 9.5 for 45 min at 35°C.
7. The eluates were lyophilized and dissolved in 20 µl of gel loading buffer (water, 10% glycerol, bromophenol blue). Samples are subjected to DNA-SDS-PAGE and visualized by autoradiography.
mutations at the p53 locus. A n n . N Y A c a d . S c i . 768, 111-128. Lin, J., Teresky, A.K., Levine, A.J. (1 9 9 5 ). Two critical hydrophobic amino acids in the N-terminal domain of the p53 protein are required for the gain of function phenotypes of human p53 mutants. O n c o g e n e 10, 2387-2390. Michalovitz, D., Halevy, O.and Oren, M. (1 9 9 1 ). p53 Mutations: Gains or losses? J . C e l l . B i o c h e m . 45 , 22-29. Mosner, J., Mummenbrauer, T., Bauer, C., Szakiel, G., Grosse, F.and Deppert, W. (1 9 9 5 ). Negative feedback regulation of wild-type p53 biosynthesis. EMBO J. 14, 4442-4449. Müller, B.F., Paulsen, D.and Deppert, W. (1 9 9 6 ). Specific binding of MAR/SAR DNA-elements by mutant p53. O n c o g e n e 12, 1941-1952. Pohl, J., Goldfinger, N., Radler-Pohl, A., Rotter, V.and Schirrmacher, V. (1 9 8 8 ). p53 increases experimental metastatic capacity of murine carcinoma cells. M o l . C e l l . B i o l . 8, 2078-2081. Shaulsky, G., Goldfinger, N.and Rotter, V. (1 9 9 1 ). Alterations in tumor development in vivo mediated by expression of wild type or mutant p53 proteins. Cancer R e s 51, 5232-5237. Soussi, T., Legros, Y., Lubin, R., Ory, K.and Schlichtholz, B. (1 9 9 4 ). Multifactorial analysis of p53 alterations in human cancer: A review. Int. J. Cancer 57, 1-9.
References Cockerill, P.N., Yuen, M.-H.and Garrard, W.T. (1 9 8 7 ). The enhancer of the immunglubulin heavy chain locus is flanked by presumptive chromosomal loop anchorage elements. J . B i o l . C h e m . 262, 5394-5397. Deppert, W., Buschhausen-Denker, G., Patschinsky, T.and Steinmeyer, K. (1 9 9 0 ). Cell cycle control of p53 in normal (3T3) or chemically transformed (Meth A) mouse cells. II. Requirement for cell cycle progression. O n c o g e n e 5, 1701-1706. Deppert, W. (1 9 9 4 ). The yin and yang of p53 in cellular proliferation. C a n c e r B i o l . 5, 187-202. Deppert, W. (1 9 9 6 ). Binding of MAR-DNA elements by mutant p53: possible implications for its oncogenic functions. J . C e l l . B i o c h e m . 62, 172-180. DeLeo, A.B., Shiku, H., Takahashi, T., John, M.and Old, L.J. (1 9 7 7 ). Cell surface antigens of chemically induced sarcomas of the mouse. I. Murine leukemia virus-related antigens and alloantigens on cultured fibroblasts and sarcoma cells: description of a unique antigen on BALB/c Meth A sarcoma. J . E x p . M e d . 146, 720-734. Dittmer, D., Pati, S., Zambetti, G., Chu, S., Teresky, A.K., Moore, M., Finlay, C.and Levine, A.J. (1 9 9 3 ). Gain of function mutations in p53. Nature Genet. 4, 42-46. Levine, A.J., Wu, M.C., Chang, A., Silver, A., Attiyeh, E.F., Lin, J.and Epstein, C.B. (1 9 9 5 ). The spectrum of
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Steinmeyer, K.and Deppert, W. (1 9 8 8 ). DNA binding properties of murine p53. O n c o g e n e 3, 501-507. Strauss, B.E.and Haas, M. (1 9 9 5 ). The region 3' to the major transcriptional start site of the MDR1 downstream promoter mediates activation by a subset of mutant p53 proteins. B i o c h e m . B i o p h y s . R e s . C o m m u n 217, 333-340. Weißker, S., Müller, B., Homfeld, A.and Deppert, W. (1 9 9 2 ). Specific and complex interactions of murine p53 with DNA. O n c o g e n e 7, 1921-1932. Zambetti, G.P.and Levine, A.J. (1 9 9 3 ). A comparison of the biological activities of wild-type and mutant p53. Faseb J . 7, 855-865.
Gene Therapy and Molecular Biology Vol 1, page 551 Gene Ther Mol Biol Vol 1, 551-580. March, 1998.
Transcription-promoting genomic sites in mammalia: their elucidation and architectural principles Jürgen Bode1, Jörg Bartsch2, Teni Boulikas3, Michaela Iber1, Christian Mielke4, Dirk Schübeler 1, Jost Seibler1, and Craig Benham5 1
GBF, National Research Center for Biotechnology, Genregulation und Differenzierung, D-38124 Braunschweig, Mascheroder Weg 1, Germany. 2 3 4 5
Entwicklungsbiologie, Universität Bielefeld, D-33501 Bielefeld, Germany. Institute of Molecular Medical Sciences, 460 Page Mill Road, Palo Alto, California 94306 USA. Department of Molecular Biology, University of Aarhus, DK-8000 Aarhus, Denmark. The Mount Sinai Med. Center, New York/Biomathematical Sciences New York, 10029 USA.
___________________________________________________________________________________________________ Corresponding author: Jürgen Bode Tel./Fax: +49 531 6181 251/262, E-mail: jbo@gbf-braunschweig.de
Summary Scaffold/matrix attached regions (S/MARs) represent a relatively novel addition to the class of cisacting DNA sequences i n the eukaryotic genome. These elements are thought t o operate via functional contacts to the protein backbone of the nucleus. S/MARs of several kilobases are found at the putative borders of several chromatin domains, and shorter elements with basically the same physicochemical properties occur i n close association with certain enhancers or i n introns. Accordingly, S/MARs can be situated either i n nontranscribed regions or within transcription units. Biological roles that have been assigned to them include insulating and chromatin domain opening functions. These activities apparently are not separable, and both are compatible with the same kind of structure. In this contribution we present a series o f recent results suggesting that S / M A R s act as topological gauges with the potential to adapt their functions to environmental stresses. We also suggest that previously noted uncertainties regarding their activities may have arisen from inadequacies in the methods that were used for their characterization. We discuss the application of new, highly controlled site specific recombination methodologies that integrate single copies into controlled genomic positions to the study of transgene and S/MAR functions in cell cultures and in transgenic organisms.
I. Organization of the eukaryotic genome The eukaryotic genome is organized on at least four levels. At the lowest level the double-stranded DNA molecule combines with octamers of core histones, wrapping around each in two left-handed superhelical turns. This produces a string of nucleosomes whose spacing is largely determined by the presence of a linker histone.
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Since these basic features emerged, evidence has accumulated that the orderly arrangement of nucleosomes can be affected by transacting factors and structural features of the DNA. In particular, DNA sequences with an intrinsic curvature or bendability prefer to be accommodated within a nucleosome, causing phased arrays even in the absence of additional proteins (reviewed by Wolffe, 1994b).
Bode et al: Architectural principles of S/MARs The nucleosome string, also called the 10 nm filament, shows a propensity to fold into a fiber with a diameter of 30 nm. This fiber in turn is organized into looped domains. Early evidence suggesting this domain structure included the observation that neither micrococcal nuclease nor restriction enzymes are able to release from nuclei soluble chromatin with a DNA chain length in excess of 75000 base pairs (Igó-Kemenes and Zachau, 1977). Around the same time the existence of topologically independent domains was established by microscopic studies of histonedepleted metaphase chromosomes (Paulson and Laemmli, 1977) and nuclei (Cook and Brazell, 1978). These studies revealed the presence of a supporting structure, the nuclear scaffold or matrix, to which DNA was periodically attached to form superhelical loops. Experiments with intercalating agents, which at low concentrations cause an expansion and at higher concentrations a contraction of the halo, were explained by dye-induced relaxation of negative superhelical loops and to their subsequent overwinding. These loops evidently were held in a way that constrained their topologies by preventing changes in their linking numbers. Attached regions of DNA were subsequently characterized by several extraction procedures (Mirkovitch et al., 1984; Cockerill and Garrard, 1986; review: Boulikas, 1995) and accordingly they were either termed scaffold- or matrix-attached regions (S/MARs). Since the same elements are recovered by various protocols, the original distinction of SAR- and MAR-elements seems no longer justified (Kay and Bode, 1994). Besides the common extraction approach there are other, supposedly milder methods aimed at detecting attachment sequences. However, these mostly fail to establish the existence of functional S/MARs (Jackson et al., 1990; Eggert and Jack, 1991; Hempel and Strätling, 1996). A critical evaluation of these experiments shows that the S/MAR elements either were not probed in their genuine transcriptional context and/or that the topological state of their domains had been perturbed by restriction (Bode et al., 1996). A careful study by Ferraro et al. (1995, 1996) used cis-diamminedichloroplatinum to form reversible crosslinks between matrix proteins and DNA in intact cells. The use of authentic S/MAR probes for Southwestern blotting strongly suggested that the separated matrix proteins in fact are the interacting partners of the S/MARs.
A. Chromatin domains and boundary elements Genes which are committed to transcription are generally accessible to the action of DNaseI (Weisbrod et al., 1982). An elevated sensitivity has been demonstrated to extend several kbp from the transcribed region until an area of lower accessibility is reached. It is tempting to
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speculate that the boundaries between these regions could be formed by S/MARs, which would prevent the topological changes within an active domain from propagating into quiescent ones (Bode et al., 1992). Examples for which this situation has been documented include the domains of the chicken lysozyme gene (PhiVan and Strätling, 1988), the human apolipoprotein gene (Levy-Wilson and Fortier, 1989), the human ß-globin cluster (Dillon and Grosveld, 1993), and the human interferon-ß gene (Bode et al., 1995). These findings led to the idea that the DNA loops defined above represent functional units within the genome, so-called chromatin domains. The group of DNA “boundary elements” that have been implicated in the functional compartmentalization of the eukaryotic genome share certain common attributes. One defining property is insulation: a boundary element placed between two cis-acting elements inhibits their interactions. When a promoter is separated from an enhancer in this way, for example, the enhancer is no longer able to interact with the transcription initiation complex at the promoter (review: Corces et al., 1995). Early work found certain sequences that exhibited insulation, although they did not appear to be S/MARs. Examples are the scs and scs´ sequences flanking the Drosophila heat shock locus (Kellum and Schedl, 1992, Vazquez et al., 1994). Although scs and scs' did not behave as S/MARs, at least in the initial assay, they have a number of properties in common with them. Each contains a large, nucleaseresistant core spanning a DNA segment that is very AT rich and flanked by DNaseI hypersensitive sites. After heat shock, both elements are primary targets for the action of topoisomerase II, which is an abundant S/MAR-associated protein (Laemmli et al., 1992). Other examples are a sequence within the gypsy transposon of Drosophila (Wolffe, 1994a), and a flanking element in the ß-globin locus of chicken (Chung et al., 1993). The latter element coincides with a constitutive hypersensitive site (HS4), and blocks the action of enhancers in a way resembling scs and scs'. Although this GC-rich sequence is similar in many aspects to the HS5-associated sequence in the human ßglobin locus, which is a S/MAR (Li and Stamatoyannopoulos, 1994), it has no S/MAR activity in vitro. If it were matrix- attached in vivo, it would have to be by a different, but possibly related, mechanism (see chapter V).
B. Structural factors affecting transgene expression levels Several factors conspire to make the expression levels of transgenes highly unpredictable when transfections are performed using conventional techniques. The two most
Gene Therapy and Molecular Biology Vol 1, page 553 important of these are positional effects and copy number effects.
and/or to the sequestering of these complexes in a heterochromatic nuclear compartment.
Position effect variegation (PEV) can be defined as a position-dependent inactivation of gene expression in a fraction of cells that generate a particular tissue. The first and best documented instance of PEV is a chromosomal rearrangement in Drosophila in which an allele of the white (w+) gene is transferred to a site close to the centromere. After this translocation its previously uniform expression becomes “variegated,” producing patches of pigmented and unpigmented cells in the eye. It is thought, but still unproven, that a pericentromeric location renders the gene susceptible to the spreading of heterochromatic condensation. PEV has also been demonstrated in yeast and in mammals for gene sequences within centromeres or close to telomeres (Dobie et al., 1997).
It is obvious that studies of PEV promise insights into the basis for heterochromatin formation and the role of higher order chromatin and chromosome structure in gene regulation. On the other hand, positional and copy number effects on transgene expression levels are serious obstacles to the straightforward application of reverse genetics. The various ways in which transgene expression patterns are affected by multiple copy integration events and inadvertent occupation of certain integration sites will be discussed further below (chapter III). Chapter IV presents a proposed transfection strategy that does not have these problems.
Variegated expression can occur in transgenes as well as in endogenous genes. As the expression level of a transgene is highly dependent on its integration site, which cannot be predetermined with conventional transfection techniques, the forces leading to PEV can cause large and unpredictable variations in expression levels. In the case of mice, it has been reported that transgene integration into pericentromeric regions is the most frequent inactivating process (Festenstein et al., 1996).
A prototype LCR has been defined upstream from the human ß-globin genes. It contains five DNaseI hypersensitive sites, each of which is a small region of 200-300 bp containing a high density of transcription factor binding sites. This LCR is absolutely required for expression, and it confers an altered chromatin structure on a region of more than 150 kbp. (Dillon and Grosveld, 1993). The existence of an LCR has also been demonstrated at the human CD2 locus (Festenstein et al., 1996) and in the chicken lysozyme gene domain. In the latter case a group of proximal regulatory sites (between -1 and -3 kb) and two distal sites (at -6.1 and -7.9 kb) are all required for high-level, position-independent expression. It follows that the collection of these separated functions together constitutes the LCR (Sippel et al. 1993, Bonifer al., 1994, 1996).
However, in some remarkable cases transgenes are expressed in an integration-site independent manner (Chamberlain et al., 1991; Greaves et al., 1989; Aronow et al., 1992; Palmiter and al., 1993; Schedl et al., 1993; Thorey et al., 1993, Neznanov et al., 1996). Attempts to identify the sequences responsible for this insulating effect have not yet led to unambiguous candidate elements. One simple explanation might be that these constructs are delimited by boundary structures (Eissenberg and Elgin, 1991). Alternatively or additionally, they may contain elements that prevent mislocalization into heterochromatic nuclear compartments. The latter class of elements has originally been termed “dominant control regions” (DCR; Collis et al., 1990) and now, more commonly, “locus control regions” (LCR; Epner et al., 1992). They are thought to form extraordinarily stable complexes with their coordinated promoters in a way that overcomes external influences. In vertebrate transfection experiments the transgenes frequently insert in large tandem arrays. In these arrays expression levels are not strictly correlated with copy number, the extreme case being where expression is completely absent. This phenomenon is termed “repeatinduced gene silencing” (RIGS) in animals (Dobie et al., 1997), and “cosuppression” in plant systems (Matzke and Matzke, 1995). It is presumed that the close repetition of sequences leads to the formation of unproductive multiprotein complexes between transcription factors
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C. Locus control elements
Many transgenic studies involving either a complete LCR or its core sequences have relied on the analysis of cells possessing more than one copy. More refined studies are possible using retroviral vectors, which enable a single copy of the transcription unit per cell to be integrated in a precise way and in the absence of selection. (This approach will be described in detail below). In retroviral transfection experiments performed to date, the LCR and its components act more like a classical enhancer than as an element dominating chromatin structure. These unexpected observations raise the question of whether an LCR can truly confer position-independent expression when present in one copy per cell (Novak et al. 1990).
II. The elusive roles of scaffold/matrix attached regions S/MARs have been observed to have both boundary functions and to act upon transcriptional rates. Due to their locations at the putative ends of chromatin domains, they have been considered as domain borders (Phi-Van and Strätling, 1988; Bode and Maass, 1988; Levy-Wilson and
Bode et al: Architectural principles of S/MARs Fortier, 1989; Dillon and Grosveld, 1993). In some systems they were found to dampen positional effects, as expected of insulating boundary elements (Stief et al., 1989; Phi-Van et al., 1990, 1996; Kalos and Fournier, 1995). Although S/MARs are clearly distinguishable from enhancers, they augment transcriptional levels by a distinct, enhancer-independent mechanism (Klehr et al., 1991, 1992; Dietz et al., 1994; Poljak et al., 1994). Conversely, in some systems enhancers are fully active only when associated with S/MAR elements (reviews: Bode et al., 1995, 1996, and section V).
A. An operational definition Originally, S/MARs were defined using only the protocols that led to their detection. These procedures involve the isolation of interphase nuclei, followed by extraction of non-matrix proteins to yield a nuclear halo. It has been shown that the total number of loops per cell can depend on details of the procedure used. For every attachment existing in vivo, several new attachments may be created in vitro as nuclei are prepared, stabilized and lysed (Jackson et al., 1990). Moreover, the matrix-S/MAR contacts of degraded, topologically unrestrained halos are accessible to competing, soluble S/MAR elements (Mielke et al., 1990; Kay and Bode, 1994, 1995; Bode et al., 1995 and references therein). These observations suggest that not all S/MARs are constitutively associated with the nuclear matrix in vivo (Dillon and Grosveld, 1993). Techniques able to assess the occupation of S/MARs in vivo are currently emerging (Ferraro et al. 1995, 1996). Although the central biological effects of S/MAR elements are clearly compatible with their affinity for the nuclear matrix, others have been harder to explain. Below we describe the more prominent properties of these elements. These diverse properties are reconciled by recent experiments that have been performed in our laboratory (section IVA). In Section V we will discuss novel transfection strategies using S/MAR elements. These have the capacity to avoid the uncertainties arising from positional and copy number effects, providing a comprehensive and unambiguous analysis of the associated functional aspects of transfected elements, including S/MARs.
B. Transcriptional augmentation S/MARs are a relatively new addition to the list of cisacting elements known to elevate transcriptional rates. In our experience the simultaneous presence of an enhancer is not required for this S/MAR activity (Klehr et al., 1991). This effect, which we call 'transcriptional augmentation', is clearly separable from prototypical enhancement since enhancers, but not S/MARs, are active in transient assays.
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But like enhancers, S/MARs act independent of orientation and independent of distance, provided this is at least several kilobases (Klehr et al., 1991 and section IVA). This activity is found for minimal, viral and cellular promoters, of both inducible and housekeeping genes. If an enhancer is also present, enhancement and augmentation factors act roughly multiplicatively (Bode et al., 1995). Although enhancers are generally believed to interact with the basal transcription machinery by looping, the experimental data supporting such a mechanism are not conclusive. To explain the correct choice of promoter, it has been suggested that the initial contacts must be checked by a tracking mechanism (Weintraub, 1993). Both looping and tracking may be modulated by the occurrence of S/MARs, although there are divergent views of how this is accomplished. On one hand, S/MARs may associate nearby enhancers to the nuclear matrix, and thereby assure their proximity to transcribed units (Boulikas, 1995). Such a mechanism could enable the formation of alternative functional units, activating their respective promoters at the matrix by a directional transfer of transcription factors. On the other hand, by acting as domain boundaries (IA), S/MARs could limit the effect of an enhancer to the domain in which it occurs. This view is compatible with the results from enhancer-blocking experiments (Bode et al., 1995; Li and Stamatoyannopoulos, 1994). Finally, S/MARs might serve as domain openers, as first proposed by Zhao et al. (1993). Although a direct correlation has been demonstrated between the binding and augmenting activities of S/MARs (Mielke et al., 1990; Kay and Bode, 1995; Allen et al. 1996), it is not clear whether nuclear matrix binding per se is sufficient to augment transgene expression in stably transfected cells (Phi-Van and Str채tling 1996). If this were the case one could use a simple in vitro binding test to predict in vivo properties. Very recent studies show that the general augmenting effect of S/MARs can be disrupted by the overexpression of certain S/MAR binding proteins (Kohwi-Shigematsu et al, 1997). A simple explanation for this phenomenon would be the interference of such a (possibly soluble) factor with genuine S/MAR-matrix contacts, but more sophisticated explanations also are possible (see the model by Scheuermann and Chen, 1989; Zong and Scheuermann, 1995). The biological effects of S/MAR elements commonly are studied by transfecting a S/MAR-reporter construct and relating its expression level to that of a S/MAR-free control that has been transfected in parallel. In animal cells, such an approach consistently leads to a significant elevation of the reporter signal. Although this usually is intuitively ascribed to cis-actions of the S/MAR, such an effect could in principle arise in several ways:
Gene Therapy and Molecular Biology Vol 1, page 555 (i) as an immediate effect of the S/MAR on transcriptional initiation. This is the proposed explanation for almost every transcriptional S/MAR effect published to date. (ii) as a copy number effect. Many (but not all) S/MAR elements tend to increase the number of integrated copies relative to S/MAR free control (Schlake, 1994). This has been ascribed to the recombinogenic nature of these sequences (Sperry et al., 1989; Bode et al., 1995, 1996). It should be noted that, due to PEV-related phenomena (Section IB), the activity of a single copy can normally not be derived by just dividing the overall expression by the number of integrated copies. (iii) as a targeting effect. Owing to the affinity of S/MARs for the nuclear matrix, which is thought to be the site of active transcription, a preferential integration of S/MAR constructs into actively transcribed regions or active nuclear compartments could occur. S/MAR free controls would not exhibit this preference.
C. Insulator functions The presence of S/MARs has been shown to be required to prevent the ectopic expression of transgenes (Sippel et al., 1993). In one case a S/MAR is needed to enable correct, hormonal gene regulation (McKnight et al., 1992, 1996). These and other results from a range of insulation experiments strongly implicate S/MARs in defining the boundaries of autonomously regulated chromatin domains. S/MARs have been studied in both animal and plant systems. As a rule, transcriptional augmentation by S/MARs is most easily seen in mammalian cells, while in plants their insulator functions are more frequently observed (Bode et al., 1995). We ascribe this difference to the fact that most plant studies are based on T-DNA vectors which, when integrated into genomic loci, yield expression levels high enough to cope with applied selection pressure (Dietz et al, 1994). In this case the maximum attainable level of gene expression does not depend on the presence of S/MARs, whose most easily observed function then becomes to stabilize an elevated (but not extreme) expression level shielded from negative and positive influences of the surroundings (Dietz et al., 1996, Bode et al., 1996). This would agree with the insulating or bordering function suggested by Mlynarova et al. (1995). After the enhancer-blocking assay (IA), described previously, copy number dependence is by far the most common principle used to detect and assay insulator functions. However, there are major difficulties inherent in this method (demonstrated by Poljak et al., 1994; Schlake, 1994). These are due, at least in part, to the fact that
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multiple copies often do not integrate at separate genomic locations, but rather as a single tandem array at a site which is unique for each cell. This induces many unexpected interactions, rearrangements and cellular 'defense mechanisms' (Mehtali et al., 1990; Kricker et al., 1992; Dorer and Henikoff, 1994; Kalos and Fournier, 1995; Dorer, 1997). For this reason there frequently is not a linear correlation between copy number and gene transcription, even in the case of low copy numbers (see IB and IIIC).
D. Intronic S/MARs S/MARs cannot simply be considered to represent static delimiters of functional domains. This is demonstrated by the detection of S/MARs within introns. Examples of single genes that are apparently divided over two domains include the genomic sequences encoding hamster DHFR (Käs and Chasin, 1987), human topo I (Romig et al., 1992), human interleukin-2 (Artelt and Bode, unpublished) the mouse immunoglobulin !- and µ-chains (Cockerill and Garrard, 1986, Cockerill et al., 1987) and a light-inducible plant gene (Stockhaus et al., 1987, Mielke et al., 1990). Studies on MPC-11 plasmacytoma cells have shown that 9% of poly(A) mRNA arises from the !-locus, whose transcription must be completed on average once every 3.2 s (Cockerill and Garrard, 1986). Since by definition intronic S/MARs are transcribed, and since they do not impede passage of RNA polymerase II, their occupation must be regulated. Functional analyses have recently revealed complex roles for S/MARs in gene expression, which can only be appreciated after structural analyses of these composite elements (section VB).
E. Current uses of S/MAR elements Retroviral delivery systems enable the efficient transfer and expression of transgenes in primary cells. Sometimes, a pitfall of these methods has been a continuous downregulation of the transgene (but see IVA). Because S/MAR elements augment transcription and stabilize its level over extended periods of time (Bode et al., 1995), they could be used to construct a new generation of expression vectors. The mechanism of retroviral replication enables one to establish a minidomain by simply introducing a S/MAR element into the 3´- LTR (Schübeler et al., 1996 and Figure 3). In this minidomain the expression unit is flanked by two S/MARs, which can stabilize transgene expression. A number of current pharmaceutical developments are exploiting this approach. Although short and apparently functional S/MAR elements can be constructed by oligomerizing certain subS/MAR motifs (core unwinding elements, see Mielke et
Bode et al: Architectural principles of S/MARs al., 1990, Bode et al., 1992 and VB), the practical use of such synthetic elements for retroviral transfection is limited by the intrinsic genomic instability within and between the multimers. One can avoid this problem by using minidomain bordering elements from different sources (Bode et al., 1995, Dietz et al., 1994). A whey acidic protein (WAP) transgene was found to be active in just 1 out of 17 lines of transgenic mice, demonstrating copy-number independent (position dependent) expression. After ligating S/MARs to the transgene, 7 of 9 lines exhibited transgene activity, and correct hormonal regulation occurred in the majority of cases (McKnight et al., 1992, 1996). These experiments show that S/MARs are able to prevent ectopic expression (Sippel et al., 1993). S/MARs have found widespread use in the manipulation of plant cells, callus cultures and complete plants. Although increased and position-independent transcription has been described by various authors, these phenomena are only linked in some cases (SchĂśffl et al., 1993, Mlynarova et al., 1994, van der Geest et al., 1994). Examples are known of plant cells transformed by Agrobacterium in which the use of S/MARs improves copy-number dependent (Breyne et al., 1992) or positionindependent (Dietz et al., 1996) expression without affecting transcriptional levels (Breyne et al., 1992). In other cases where microprojectile bombardment was used, transcription was dramatically raised independent of copy number (Allen et al., 1993, 1996). These differences may, in part, be due to the gene transfer technique used. The first method tends to target transcriptionally competent sites, while random positions are hit in the second one (Dietz et al., 1994).
III. Problems associated with reverse genetics The level of in vivo expression of a transfected gene may be quite different from that which occurs when the same gene is in its natural context. Although transient assays in cultured cells have been used in the past to characterize tissue-specific enhancers, their potential is limited because the actions of these and other cis-acting elements depend sensitively on chromosomal context. Proximity of enhancers to S/MAR elements provides a paradigm example of context-specific behavior (reviews: Bode et al., 1995, 1996). These observations underscore the difficulty of establishing a dependable approach to the study of gene function. The analysis of gene expression must be performed in a native chromatin context. However, this approach is complicated by a number of problems arising from the following facts (section IIIA-C).
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A. Need for a selection marker Since only a small percentage of cells incorporate foreign DNA after transfection, selection for a drug resistance marker is required to isolate those cells that harbor the construct. This marker has to be expressed at a threshold level, which creates a selection bias against low producers (Blasquez et al., 1992). Positive selection markers are constitutively expressed, which can seriously interfere with the transcription even of remote genes. An extreme example has been reported of a gene that became completely deregulated by a selection marker which was placed 50 kbp away (Fiering et al., 1993). Further, some prokaryotic vector sequences have been found to inhibit (Palmiter and Brinster, 1986) or to promote gene expression (Seibler and Bode, 1997).
B. Integration occurs at random Since there is no evidence for site-directed insertion, the generation of random chromosomal breaks is thought to be the rate-limiting step of the prevailing integration mechanism. This would explain the predominance of unique integration sites in mammalian cells and the observation that certain (but not all; see Mielke et al., 1990) cell types prefer linearized over circular templates for integration (Palmiter and Brinster, 1986). Random integration can subject transgene expression to unwanted local influences (position effects) resulting from their proximity to regulatory elements or heterochromatic regions (IB).
C. Multiple copy integration If DNA is introduced by standard transfection techniques or by microinjection, multiple gene copies are usually integrated at a single site. This is probably due to homologous recombination events occurring among the transfected molecules (Phi-Van and Strätling, 1996). There are several ways in which this tandem arrangement can alter the effect of an element from what it would be as a single copy. One usually cannot determine how many copies of a gene are functional templates, or whether different copies perform different functions (Oancea et al., 1997). Tandem repetition of promoter elements could trigger the formation of multiprotein complexes between transcription factors, with largely unpredictable consequences (IB). It also may lead to a more than proportional effect of a cis-acting regulatory sequence, such as a S/MAR (Stief et al., 1989). More typical are shutoff processes occurring with time. These have been associated with methylation followed by mutagenesis (Mehtali et al., 1990; Dorer, 1997) and/or a genomic instability resulting from the continuous loss of members of the array (Palmiter et al., 1982; Weidle et al., 1988). As a
Gene Therapy and Molecular Biology Vol 1, page 557 consequence, expression levels cannot be expected to be
proportional to copy number (Figure 1).
Figure 1. Inadequacy of conventional gene transfer techniques. Conventional transfection techniques lead to multi-copy integration events, usually at a single site and in tandem, head-to-tail orientation. Whether all members of a multigene complex are transcibed at the same rate is an unsolved question
While a tandem head-to-tail integration at a given site is considered typical, more complicated integration forms were recently demonstrated for single S/MAR constructs (Phi-Van and Str채tling, 1996). Most of the copies were colocalized as usual, but they clearly differed regarding their relative orientation: whereas head-to-head (hh) and head-totail (ht) were preferred, hh plus tt and ht plus tt tandems were strongly disfavored and exclusive tt integrations were lacking altogether. It may be speculated in this case that the unequal ends, which contained a S/MAR at their 5' end and a unique vector sequence at their 3' terminus, were responsible for this effect. This nonrandom distribution could result from preintegration ligations of two molecules. Alternatively, the close juxtaposition of two identical copies could induce inversions. The recombinogenic nature of S/MAR elements (Sperry et al. 1989) might add to the complications associated with conventional gene transfer techniques.
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In conclusion, with current transformation methods, insertion of DNA into the genomes occurs at random, and in the case of plant systems in many instances at multiple sites. The associated position effects, copy number differences and multigene interactions can make conventional gene expression experiments difficult to interpret.
1. An immediate solution: gene transfer by electroporation can be optimized Classical gene transfer experiments are based on the cotransfer of a reporter and a selector construct which are coprecipitated by Ca++-phosphate and taken up by endocytosis (transfection). As an alternative, DNA can be transferred from solution by transiently permeabilizing the cells in an electric field (electroporation). This technique is most efficient if the reporter and the selector gene are
Bode et al: Architectural principles of S/MARs physically linked in a single construct. For some cell lines the electrical parameters and cell survival rates can be optimized in a way that yields predominantly single copy integration events (Mielke et al., 1990, 1996; J. Bartsch, unpublished). This technique eliminates the unpredictable effects of multicopy integration. This method has been used to compare expression levels of S/MAR-constructs to those of S/MAR-free controls. Representative data are found in Figure 2. The average level of expression from a S/MAR-free luciferase control (Lu: 85 700 light units) was increased eightfold if an upstream S/MAR element was present (E-Lu: 698 000 light units), and 26-fold if it was transfected as a minidomain with upstream and downstream S/MAR elements attached (E-Lu-W: 2 253 000 light units). These results are similar to those previously reported (Klehr et al., 1991, 1992; Bode et al., 1995, 1996; Phi-Van et al., 1990; Poljak et al., 1994). This analysis has been extended to individual clones to derive information about the insulator function of S/MARs that is difficult to obtain by either the enhancer-blocking assay (IA) or the copy number dependence of expression (section IIC). Since expression levels vary by three orders of magnitude, they have been plotted in F i g u r e 2 on a logarithmic scale. The largest inter-clone variation of expression was 340 fold, found for the S/MAR-free control. A single S/MAR element reduces the variation to
Figure 2. Insulator function of S/MARs demonstrated at the single copy level A luciferase-neomycin resistance construct was introduced into CHO cells either as a S/MAR-free control (Lu), flanked by an upstream S/MAR element (E-Lu) or by as a minidomain flanked by an upstream and a downstream S/MAR (E-Lu-W). Electroporation of these constructs resulted in 10 clones (Lu), 20 clones (E-Lu) and 90 clones (E-Lu-W), resp.. 9-10 clones with a single integrated copy of each construct (solid bars) and 2-3 clones with 2-3 copies (hatched bars) were selected for determinations of the luciferase expression level. Average luciferase levels (light units) were 86 E3 +/-124% (Lu), 590E3 +/-39% (ELu), 2273E3+/-14% (E-Lu-W).
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4.6 fold. This E-Lu constructs has a single insulator at its 5' end and could, in principle, experience position effects acting through its â&#x20AC;&#x153;openâ&#x20AC;? 3' terminus. If the domain is closed by a second S/MAR element (E-Lu-W) the variation between clones is reduced to a factor of two. These data are in general agreement with a recent study by Kalos and Fournier (1995), who demonstrated that in the presence of both apolipoprotein B and S/MARs the expression of a test construct was increased from the detection limit to at least 200 fold higher levels. At the same time the variation between the clones decreased from >10-fold to <3-fold. For technical reasons these studies were restricted to a small number of clones, and apparently they have not been generally accepted (see Wang et al., 1996). We believe that the combined results of our studies strongly support the notion that S/MARs can enable position independent expression of transfected genes. Whether this is due to insulator function, as currently believed, is not clear. Ours is the first study to demonstrate the dramatic influence even of a single S/MAR, which would also be compatible with a dominant (domain opener) effect in case of the E-Lu construct. This information cannot be derived from any assay based on the presence of several copies (Figure 1), since in the common case of tandem integration each individual member but one will be an effective double-S/MAR construct (...E-Lu-E-Lu...).
Gene Therapy and Molecular Biology Vol 1, page 559
Figure 3. Use of retroviral vectors Because those retroviral genes that are necessary for retrovirus replication have been replaced by a reporter gene (secretory alkaline phosphatase, SEAP) and a selection marker (fusion gene from Hygromycin-B-phosphotransferase and HSVThymidine kinase, HygTK), these functions have to be provided in trans by a packaging cell line. These helper cells produce infectious retroviral particles which can be used to transfer the construct into the recipient cell. After reverse transcription of viral RNA, the transgene is stably linked with the genome (provirus state). Promoter and terminator functions reside in the long terminal repeats (LTRs). Moderate manipulation of the LTRs is compatible with their function and the introduction of S/MAR elements (Mielke et al., 1996) and Flp-recognition target sites (FRTs) has been described (Sch체beler and Bode, 1997; see also section IVA).
Interestingly, the properties documented in our study appear to be independent of the source of the S/MAR. Elements of human origin (E) behave in our system like their counterparts from plants (W), provided that they both show a corresponding affinity for the scaffold in the in vitro test system (cf. also Dietz et al., 1994). To date it has not been possible to separate the insulation and augmentation properties of S/MARs (Phi-Van and Str채tling, 1996) which both require a threshold length of DNA in excess of about 300 bp. Together, these results suggest that the basic functions of insulation and transcriptional augmentation are widely conserved, and are based on structural features which are recognized by ubiquitous proteins rather than by specialized factors. We note that five clones in our experimental series contained more than a single copy (hatched bars in Figure 2). Since their expression is in the same range found for the single copy integrates, a standard copy-number dependence test (IIC) would have failed to reveal any evidence for an insulator function in this system. This reemphasizes the fact that any test of sophisticated S/MAR functions must use single copy integrants.
IV. Advanced approaches: A progress report While the electroporation method has effectively eliminated the detrimental effects of multicopy integrations, the other critical problems (numbered (i) to (iii) in section IIB) still need to be addressed. The 559
following studies describe recent developments in this direction. They allow the integration of single, intact copies, in some cases without the cotransfer of vector sequences. They also are aimed at preventing any integration bias which may arise from the biochemical properties of S/MAR elements (criterion 3 in IIB).
A. What retroviruses can tell us We used a retroviral vector infection procedure as it facilitates the introduction of single copies with strictly defined ends, the long terminal repeats (LTRs). For these studies we have constructed an expression cassette consisting of a reporter gene (SEAP, secretory alkaline phosphatase) and a selector gene (PAC, puromycin Nacetyltransferase). Both members of this bi-cistronic cassette are synchronously expressed owing to the fact that an internal ribosome entry site (IRES) enables the translation of the second cistron. To obtain an infectious retrovirus particle, accessory functions have to be provided in trans. This is done by first transfecting the construct into a helper cell line (Psi2). The helper cells contribute the gag, env and pol functions, which package the viral mRNA and secrete the virus into the medium. These viruses can be harvested and used for an infection of the ultimate target cells. There copies of the viral mRNA are reverse-transcribed into DNA. During this process the sequence of the 3'-LTR is copied to form an identical 5'LTR (Figure 3). Elements cloned into the 3'-LTR (marked by a half arrow) will then appear also in the 5'terminus. This strategy has been used both to introduce a
Bode et al: Architectural principles of S/MARs minidomain (S/MARs at both ends, see section IIE), and to generate two Flp recombinase target sites which are recognized and recombined by the Flp enzyme (Sch端beler et al, 1997 and section IVB). Before making extensive use of these possibilities, we decided to thoroughly characterize the sites of provirus integration. As an initial step, the identical construct was introduced into target cell by standard transfection, electroporation and infection techniques. Expression levels of the SEAP reporter were compared and related to the number of integrated copies (Figure 4). While the overall expression level turned out to be mostly independent of the procedure used for transferring the vector, the per-copy level for transfection is clearly inferior to electroporation and electroporation in turn is inferior to infection. Only infection guarantees the incorporation of single copies which remain stable over extended time intervals (Sch端beler et al, 1997 and unpublished). This indicates that there is no gradual inactivation due to methylation. If such an event were to occur, selection pressure could be applied to eliminate it.
1. Anatomy of retroviral integration sites It is the prevailing view that transcriptionally active genomic regions and regions associated with DNase I
Figure 4. Transcriptional properties of transgenes introduced by three different gene transfer techniques. A single construct (pM5sepa) was introduced into NIH3T3 murine embryo fibroblasts by three gene transfer techniques. pM5sepa contains a reporter (SEAP, secretory alkaline phosphatase) and a selector gene (PAC, puromycin Nacetyltransferase) linked by an internal ribosomal binding site (IRES) to enable coupled expression of both cistrons. Solid bars represent total expression from an entire pool of PAC-resistant clones. Hatched bars refer to expression levels divided by copy numbers.
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hypersensitive sites are preferred by the integration machinery of a retrovirus (Rohdewohld et al., 1987). We wanted to study the architecture of these integration sites in order to obtain information about the factors which mediate a consistently high and stable level of expression. Until recently, most methods for isolating proviral flanking sequences involved plasmid rescue. This approach led to the identification of a small number of highly preferred integration targets (Shih et al., 1988). Some of these results could not be confirmed by subsequent PCRbased techniques that were developed to study integration without prior selection by molecular cloning. This has led to a critical re-evaluation of the prevailing views (WithersWard et al., 1994). We have applied retroviral vectors in conjunction with inverse PCR techniques to reconstruct a number of these sites for further characterization. As in many previous studies, the recovered integration sites conformed to no obvious consensus sequence. This suggests that the site of integration into the host genome may be determined by other factors, such as DNA secondary structure and/or host proteins. While the retroviral integrase performs the central cutting and joining steps as part of a 160 S nucleoprotein complex, the final resolution steps require host functions which may lead to a further selection among a number of initial target sites.
Gene Therapy and Molecular Biology Vol 1, page 561
.... S/MAR-type DNA: -recombinogenic -supporting transcription
.... bent DNA -no positioned nucleosomes (Boulikas, unpublished) -backbone strain retrievable for unpairing (Ramstein & Lavery, 1988)
Figure 5 . Architecture o f h i g h l y e x p r e s s i n g g e n o m i c s i t e s w h i c h are t h e preferred t a r g e t s for r e t r o v i r a l i n t e g r a t i o n . Integration occurs into S/MAR type DNA which is flanked by bent segments. Bending prevents the association of nucleosomes as to keep these sites accessible. It is therefore different from the type of curvature which accommodates nucleosomes (Mielke et al., 1996).
Remarkably, all investigated examples conformed to a unique pattern (Mielke et al., 1996), summarized in Figure 5. All integration events occurred into scaffold/matrix attached regions (S/MAR-elements) which were flanked by DNA that was bent in a way that discourages nucleosome assembly (Boulikas et al., unpublished). These S/MARs belong to a novel class that does not conform to the AT-rich prototype. On the other hand they exhibit most of the in vitro and in vivo properties of S/MARs. Their binding is competed by ssDNA, and they induce significant transcriptional augmentation (as opposed to enhancement) if they are investigated as parts of mammalian expression vectors. We have suggested that the entire insertion process might be guided by the nuclear matrix, which also could provides the enzymatic functions for the final steps of the integration process (Mielke et al., 1996). Integration into S/MARs may also be ascribed to their recombinogenic properties while the selection of non AT-rich subtypes seems to be directed by the mechanism of integration. In summary, retrovirus integration occurs at sites which are available due to their lack of nucleosomes, and which show structural and functional features reminiscent of S/MARs (see analyses under VB/C).
2. S/MAR effects on transcriptional initiation and elongation At this stage of our investigation it was unclear if retroviral integration sites were appropriate for the study of S/MAR constructs, as S/MAR functions also resided in
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their immediate vicinity. But these tools permit integration into a subclass of genomic sites whose selection is dictated by the retroviral integration machinery, not by the presence or absence of the S/MARs within the vectors (criterion 3 under IIB). We inserted an 800 bp S/MAR element from the upstream border of the human interferon-Ă&#x; domain into the vector shown in Figure 4. This element was cloned into various positions, both within and outside a transcribed region of 4.3 kb. Insertion into the 3'-LTR yielded a minidomain (two flanking S/MAR elements) according to the scheme in Figure 3. This study revealed a range of unexpected S/MAR effects that were obscured when the same constructs were introduced by transfection (SchĂźbeler et al., 1996). The most striking observation was that at a distance of about 4 kb, the S/MAR supported transcriptional initiation whereas at distances below 2.5 kb transcription was essentially shut off. Controls proved the functionality of all constructs in the transient expression phase, and ruled out any influence of S/MAR position on transcript stability. Moreover, no pausing or premature termination was observed within these elements. One interpretation of these results is presented in Figure 6. This assumes that S/MARs are kept in a single-stranded state by association with ssDNA binding proteins. This "unwound" structure is able to facilitate the progression of an approaching polymerase if the buildup of positive supercoils is sufficient for breaking the contacts between the single-strands and the binding proteins (transition D2 -> D2.1). If positive superhelicity is minor,
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Figure 6 . S c a f f o l d / M a t r i x attached r e g i o n s act upon transcription in a contextdependent manner. S/MARs can be trapped in vivo as a single-stranded structure by chloroacetaldehyde (CAA). It is hypothesized that single strands are kept apart by ssDNA binding proteins (triangles). Unconstrained positive supercoils arising from a S/MARscaffold association are ultimately removed by the action of topoisomerases (B, C). If the S/MAR is located immediately downstream from a transcription initiation site, the polymerase cannot pass the attachment point as the buildup of positive superhelicity is insufficient to rupture S/MAR-scaffold contacts (D1). If it is situated further upstream the contacts can be broken by the approaching polymerase and positive superhelicity can be relaxed as it is compensated be the now unconstrained unwound DNA structure of the S/MAR segment (complementarity of plectonemic and paranemic structures, see Yagil, 1991). Other polymerases can initiate in the wake of the first one.
B. A novel concept: genomic reference integration sites
binding of the ssDNA is strong enough to prevent a rotation of the DNA helix about its axis, which inhibits the progress of the polymerase(state D1). This model explains the bordering and insulator functions of a S/MAR, which will prevail if the element is long enough. It also explains the observation that, in principle, a polymerase can progress through a short S/MAR, and even benefit from the fact that S/MARs are repositories of unwound DNA. This may explain the essential fact that transcriptional augmentation is only found after anchoring the construct in the genome of the host cell. It is unnecessary to postulate the existence of different S/MAR types to perform bordering and augmenting functions. In our experiments both functions are provided by an intermediate-size S/MAR isolated from the center of an extended putative domain border.
The study of cis-acting regulatory elements is often confounded by the variability of gene expression among independent transformants. This variability is ascribed to chromosomal position effects (PEV, see IB) at the sites of transgene integration. Inserting single copy test constructs into the same genomic target would control these effects and facilitate valid comparisons of expression levels. Targeting a given site via homologous recombination, though successful in fungal and some animal systems, is not always practical because it occurs at very low frequencies compared with the high background of illegitimate recombination events.
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Figure 7. Reactions catalyzed by site-specific recombinases: Excision/integration and inversion Excision is the consequence of removing a stretch of DNA which is flanked by two equally-oriented FRT sites; the reaction is mediated by a crossover between these sites. In principle, this reaction is reversible in the sense that a circular vector can be accommodated at a genomic site carrying an FRT tag. The native role of Flp recombinase in yeast is the inversion of the origin of replication region on the 2 plasmid which is localized between two inversely-oriented FRTs (right-hand part). A full Flp-recognition target site consists of three 13 bp repeats and an 8 bp spacer within 48 bp of DNA (bottom). Each of the 13 bp repeats represents an Flp binding element (FBE) and the spacer is the region in which single strand cuts (vertical arrows) are introduced in preparation of the crossover and resolution steps. There is no physical contact of the recombinase to the spacer which determines the polarity and identity of the site. Hence, spacer mutants will be recombined with an identical FRT site but not with a wild type FRT (Schlake and Bode, 1994). For convenience, the FRT-site is symbolized by an half arrow.
Flp recombinase from the 2 plasmid of Saccharomyces cerevisiae can be introduced into mammalian cells to perform site-specific integration reactions (O´Gorman et al., 1991). This enzyme excises any piece of DNA that is flanked by two Flp-recognition target (FRT-) sites of identical orientation (Walters et al., 1996). Even more important in the present context, it also performs the reverse reaction of integrating an FRTlabelled circular vector into an FRT-tag placed in the genome (Figure 7; cf. Schlake and Bode, 1994 and references therein). This enables site-specific integration while avoiding the problems of earlier methods. Related approaches are being developed on the basis of the Cre/loxP1system of bacteriophage P1 which is mostly 563
used for excision-type reactions and with an increasing number of other members of the Int recombinase family (reviews: Kilby et al., 1993; Sauer, 1994). Suitable sites for integration can be prepared by introducing an FRT-tagged construct into an endogenous locus with known properties. This is only possible in systems permissive of homologous recombination, and for loci that are redundant and constitutively expressed such as the histone locus in mice (Wigley et al., 1994). Alternatively, the construct can be transferred by electroporation (section IIIC1). Among the multiple clones that result, those integrants will be chosen which mediate a high and consistent expression in the absence of continued selection pressure. This will guarantee that the respective
Bode et al: Architectural principles of S/MARs site does not become inactivated by heterochromatization, by DNA methylation, or by being situated in a locus that is genetically unstable. Both procedures are initially laborious, but screening has only to be performed once. As an additional advantage of the approach, if an FRT-tagged construct tends to form head-to-tail multimers, these concatemers will be reduced by a continuous action of the recombinase which (according to F i g u r e 7 ) will excise pieces of DNA that are flanked by equally-oriented sites (Lakso et al., 1996). If the individual vector contains just one of these sites, ideally the excision will continue until a single, intact copy is left.
Strand cutting can be initiated when one individual FRT site is occupied by two Flp monomers. This â&#x20AC;&#x153;transhorizontalâ&#x20AC;? cleavage mode does not require the presence of a synaptic complex. Each monomer of the recombinase has only a partial active site and contributes to the formation of a full active center by donating the catalytic tyrosine to the Arg-His-Arg cleft of the partner that is bound across from it on the other side of the spacer. This mechanistic property of Flp guarantees that during the two-step strand transfer of a complete recombination reaction, the activation of one pair of active sites is coupled to the disassembly of the other (Kwon et al., 1997).
For a convenient characterization of expression parameters, it is advantageous first to introduce a reporter gene which can be monitored easily. Since integration can be reversed by a second pulse of Flp activity, the remaining FRT site is then open for other rounds of integration during which any gene of interest can be directed to the pre-defined locus. The general validity of this concept was demonstrated using the Cre/loxP system (Fukushige and Sauer, 1992). However, the straightforward application of the scheme in Figure 7 faces a number of problems which will be discussed after describing some molecular features of site specific recombinases, exemplified by Flp/FRT.
Synapsis is mediated by protein-protein interactions between the bound recombinase molecules. In this way the paired strand cleavage steps become coordinated (Figure 8). This underlines the importance of the synaptic complex, which channels the chemistry of strand breakages so that recombination, not self-healing, is the final result. After the initial strand breaks, strand transfer, and ligation, a Holliday intermediate is formed. Homology permits the Holliday junction to undergo branch migration and isomerization, during which the crossover strands and the helical strands switch functions. This isomerization probably results from a preference for one pair of stacking isomers over another (Li and al., 1997).
1. Mechanism of recombination: design and function of FRT sites
2. The homology checkpoints
The amino acid sequence of Flp bears no resemblance to any known DNA binding motif. The full 48 bp FRT site consists of three individual 13 bp Flp binding elements (FBE a-c), two of which form an inverted repeat around an 8 bp spacer. The third 13 bp element (c) represents a direct repeat and is separated from b by a single base pair. While most reactions are also possible with a minimal 34 bp site consisting of the inverted repeat and the spacer, the integration reaction appears to benefit from the presence of the extra FBE (Lyznik et al., 1993). Association of Flp with its site causes bending of two types. While each Flp monomer introduces an individual bend of 40o; a larger bend (>140o ) is caused by the interaction of the two Flp monomers across the spacer. This is a consequence of strong interprotomer contacts which occur despite the fact that the protein binding sites lie on opposite faces of the DNA. The Flp monomer bound by the third element does not participate in these strong cooperative interactions, which require flexibility of the spacer. This spacer becomes single stranded upon Flp binding (Kimball et al., 1995).
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A central requirement for recombinations catalyzed by members of the INT family is an absolute homology between the partner substrates in a strand-exchange reaction. This implies a DNA-DNA interaction at some point in the reaction. Recent evidence suggests that homology is not checked before strand cleavage, so the first strand transfer can occur in spite of one or more mismatches. Subsequently there are two homology checkpoints. First, the strand-joining step requires complementary base pairing to orient the 5-OH group for its attack on the phosphotyrosyl bond at the cleavage point. Second, the branch-migration event and associated isomerization of the recombination complex require homology (see insert to Figure 8).
3. Construction and use of reference integration sites An early application of the Flp/FRT technology investigated chromatin domains in situ ( Figu re 9A). It was based on the assumption that, in a single-S/MAR construct, the reporter remains sensitive to influences from the surrounding chromatin. We transfected the S/MARLuciferase-FRT cassette and subsequently isolated a series
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F i g ur e 8 . Fo rma tio n a n d re s o lu tio n o f t he Holliday s tr uc tur e dur ing the ac tion of Flp r e c om bi nase. Four molecules of the recombinase (ellipses) participate in site specific recombination which are bound next to the crossover region (8 bp spacer). After single strand cuts are introduced into the recombining partner, a first strand transfer step occurs, followed by ligation. During a branch migration process, which depends on absolute homology of the interacting strands, the end of the spacer is reached. During or following this process an isomerization is thought to produce a structure in which the crossover and noncrossover strands are switched. The structure is resolved by two more single strand cuts and ligation. Insert: Relative movement of two (more extended) homologous helices during branch migration.
of clones with widely different expression characteristics. After closing the domain by Flp-mediated targeting of a second S/MAR element to the endogenous site, clones were isolated and the levels of luciferase expression were compared to the initial ones. Among the recovered clones there was only a minority showing an augmented luciferase activity. When a second pulse was applied to these to reexcise the second S/MAR, the old expression characteristics were re-established. These clones underwent the modification-demodification cycle depicted in Figure 9A (Bode et al., 1996). The majority of them did not permanently acquire the second S/MAR element, but rather gained hygromycin resistance due to a faulty integration of the circular FRT-S/MAR-HygTk vector. In other cases integration of the circular Flp expression vector occurred, leading to a permanent base level of recombinase activity (Iber, 1997). These results underline a major problem with the Flp and Cre recombinase systems: because the
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reactions are reversible and excision is highly favored (being an intramolecular process), they do not allow one to control the direction of recombination. This approach did therefore not permit the investigation of a large number of events to establish an insulation function of S/MARs, especially at sites with an initially mediocre expression. Nevertheless, it provided the clear demonstration of a transcriptional augmentation at a singular integration reference site. This experiment is therefore considered additional evidence that augmentation is - at least in part - due to a cis-effect of S/MARs on transcriptional initiation. At present the fastest progress in the field is expected from a simple reversal of the above strategy (Figure 9B): A complete minidomain is constructed, and the flanking S/MAR elements are removed by the successive action of two different site specific recombinases. For this approach
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F i g u r e 9 . E n g i n e e r i n g t h e g e n o m e : A d d i t i o n , e x c i s i o n and e x c h a n g e r e a c t i o n s catalyzed b y F l p recombinase A . Circular vectors can be integrated at a genomic locus that has been tagged with an identical site. The reaction has to be driven by an excess of the vector and will easily reverse if Flp activity persists after its dilution or degradation. The depicted experiment has been used to complete an artificial domain by the addition of a second S/MAR element (Bode et al., 1996) B . Excision of boundary elements by site-specific recombinases (Flp, Cre) can be used to decompose an artificial domain in a stepwise fashion. The upstream S/MAR is surrounded by FRT sites and is removed by the action of Flp recombinase. In a related reaction, the downstream S/MAR can be excised by Cre recombinase acting upon the lox sites. C . An expression cassette (gene 1) that is surrounded by a wild type FRT site and an FRT linker-mutant can be exchanged for a cassette (gene 2) with an analogous set of sites. This double-reciprocal crossover reaction will delete the plasmid sequences contained in the circular gene 2 vector and will hence result in a so called “clean exchange”.
fluorescence a population of cells was obtained in which excision had occurred with over 90% efficiency. This underscores the fact that excision is a spontaneous event. In a separate step, the downstream S/MAR was removed by Cre recombinase. Here, Cre was expressed stably and expressors were selected via hygromycin resistance, generated by a cotransfected selection marker. The results of a pilot experiment on a cell population, generated by electroporation as for Figure 2 (thereby mostly consisting of single copy constituents), are shown in Figure 10. These data show a moderate effect (25%
we initially used the same 800 bp S/MAR element in both the 5´ and 3´ positions so the results could be related to the position of the element without the need to consider different S/MAR structures. The upstream S/MAR was surrounded by two FRT sites, hence could be excised by a pulse of Flp recombinase. Similarly, the downstream S/MAR was surrounded by two loxP sites, and could be excised by Cre recombinase. Selection of cells which had received the Flp construct was facilitated by a bicistronic vector encoding Flp (first cistron) and GFP (greenfluorescent protein, second cistron). By sorting for green 566
Gene Therapy and Molecular Biology Vol 1, page 567 decrease of expression) for the removal of the 5´ S/MAR and a strong one (85%) for the removal of the corresponding 3´ element. It is noted that these findings agree with the model of S/MAR action depicted in Figure 6 (transition D2 -> D2.1): the unwound structure stored in a S/MAR element downstream from the site of transcriptional initiation can be utilized to release the positive superhelical strain that accumulates in front of a transcribing polymerase. Recent experiments by Wang and Dröge (1996) have demonstrated the persistence of supercoiling even in the presence of topoisomerase activities which resolve topological problems on a longer time scale.
The experiments outlined in Figure 8B will permit an extended series of tests for the properties of individual clones, even at the single cell level. This possibility arises from the use of the LacZneo gene (Walters et al. 1995) the product of which confers neomycin resistance and at the same time allows fluorescence-activated cell sorting (FACS analysis) of clones according to their lacZ expression level. In another system it was shown that integration occurs next to the centromere in up to 50% of clones, which causes position-effect variegation (PEV) unless the chromatin is either kept open by an enhancer-
Figure 10. Probing the activity of S/MARs in situ: excision of the 5´-S/MAR and the 3´-S/MAR from an a r t i f i c i a l m i n i d o m a i n . The experiment followed the outline given in Figure 9 and is based on two identical 800 bp S/MAR elements flanking a LacZ/neo fusion gene. Excision of the 3´-S/MAR by Cre results in a larger effect than excision of the 5´ element.
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Gene Therapy and Molecular Biology Vol 1, page 568 between this and the wild-type site becomes less probable favoring the forward (integration) reaction (Senecoff et al., 1988; Araki et al., 1997). Unfortunately, when compared with wild type FRTs, point mutations also lead to a reduced recombination between the LE and RE sites, resp., and there are even cases where the enhanced stability conferred upon the integrated molecule was outweighed by the far fewer integration events which resulted from an inefficient forward reaction.
like function (Festenstein et. al., 1996) or flanked by insulator-type elements. Both the domain-opener (Zhao et al. 1993) and the insulator functions (Stief et al., 1989; Phi-Van et al., 1996) that have been ascribed to S/MARs should prevent PEV. We hope to trace these functions by successively eliminating the 5´- and the 3´- elements. The effect of each of these deletions on the decay of expression will be traced over an extended period of time (cf. Walters et al., 1996).
There are no identified protein-DNA interactions in the 8 bp spacer sequence of an FRT site. At least six (and possibly all) of these bases can be changed without destroying Flp binding activity. Such changes produce a mutant site that will recombine with a second mutant site of the same composition, but not with one having a different spacer sequence (Schlake and Bode, 1994). We have indicated above (section IVB2) that this is an immediate consequence of the homology check points occurring during the branch migration step of the recombination cycle.
4. The equilibrium problem and its solutions The simple integration/excision system of Figure 9A has one major drawback, caused by the reversibility if the recombination reactions. Since intramolecular excision is kinetically favored over bimolecular integration, insertion products are inherently unstable in the presence of recombinase (Seibler and Bode, 1997). As an example, we have reported the facile excision of retroviral sequences between FRT sites which were strategically placed into the long terminal repeats (LTRs) of a provirus (Schübeler et al, 1997; cf. also Figure 3). Reversal of this step, i.e. use of the remaining site for re-integration, proved to be unfeasible. We have recently shown that this goal can be achieved by applying a very stringent selection system (integration of a promoter- and ATG-free cassette next to a preexisting promoter and translation-initiation site) and by maintaining an open chromatin structure around the target site (Seibler et al, submitted).
Based on these results we have studied the feasibility of a cassette exchange reaction mediated by Flp (RMCE concept, see Figure 8C and Seibler and Bode, 1997). We demonstrated that the double-reciprocal crossover events occurring between FRT couples of identical composition enable the efficient substitution of a recombination target flanked by a wild-type FRT site and an FRT- mutant for another cassette designed in the same way. Since RMCE is a true equilibrium reaction (both the forward and the reverse reaction are bimolecular processes) it proceeds to near completion if the exchange plasmid can be provided at a sufficiently high excess (Seibler and Bode, 1997). So far, the RMCE concept not only provides the most efficient solution for the equilibrium problem but it also enables a “clean” replacement of one expression cassette for another in the sense that prokaryotic vector parts can be deleted during the exchange step by an appropriate placing of the wild type FRT site (F) and the FRT mutant (Fn)
Several measures have been taken to limit the activity of the recombinase to a time interval where a high concentration of the circular exchange vector drives the integration. Conventionally, this is done by generating a pulse of Flp activity from an appropriate concentration of a transiently expressed construct. Since in many cases the transient expression phase is followed by the integration/stable expression of the vector, recombinasemRNA or -protein has been used instead. In another approach the recombinase gene has been placed under the control of an inducible vector (Logie and Stewart, 1995). Alternatively, one could abolish recombinase activity following integration by directing the integration event so that it separates the promoter from its coding sequence (Kilby et al., 1993). Another strategy is to use mutant target sites. For both loxP and FRT exact 13 bp inverted repeats are the recombinase binding sites, which implies a stringent sequence requirement. If a point mutation is introduced in one of the repeats, a recombination between the site with a mutation in the left element (LE) and another site with a mutation in the right element (RE) would yield two recombination product sites, i.e. a wildtype one and one with mutations in LE and RE. Since the LE plus RE site has a dramatically reduced affinity for the recombinase, a subsequent excisional recombination
5. Outlook The ability to perform a clean exchange of one expression cassette for another provides an entirely new way to gently manipulate the genome. In its most stringent form this approach requires that expression patterns remain unperturbed, as assessed by the simultaneous transcription of a selection marker which has to be removed in a second step. This goal can be achieved by a novel two-step strategy called "tag-and-exchange" (Askew et al., 1993) or double replacement (Stacey et al., 1994). In our modification of the concept, an expression cassette carrying the HygTk positive/negative selection marker is introduced in step 1. In the appropriate cell types, this can either be achieved by an 3-type homologous
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Gene Therapy and Molecular Biology Vol 1, page 569 recombination which substitutes an endogenous gene for the HygTk- “tag” or by a random integration followed by the selection of suitable integrants. The presence of the tag can be assessed because the HygTk gene product mediates resistance to hygromycin. Following the outline in Figure 9C, the HygTk tag is removed in step 2 by an exchange reaction utilizing the RMCE principle. Successful events can be screened for the absence of the HygTk cassette (negative selection). This is done in the presence of ganciclovir which is converted to a toxic compound by the thymidine kinase activity of the HygTk fusion gene product. As a result, the initial HygTk tag serves as a selection marker in both steps of the procedure, obviating the need for a marker on the final DNA. This procedure can be performed in embryonic stem (ES-) cells with high efficiency since this cell type does not spontaneously integrate circular DNA. In this way a circular Flp expression construct can be provided at high enough concentrations to generate sufficient recombinase activity. Although it can disappear by dilution or degradation, it will not be incorporated into the genome by an unspecific integration event. An excess of the circular exchange plasmid also can be used whereby the specific exchange mediated by the two sets of Flp sites becomes the favored pathway (Seibler et al, submitted). The combination of targeted gene modification and production of animals derived from ES cells has established a powerful method for studying gene function in the developing animal. Genes can be disrupted, inserted, or modified in the ES cell genome, and the altered cells can be used to generate chimaeric animals. If ES cells contribute to the germ line, chimeras can be outcrossed to produce progeny that are heterozygous or homozygous for the genomic modification. Therefore, using the tag-andexchange concept in combination with the RMCE technology the way is open to exchange an endogenous gene for an analogue carrying gentle mutation(s). Since the method avoids any further modification at this particular locus, the effect of the mutation will become immediately obvious.
V. Can S/MAR functions be derived from sequence information?
their presence with the occurrence of sequence and structural motifs which have subsequently been used to develop algorithms for the prediction of S/MARs from sequence data.
A. Six prominent rules The predictive scheme introduced by Krawetz and colleagues (Kramer et al., 1996; Singh et al., 1997) and Boulikas (1993a,b) makes use of six features which in various combinations confer an affinity for the nuclear matrix or scaffold: (i) DNA replication occurs in association with the matrix, so sites of matrix attachment share certain AT-rich tracts with homeotic protein recognition sites (including several ATTA and ATTTA tracts) and origins of replication. (ii) A number of genes contain TG-rich sequences in their 3'-UTRs which can be S/MARs (Boulikas et al, 1996). (iii) Intrinsically curved DNA occurs within or near several S/MARs, although curvature or bending is no prerequisite for S/MAR activities in vitro (von Kries et al., 1990). ( i v ) Certain dinucleotides, TG, CA or TA, that produce a kink when separated by 2-4 or 9-12 nucleotides, are prominent features of some S/MARs (Boulikas, 1993a). ( v ) Topoisomerase II consensus sequences and cleavage sites are concentrated at sites of nuclear attachment. These have been used to excise complete chromatin domains (Targa et al., 1994). Although this enzyme responds to topology rather than to a strict consensus, the presence of Drosophila and vertebrate consensus sequences have served as important criteria for the prediction of S/MARs. (vi) Many S/MARs contain significant stretches of AT-rich sequences and both the occurrence of An runs (Käs et al., 1993) and (AT)n tracts (Bode et al., 1992) has been implicated in S/MAR functions. These six patterns have been used to define a set of decision rules with which DNA sequences can be searched to find regions having S/MAR potential. Several examples were published which show a reasonable correspondence between these predictions and wet-lab results (Kramer et al., 1996; Singh et al., 1997; Krawetz and Bode, unpublished).
B. Stress-induced duplex destabilization (SIDD)
S/MARs are polymorphic and appear to be distributed throughout the eukaryotic genome. They are specific for eukaryotes, as demonstrated by the observation that S/MAR-scaffold interactions cannot be disrupted by an up to 60,000-fold excess of double-stranded bacterial DNA (Kay and Bode, 1994, 1995). Although prototype S/MARs are AT-rich, they do not share sufficient sequence similarity to allow cross-hybridization (Gasser and Laemmli, 1987; Phi-Van and Strätling, 1990). Biologists have physically identified S/MARs and tried to correlate
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The above criteria indicate that S/MAR activity may be related to structural or topological features which are not strictly linked to primary sequence. Chemical probing and 2D gel analyses of S/MAR constructs under superhelical tension revealed that these elements readily relieve strain by becoming stably base-unpaired. In all cases, unpairing could be shown to initiate at a nucleation site, the core unwinding element (CUE), then extend to a wider region (Figure 11). These observations have led to the suggestion that S/MARs contain efficient base-unpairing
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Figure 11. Stress-induced structures in particular double stranded DNA sequences. Increasing negative superhelical densities lead to base unstacking which initiates at core unwinding elements (CUEs). This process is followed by a more extensive strand separation which finally involves an entire base-unpairing region (BUR). The energy stored in the open structures may be retrieved by nearby cruciform-, Z-DNA- or triplex-forming sequences. CUEs and BURs can be trapped by chloroacetaldehyde (CAA) which forms etheno-derivatives with cytosine and adenosine bases that are located in single-stranded regions. The figure shows a derivatized adenine which is no longer able to base pair.
shown in Figure 12 for several S/MARs integrated into plasmids. These are an 800 bp fragment from the 7 kb S/MAR upstream from the human interferon-ß (huIFN-ß) gene (Bode and Maass, 1988; element IV in Mielke et al., 1990), an inactive mutant of that sequence, and the immunoglobulin µ-chain enhancer-associated S/MAR.
regions (BURs). Since single-stranded character could also be found at S/MARs in living cells, it is possible that duplex destabilization mediates at least some of their functions (Bode et al, 1992, 1995, 1996). This hypothesis is supported by observations that base-unpairing properties correlate with the strength of binding of S/MARs to nuclear scaffold/matrix preparations in vitro, and to the potential of these elements to augment transcriptional initiation rates in vivo (Mielke et al., 1990, Bode et al., 1992, see also Allen et al., 1996).
For these analyses the S/MAR sequences were placed in the pTZ-18R plasmid. This is the same plasmid as was used to experimentally determine the reactivity of the huIFN-ß S/MAR and ist mutant with the single-strand specific reagent chloroacetaldehyde (CAA) (Bode et al., 1992 and Figure 11). In all cases a superhelix density of -0.05 was used, simulating the conditions existing in a bacterial plasmid (Benham et al., 1997). In the resulting destabilization plot a value near zero indicates an
We have recently put these hypotheses to a critical test by calculating the stress-induced duplex destabilization (SIDD) profiles for prototype S/MARs for which chemical reactivity data were already available. Sample results are
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Gene Therapy and Molecular Biology Vol 1, page 571 essentially completely destabilized base pair, which is predicted to denature with almost no input of additional free energy. But partial destabilization, indicated by
intermediate energy values, may also be important, as it may enable protein binding or other events to occur.
Figure 12. Stress-induced duplex destabilization (SIDD-)profiles for prototype S/MARs and a mutant. A. 800 bp fragment from the huIFN-Ă&#x; upstream S/MAR shown in a vector backbone. The S/MAR insert is indicated by the horizontal bar and the CUE is marked by an asterisk. The peaks at map positions 2.2, 3.2 and 3.7 kb are destabilized sites at the amp terminator, amp promoter and f1ori of the vector backbone B . same as A but after mutagenesis of the CUE (light asterix) C. SIDD profile for the murine IgH-enhancer-S/MAR sequence, superimposed on CAA- modification data according to KohwiShigematsu and Kohwi (1990, 1997). Sites for some S/MAR-binding proteins have been added (from Dickinson et al., 1992).
The calculated destabilization profiles show some distinct features which recurred in all other analyses performed to date: (i) Those parts of the sequence derived from the original plasmid are generally stabilized by 8-10 kcal/mole. However, there are three sharp and well separated minima between 2.0 and 3.7/0 kbp which correspond to the terminator and promoter of the "-lactamase gene, resp. and to the f1 origin. All these elements have been the subject of earlier analyses (Figures 2 and 3 in Benham, 1993). For our purposes they serve the role of well defined internal standards; (ii) in striking contrast to the prokaryotic part, the S/MAR sequence (present between 0.2 and 1.0 map units) is chaotically destabilized exhibiting a characteristic succession of minima with a spacing of 200-400 bp over its entire length. Such a modular design is thought to be related to function and may thereby be of diagnostic value (see the analyses by Okada et al., 1996);
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(iii) a core unwinding element (AATATATTT in this case), mapped by chemical labeling techniques to position 0.72 occurs at one of the most destabilized sites on the molecule (Bode et al., 1992); The "-lactamase-associated sites mentioned above were among the first for which local denaturation had been demonstrated by nuclease digestion on superhelical pBR322 DNA (Kowalski et al., 1988). The free-energy parameters governing these transitions have been calculated from these experimental results (Benham, 1992). The plasmid-derived peak centered at position 3.7/0 coincides with the f1 ori which, in the context of this multipurpose plasmid, enables the generation of single stranded DNA after superinfection with a helper phage. Although the f1ori is too short to constitute a S/MAR per se, we have found that it contributes synergistically to scaffold binding in the presence of other S/MAR-sequences (Figure 1 in Mielke et al., 1990).
Bode et al: Architectural principles of S/MARs pCL (Figure 12A) and pCLmut (Figure 12B) contain the unwinding core of the huIFN-ß S/MAR in its wild type and mutagenized form, respectively. A critical comparison shows that the core unwinding element which is stabilized by less than 1 kcal/mol in pCL reaches 8 kcal/mole after mutagenesis, again in perfect agreement with the chemical reactivity data (Bode et al, 1992). We also have calculated destabilization profiles for an extended series of S/MAR elements whose relative binding strengths have been established by S. Michalowski and S. Spiker (in preparation). A prediction of the relative binding strength of these elements was obtained by relating the area covered by these S/MARs in the SIDD profile to the area covered by the ampr- related peaks. The results agreed well with the experimental data (correlation coefficient 0.89). This was better than predictions based on the occurrence of other criteria, such as A-boxes (0.58), AT richness (0.77) and even the occurrence of a motif common to this particular set of sequences (0.81). Evidence from several laboratories shows that some S/MARs cohabit with enhancers (Gasser and Laemmli, 1986). This association is particularly intriguing, as S/MARs have the capacity to augment transcription via a non-enhancer mechanism (IIB). The most thoroughly studied examples are the immunoglobulin !- and µchain intronic enhancers, which are associated with one and with two distinct S/MAR elements, respectively. (Cockerill and Garrard, 1986a,b ; Cockerill et al., 1987). They function in domain opening (Zhao et al., 1993; Bode et al., 1996), which operates during embryonic development (Jenuwein et al., 1993, 1994, 1997; Forrester et al., 1994; Oancea et al., 1997). A regional demethylation occurs in a process that relies on several cisacting modules, including the S/MAR (Lichtenstein et al., 1994; Kirillov et al., 1996; Jenuwein et al., 1997). While any S/MAR sequence appears to be able to function in this reaction, tissue specificity is contributed by the intronic enhancer (Kirillov et al., 1996). For the µ-chain intronic enhancer (Figure 12c) Kohwi-Shigematsu & Kohwi (1990, 1997) have demonstrated an overlap between specific protein binding sites and locations that become stably and uniformly unpaired when this region is subjected to torsional stress. Prominent destabilized sites coincide with both the 3´- and the 5´-S/MAR which have been characterized by Cockerill et al., 1987 and Mielke et al., 1990 (cf. elements XVIl and XVIr). We also have evaluated the destabilization properties of the 992 bp XbaI fragment (Figure 12c) by computation. A striking tripartite destabilization profile occurs in the insert region, in which the (stable) enhancer is bounded by two strongly destabilized flanks. The latter regions coincide with the S/MARs, and also with the regions accessible to
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CAA. The precision of this analysis becomes evident from the fact that the unwinding feature which initiates at a core unwinding element, AATATATTT, then spreads in the 5´ direction and is stalled at the enhancer border (KohwiShigematsu and Kohwi, 1990), is precisely predicted. This directional preference is not readily explained by the mere A+T-contents of neighboring sequences which are both 70%. In contrast to this, the unwinding region 5´ of the enhancer does not influence neighboring regions, i.e. it shows an all or none reactivity towards CAA. This property is reflected by the two steep flanks bordering the CAA-reactive region in the destabilization profile.
C. Occurrence of secondary structures These results raise the question of whether the presence of long stretches of base-unpaired or destabilized duplex DNA is sufficient to account for S/MAR activities, or if these are modulated by alternative stress-induced structures. Schroth and Ho (1995) have demonstrated that strong cruciform forming sequences (inverted repeats, IR) occur at relatively high frequency in yeast (1/19700 bp) and humans (1/41800 bp) whereas triple-helix promoting sequences (mirror repeats, MR) are abundant only in humans (1/49400 bp). While eukaryotic IRs are very A+T-rich, prokaryotic ones have a relatively high G+C-content and occur almost exclusively in transcription termination sites. Base composition is important because cruciforms form more easily in AT-rich sequences. Since strong cruciform and triplex DNA forming sequences are not abundant in the E. coli genome, these results suggest they may have specific roles in eukaryotes, where they are concentrated in S/MARs and in ORIs (Boulikas & Kong, 1993; Boulikas, 1995; Mielke et al., 1996 and below). Still other observations hint at a regulatory role of direct repeats in the replication of eukaryotic genomes. Direct repeats are also common in S/MARs (Opstelten et al., 1989, Mielke et al., 1996). Among the possible supercoiled-induced alternate DNA structures, triplexes are now felt to be the best candidates for serving a role in gene expression, as their requirements for specific environmental conditions and negative supercoiling are the least stringent (Palacek, 1976, 1991). After strand separation, the experimentally best characterized structural change in a negatively supercoiled DNA is the C-type cruciform extrusion. This transition initiates with a coordinated opening of many base pairs to form a large bubble that can be trapped by a single-strand specific reagent like chloroacetaldehyde (F igure 11). As a prelude to opening, the base pairs must be unstacked. This partial relaxation already mediates reactivity to osmium tetroxide, which allows the researcher to visualize subsequent intermediates on the extrusion pathway (Furlong et al., 1989).
Gene Therapy and Molecular Biology Vol 1, page 573 For C-type extrusions, AT-rich BURs may be positioned at the center of a cruciform as in F i g u r e 1 1 . These also could be responsible for a coordinate destabilization of a large domain in the supercoiled DNA, and thereby increase the probability that more distant sites "harvest" the energy stored in these structures. In this case a BUR could be separated from a stress-induced non-B structure, and might be recognized and thereby stabilized by certain scaffold proteins. Clearly, this point needs clarification and it can only be approached when the appropriate experiments have been performed to determine the precise energetics for various alternatives under the same environmental conditions. For the time being, analyses have to be restricted to the sequence features which would be compatible with secondary structure formation in and around the destabilized regions.
1. Potential cruciforms in S/MAR-type sequences
element of the same extension (H. RĂźhl, unpublished). An SIDD analysis of the intron reveals a succession of peaks, one of which represents the actual site of integration. While a more extensive in vitro analysis of the S/MAR potential along the entire sequence is in progress (C. Mielke, unpublished), the question arises whether this particular minimum was preferred by incidence or whether there are signals superimposed to it which might have attracted the retroviral integration machinery. It is demonstrated in Figure 13 that integration had again occurred at the tip of a potential stem loop structure i.e. between the two guanidine residues shown in bold print. While none of these analyses can yet be considered proof for the existence or relevance of these structures (VC), it is certainly tempting to consider their contribution to explain the range of properties associated with S/MAR-type sequences (IIB-E).
VI. Conclusions and perspectives
Several independent lines of evidence support the idea that cruciform structures might be enriched in S/MARs (Boulikas, 1993, 1995): First, hnRNA, a component of the nuclear matrix, is anchored by regions that correspond to DNA inverted repeats. That these features direct origins of replications and S/MARs to the nuclear matrix is suggested by the results of a recent random cloning experiment which found a number of elements that were significantly enriched in IRs. In 77 kb of the human Ă&#x;globin locus 22 potential cruciforms have been found, some of which coincide with S/MAR sequences. The recognition of these sequences could be due to several established protein components of the matrix (review: Bode et al., 1996), among these HMG1 and a number of transcription factors. Using a computer program for a search of regions with the sequence requirements for cruciform formation, we noted an extended inverted repeat in the human interferon-Ă&#x; upstream S/MAR. The corresponding stem-loop structure (F i g u r e 1 3 ) would expose the core unwinding element (ATATTT) on the tip of a loop. We have recently determined, that retroviruses prefer S/MAR-type sequences for their integration into the host's genome (F i g u r e 5 ). Within these sequences we noted a high proportion of direct and inverted repeats (Mielke et al., 1996). When we calculated the corresponding SIDD profiles there were again several coincidences between stabilization minima and potential stem-loop structures (see Int-19 and Int-26 in Figure 13). A much investigated retroviral integration event which leads to an extensive deregulation of the collagen(I) expression is localized in the gene's first intron (Breindl et al., 1984). As in the other reported cases, a 300 bp sequence around this site behaves as a prototype S/MAR
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S/MARs are typically found at the borders of eukaryotic gene domains. A recent compilation (Boulikas, 1995) covers 50 well characterized S/MAR-elements for mammalian, rodent, chicken, Drosophila and plant genes defining domains of 5-400 kb (average size 60 kb). Since there is an inverse relation between domain size and potential gene activity and a frequent association of enhancers with S/MARs, the study of S/MAR organization yields valuable initial information about the nature and expression of those genes that are associated with them. The human genome project is aimed at the localization of all 50-100000 human genes. Progress depends, to a large extent, on the availability of markers that are polymorphic, very common (1E5 to 1E6 copies per genome) and evenly dispersed. Recovery of all S/MARs from human chromosome 19 by Nikolaev et al. (1996) has shown that these are indeed individual and do not belong to any family of repeated sequences (IIB-D, VB). These attributes could make S/MARs valuable genomic markers in sequencing projects. Many S/MAR-related functions seem to depend on particular DNA structures (Boulikas 1995, Singh et al., 1997) which are recognized by distinct sets of single- or double-strand specific binding proteins (Bode et al, 1996). Their pronounced base unpairing character (Bode et al., 1992) together with a possible propensity subsequently to form non-B DNA structures under superhelical tension (Boulikas 1993) may explain the observation that some S/MARs coincide with recombination hot spots (Sperry et al., 1989, Kohwi and Panchenko, 1993). Recently investigated examples include sites of translocation in the human type I interferon gene cluster (Pomykala et al., 1994; Diaz 1995) and at the MLL breakpoint cluster region
Gene Therapy and Molecular Biology Vol 1, page 574
Figure 13. Is base-unpaired DNA stabilized by secondary structure formation? The figure shows sequences at the sites of duplex destabilization which have the potential to yield cruciforms. The CUE (ATATTT) of the human interferon upstream S/MAR is localized at the top of a potential stem-loop structure. Related sites are also the targets for retrovirus integration which is exemplified by a Moloney murine leukemia integration site (Breindl et al., 1984) and by two targets for a retroviral vector (Mielke et al., 1996).
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(Broeker et al., 1996a, b), all of which occur within S/MARs. Currently several projects address the question of whether S/MAR elements, besides structuring the human genome, might also give rise to the extreme instability of these loci. Until recently, the identification of genomic segments associated with the nuclear matrix has essentially relied on biochemical strategies. Sequence searches for S/MARs have met with serious difficulties since, although several characteristic motifs are known, no true consensus is apparent (Boulikas, 1993, Kramer & Krawetz, 1995, Kramer et al., 1996). The novel, structure-related approach that is discussed above (VB-C) suggests that it may be possible to recognize S/MARs on the basis of subtle underlying properties of their sequences. This would allow rapid progress in the localization of functional genes and some of their associated regulatory features (Benham et al., 1997). The availability of an increasing number of S/MAR elements and the characterization of both their common and individual properties will provide valuable insights regarding genomic organization and regulation. In the emerging fields of improving agricultural crops, and human gene therapy the inclusion S/MARs that regulate chromatin structure in transgene constructs appears of immediate use to obtain consistent and authentic expression patterns. Any of these protocols relies on the efficiency of DNA delivery as well as on expression properties. These are profoundly influenced by the nature of the insertion site and the presence of DNA elements with the potential to overcome chromosomal position effects. Site specific recombination systems are being developed which will ultimately allow successive rounds of transformation with different genes inserted into the same locus. This locus could either be an endogenous site which can be targeted without interrupting central genomic functions or a site which has been constructed in situ by the inclusion of elements which define an autonomously regulated gene domain (IVB-C).
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Bode et al: Architectural principles of S/MARs Kohwi-Shigematsu T., and Kohwi Y. (1 9 9 0 ). Torsional stress stabilizes extended base-unpairing in DNA flanking the immunoglobulin heavy chain enhancer. B i o c h e m i s t r y 29, 9551- 9560. Kohwi-Shigematsu T., and Kohwi Y. (1 9 9 7 ). High unwinding capability of matrix attachment regions and ATC-sequence context-specific MAR-binding proteins. in Nuclear S t r u c t u r e a n d G e n e E x p r e s s i o n , (Berezney R and Stein G, Editors), pp111-114, Academic Press. Kohwi-Shigematsu, T., Maass K, and Bode J (1 9 9 7 ). A thymocyte factor, SATB1, suppresses transcription of stably integrated MAR-linked reporter genes. B i o c h e m i s t r y , 36, 12005-12010. Kowalski D., Natale D. A., and Eddy M. J. (1 9 8 8 ). Stable DNA unwinding, not "breathing" accounts for singlestrand- specific nuclease hypersensitivity of specific A+Trich sequences. P r o c N a t l A c a d S c i U S A 85, 94649468. Kramer J. A., Singh G. B., and Krawetz S. A. (1 9 9 6 ). Computer- assisted search for sites of nuclear matrix attachment. G e n o m i c s 33, 305-308. Kricker M. C., Drake J. W., and Radman M. (1 9 9 2 ). Duplication- targeted DNA methylation and mutagenesis in the evolution of eukaryotic chromosomes. P r o c N a t l Acad Sci USA 89, 1075-1079. Kwon H. J., Tirumalai R., Landy A., and Ellenberger T. (1 9 9 7 ). Flexibility in DNA recombination: Structure of the lambda integrase catalytic core. S c i e n c e 276, 126131. Laemmli U. K., Kaes E., Poljak L., and Adachi Y. (1 9 9 2 ). Scaffold-associated regions: cis-Acting determinants of chromatin structural loops and functional domains. Curr Opin Genet Dev 2, 275-285. Lakso M., Pichel J. G., Gorman J. R., Sauer B., Okamoto Y., Lee E., Alt F. W., and Westphal H. (1 9 9 6 ). Efficient in vivo manipulation of mouse genomic sequences at the zygote stage. P r o c N a t l Acad S c i USA 93, 58605865. Levy-Wilson B., and Fortier C. (1 9 8 9 ). The limits of the DNAse I-sensitive domain of the human apolipoprotein B gene coincide with the location of chromosomal anchorage loops and define the 5' and 3'-boundaries of the gene. J B i o l C h e m 264, 21196- 21204. Li Q., and Stamatoyannopoulos G. (1 9 9 4 ). Hypersensitive site 5 of the human Beta locus control region functions as a chromatin insulator. B l o o d 84, 1399-1401. Li X. J., Wang H., and Seeman N. C. (1 9 9 7 ). Direct evidence for Holliday junction crossover isomerization. B i o c h e m i s t r y 36, 4240-4247. Lichtenstein M., Keini G., Cedar H., and Bergman Y. (1 9 9 4 ). B cell-specific demethylation: A novel role for the intronic kappa chain enhancer sequence. C e l l 76, 913923.
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Gene Therapy and Molecular Biology Vol 1, page 581 Gene Ther Mol Biol Vol 1, 581-590. March, 1998.
Synthetic concatemers as artificial MAR: importance of a particular configuration of short AT-tracts for protein recognition Ken Tsutsui Department of Molecular Biology, Institute of Cellular and Molecular Biology, Okayama University Medical School, 2-5-1 Shikatacho, Okayama 700, Japan
_____________________________________________________________________________________ Correspondence to: Ken Tsutsui, Tel: 086-235-7386, Fax: 086-224-5150, E-mail: tsukken@cc.okayama-u.ac.jp
Summary The matrix attachment region (MAR), a class of sequences involved in organization of genomic DNA into looped superhelical domains, i s also believed t o be important i n the regulation o f nuclear functions such as DNA replication, transcription, and recombination. The association of MARs to the nuclear matrix is probably mediated by a variety of proteins which selectively bind to MAR. Because o f the complex nature o f MAR-protein interactions, i t i s s t i l l difficult t o t e l l , from sequence information alone, whether or not a particular DNA region is a MAR. An abundant nuclear protein, termed SP120, is one of the major MAR binding proteins identified so far that selectively binds to AT-rich MARs of different origins i n v i t r o . We have recently found that SP120 also binds to a GCrich concatemer synthesized by random ligation of a short duplex oligonucleotide. Although the result seemed rather paradoxical at first, subsequent experiments with sequence-manipulated concatemers indicated that intactness of short homopolymeric AT-tracts harbored in the concatemer and a particular pattern of their distribution within it are prerequisites for the binding.
B. Characteristic features of MAR
I. Introduction A. Short history Presence of a long-range organization in nuclear DNA was originally proposed in early studies analyzing the sedimentation behavior of detergent-lysed cells in a solution containing intercalating agents (Cook and Brazell, 1975; Ide et al., 1975). The interphase chromatin DNA appeared to be folded into supercoiled loop domains through its attachment to a subnuclear skeletal structure, which was later isolated and characterized as the nuclear matrix (Berezney and Coffey, 1977) or nuclear scaffold (Mirkovitch et al., 1984). This model acquired further support when characteristic DNA regions were discovered at the attachment sites (Cockerill and Garrard, 1986; Mirkovitch et al., 1984). Such DNA regions, designated SAR or MAR, have been allocated in many cloned genes and implicated in the regulation of gene expression and other nuclear processes (reviewed in Gasser et al., 1989; Zlatanova and van Holde, 1992; Boulikas, 1995).
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Comparison of MAR sequences identified thus far revealed some characteristic features. 1) Relatively long DNA region is required for the attachment and very few MARs are shorter than 300 bp. 2) MARs are usually located in noncoding regions. 3) With a few exceptions MARs are rich in adenines (A) and thymines (T); A+T content of a typical AT-rich type MAR is higher than 65%. 4) Regulatory sequences are frequently present in the vicinity of MAR or cohabit with MAR. These include replication origins, transcriptional enhancers and promoters. 5) Other sequence motifs like AT-tracts, DNA unwinding elements, clustered recognition sites for various transcription factors, inverted repeats or palindromes, cleavage consensus for DNA topoisomerase II, curved/kinked DNA motifs, left handed and triple helical structures, and DNase I-hypersensitive sites are frequently associated with MARs (Boulikas, 1995). 6) MARs appear to function beyond species barrier. For instance MARs isolated from yeast can specifically bind to the nuclear matrix prepared from mouse cells.
Tsutsui: SP120 interacts with short homopolymeric AT-tracts
C. SP120 and other MAR binding proteins Since MAR sequences are heterogeneous in nature, proteins interacting with MAR can also be heterogeneous. Indeed, several nuclear proteins including ARBP (von Kries et al., 1991), SATB1 (Dickinson et al., 1992), lamin B1 (LudĂŠrus et al., 1992), SAF-A (Romig et al., 1992), nucleolin (Dickinson and Kohwi-Shigematsu, 1995), histone H1 (Izaurralde et al., 1989), and DNA topoisomerase II (Adachi et al., 1989) have already been shown to bind MAR with considerable affinity. We have identified an MAR binding protein, termed SP120, which is associated with the nuclear matrix (Tsutsui et al., 1993). Sequence determination of cDNA for the rat SP120 showed that the protein is a homologue of human hnRNP U protein (Kiledjian & Dreyfuss, 1992), one of the major components of the heterogeneous nuclear RNA-protein complex (hnRNP). U protein had been characterized as an RNA binding protein by UV-induced cross-linking experiments in vivo (Dreyfuss et al., 1984) and suggested to be involved in the processing of pre-mRNA (Portman and Dreyfuss, 1994). Therefore, SP120/hnRNP U protein is a multifunctional protein operating in those nuclear processes. Using partial cDNA segments expressed in E. coli, a putative MAR binding site of SP120 has been located within the C-terminal tail region of 171 amino acids, designated RG domain, that contains 16 repeats of Arg-Gly dipeptide (Tsutsui et al., unpublished).
D. Aims of this study Molecular interactions involving MAR at the base of chromatin loops can be quite complex and probably proteinprotein interactions would also be important in the stabilization of "anchorage complex". What determines the binding specificity of MARs toward the matrix, therefore, is a difficult question to answer. However, it would be reasonable to assume that relatively small numbers of proteins play a key role to recognize MAR sequences and serve as a nucleation center or a platform for subsequent association of additional proteins. To evaluate this model, sequence motifs recognized by these MAR binding proteins must be nailed down within individual MARs. The proteins listed above are abundant nuclear proteins and expressed ubiquitously except for SATB1 which is specific to thymus. Although consensus motifs for the binding of ARBP (Buhrmester et al., 1995) and SATB1 (Dickinson et al., 1992) have been proposed, those for the other proteins are less characterized. Before searching for the sequence motifs recognized by SP120, we first tried to identify essential regions within a representative MAR reported previously. In the first part of this article, relative affinities of subfragments from the mouse Ig! MAR are analyzed in an in vitro binding reaction with isolated nuclear matrix enriched with SP120. The
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results suggest an interesting possibility that the MAR activity is elicited through a synergistic interaction of two subdomains separated no farther than 300 bp (Okada et al., 1996). The second part describes experiments with purified SP120 and synthetic concatemers, which suggested that multiple A n/Tn tracts positioned in a particular configuration are essential for the recognition by SP120.
II. Analysis of subdomain structures in natural MAR A. Methods for assessment of MAR activity All the data presented here are based on the in vitro binding assay in which a preferential binding of end-labeled MAR fragments to isolated nuclear matrix is measured in the presence of a large excess of unlabeled competitor DNA (Tsutsui et al., 1993). The matrix preparation used here is similar to the "nuclear scaffold" in that histones were extracted with lithium diiodosalicylate after stabilization of nuclei with Cu 2+ (Tsutsui et al., 1988). SP120 is highly concentrated in the matrix, being a single major MAR binding protein detectable by southwestern blotting. The 32Plabeled DNA fragments bound to the matrix was directly counted in a scintillation counter or analyzed by electrophoresis in 5% polyacrylamide gels after digesting the matrix with proteinase K. The radioactive DNA bands were quantified by densitometric scanning of autoradiograms.
B. The intronic MAR of mouse Ig gene The MAR localized within the mouse immunoglobulin k gene is a typical example of AT-rich MARs except for its intronic location, consisting of a stretch of DNA (370 bp) with high A+T content (70%). This MAR is interesting in functional aspects since, together with the nearby transcriptional enhancer, it has been shown to be required as a cis element for the enhanced expression (Xu et al., 1989), demethylation (Lichtenstein et al., 1994), and somatic hypermutation (Betz et al., 1994) of Ig! transgenes. As shown in F i g u r e 1 , numerous short stretches of consecutive A's and T's are contained in the MAR. Importance of AT-tracts for protein recognition has been noticed in previous studies (Adachi et al., 1989; Käs et al., 1989). Comparison of sequences for known MARs strongly suggested that these tracts (referred to as A- or T-patches hereafter) are a characteristic landmark for MARs. Another interesting observation is that the occurrence of both patches appears to be essential for the MAR activity since a biased abundance of T-patches, for instance, shows a poor correlation.
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Figure 1. Short homopolymeric AT-tracts cluster in the intronic MAR of mouse immunoglobulin ! gene. Homopolymeric stretches (n"3) of adenines (A-patch) and thymines (T-patch) are highlighted by light shade and dark shade, respectively. Cleavage sites for the restriction enzymes used in this study are indicated. Figure 2. Relative binding affinities of restriction fragments to the nuclear matrix. Ig! MAR was cut with the enzymes shown on the left side (also abbreviated on the line map. A, AluI; S, SspI; M, MboII; Dd, DdeI; Dr, DraI). Unlabeled internal fragments are omitted from the figure. Fragments with high, low, and negligible (1%>) affinities are represented by filled, hatched, and open bars, respectively. The boxed figures stand for percentages of input fragments bound to the matrix.
input is retained on the matrix. Both fragments generated by a cleavage at the unique MboII site show a much reduced, but still significant binding of 5%. This level of binding can be easily overlooked but it has a prime importance as a contri-
C. Ig MAR is composed of two elementary subdomains As the minimal region required for the binding activity of this MAR had not been analyzed, we first compared the binding affinities of several restriction fragments (summarized in Figure 2). Under the experimental conditions used here, about 20% of the full length MAR 583
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Figure 3. Effects of dimerization of MAR fragments. (a) Self-dimerization of the MboII fragments. (b) Dimerization of the MboII-AluI fragment. pMM22 is a control construct with an extension of nonMAR segment derived from the # lactamase gene.
bution from elementary local structures, because the binding activity can be largely restored by their self-dimeriza-tion (see below). Further fragmentation of the MboII 3'-fragment suggests that the region between the MboII site and the downstream AluI site is required for the residual binding. Does the large decrease in the affinity after cleavage with MboII simply imply that the whole intact MAR is essential for the high affinity? The experiment shown in Figure 3a reveals, however, that this is not the case. Self-dimerization of the 5' half (S) or 3' half (L) of Ig! MAR greatly increases their affinity to the matrix. This effect is more evident with the latter half; the dimer fragment (2L) shows even greater affinity than the original MAR.
enhancement or synergism is observed (Figure 3b). This effect is not due to the doubled fragment length since a control experiment with the monomer fragment extended by a nonMAR sequence (A+T=50%) showed no effect.
D. Synergism between the subdomains The MboII-AluI segment may thus serve as a minimal sequence element that confers MAR-like features upon any DNA, provided that it is duplicated in the same molecule. This possibility can be tested by inserting nonMAR spacers of varying length between the monomer units of the dimer clone (Figure 4). As expected, DNA fragments flanked by the duplicated elements are strong MARs as long as they are
The binding experiments with restriction fragments (Figure 2) suggested that the 3' portion of L fragment is dispensable. When the DNA segment between the MboII site and the 3' AluI site is dimerized , a similar level of 584
Gene Therapy and Molecular Biology Vol 1, page 585 not separated by more than 300 bp. As the spacer length exceeds 500 bp, however, the binding affinity decreases abruptly and levels off to the monomer level at about 3 kb. Therefore, the synergistic effect does not propagate beyond 500 bp. It appears that each subdomain is complexed with a subset of matrix proteins and a cooperative interaction between these complexes contributes to the increased affinity. Obviously, the complexes should be positioned within 300 bp for the interaction to occur. This excludes a simple DNA-looping-out mechanism. The bipartite subdomain organization could be a general feature of ATrich MARs. Similar observations have been reported previously on the Drosophila histone gene MAR (Gasser and Laemmli, 1986) and chicken lysozyme 5' MAR (von Kries et al., 1991). This principle may also be applied to the Drosophila fushitarazu gene MAR (Tsutsui et al., unpublished).
130 bp) has been identified in the Ig! MAR, next approach would be to compare the affinities of mutated subdomain sequences in which AT-patches are disrupted one by one. Before completion of this type of experiments, however, another effective strategy was suggested from a rather serendipitous finding. In a purification protocol for a transcription factor IRF-2 using a DNA-affinity resin, a nuclear protein of 120 kDa copurifies with IRF-2 (Vaughan et al., 1995). Although this is actually a contaminating protein, it shows a strong affinity to the concatemerized recognition sequence for IRF-2 as revealed by southwestern blotting. The sequence shown in Figure 5a resides in the promoter region of human histone H4 gene but no MAR has been identified nearby. When the sequence is mutated by an insertion of A/T-pair (Figure 5b), the binding of IRF-2 is abolished whereas the 120 kD protein persists to bind, indicating that this protein has less stringent sequence requirements. An interesting feature of the IRF-2 recognition sequence is the presence of two AT-patches in a GC-rich context (A+T content of the mutant 26-mer is only 42%). When the mutant duplex is ligated through its sticky ends, two alternative orientations are expected for each monomer. Inverted ligation between neighboring monomer units results in the generation of both A- and T-patches on the same strand. This implies a variegated presentation of AT-patches in the ligation product, which is also a characteristic feature of natural MARs. Based on these observations we suspected that the protein might be SP120.
B. The concatemer interacts with SP120
Figure 4 . Attenuation of the synergism between MAR subdomains as a function of increasing distance. The dimerized MboII-AluI fragments (fat arrows) were separated by insertion of nonMAR spacers derived from plasmid pUC18 (thick lines).
III. Synthetic concatemers as an artificial MAR A. Initial hints The frequent occurrence of AT-patches in MARs suggests that they interact directly with MAR binding proteins including SP120. Once the elementary subdomain (about 585
Before the synthesis of concatemer to test its possible binding with SP120, one modification was made on the mutant sequence (Figure 5c). This base change introduces restriction sites only between the monomer units ligated in inverted orientations (Figure 5d), enabling easy confirmation of the ligation product and cleavage of concatemers at specific sites. As expected, the concatemer synthesized by ligating the oligomer duplex shown in Figure 5c exhibits a strong binding to SP120 on southwestern blots. Poly(dA)â&#x20AC;˘poly(dT), a synthetic duplex DNA frequently used as a competitor for AT-rich MARs, competes the binding effectively, indicating that the concatemer behaves like AT-rich MARs despite its low AT content. When the concatemer is digested with either BglII or FbaI, the binding decreases to a basal level. This is consistent with the predicted importance of both AT-patches in the concatemer binding. The concatemer immobilized on Sepharose beads selectively binds SP120 in nuclear extracts and can be used as an affinity resin to purify the protein. The specific binding of the concatemer was confirmed by a nitrocellulose filter
Gene Therapy and Molecular Biology Vol 1, page 586
Figure 5. Sequences of oligonucleotide duplexes for concatemer synthesis. AT-patches are shaded. (a) recognition motif of IRF-2 (wild type sequence). (b ) mutant sequence. (c ) modified mutant. (d) structure of a representative concatemer containing all possible orientations of neighboring monomer units (bars). Dark shaded areas in the bar represent AT-patches. Restriction sites created after ligation are also indicated. The upper strand in (c ) containing T-patches are designated "A", and the complementary lower strand, "B". Using this definition, the concatemer sequence can be described either as AABBA (upper strand) or as BAABB (lower strand).
activity is accounted for by only a subset of RC molecules with a particular pattern of patch distribution that are contained in the heterogeneous population.
binding assay with purified SP120. The dissociation constant estimated from these data is in the same order as Ig! MAR (about 3 nM).
D. Patch mutants
C. Concatemers with defined monomer orientations
The possible involvement of AT-patches in the protein recognition can be tested more directly with RCs with altered patches. Three categories of mutant sequences were designed and RCs were synthesized by random ligation of mutant oligos (Figure 7). In the "patch disruptants", both T 3- and T4-patches are disrupted by replacing T's with G/C or A. In the "patch convertant", only the T4-patch is converted to an A4-patch. All these concatemers show a marginal binding to SP120, indicating that the intactness of AT-patches are indeed required for the binding.
Use of synthetic concatemers as an artificial MAR is an extremely powerful strategy to delineate sequence requirements for the recognition by SP120. Easiness of obtaining mutated sequences is an obvious advantage. Inaddition, the GC-rich background of the concatemer should provide a highly informative environment to assess the importance of AT-rich motifs in it. Orientation of monomer units in the concatemer described above are randomized because of the palindromic nature of the overhang or sticky ends. In the following sentences this type of concatemer is referred to as "RC" for random concatemer. It is possible to synthesize other types of concatemers simply by manipulating the overhang sequences; "DC" and "AC" representing direct and alternating orientations, respectively (Figure 6). Also here, the terminal sequences were designed so as to introduce junctional restriction sites.
In the "phase mutants", the two T-patches are either moved toward the center without changing the spacing, or the distance between them are elongated. Distribution pattern of AT-patches in these concatemers is different from that in the wild type RC. Although a decreased binding is observed with these mutants, a significant level of binding persists. An important conclusion from this experiment is that functional patches are homopolymeric and patches with mixed A's and T's have no contribution to the binding. The decreased affinity of the patch convertants, whose A/T patch ratio is the same as in wild RC, suggests again the distribution pattern of patches is the determinant factor for successful binding.
Comparison of affinities of these concatemers to purified SP120 ,using a filter binding assay or southwestern blotting, gives an unequivocal result; only RC shows a significant binding. It should be emphasized that all the concatemers have identical AT content and the same number of ATpatches. Thus, the result strongly suggests that the binding 586
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Figure 6. Synthesis of concatemers with defined monomer orientations. Complementary oligonucleotide pairs shown in the left column were annealed and ligated. Junctional restriction sites created in the concatemers are shown in shaded boxes and expected restriction fragments are shown in the right column. Structure of the concatemers were confirmed by fragmentation analysis with these enzymes.
E. Hexamer is the shortest concatemer with binding activity Under the ligation conditions used here, the resulting RCs are heterogeneous in size, ranging from 50 bp (dimer) to over 2,000 bp. It is anticipated that longer RC binds more tenaciously to SP120. To determine the minimal size of RC capable of interacting with SP120 with significant affinity, RC was size-fractionated in agarose gels and oligoconcatemers were extracted from the gel. When individual oligomers were subjected to the filter binding assay, a significant binding was observed with oligomers longer than hexamer but not with shorter ones (dimer to pentamer).
Figure 7. Oligonucleotides used for the synthesis of patch mutants. Only the T-patch strands are shown. Positions of Tpatches and altered bases are highlighted.
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This result suggests that 156 bp is the minimal length to harbor all essential AT-patches supporting the MAR activity. Active hexamer units may be equivalent to the elementary subdomains of Ig! MAR, and when they are duplicated in RC, synergistic interaction between them should largely promote the binding. Interestingly, the size of the hexamer is comparable to that of the Ig! MAR subdomain (about 130 bp). In light of available evidence it should be possible to determine the configuration of essential AT-patches that confers MAR activity to RC. One way to do this is to clone the heterogeneous concatemer in a plasmid vector and examine the binding activity of each clone. Subsequent alignment of a battery of sequences for active concatemers should reveal the pattern of essential patches. Although we have made several trials with different cloning systems, no clones with desired inserts have been obtained probably due to the instability of repetitive sequences in host bacteria.
Tsutsui: SP120 interacts with short homopolymeric AT-tracts Therefore, we decided to synthesize all possible hexaconcatemers by a similar strategy as described above. There exist 36 different hexamers theoretically (F i g u r e 8 ). To synthesize all the hexamers, 26 different 26-mer oligonucleotides with complementary overhangs are required. Six sets of complementary oligos are first annealed pairwise and then mixed and ligated. The resulting concatemer is heterogeneous in size but the monomer units are ligated in desired orientations and BamHI sites are introduced at every 6 units. Digestion with BamHI releases a hexamer which can be purified by polyacrylamide gel electrophoresis. Experimental results with hexamers are not shown here since the work along this line is still under way.
F. MAR organization and mode of interaction with proteins Based on the results presented in this article, I propose here the following principles for MAR organization. 1) MAR activity is exerted by a synergistic interaction between at least two subdomains located no farther than 300 bp apart. 2) Each subdomain contains essential AT-patches positioned with particular intervals. The latter principle was deduced from the binding experiments with concatemers and purified SP120. This could be generalized, however, to any MARs because a similar mode of MAR-protein interaction appears to involve other abundant MAR binding proteins, e.g., histone H1 and topoisomerase II. It is possible that essential AT-patches are the sites interacting directly with MAR binding proteins. The Arg, Gly-rich C-terminal domain of SP120 may selectively recognize AT-patches by its insertion into the small groove of DNA which is significantly narrowed at homopolymeric AT-tracts. As this type of interaction is usually weak, multiple contacts between MAR and proteins would be essential for a stable complex to form. The complex can be formed either by disordered aggregation of proteins or by organized interaction between protein molecules (F igure 9). In the latter case the MAR subdomain is likely to be folded in a particular way. This model is also consistent with the ordered positioning of AT-patches in the subdomain. It should be pointed out finally that other factors like bent structure or inverted repeats might also be involved since these motifs are indeed enriched in the concatemers. We have not done any systematic experiments to test this possibility.
IV. Prediction of MAR from sequence information As genome projects on human and other species advance, sequence databases are increasingly flooded with uncharacterized genomic sequences. Since MAR is related to many important nuclear functions, it would greatly facilitate 588
the prediction of functional organization of unknown genomic regions if one could predict the precise genomic location of MARs by using computer-assisted search methods. This information may also be useful in gene manipulation technologies including gene therapy. The common strategy adopted in recent works along this line is to search for a variety of sequence patterns that are known to occur frequently in MARs, e.g., AT- or GT-rich short tracts, curved or kinked DNA motifs, or topoisomerase II cleavage motifs. The statistical significance calculated for observed frequencies of these patterns are then combined to derive a probability function ("MAR potential") which is computed over the region of interest using a sliding window algorithm (Singh et al., 1997). This survey protocol successfully detected known MARs in several genomic regions. We have applied the MAR organization principle formulated above to predict MARs in various DNA regions containing known MARs and obtained satisfactory results with considerably small incidence of false negatives and false positives. The method also predicted MARs in some genomic regions where presence of MAR had not been assessed experimentally. The MAR activity of candidate regions was confirmed later by binding assays with isolated nuclear matrix (Tsutsui et al., unpublished).
Acknowledgments I thank Drs. Shoshiro Okada and Kimiko Tsutsui for their contributions to this work, and Mika Ishimaru for technical assistance.
References Adachi, Y., Käs, E., and Laemmli, U.K. (1 9 8 9 ) Preferential, cooperative binding of DNA topoisomerase II to scaffoldassociated regions. EMBO J. 8, 3997-4006. Berezney, R., and Coffey, D.S. (1 9 7 7 ) Nuclear matrix. Isolation and characterization of a framework structure from rat liver nuclei. J . C e l l B i o l . 73, 616-637. Betz, A.G., Milstein, C., González-Fernández, A., Pannell, R., Larson, T., and Neuberger, M.S. (1 9 9 4 ) Elements regulating somatic hypermutation of an immunoglobulin kappa gene: critical role for the intron enhancer/matrix attachment region. C e l l 77, 239-248. Boulikas, T. (1 9 9 5 ) Chromatin domains and prediction of MAR sequences. I n t . R e v . C y t o l . 162A, 279-388. Buhrmester, H., von Kries, J.P., and Strätling, W.H. (1 9 9 5 ) Nuclear matrix protein ARBP recognizes a novel DNA sequence motif with high affinity. B i o c h e m i s t r y 34, 4108-4117. Cockerill, P.N., and Garrard, W.T. (1 9 8 6 ) Chromosomal loop anchorage of the kappa immunoglobulin gene occurs next to the enhancer in a region containing topoisomerase II sites. C e l l 44, 273-282.
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Tsutsui: SP120 interacts with short homopolymeric AT-tracts Figure 8. List of all possible hexaconcatemers. Monomers with the alternative orientations are shown by red and yellow bars. T- and A-patches are painted dark blue and green, respectively. Figure 9. Models for organization of MAR subdomains by interacting proteins. (a) Complex formation by disordered aggregation of proteins. (b ) Formation of an organized complex aided by specific protein-protein interactions. AT-patches are shown by dark disks and proteins by shadowed spheres.
Cook, P.R., and Brazell, I.A. (1 9 7 5 ) Supercoils in human DNA. J . C e l l S c i . 19, 261-279.
Binding of matrix attachment regions to lamin B1. C e l l 70, 949-959.
Dickinson, L.A., Joh, T., Kohwi, Y., and Kohwi-Shigematsu, T. (1 9 9 2 ) A tissue-specific MAR/SAR DNA-binding protein with unusual binding site recognition. C e l l 70, 631-645.
Mirkovitch, J., Mirault, M.-E., and Laemmli, U.K. (1 9 8 4 ) Organization of the higher-order chromatin loop: specific DNA attachment sites on nuclear scaffold. C e l l 39, 223232.
Dickinson, L.A., and Kohwi-Shigematsu, T. (1 9 9 5 ) Nucleolin is a matrix attachment region DNA-binding protein that specifically recognizes a region with high base-unpairing potential. M o l . C e l l . B i o l . 15, 456-465. Dreyfuss, G., Choi Y.D., and Adam S.A. (1 9 8 4 ) Characterization of heterogeneous nuclear RNA-protein complexes in vivo with monoclonal antibodies. M o l . C e l l . B i o l . , 4, 1104-1114. Gasser, S.M., and Laemmli, U.K. (1 9 8 6 ) The organization of chromatin loops: characterization of a scaffold attachment site. EMBO J. 5, 511-518. Gasser, S.M., Amati, B.B., Cardenas, M.E., and Hofmann, J.F.X. (1 9 8 9 ) Studies on scaffold attachment sites and their relation to genome function. I n t . R e v . C y t o l . 119, 5796. Ide, T., Nakane, M., Anzai, K., & Andoh, T. (1 9 7 5 ) Supercoiled DNA folded by non-histone proteins in cultured mammalian cells. Nature 258, 445-447. Izaurralde, E., Käs, E., and Laemmli, U.K. (1 9 8 9 ) Highly preferential nucleation of histone H1 assembly on scaffoldassociated regions. J . M o l . B i o l . 210, 573-585. Käs, E., Izaurralde, E., and Laemmli, U.K. (1 9 8 9 ) Specific inhibition of DNA binding to nuclear scaffolds and histone H1 by distamycin. The role of oligo(dA).oligo(dT) tracts. J . M o l . B i o l . 210, 587-599. Kiledjian, M., and Dreyfuss, G. (1 9 9 2 ) Primary structure and binding activity of the hnRNP U protein: binding RNA through RGG box. EMBO J. 11, 2655-2664. Lichtenstein, M., Keini, G., Cedar, H., and Bergman, Y. (1 9 9 4 ) B cell-specific demethylation: a novel role for the intronic kappa chain enhancer sequence. C e l l 76, 913-923. Ludérus, M.E., de Graaf, A., Mattia, E., den Blaauwen, J.L., Grande, M.A., de Jong, L., and van Driel, R. (1 9 9 2 )
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Okada, S., Tsutsui, K., Tsutsui, K., Seki, S., and Shohmori, T. (1 9 9 6 ) Subdomain structure of the matrix attachment region located within the mouse immunoglobulin ! gene intron. B i o c h e m . B i o p h y s . R e s . C o m m u n . 222, 472-477. Portman, D.S., and Dreyfuss, G. (1 9 9 4 ) RNA annealing activities in HeLa nuclei. EMBO J. 13, 213-221. Romig, H., Fackelmayer, F.O., Renz, A., Ramsperger, U., and Richter, A. (1 9 9 2 ) Characterization of SAF-A, a novel nuclear DNA binding protein from HeLa cells with high affinity for nuclear matrix/scaffold attachment DNA elements. EMBO J. 11, 3431-3440. Singh, G.B., Kramer, J.A., and Krawetz, S.A. (1 9 9 7 ) Mathematical model to predict regions of chromatin attachment to the nuclear matrix. N u c l e i c A c i d s R e s . 25, 1419-1425. Tsutsui, K., Tsutsui, K., & Muller, M.T. (1 9 8 8 ) The nuclear scaffold exhibits DNA-binding sites selective for supercoiled DNA. J B i o l C h e m 263, 7235-7241. Tsutsui, K., Tsutsui, K., Okada, S., Watarai, S., Seki, S., Yasuda, T., and Shohmori, T. (1 9 9 3 ) Identification and characterization of a nuclear scaffold protein that binds the matrix attachment region DNA. J . B i o l . C h e m . 268, 12886-12894. Vaughan, P.S., Aziz, F., van Wijnen, A.J., Wu, S., Harada, H., Taniguchi, T., Soprano, K.J., Stein, J.L., and Stein, G.S. (1 9 9 5 ) Activation of a cell-cycle-regulated histone gene by the oncogenic transcription factor IRF-2. Nature, 377, 362-365. von Kries, J.P., Buhrmester, H., and Strätling, W.H. (1 9 9 1 ) A matrix/scaffold attachment region binding protein: identification, purification, and mode of binding. C e l l 64, 123-135.
Gene Therapy and Molecular Biology Vol 1, page 591 Xu, M., Hammer, R.E., Blasquez, V.C., Jones, S.L., and Garrard, W.T. (1 9 8 9 ) Immunoglobulin kappa gene expression after stable integration. II. Role of the intronic MAR and enhancer in transgenic mice. J . B i o l . C h e m . 264, 21190-21195. Zlatanova, J.S., and van Holde, K.E. (1 9 9 2 ) Chromatin loops and transcriptional regulation. C r i t . R e v . E u k a r . G e n e Expr. 2, 211-244.
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Gene Therapy and Molecular Biology, Vol 1, page 591 Gene Ther Mol Biol Vol 1, 591-598. March, 1998.
Replicon map of the human dystrophin gene: asymmetric replicons and putative replication barriers Lilia V. Verbovaia1,2 and Sergey V. Razin1,3 . 1Institute of Gene Biology RAS, Vavilov St. 34/5, 117334 Moscow, Russia. 2International Centre for Genetic Engineering and Biotechnology, Padriciano 99, I-34012 Trieste, Italy. 3Institut Jack Monod, 2, place Jussieu-tour 43, 75251 Paris, CEDEX 05, France.
_________________________________________________________________ Correspondence to: Sergey V. Razin Tel: +7-095-135 97 87; Fax: +7-095-135 41 05; E-mail: razin@mx.ibg.rssi.ru
Summary Using the replication direction assay and oligonucleotide probes designed on the basis of the known exon sequences of the human dystrophin gene we have made a replicon map of this giant gene. It has been found that dystrophin gene is organized into at least six replicons ranging in size from 170 to more than 500 kb. One of the replicon junctions (sites of replication termination) was mapped in intron 44, i.e. roughly in the same area where the major recombination hot spot is located. It is also worth mentioning that the central part of the dystrophin gene (exons 8 48) is organized into relatively short symmetrical replicons surrounded by two extended regions of apparently unidirectional replication (exons 1 - 8 and exons 49 - 64). These observations suggest for the first time that there should be certain signals for the termination of replication in euchromatic areas of the genome of higher eukaryotes. Furthermore, it may be concluded that the replication of the central part of dystrophin gene must be completed much faster than the replication of its ends. This may induce some topological stresses resulting in an increased rate of chromosomal rearrangements within this gene. The experimental approach used in our study may be helpful for fast analysis of the replication structure of other areas of the human genome provided that these areas are saturated with STS markers.
Although these sites can be mapped using the analysis of replication polarity (see below and also Handeli et al., 1989), it is possible that their positions are determined simply by a distance from the replication origins and by the speed of replication forks progression. Such is indeed the case in the simian virus 40 circular genome, as the insertion in one arm of the SV-40 replicon of a DNA sequence element retarding the progression of the replication fork was found to cause a displacement of the replication termination site in the direction of the more slowly moving replication fork (Rao et al., 1988; Rao, 1994). In yeast cells the termination of replication does not occur at specific places determined (at least in nonnucleolar regions) by any specific DNA sequence element. It appears to be a consequence of converging of the replicating forks within a relatively broad region (Zhu et al., 1992). At the same time, some DNA sequences pausing the replication forks progression (such as the transcription termination signal for RNA polymerase I) were reported to serve as preferential sites of replication
I. Introduction The human dystrophin gene is the largest gene so far identified and characterized. It extends over 2 mb on the short arm of the X-chromosome (Burmeister et al., 1988). This gene frequently undergoes different rearrangements causing Duchenne or Becker muscular dystrophy (Wapenaar et al., 1988; Den Dunnen et al., 1989; Blonden et al., 1991). Analysis of the replication structure of the dystrophin gene may give new insight into the mechanisms of this gene rearrangement as it seems probable that at least some recombination events occur in connection with DNA replication. It has long been shown that the genome of higher eukaryotes is replicated as a set of quazi-independent replication units (replicons). Each replicon seems to possess a specific site (or area) where the replication starts (for a review see Hamlin, 1992; DePamphilis, 1993; Hamlin and Dijkwel, 1995). As far as the sites of termination of DNA replication (i.e. replicon junctions) are concerned, the situation seems to be less clear.
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Verbovaia and Razin: Replicon Map of the Human Dystrophin Gene termination in yeast and mammalian cells (Umek et al., 1989; Kobayashi et al., 1992; Little et al., 1993). One may be surprised to realise how little we know about replication structure of DNA of higher eukaryotes. Even the average size of replicons constitutes a matter of discussion. The common view is based on the results of DNA fiber radioautography studies carried out more then 20 years ago. These studies lead to a conclusion that DNA of higher eukaryotes is organized in clusters of simultaneously working replicons. The size of individual replicons within a cluster was estimated as 50 to 300 kb (Huberman and Riggs, 1966, 1968; Callan, 1974; Stubblefield, 1974; Edenberg and Huberman 1975; Painter, 1976). This common interpretation of the DNA fiber radioautography data was, however, questioned by Liapunova and coauthors who presented arguments for the much larger size of replicons (150-900 kb) in mammalian cells and for the absence of replicon clusters (Yurov Yu. B. and Liapunova, 1977; Liapunova, 1994). Several procedures for mapping replication origins in
mammalian genome have been developed recently (for review see Hamlin, 1992; Vassilev and DePamhilis, 1992; DePamphilis, 1993; Hamlin and Dijkwel, 1995). However, most of these procedures are not suitable for the analyzis of replication structure of large genomic areas. Only one modern protocol, namely that based on the determination of the polarity of leading DNA strand synthesis (Handeli et al., 1989; Burhans et al., 1991) may be used for this purpose as it is relatively simple and permits the approximate positions of both replication origins and termination sites to be mapped. Here we are presenting a replicon map of the dystrophin gene constructed using the replication direction assay. It has been found that this gene is organized into at least six replicons ranging in size from 170 to more than 500 kb. One of the replicon junctions (sites of replication termination) was mapped in intron 44, i.e. roughly in the same area where the major recombination hot spot is located (Wapenaar et al., 1988; Den Dunnen et al., 1989; Blonden et al., 1991). The experimental approach used in our study (utilization of oligonucleotide probes in the replication direction assay) may be helpful for fast analysis of the replication structure of other areas of the human genome provided that these areas are saturated with STS markers.
II. Results A. Mapping approach Determination of the polarity of leading DNA strands synthesis became possible due to the demonstration that the inhibition of protein synthesis in proliferating cells preferentially suppresses the synthesis of the discontinuous (lagging) DNA strand. Hybridization of the nascent DNA synthesised under these condition with strand-specific probes can thus be used to assay the polarity of leading DNA strand synthesis (Handely et al., 1989; Burhans et al., 1991). The principle of the abovedescribed mapping protocol is illustrated in Fig. 1. Although the mechanism of imbalanced synthesis of leading and lagging DNA strands in the presence of protein synthesis inhibitors is still not known, the validity of the approach has been verified in experiments with different genomic areas (Handely et al., 1989; Burhans et al., 1991; Kitsberg et al., 1993) and can hardly be questioned. It was originally proposed to use as strandspecific probes for the replication direction assay the RNA chains transcribed in opposite directions from the same DNA fragment (Handely et al., 1989). Naturally these probes could be made only after cloning of the necessary DNA fragment in an appropriate vector. In order to facilitate the mapping protocol we have developed conditions for using 20-mer oligonucleotides as strand-specific probes. To test the approach we have analysed the direction of replication forks movement within the domain of chicken alpha-globin genes (Verbovaia and Razin, 1995). The results obtained were
Figure 1. A scheme illustrating the experimental procedure used to determine the polarity of leading DNA strand synthesis. The nascent DNA chains in a replication loop are shown by thick arrows. Short arrows show ligated Okazaki fragments (synthesised before addition of emetine). The scheme is based on the data of Burhans et all. (1991) who have demonstrated that emetine induce imballanced DNA synthesis. Although based on a wrong assumption, the protocol for determining the polarity of leading DNA synthesis was developed two years earlier by Handeli et al. (1989).
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Gene Therapy and Molecular Biology, Vol 1, page 593 in perfect agreement with the previously published data on mapping the replication origin in this domain. Oligonucleotide probes can be easily washed out from the filters and the same filters with immobilized nascent and total DNA from cells treated with emetine or other inhibitor of protein synthesis can be used sequentially in a number of hybridization experiments. To study the replication structure of human dystrophin gene we have used HEL 92.1.7 cells derived from a male patient as it was not clear whether the replication structures of the active and non-active copies of the X-chromosome in female cells were identical. The cells were cultivated for 18 h in presence of emetine and 5-bromo-2'-deoxyuridine (BrdU) exactly as described by Handeli et al. (1989). (See also Methods section in the end of this paper). The DNA was then isolated, denatured, sheared to about 1 kb fragments and nascent DNA chains containing BrdU were separated from the bulk DNA by double immunoprecipitation, as described previously (Vassilev and Russev, 1988). Equal amounts (2 Âľg) of total DNA and nascent DNA from emetine-treated cells were immobilised on nylon filters and hybridized with oligonucleotide probes representing complementary DNA chains. In order to exclude the possibility of artefacts due to the uneven sorption of DNA on filters, each filter was sequentially hybridized to probes derived from both strands and each pair of probes was hybridized to at least two different filters. In all cases the results of these four hybridization experiments confirmed each other. A typical example is shown in Fig. 2. Two similarly prepared filters with immobilized nascent and total DNA were hybridized to the "lower chain" and the "upper chain" probes derived from the sequence of the brain promoter of the dystrophin gene (here and further we use
the designation "upper chain" for the chain which is transcribed into dystrophin pre-mRNA). This experiment has demonstrated preferential hybridization of the "upper chain" probe to the nascent DNA. After exposure, the probes were washed off the filters and the "upper chain" probe was hybridized to the filter previously hybridized to the "lover chain" probe and vise versa. Again, preferential hybridization of the "upper chain" probe with the nascent DNA was observed. The asymmetry of hybridization of the "lower chain" and the "upper chain" probes to the nascent DNA remained visible even after high-stringency wash (wash with 0.1X SSC-0.1%SDS for 15 min at 420C instead of normally used wash with 1X SSC for 15 min at 420C).
B. Mapping of replication units within the dystrophin gene To assay the polarity of replication of different parts of the dystrophin gene we prepared 36 pairs of oligonucleotide probes (Table I). Some of these probes were made on the basis of the previously described primers for STS markers (Coffey, et al., 1992). These probes are referred to by their name in the original publication (Coffey, et al., 1992) with the number of a corresponding exon indicated in parentheses. Other oligonucleotide probes were designed on the basis of the known primary structure of dystrophin mRNA (Koenig et al., 1987) and the exon-intron structure of the dystrophin gene (Roberts et al., 1993). These probes are referred to by the number of a corresponding exon. Approximate positions of the probes on the physical map of the dystrophin gene are shown in Fig. 3A. The results of hybridization of the whole set of strandspecific probes with total DNA and nascent DNA samples enriched in leading strands are shown in Fig. 3 B. The polarity of the leading DNA strand synthesis was found to switch eleven times within the area under study. Keeping in mind the fact that the replication forks meet at the termination sites and move in opposite directions from the replication origins one can say that the area under study contains 5 replication origins and 6 termination sites. The first of the termination sites is located between the brain and muscle promoters. Indeed, the brain promoter (R24 probes) is replicated in the direction of dystrophin gene transcription, while the muscle promoter (R22(E1) probes) and exons 2 to 7 (probes R12(E2), R13(E3) and R7(E7)) are replicated in the direction opposite to the direction of transcription. This conclusion follows from preferential hybridization of the nascent DNA leading strands with the "upper chain" probe of the R24 pair and with the "lower chain" probes of the R22(E1), R12(E2), R13(E3) and R7(E7) pairs, as shown schematically in Fig. 4. The next switch in replication polarity occurs between exons 7 and 8. This is a switch from the minus chain to the plus chain which is indicative of the presence of a replication origin between probes R7(E7) and R2(E8) (see the scheme in Fig. 4). Similar considerations make it possible to
Figure 2. Reciprocal hybridization of "lower chain" and "upper chain" oligonucleotide probes from the dystrophin gene brain promoter with nascent (nc) and total (tot) DNA immobilized on two similarly prepared filters. The filter "a" was first hybridized to the "lower chain" probe and then, after exposure and dehybridization, to the "upper chain" probe. The filter "b" was first hybridized to the "upper chain" probe and then, after exposure and dehybridization, to the "lower chain" probe. Note the preferential hybridization of the nascent DNA with the "upper chain" probe in both cases.
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Verbovaia and Razin: Replicon Map of the Human Dystrophin Gene conclude that the replication origins are located between exons 28 and 29, between exons 43 and 44, between exons 46 and 48 and between exons 64 and 68. The replication termination sites are located between probes
87-1 and 87-15, between exons 40 and 43, between exons 44 and 45, between exons 48 and 49 and between exons 70 and 75.
Table I. Oligonucleotide probes used for determination of the dystrophin gene replication structure.
____________________________________________________________________________________________ Names of Nucleotide sequence of Nucleotide sequence of probes the probe from the "upper" chain the probe from the lower chain ____________________________________________________________________________________________ R24 CTTTCAGGAAGATGACAGAATC GATTCTGTCATCTTCCTGAAAG R22(E1) CTTTCCCCCTACAGGACTCAG CTGAGTCCTGTAGGGGGAAAG R12(E2) GAAAGAGAAGATGTTCAAAAG CTTTTGAACATCTTCTCTTTC R13(E3) GGCAAGCAGCATATTGAGAAC GTTCTCAATATGCTGCTTGCC R7(E7) CTATTTGACTGGAATAGTGTG CACACTATTCCAGTCAAATAG R2(E8) CCTATCCAGATAAGAAGTCC GGACTTCTTATCTGGATAGG R14(E11) GTACATGATGGATTTGACAGC GCTGTCAAATCCATCATGTAC 87-1 CTATCATGCCTTTGACATTCCA TGGAATGTCAAAGGCATGATAG 87-15 ATAATTCTGAATAGTCACA TGTGACTATTCAGAATTAT R21(E25) CAATTCAGCCCAGTCTAAAC GTTTAGACTGGGCTGAATTG R25(E27) GCTAAAGAAGAGGCCCAAC GTTGGGCCTCTTCTTTAGC E28 GTTTGGGCATGTTGGCATGAG CTCATGCCAACATGCCCAAAC E29 TGCGACATTCAGAGGATAACC GGTTATCCTCTGAATGTCGCA E31 GGCTGCCCAAAGAGTCCTGTC GACAGGACTCTTTGGGCAGCC R16(E33) GTCTGAGTGAAGTGAAGTCTG CAGACTTCACTTCACTCAGAC E35 GAAGGAGACGTTGGTGGAAGA TCTTCCACCAACGTCTCCTTC R31(E39) CAACTTACAACAAAGAATCACA TGTGATTCTTTGTTGTAAGTTG R8(E40) GGTATCAGTACAAGAGGCAG CTGCCTCTTGTACTGATACC E43 GTCTACAACAAAGCTCAGGTCG CGACCTGAGCTTTGTTGTAGAC E44 GACAGATCTGTTGAGAATTGC GCATTTCTCAACAGATCTGTC R18(E45) CTCCAGGATGGCATTGGCAG CTGCCAATGCCATCCTGGAG R4(E46) ATTTGTTTTATGGTTGGAGG CCTCCAACCATAAAACAAAT E48 GTTTCCAGAGCTTTACCTGA TCAGGTAAAGCTCTGGAAAC E49 ACTGAAATAGCAGTTCAAGC GCTTGAACTGCTATTTCAGT E50 GAAGTTAGAAGATCTGAGCTC GAGCTCAGATCTTCTAACTTC E53 CAGAATCAGTGGGATGAAGTA TACTTCATCCCACTGATTCTG E54 CCAGTGGCAGACAAATGTAG CTACATTTGTCTGCCACTGG E55 TGAGCGAGAGGCTGCTTTGG CCAAAGCAGCCTCTCGCTCA R20(E56) GGTGAAATTGAAGCTCACAC GTGTGAGCTTCAATTTCACC E60 ACTTCGAGGAGAAATTGCGC GCGCAATTTCTCCTCGAAGT E61 GCCGTCGAGGACCGAGTCAG CTGACTCGGTCCTCGACGGC E64 ACTCCGAAGACTGCAGAAGG CCTTCTGCAGTCTTCGGAGT E68 TAAGCCAGAGATTGAAGCGG CCGCTTCGATCTCTGGCTTA E70 ACATCAGGAGAAGATGTTCG CGAACATCTTCTCCTGATGT E75 CTGCAAGCAGAATATGACCG CGGTCATATTCTGCTTGCAG R5(E79) CAGAGTGAGTAATCGGTTGG CCAACCGATTACTCACTCTG
Figure 3 (Following page). Determining replication polarity within the dystrophin gene. (A) A scheme illustrating the exon-intron structure of the dystrophin gene and the results of determination of replication polarity. On the map of the dystrophin gene the exons are shown by vertical dark bars. Each tenth exon is indicated by the number. Positions of the brain and muscle promoters are shown by arrows above the map. The results of the analysis of replication direction are shown below the map. The vertical bars indicate the positions of the probe pairs used to assay the replication polarity. The direction of replication determined by hybridization of nascent DNA with each of the probe pairs is shown by horizontal arrows. Approximate positions of the origins (ori) and termination sites (t ) are indicated above the arrows. (B) Hybridization of strand-specific probes with total DNA (tot) and nascent DNA (nc) from emetine-treated cells. The names of the probe pairs are indicated above the autoradiographs. "-" and "+" indicate the results of hybridization with probes derived from the lower and the upper chains, respectively.
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C. The replication structure of the dystrophin gene and recombination hot-spots It may be of interest that one of the replication junctions (termination sites) identified in the present study is located in intron 44, i. e. roughly colocalizes with the main recombination hot-spot in the dystrophin gene (Wapenaar et al., 1988; Den Dunnen et al., 1989; Blonden et al., 1991). Although the significance of this colocalization (if any) is not presently clear, it is worth mentioning that in prokaryotic cells the sites of replication termination have long been known to constitute recombination hotspots (Bierne et al., 1991; Horiuchi et al., 1994; Horiuchi et al., 1995). According to one of the models, the replication fork posed at a termination site is a weak point on DNA where a doublestranded-break may occur with a high probability (Horiuchi et al., 1995; Michel et al., 1997). Some data suggest that a similar mechanism may account for the formation of recombination hot-spots also in eukaryotic cells (Horiuchi et al., 1995). In agreement with this idea it was demonstrated that pausing of the replication machinery by certain DNA secondary structures, DNA damage or DNA-protein interaction cause an increase in the rate of DNA rearrangements (Bierne and Michel, 1994). It is known that in eukaryotic cells finalization of DNA replication (juncture of neighbouring replicons) is a relatively slow process. During this step the replication forks retain single-stranded regions which can be relatively easy converted into double-stranded breaks. Furthermore, merging of replicons depends on the reactions catalysed by DNA topoisomerases which seem to be able under certain conditions to carry out illegitimate recombination of DNA strands and hence to introduce deletions and insertions into DNA (Gale and Osheroff, 1992; Shibuya et al., 1994; Henningfeld and Hecht, 1995; Bierne et al., 1997). An interesting feature of the replication structure of dystrophin gene is that the central part of the gene (exons 8 - 48) is organized into relatively short symmetrical replicons which are surrounded by two extended regions of apparently unidirectional replication (exons 1 - 8 and exons 49 - 64). Assuming that the rate of replication forks progression is the same in all replicons, it may be concluded that the replication of the central part of the gene must be completed much faster than the replication of its ends. This may cause some topological stresses resulting in an increased rate of chromosomal rearrangements within the dystrophin gene.
Figure 4. A scheme illustrating the interpretation of the results of hybridization of strand-specific probes with DNA samples enriched in nascent DNA leading strands. The upper chain and the lower chain probes are designated correspondingly by "+" and "-".
III. Discussion A. The size of replicons The present study has demonstrated for the first time that a single gene may be organized into several replicons. The average size of replicons mapped within the area under study constitutes 500 kb (with variations from 170 to 1000 kb). This finding contradicts to the common view that the average sizes of replicons in mammalian cells are from 50 to 300 kb. However our observations are in perfect agreement with the estimations of replicon sizes made by Liapunova and Yurov (reviewed by Liapunova, 1994). Furthermore, analysis of the temporal order of DNA replication in the H-2 mouse majour histocompactibility complex also suggested that mammalian replicons are larger then 300 kb (Spack et al., 1992). Similar conclusion follows from the results published by Bickmore and Oghene (1996).
B. Asymmetrical replicons and replication barriers. The results of the present study demonstrate that in the human genome the replicons may be asymmetrical. Indeed, an extended (500 kb) region including exons 49 64 seems to be replicated unidirectionally. The opposite arm of the same replicon is relatively small (less than 100 kb). It is possible that the left end of the dystrophin gene (500 kb DNA stretch) is also replicated unidirectionally. At least all exons scattered along this region are replicated in the same direction. Some of the replication termination sites mapped in the present study are not located at the middle of the distance between two neighbouring origins. This suggests that there should be some specific signals determining positions of termination sites. Up to now the replication barriers of this kind were observed only in yeast and mammalian ribosomal genes clusters (Umek et al., 1989; Kobayashi et al., 1992; Little et al., 1993).
IV. Methods A. Cell culture. Human erythroleukemia cells HEL 92.1.7 were purchased from the American Type Culture Collection. The cells were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum.
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B. Isolation of DNA samples enriched in nascent DNA leading strands.
Blonden LAJ, Grooyscholten PM, Den Dunnen JT, Bakker E., Abbs S, Bobrow M, Boehm C, van Broeckhoven C, Baumbach L, Chamberlain J, Caskey CT, Denton M, Felicetti L, Gallusi G, Fischbeck KH, Francke U, Darras B, Gilgenkrantz H, Kaplan J-C, Hermann FN, Junien C, Boileau C, Liechti-Gallati S, Lindlof M, Matsumoto T, Niikawa N, Muller CR, Poncin J, Malcolm S, Robertson E, Romeo G, Colone AE, Scheffer H, Schroder E, Schwartz M, Verellen C, Walker A, Worton R, Gillard E and Van Ommen GJB (1991) 242 Breakpoints in the 200-kb deletion-prone P20 region of the DMD gene are widely spread. Genomics 10, 631-639.
To induce imbalanced synthesis of nascent DNA strands, exponentially growing cells were treated with emetine, as described previously (Handeli et al., 1989; Burhans et al., 1991). Emetine was added to the conditional medium up to a concentration of 2 µM. This was followed (after 15 min incubation) by the addition of 5-bromo-2'-deoxy-uridine (10 µg/ml) and 3 H deoxy-cytidine (2 µCi/ml). The cells were cultured in this medium for 16 h. Then they were collected and their DNA was isolated. After shearing (to give fragments with an average size of 1 kb) and denaturation of the DNA, the BrdU-labelled nascent DNA chains were separated from the bulk DNA by double immunoprecipitation, as described previously (Vassilev and Russev, 1988).
Burhans WC, Vassilev LT, Wu J, Sogo JM, Nallaseth F and DePamphilis ML (1991) Emetine allows identification of origins of mammalian DNA replication by imbalanced DNA synthesis, not through conservative nucleosome segregation. EMBO J. 10, 4351-4360. Burmeister M, Monaco AP, Gillard EF, Van Ommen G-F, Affara NA, Ferguson-Smith MA, Kunkel LM and Lehrach H (1988) A 10-megabase map of human Xp21 including the Duchenne muscular dystrophy gene. Genomics 2, 189-202.
C. Immobilization of DNA on nylon filters and hybridization experiments. Equal amounts (2 µg) of the nascent and bulk DNA were immobilized on Hybond-N+ nylon filters (Amersham) using a Bio-Dot SF microfiltration unit (Bio-Rad). The equivalency of immobilization of all probes was verified by hybridization with 32P-labelled human repeated sequence of alu type. The oligonucleotides were labelled with !32P-ATP using T4 phage polynucleotide kinase, as described previously (Maniatis et al., 1982). Hybridization was carried out in a Rapid Hyb solution (Amersham) for 1 h at 42 0 C. After hybridization, the filters were washed one time in 5XSSC - 0.1% (w/v) SDS solution for 20 min at room temperature and two times (15 min each) in 1X SSC - 0.1% (w/v) SDS solution at 42 0 C. Then the filters were exposed to the Kodak film at -75 0 C with an intensifying screen (Dupont). For dehybridization of the radioactive probes the filters were incubated in 0.4 M NaOH solution for 30 min at 45 0 C. Then they were neutralized (15 min at room temperature) in the following solution: 0.1X SSC - 0.1%(w/v)SDS - 0.2M TrisHCl (pH 7.5).
Callan HG (1974) DNA replication in the chromosomes of eukaryotes. Cold Spring Harb. Symp. Quant. Biol. 38, 195204. Coffey AJ, Roberts RG, Green ED, Cole CG, Butler R, Anand R, Giannelli F and Bentley DR (1992) Construction of a 2.6-mb contig in yeast artificial chromosomes spanning the human dystrophin gene using an STS-based approach. Genomics 12, 474-484. DePamphilis ML (1993) Eukaryotic DNA replication: anatomy of an origin. Annu. Rev. Biochem. 62, 29-63. Den Dunnen JT, Grootscholten PM, Bakker E, Blonden LAJ, Ginjaar HB, Wapenaar MC, van Paassen HMB, van Broeckhoven C, Pearson PL and Van Ommen GJB (1989) Topography of the Duchenne muscular dystrophy (DMD) gene: FIGE and cDNA analysis of 194 cases reveals 115 deletions and 13 duplications. Am. J. Hum. Genet. 45, 835-847. Edenberg HJ and Huberman JA (1975) Eukaryotic chromosome replication. Annu. Rev. Genet. 9, 245-284. Gale KC and Osheroff N (1992) Intrinsic intermolecular DNA ligation activity of eukaryotic topoisomerase II. Potential roles in recombination. J. Biol. Chem. 267, 12090-12097.
Acknowledgements
Gorecki DC, Monako AP, Derry JML, Walker AP, Bernard EA and Bernard PJ (1992) Expression of four alternative dystrophin transcripts in brain regions regulated by different promoters. Hum. Mol. Genet. 1, 505-510.
This work was supported by grant N 097 from the Russian State Program "Frontiers in Genetics", by the grant 96-0449120 from the Russian Foundation for Support of Fundamental Science and by the ICGEB grant CRP/RUS 93-06 to S.V.R.
Hamlin JL (1992) Mammalian origins of replication. BioEssays 14, 651-659.
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Bickmore WA and Oghene K (1996) Visualizing the spatial relationships between defined DNA sequences and the axial region of extracted metaphase chromosomes. Cell 84, 95-104.
Handeli S, Klar A, Meuth M and Cedar H (1989) Mapping replication units in animal cells. Cell 57, 909-920.
Bierne H, Ehrlich SD and Michel B (1991) The replication termination signal terB of the Escherichia coli chromosome is a deletion hot spot. EMBO J. 10, 2699-2705.
Henningfeld KA and Hecht SM (1995) A model for topoisomerase I-mediated insertions and deletions with duplex DNA substrates containing branches, nicks, and gaps. Biochemistry 34, 6120-6129.
Bierne H and Michel B (1994) When replication forks stop. Mol. Microbiol. 13, 17-23.
Horiuchi T, Fujimura Y, Nishitani H, Kobayashi T and Hidaka M (1994) The DNA replication fork blocked at the Ter site may be an entrance for the RecBCD enzyme into duplex DNA. J. Bacteriol. 176, 4656-4663.
Bierne H, Ehrlich SD and Michel B (1997) Deletions at stalled replication forks occur by two different pathways. EMBO J. 16, 3332-3340.
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Verbovaia and Razin: Replicon Map of the Human Dystrophin Gene Horiuchi T, Nishitani H and Kobayashi T (1995) A new type of E. coli recombination hotspot which requires for the activity both DNA replication termination events and the Chi sequence. Adv. Bioph. 31, 133-147.
Rao BS (1994). Pausing of simian virus 40 replication fork movement in vivo by (dG-dA) .(dT-dC) tracts. Gene 140, 233237. Roberts RG, Coffey AJ, Bobrow M and Bentley DR (1993). Exon structure of the human dystrophin gene. Genomics 16, 536538.
Huberman JA and Riggs AD (1966) Autoradiography of chromosomal DNA fibers from Chinese hamster cells. Proc. Natl. Acad. Sci. USA 55, 599-606.
Shibuya ML, Ueno AM, Vannais DB, Craven PA and Waldren CA (1994) Megabase pair deletions in mutant mammalian cells following exposure to amsacrine, an inhibitor of DNA topoisomerase II. Cancer Res. 54, 1092-1097.
Huberman JA and Riggs AD (1968) On the mechanism of DNA replication in mammalian chromosomes. J. Mol. Biol. 32, 327341. Kitsberg D, Selig S, Keshet I and Cedar H (1993) Replication structure of the human "-globin gene domain. Nature 366, 588-590.
Spack EG, Lewis ED, Paradowski B, Schimke RT and Jones PP (1992) Temporal order of DNA replication in the H-2 major histocompactibility Complex of the mouse. Mol. Cell. Biol. 12, 5174-5188.
Kobayashi T, Hidaka M, Nishizawa M and Horiuchi N (1992) Identification of a site required for DNA replication fork blocking activity in the rRNA gene cluster in Saccharomyces cerevisiae. Mol. Gen. Genet. 233, 355-362.
Stubblefield E (1974) The kinetics of DNA replication in chromosomes. In The cell nucleus, H. Busch, ed. (Academic Press, New York), vol. 2, pp. 149-162. .
Koenig M, Hoffman EP, Bertelson CJ, Monaco AP, Feener C and Kunkel LM (1987) Complete cloning of Duchenne muscular dystrophy (DMD) cDNA and preliminary genomic organization of the DMD gene in normal and affected individuals. Cell 50, 509-517.
Umek RM, Linskens MHK, Kowalski D and Huberman J (1989) New beginnings in the studies of eukaryotic DNA replication origins. Biochim. Biophys. Acta 1007, 1-14. Vassilev L and Russev G (1988). Purification of nascent DNA chains by immunoprecipitation with anti BrdU antibodies. Nucl. Acids Res. 16, 10397.
Liapunova NA (1994) Organization of replication units and DNA replication in mammalian cells as studied by DNA fiber radioautography. Int Rew. Cytol. 154, 261-308.
Vassilev LT and DePamhilis ML (1992) Guide to identification of origins of DNA replication in eukaryotic cell chromosomes. Crit. Rev. Biochem. Mol. Biol. 27, 445-472.
Little RD, Platt TH, Schildkraut CL (1993) Initiation and termination of DNA replication in human rRNA genes. Mol. Cell. Biol. 13, 6600-6613.
Verbovaia L and Razin SV (1995) Analysis of the replication direction through the domain of #-globin-encoding chicken genes. Gene 166, 255-259.
Michel B, Ehrlich SD and Uzest M (1997) DNA double-strand breaks caused by replication arrest. EMBO J. 16, 430-438.
Wapenaar MC, Kievits T, Hart KA, Abbs S, Blonden LAJ, den Dunnen JT, Grootscholten PM, Bakker E, Verellen-Dumoulin Ch, Bobrow M, van Ommen GJB and Pearson PL (1988). A deletion hot spot in the Duchenne muscular dystrophy gene. Genomics 2, 101-108.
Maniatis T, Fritsch EF and Sambrook J (1982) Molecular cloning: a laboratory manual. (Cold Spring Harbor, New York: Cold Spring Harbor Laboratory). Painter RB (1976). Organization and size of replicons. In Handbook of Genetics, R.C. King, ed. (Plenum, New York). vol. 5, pp. 169-186..
Yorov YB and Liapunova NA (1977) The units of DNA replication in the mammalian chromosomes: evidence for a large size of replication units. Chromosoma 60, 253-267.
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Initiation of DNA replication at the rat aldolase B locus â&#x20AC;&#x201D;An overlapping replication?â&#x20AC;&#x201D;
set of
DNA
elements
regulates
transcription
and
Ken-ichi Tsutsumi and Yunpeng Zhao Institute for Cell Biology and Genetics, Faculty of Agriculture, Iwate University, Ueda, Morioka, Iwate 020, Japan ________________________________________________________________________________________________ Correspondence to: K. Tsutsumi, Fax: + 81 -19 621 6243, E-mail: kentsu@iwate-u.ac.jp
Summary In higher eukaryotes, DNA replication initiates at multiple s i t e s on each chromosome. Positioning and firing of the replication origins are not fixed, but different and selected origins may i n i t i a t e a t d i f f e r e n t t i m e s i n a s i n g l e c e l l c y c l e o f p a r t i c u l a r c e l l s t h r o u g h a range o f complex mechanisms controlling, for example, cell differentiation. The origin region at the rat aldolase B locus (ori A1) has been found to encompass the promoter which governs liver-specific transcription. Ori A1 is, thus, thought to be a suitable target in investigating causal relationships among those under control o f c e l l differentiation, i . e . , firing or silencing o f the origin, c e l l type-specific regulation of transcription, and positioning of nearby origins. In this article, we summarize our approach to elucidate such relationships. We describe sequence-dependent replication from ori A1, overlapping o f essential regions required for replication and transcription, c e l l cycle-regulated binding of factors to the essential region, and then chromosomal state of the ori A1 region in the nucleus.
I. Introduction Initiation of DNA replication is one key control point in a process of cell cycle progression. In eukaryotic cells, each chromosome contains multiple replication origins which are activated in a stringently controlled temporal and spatial order during S phase. In addition, not all origins are fired for genome duplication in a single cell cycle, some origins are generally used while others are not (DePamphilis, 1993a; Coverley and Laskey, 1994; Hamlin et al., 1995; Stillman, 1996). Which origins are selectively used and when they are activated are, however, not fully understood. The selection and positioning of origins on the chromosomes might be controlled by multiple regulatory processes such as developmental program, transcription activity of nearby genes, and firing of nearby origins (James and Leffak, 1986; Wolffe and Brown, 1988; Trempe et al., 1988; Leffak and James, 1989; Fangman and Brewer, 1992). Evidences have been accumulating that cis-elements for transcription promote replication activity as well (for review see DePamphilis, 1993b). For example, transcription factors AP1 (Guo and DePamphilis, 1992), NF1 (Mul and Van der Vliet, 1992), Oct1 (O'Neill et al., 1988) and c-Jun (Ito, Ko et al., 1996) strongly stimulate 599
virus DNA replication. In cellular chromosomes, origin region or regulatory region for DNA replication often encompasses transcriptional promoter or contains ciselements for transcription (Vassilev and Johnson, 1990; Ariizumi et al., 1993; Taira et al., 1994; Tasheva and Roufa, 1994; Zhao et al., 1994). Mutant human cells having deletions at either near the promoter (Kitsberg et al., 1993) or locus control region (LCR) (Aladjem et al., 1995) of the !-globin locus fail to initiate DNA replication from the origin located within the !-globin locus. On the contrary, several studies suggested that transcription and DNA replication are antagonistic events. A head-on collision between a replication fork and transcribing RNA polymerase complex arrests or pauses replication in yeast (Brewer et al., 1992; Deshpande and Newlon, 1996), in a similar way to E. coli (Liu et al., 1993; Liu and Alberts, 1995). Origin recognition complex (ORC), the eukaryotic replication initiator, represses transcription of certain genes in yeast (Bell and Stillman, 1992; Micklem et al., 1993; Foss et al., 1993; Bell et al., 1995), although the role of ORC in the repression was recently shown to be separable from its role in replication initiation (Fox et al., 1997).
Tsutsumi and Zhao: Initiation of DNA replication at the rat aldolase B locus A similar inverse correlation occurs in Xenopus ribosomal RNA genes, in which replication initiation is specifically repressed within transcription units and limited to nontranscribed regions, whereas the replication randomly initiates throughout the transcribed and nontranscribed sequences in early embryos (Hyrien et al., 1995). Taken together, these observations strongly suggest a tight link between transcription regulation and positioning or firing of replication origins in the chromosomal context. However, how transcription and replication interact with each other, and what biological system(s) the interaction is involved in mammalian cells are still unknown. In this review, we describe our approach to characterize replication origin at the rat aldolase B locus. We also discuss on DNA elements required for replication initiation and on the possible correlation between regulatory systems of replication and transcription.
II. The rat aldolase B gene: function, structure and liver-specific expression Before focusing on replication origin, we start by briefly describing about the gene coding for aldolase. Aldolase is an enzyme acting on fructose-1,6-bisphosphate metabolism in a processes of glycolysis and gluconeogenesis. The enzyme is a tetrameric protein composed of a combination of three different subunits, A, B and C which are distributed in different tissues and organs in animals (Horecker et al., 1972). Expression of the gene encoding the aldolase B subunit (AldB) is cell type-specific and is under control of cell differentiation; the gene is preferentially expressed in the liver, kidney and jejunal mucosa in adult animal, but the expression is repressed at an early fetal stage and in dedifferentiated hepatocellular carcinomas (Horecker et al., 1972; Numazaki et al., 1984; Tsutsumi et al., 1985; Sato et al., 1987). Studies on the mechanisms operating in such a regulated expression revealed the importance of at least four cis-elements (sites A, B, C, and D) on the proximal 200 bp promoter in the liver-specific transcription, to which a number of regulatory factors interacts cell type-specifically or ubiquitously (F i g . 1 ). For example, bindings of HNF1 to site A, AlF-B or NF-Y to site B, and C/EBP or AlF-C to site C seem to confer liver-specific transcription (Tsutsumi et al., 1989; Ito, Ki et al., 1990; Raymondjean et al., 1991; Gregori et al., 1993; Tsutsumi et al., 1993; Yabuki et al., 1993; Gregori et al., 1994). Site D acts as a silencer-like element upon transfection, but its role is still unknown (Gregori et al., 1993). On the other hand, in AldB non-expressing rapidly dividing cells, a different set of factors bind to these sites though their functions are not fully known, e.g., site B and site C bind growth-inducible factors Ryb-a (Ito, Ki et al., 1994) and an alternate type of AlF-C (Yabuki et al., 1993), respectively (discussed later). Thus, various set of regulatory factors interact with the proximal 200 bp promoter, determining cell type-specificity and the level of transcription. 600
III. An initiation region of DNA replication encompasses promoter of the AldB gene in AldB non-expressing rat hepatoma cells Regulated state of eukaryotic genes is achieved through the assembly of specialized, heritable chromatin structure with confined domains on chromosomes, which might have causal relationship with locus control region (LCR), insulator, nuclear matrix-association, and positioning of replication origins etc. It is also suggested that regulatory pathways that govern transcription and initiation of DNA replication affect each other or cross-talk (DePamphilis, 1993b; Hamlin et al., 1995; Stillman, 1996). Based on these considerations, we initially thought that transcriptional repression of the AldB gene in rapidly dividing cells, such as fetal liver and hepatoma cells, might somehow relate to initiation of replication. For a step toward understanding the functional and positional relationship between transcription and replication, we tried to identify the replication initiation region nearest to the AldB gene. To locate initiation region of replication, newly replicated DNA chains were labeled with bromodeoxyuridine (BrdU) using synchronously cultured rat hepatoma dRLh84 cells. BrdU-substituted DNA has higher density as compared to unsubstituted parent DNA and can be separated by ultracentrifugation through CsCl density-gradient. Cells arrested at G1/S boundary by double-thymidine-block were released from the arrest to enter S phase, and cultured in a fresh medium containing BrdU. After various time period, BrdU-labeled DNA was prepared by CsCl isopycnic centrifugation after digestion with an appropriate restriction enzyme, and hybridized with probes corresponding to various regions in and around the AldB gene region. These experiments showed that (i) replication of the AldB gene region starts at mid-S phase, and (ii) the initiation region locates near or within the AldB gene region. Further analysis of the newly replicated short DNA fragments prepared by alkaline sucrose density-gradient centrifugation revealed that (iii) the initiation region expands about 1.5 Kb or less, which encompasses transcription promoter of the AldB gene (F i g . 1 ) (Zhao et al., 1994).
IV. Specific sequence is required for replication of plasmids carrying the AldB origin fragments In vivo analyses of newly replicated DNA identified an origin region of DNA replication which encompassed promoter of the AldB gene. Since several mammalian origins have been reported to possess activities to replicate autonomously (for example, Ariga et al., 1987; McWhinney and Leffak, 1990; Wu et al., 1993), we tried to examine whether the AldB origin fragment promotes replication in a plasmid form upon transfection into mammalian cells. For this purpose, large DNA fragments ranging from 4 Kb to 6.3 Kb derived from the AldB origin
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F i g . 1 . Two origin regions A1 and A2 in the vicinity of the AldB locus in rat. Vertical lines represent EcoRI sites and the lengths of the EcoRI fragments are shown in Kb below the map. Lower panel shows structure off the promoter within the replication origin (A1) region. Transcription factors that interact with the promoter are also shown. Bent arrow indicates position and direction of the AldB gene transcription.
Fig. 2. Replication of a plasmid carrying origin fragment (A1) in transfected cells. Plasmids carrying in vivo origin fragments from - 5.7 Kb to + 0.625 Kb (pBOR6.3) or from - 0.675 to + 0.263 Kb (pBOR0.94) were cotransfected with pUC19 or pBOR6.3, respectively, into Cos-1 cells. After the transfected cells were cultured for 72 hr in the presence of BrdU, low-molecularweight DNA was extracted, digested with EcoRI, and fractionated by CsCl isopycnic ultracentrifugation. DNA in each fraction was separated on an 1% agarose gel, transferred onto a nylon membrane, and hybridized with a random-primed, 32 P-labeled pUC19 DNA fragment as a probe. LL (light-light ) DNA, HL (heavy-light) DNA, and HH (heavy-heavy) DNA indicate unsubstituted, hybrid, and fully substituted DNAs, respectively (see text).
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F i g . 3 . The 200 bp AldB gene promoter is essential for initiation of replication in transfected cells. Various deletion constructs shown in the left panel were transfected together with pBOR6.3 as an internal control and processed as in F i g . 2 . Replication efficiencies were based on the amounts of HH and HL DNAs in total (HH, HL, and LL) DNAs, and values were expressed relative to the activity of pBOR6.3.
region were inserted into plasmid pUC19, and assayed for autonomous replication in Cos-1 cells based on semiconservative BrdU-substitution of replicating DNA chain. As briefly mentioned above, incorporation of BrdU into replicating DNA increases density of the DNA chain (designated as H chain) and causes it to band at a density higher than that of unsubstituted DNA chain (L chain) in CsCl density-gradient. The transfected cells were grown in the presence of BrdU, then low-molecular-weight DNAs were extracted by the procedure described by Hirt (Hirt, 1967). Newly-replicated, BrdU-labeled DNA was fractionated by CsCl isopycnic ultracentrifugation after digestion with a restriction enzyme, and then subjected to Southern blot hybridization. F i g u r e 2 shows a typical example of such a replication assay, using a plasmid (designated as pBOR6.3) bearing a 6.3 Kb fragment extending from - 5.7 Kb to +625 bp in bacterial plasmid vector pUC19. In this case, double-stranded DNA with both strands being replaced by BrdU (HH DNA) appeared in addition to HL and LL DNAs. This means, considering from the semiconservative replication, that at least two rounds of replication had occurred. A negative control plasmid pUC19 co-transfected with pBOR6.3 did not initiate replication, indicating that the observed replication depends on DNA fragment inserted. Plasmid containing the 0.94 Kb fragment from - 675 bp to + 263 bp (pBOR0.94) exhibited similar activity as compared to cotransfected pBOR6.3 (F i g . 2 , lower panel). Thus, the 0.94 Kb fragment extending from -675 bp to +263 bp seems to have minimum essential components for autonomous
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replication. Such replication assays were carried out using various deletion constructs and compared their activities to replicate. The results indicated that a 200 bp fragment extending from -200 bp to -1 bp is indispensable to replication initiation, since deletion of the fragment abolished replication activity (F i g . 3 ). The 200 bp fragment alone could not direct replication. But it restored replication activity when ligated to either upstream (about 500 bp) or downstream fragment (about 300 bp). This observation makes the origin architecture rather complicated. However, since both flanking regions share no similar sequence, it is not conceivable that the same replication elements are present in these two regions. Although entirely unknown at present, one explanation for this may be the presence of different auxiliary elements in each flanking region, both of which have similar activities for replication initiation when they cooperate with the 200 bp sequence (F i g . 4 ). So far, controversial observations concerning plasmid replication in mammalian cells have been reported (reviewed by Coverley and Laskey, 1994). Several of them pointed out, for example, that the length of the DNA template is crucial, rather than sequence; even bacterial DNA in origin-depleted mammalian virus vector replicated in mammalian cells (Krysan and Calos, 1991; Heinzel et al., 1991; Krysan et al., 1993). Further, the fact that no consensus sequences for origins have been found would support the idea.
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F i g . 4 . Three important regions in the predicted origin region. A to D in filled box represent cis-elements for transcription (see F i g . 1 ). PPu and A/T indicate purin-rich element having binding site for a factor Pur " (PUR consensus sequence) and A/T-rich sequence, respectively. Numbers indicate positions in bp relative to the transcription start site.
In the case of the AldB origin, however, we prefer that specific sequence elements rather than length is required for replication (Zhao et al., 1997). Plasmids used in our replication assays do contain none of mammalian virus DNA sequence, origin of virus DNA, and binding sites for virus T antigen which would activate replication to some extent, so that the observed replication might depend on the AldB origin sequence and is free from initiation machinery for virus DNA replication. Probably, the possibility that quite diverse sets of specific sequence elements can promote firing each of the multiple potential origins on chromosomes might be one of the explanations why no apparently conserved sequence is found in the limited numbers of mammalian origins so far identified (for review, see Stillman, 1996). Indeed, some eukaryotic origins are reported to require sequence-specific interaction of factors to drive initiation (Caddle et al., 1990, Dimitrova et al., 1996). Several transcription factors have been shown to be activators of replication initiation (Li and Botchan, 1993, He et al., 1993, DePamphilis, 1993a). In addition, a 28 kDa factor found in HeLa cell nuclei binds a purin-rich sequence (PUR consensus sequence, discussed later), which is conserved in several origins from yeast, hamster and human to serve initiation of replication as a sequencespecific helix-destabilizing factor (Bergemann and Johnson, 1992). In this view, the AldB origin region has the structural features for potential origins (DePamphilis, 1993b). The 200 bp region at the AldB promoter contains binding sites 603
for multiple transcription factors, the above mentioned PUR consensus sequence (discussed later), and an A/T-rich sequence (Tsutsumi et al., 1989, Zhao et al., 1994).
V. Cell cycle-regulated factors bind to the 200 bp region in the AldB origin We next intended to know whether the 200 bp region binds factors from hepatoma cells (dRLh 84), in which this region was shown to be centered on an initiation region of chromosomal DNA replication (Zhao et al., 1994)). Within this 200 bp proximal promoter, at least four important cis-elements ( sites A, B, C and D) have been shown to confer tissue- and developmentally specific transcription. Site-A binds both a liver-specific factor HNF-1 and its competitive antagonist HNF-3 (Tsutsumi et al., 1989; Ito, Ki et al., 1990; Raymondjean et al., 1991; Gregori et al., 1993; Gregori et al., 1994), site-B (a CCAAT motif) binds factors AlF-B, NF-Y and a growthinducible factor Ryb-a (Tsutsumi et al., 1993; Gregori et al., 1994; Ito, Ki et al., 1994). Site-C binds to C/EBP, DBP and a novel helix-loop-helix protein AlF-C (Yabuki et al., 1993; Gregori et al., 1993; Gregori et al., 1994; and Yabuki et al., manuscript in preparation). Therefore, it is very interesting to examine whether or not binding of the factors to the 200 bp region is under control of cell proliferation and cell cycle progression. For this purpose, growth cycle of rat hepatoma dRLh84 cells was synchronized by double thymidine block (synchrony was monitored by flow cytometry), and their nuclear extracts
Tsutsumi and Zhao: Initiation of DNA replication at the rat aldolase B locus were prepared every 2 hr after entering S phase. The nuclear extracts were then subjected to gel electrophoretic mobility shift assay using oligonucleotides corresponding to sites A, B, C, and PPu ( polypurin sequence containing PUR consensus) as probes. Results showed that in quiescent cells binding activities to these sites were considerably low as compared with those in growing cells. Site A- and site C-binding activities exhibited similar patterns showing veritable cell cycle regulation. Namely, the activities reach a maximal levels at around G1/S boundary, then gradually decrease to the lowest level at late M to early G1 phase. Activity to bind site PPu increases toward S phase. In contrast, site B-binding activity showed only weak change throughout the cell cycle. Thus, in AldB non-expressing hepatoma cells, sites A, C and PPu bind cell cycle-regulated factors whose activities increase prior to S phase while site B binds a factor independently of cell cycle phases. At present, precise characters of these factors are unknown. However, considering from tissuespecific or ubiquitous pattern of expression of the factors that bind to the promoter, site A might bind a factor HNF3 in the hepatoma cells, since another factor HNF1 is not present in these cells (Kuo et al., 1991). Similarly, sites B and C are thought to bind Ryb-a and AlF-C, respectively, because these factors are rather enriched in rapidly growing cells such as fetal liver cells, and induced by growth signals such as partial hepatectomy or serum-stimulation of cultured cells (Yabuki et al., 1993; Ito et al., 1994; and Yabuki et al., manuscript in preparation). Since site PPu has PUR consensus sequence, it might bind a factor similar to that binds to PUR consensus, i.e., Pur " (Bergemann and Johnson, 1992). The factor Pur " seems to act as a sequence-specific helix-destabilizing
factor, and thus implicating in its involvement in initiation of replication. Recently this factor was shown to associate with the retinoblastoma protein Rb, and thus Rb might modulate binding of Pur " to its recognition site on DNA (Johnson et al., 1995). These results implied a positive correlation between binding of factors to the 200 bp region and the onset of DNA replication. In dedifferentiated dRLh84 cells, transcription promoter of the AldB gene is completely inactivated, and instead, used as a replication origin (see hypothetical model in F i g . 5). One interesting speculation is that preferential binding of the above mentioned factors in AldB non-expressing cells, instead of those usually bind in the liver and activate the AldB gene, leads to repress transcription and consequently promote replication initiation in a cell cycle-dependent manner. Similar observations were reported for Xenopus chromosome where embryo-specific origins are inactivated with concomitant activation of nearby transcription units (Hyrien et al., 1995), and for plasmids carrying a human replication origin that inhibition of replication depends on the level of promoter activity (Haase et al., 1994).
VI. Chromosomal state of the AldB origin/promoter region We have discussed above on identification, sequence requirement, and cell cycle-dependent protein-binding of the replication origin region. In this section, we will focus on the chromosomal state at the AldB origin/promoter region in relation to transcription activity, liver cell proliferation, and development.
F i g . 5 . Summary of cell cycle- and growth-dependent binding of factors to the AldB gene origin/promoter region. Details are described in the text.
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F i g . 6 . Alteration of chromosomal state at the origin/promoter region during fetal liver development. Horizontal and curved arrows indicate positions and directions of the AldB gene. Vertical arrows marked HS represent DNase I hyper sensitive site. CpG sites which were heavily methylated are shown as Me. Boxes represent promoter regions.
The AldB gene transcription in rat liver is repressed in the fetal stage until around 16th day (day 16) of gestation. Thereafter, transcription of the gene is drastically activated in the following two to three days (Numazaki et al., 1984). As expected, the activation during this fetal stage accompanies the alteration of chromosomal state of the AldB gene (see hypothetical model in F i g . 6). Comparison of the chromatin structure among those in the livers at the stages when the gene is repressed (day 14), just being activated (day 16), and fully activated (adult) revealed several distinct features for the repressed state (Daimon et al., 1986; Tsutsumi et al., 1987; Ito, Ki et al., 1995; Kikawada et al., unpublished data). Namely, the chromatin had two DNase I-hypersensitive sites II-a and II-b at day 14; the former site disappeared with concomitant activation of the gene. The region around the transcription start site was considerably resistant against DNase I digestion as the fragment derived from the DNase Ihypersensitive sites remained almost intact even after digestion with higher concentration of DNase I; with such a concentration of DNase I the chromatin at later stages was very unstable and was cut into pieces. It was also shown that two CpG sites in HhaI and HpaII sequences near
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transcription start site are hypermethylated at the repressed stage while those in adult liver are hypo-methylated. DNase I hypersensitive sites as described above might be a reflection of nuclear matrix association, since the matrix contains topoisomerase II whose cleavage sites in vivo are often found in DNase I hypersensitive regions (Poljak and K채s, 1995). Indeed, several consensus sequences for topoisomerase II cleavage site (Sander and Hsieh, 1985) were found around the DNase I-protected and hypersensitive regions, in addition to clusters of A- or Trich stretches which are often found in matrix-associated DNA (Gasser and Laemli, 1986) (Fig. 6). With this respect, further analyses using cultured cells encapsulated into agarose beads were carried out to define matrixassociated region. The results suggested that in proliferating, AldB non-expressing cells the DNase Iprotected region in the AldB chromatin, i.e., promoter/origin region, is within the matrix-associated region (MAR) (reviewed by Boulikas, 1995). It is not surprising to consider that the origin region is attached to nuclear matrix where replication initiates in proliferating cells, since MAR has been thought to be, for example, an origin of replication (Amati and Gasser, 1988), a boundary of DNA loops demarcating a regulatory domain including a
Tsutsumi and Zhao: Initiation of DNA replication at the rat aldolase B locus set of transcription units (Laemli et al., 1992), and transcription enhancer elements (Gasser and Laemli, 1986). Based on these observations, it would be considered that the alteration of chromatin state of the AldB gene promoter/origin region during fetal liver development reflects a change in the organization of functional domains in chromosome. If so, positioning of replication origins and chromosomal domain, for example, might differ between AldB expressing and non-expressing cell nuclei. In this regard, we recently found another origin region at more than 40 Kb downstream of the promoter/origin region in rat hepatoma cells. Both origins are fired in AldB nonexpressing hepatoma cells, whereas the downstream origin was not used in AldB expressing differentiated hepatoma cells, suggesting different organization of chromosomal domains (Miyagi et al., unpublished observation).
VII. Perspectives Here, we have described that the transcription promoter of the aldolase B gene is centered on an initiation region of DNA replication in rat hepatoma cells in vivo. Within the origin region, the 200 bp promoter fragment extending from -200 bp to -1bp was indispensable to autonomous replication when assayed by transfection of plasmids bearing various origin fragments. Since the 200 bp fragment alone did not confer replication, the fragment is thought to cooperate with the flanking sequences to play an important role in initiation of replication. The 200 bp promoter consists of multiple cis-elements for liver-specific transcription. In rat hepatoma cells, in which the AldB gene is completely inactivated, protein factors bound to the cis-elements in a cell cycle- or growth-regulated manner, suggesting the involvement of the 200 bp region in regulation of replication initiation. Thus, the promoter of the AldB gene has dual roles in regulation of both liverspecific transcription and initiation of replication. The results, however, do not confine actual "start point" of replication. The 200 bp region, for example, could not necessarily be a start point but rather be an auxiliary element. Further, whether the start point resides at a single location or distributes throughout the origin/promoter region is unknown. These points remain to be elucidated. Firing of replication origin either at or neighboring the AldB locus, or both, might influence the transcriptional state of the gene, since these biological reactions in a single specialized DNA domain are together regulated according to a DNA loop model, a chromosome model of topologically independent DNA loop domains which are separated by periodic association with nuclear matrix (Laemli et al., 1992). Concerning to this, the observation that the origin/promoter region is attached onto nuclear matrix in vitro and in vivo implies the importance of the region in assembly of functional domains in a chromosome, since MAR has been postulated to act as a replication origin and, in addition, as an insulator-like element in that it reduces position effect in transgenic mice (for example, McKnight et al,, 1992). Thus, we think that repression and activation of the AldB gene might reflect positioning of 606
replication origins and alteration of domain structure in chromosomes. In fact, as mentioned earlier, usage of the two origins in the vicinity of the AldB locus differs between the AldB expressing and non-expressing cells. It would be quite important to know how transcription and replication reflect each other in the chromosomal context, since the mechanism that govern such a causal relationship might be involved in cell differentiation and development. For this purpose, the AldB gene promoter/replication origin would be one of the suitable targets.
Acknowledgment This work was supported in part by a Grant-in-Aid for Scientific Research on Priority Areas and a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan. We greatly acknowledge Drs. K.Ishikawa, R.Tsutsumi, M.Yamaki, Y.Nagatsuka and K. Ito for their collaboration, help, discussions and encouragement.
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Micklem, G., Rowley, A., Harwood, J., Nasmyth, K., and Diffley, J.F.X. (1 9 9 3 ) Yeast origin recognition complex is involved in DNA replication and transcriptional silencing. Nature 366, 87-89. Mul, Y.M., and Van der Vliet, P.C. (1 9 9 2 ) Nuclear factor I enhances adenovirus DNA replication by increasing the stability of a preinitiation complex. E M B O J . 11, 751760. Numazaki, M., Tsutsumi, K., Tsutsumi, R. and Ishikawa, K. (1 9 8 4 ) Expression of aldolase isozyme mRNA species in fetal liver. Eur. J. Biochem. 142, 165-170. O'Neil, E.A., Fletcher, C., Burrow, C.R., Heinz, N., Roeder, R.G. and Kelley, T.J. (1 9 8 8 ) Transcription factor OTF-1 is functionally identical to the DNA replication factor NFIII. S c i e n c e 241, 1210-1213. Poljak, L. and K채s, E. (1 9 9 5 ) Resolving the role of topoisomerase II in chromatin structure and function. T r e n d s C e l l B i o l . 5, 348-354. Raymondjean, M., Pichard, A. -L., Gregori, C., Ginot, F., and Khan, A. (1 9 9 1 ) Interplay of an original combination of factors: C/EBP, NFY, HNF3 and HNF1 in the rat aldolase B gene promoter. N u c l . A c i d s R e s . 19, 6145-6153. Sander, M. and Hsieh, T. (1 9 8 5 ) Drosophila topoisomerase II double-strand cleavage: analysis of DNA sequence homology at the cleavage site. N u c l . A c i d s R e s . 13, 1057-1071. Satoh, J., Tustusmi, K., Ishikawa, M. and Ishikawa, K. (1 9 8 6 ) Dietary regulation of aldolase isozyme expression in rat intestinal mucosa. A r c h . B i o c h e m . B i o p h y s . 254, 116-123. Stillman, B. (1 9 9 6 ) Cell cycle control of DNA replication. S c i e n c e 274, 1659-1664. Taira, T., Iguchi-Ariga, S.M.M., and Ariga, H. (1 9 9 4 ) A novel DNA replication origin identified in the human heat shock
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Vassilev, L., and Johnson,E. M. (1 9 9 0 ) An initiation zone of chromosomal DNA replication located upstream of the cmyc gene in proliferating HeLa cells. M o l . C e l l . B i o l . 10, 4899-4904. Wolffe, A.P., and Brown, D.D. (1 9 8 8 ) Developmental regulation of two 5S ribosomal RNA genes. S c i e n c e 241, 1626-1632. Wu, C., Friedlander, P., Lamoureux, C., Zannis-Hadjopoulos, M. and Price, G.B. (1 9 9 3 ) cDNA clones contain autonomous replication activity. B i o c h i m . B i o p h y s . Acta 1174, 241-257. Yabuki, T. Ejiri, S., and Tsutsumi, K. (1 9 9 3 ) Ubiquitous factors that interact simultaneously with two distinct ciselements on the rat aldolase B gene promoter. B i o c h i m . B i o p h y s . A c t a 1216, 15-19. Zhao, Y., Miyagi, S., Kikawada, T., and Tsutsumi, K. (1 9 9 7 ) Sequence requirement for replication initiation at the rat aldolase B locus implicated in its functional correlation with transcriptional regulation. B i o c h e m . B i o p h y s . R e s . Commun. 237, 707-713. Zhao, Y., Tsutsumi, R., Yamaki, M., Nagatsuka, Y., Ejiri, S., and Tsutsumi, K. (1 9 9 4 ) Initiation zone of DNA replication at the aldolase B locus encompasses transcription promoter region. N u c l . A c i d s R e s . 22, 5385-5390.
Gene Therapy and Molecular Biology Vol 1, page 609 Gene Ther Mol Biol Vol 1, 609-612. March, 1998.
TARgeting the human genome to make gene isolation easy Michael A. Resnick, Natalay Kouprina, and Vladimir Larionov Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, NIH, P.O. Box 12233, 110 Alexander Dr., Research Triangle Park, NC 27709 __________________________________________________________________________________________________ Correspondence to: Michael A. Resnick, Tel: 919 541-4480; Fax: 919 541-7593, E-mail: resnick@niehs.nih.gov
Summary Considerable information is now available about the human genome and expressed sequences have b e e n i d e n t i f i e d f o r m o s t g e n e s . U n t i l r e c e n t l y t h e r e w a s n o o p p o r t u n i t y t o specifically isolate g e n e s o r s p e c i f i c c h r o m o s o m a l r e g i o n s f r o m g e n o m i c D N A . We have utilized transformationassociated recombination (TAR) in yeast to isolate genes and specific regions from total human DNA. This has been demonstrated by the direct isolation of complete copies of rDNA, BRCA1, BRCA2 and HPRT genes with high fidelity as yeast artificial chromosomes (YACs). We propose that there are many utilities of TAR cloning including gene therapy and diagnostics.
The Human Genome Project has made great strides in the decade since its inception including the cloning of most of the chromosomal DNA, the identification of unique sequences (sequence tags sites, STS's) approximately every 150 kb and the sequencing of short regions of almost all the expressed genes (expressed sequence tags, EST's). The project is ahead of schedule in that most of the genome will be sequenced within the next 5 to 10 years. In addition to understanding chromosome organization, this vast amount of information is leading to the isolation of genes that correspond to specific diseases, particularly through positional cloning. Furthermore, the genetic makeup of humans is better understood because of sequence relatedness between species. Until now there has been little opportunity to utilize the information being generated to isolate specific large regions (i.e., greater than 10 to 20 kb) or genes directly from total genomic material. Virtually all cloning of chromosomal DNA from humans, or any organism, has involved the isolation of random DNA fragments into vectors through several steps of enzymatic treatment plus ligation and the subsequent transfer into the desired bacterial or yeast host. The isolation of specific DNAs would provide a variety of opportunities, including studies of human polymorphisms, clinical diagnosis, gene therapy and the filling-in of gaps in sequenced regions. However, the only available enrichment procedure has been the physical isolation of entire chromosomes (McCormick et al., 1993). Even then, the subsequent cloning of human DNAs has involved random DNA fragments.
Over the past year a new approach has emerged that is providing for the specific isolation of genes and regions directly from total human DNA. The approach draws upon several features of the yeast Saccharomyces cerevisiae. The first is that during transformation, yeast can take up several small and large molecules (Rudolph et al., 1985; Larionov et al., 1994). Secondly, intermolecular, as well as intramolecular, recombination is highly efficient during transformation between homologous, as well diverged DNAs (Larionov et al., 1994; Ma et al., 1987; Mezard et al., 1992). This includes double-strand break recombination between broken molecules. Thirdly, human DNA contains sequences (about 1 per 20-30 kb) that can function as origins of replication (ARS-autonomously replicating sequence) in yeast (Stinchcomb et al., 1980). These features have provided for the development of a novel method based on transformation-associated recombination (TAR) to target the isolation of specific DNAs from total human DNAs. As described in Figure 1, genomic DNA is presented to yeast along with a molar excess of vector containing a selectable marker, a centromere (CEN) to assure production of a single copy of the cloned material and targeting sequence hooks A and B (the original circular plasmid is linearized at a site between A and B). [The TAR procedure simply involves the presentation of gently prepared human DNA, originally isolated in low-melt agarose plugs, to competent yeast spheroplasts along with vector DNA.]
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Figure 1 . Model of TAR cloning to generate circular YACs. Human DNA is taken up by a yeast cell along with linearized vector DNA. The vector contains a centromere and a marker for selection. If the human DNA contains segments corresponding to the segments--hooks--A and B on the plasmid, recombination will lead to the establishment of a circular YAC. Propagation of the YAC depends on the presence of a yeast ARS-like sequence in the human DNA. The various blocks could be diverged repeats, such as Aluâ&#x20AC;&#x2122;s or LINES.
libraries containing large fragments of chromosomal DNA (Larionov et al., 1996). Subsequently, the original scheme--which does not involve restricting or ligating DNA-- was modified to yield circular YACs (Larionov et al., 1996b) as described in Figure 1. The efficiency and selectivity of TAR cloning was initially demonstrated by the specific isolation of human DNA from a radiation hybrid rodent cell line containing a 5 Mb human chromosome fragment that had the Ku80 gene (Larionov et al., 1996b). A circularizing TAR vector was used that had the same human Alu for the targeting A and B hooks (see Figure 1 and Table 1). Approximately 25% of the transformants for the vector marker had YACs containing human DNA and most were greater than 150 kb. Based on the relative number of YACs isolated containing rodent DNA, this corresponded to a nearly 5000-fold enrichment (Larionov et al., 1996b) over the 0.1% human DNA present in the hybrid cells. These results led to the demonstration that TAR cloning could be used to isolate a specific human gene (Larionov et al., 1997), the breast cancer gene BRCA2. Although it had been sequenced, no complete BRCA2 gene had been isolated either as a YAC or a BAC (a bacterial artificial chromosome in E. coli). To do this, the TAR vector with hooks of approximately 500 bp each of the promoter sequence and the noncoding region of the last exon (see Figure 1) was presented to yeast cells along with total DNA isolated from human fibroblasts. About 1 in 300 transformants (Larionov et al., 1997 and unpublished) selected for the vector marker also contained
The minimum size of the hooks required for TAR cloning appears to be less than 150 bp (Larionov et al., 1996). If a human fragment containing a sequence A' and B' winds up in the same cell as the vector, recombination between the cut plasmid and the fragment will generate a circular yeast artificial chromosome (YAC) which can be selected using the plasmid marker. Because the plasmid has no yeast replication origin, sequences in human DNAs capable of functioning as A R S 's in yeast provide for the propagation of the YAC. Thus, the isolation of human DNA is essentially accomplished by marker rescue through recombination. The generation of YACs with large human segments was proposed to be due to preferential double-strand break repair at or near ends of molecules rather than internal regions (Larionov et al., 1996a). [The original model for double-strand break repair (Resnick, 1976) has now had many applications and refinements that extend from the repair of radiation-induced breaks, natural breaks and gap repair of incoming molecules (Orr-Weaver et al., 1983) to gene replacement in mammalian cells (Cappechi, 1988) and the development of knockout mice and now TAR cloning.] The opportunity to TAR clone human DNA was suggested from experiments (Larionov et al., 1994) in which it was shown that during transformation there was efficient recombination between an incoming plasmid with an Alu and an incoming human yeast artificial chromosome (YAC) that contained several Alu's . With this in mind, transformation-associated recombination was explored as an alternative means of generating linear YAC 610
Gene Therapy and Molecular Biology Vol 1, page 611 Table 1. Specific isolation of human DNA by TAR cloning DNA cloned and source
Hook A
Hook B
100 Mb Chromosome 16 in a monochromosomal hybrid [6,7]
consensus ALU
BLUR13 ALU
5 Mb Ku80 in a radiation hybrid [7]
consensus ALU
BLUR13 ALU
43 kb rDNA unit in total human DNA [10]
non transcribed spacer
BLUR13 ALU
90 kb BRCA2 in total human DNA [4]*
5' upstream sequence
3' downstream sequence
5' upstream sequence
3' downstream sequence 3' downstream sequence
82 kb BRCA1 in total human DNA *
(unpublished)
70-350 kb HPRT in total human DNA * (unpublished)
BLUR13 ALU
* Up to 1% of the yeast transformants had the gene of interest. The genes were identified through pooling of transformants, PCR analysis, followed by isolation of clones.
F i g u r e 2 . A TAR cloning cycle for the specific isolation of human DNA and its reintroduction into mammalian cells. Human DNA can be specifically isolated in yeast by TAR cloning, modified for transfer to bacteria and then transferred to mammalian cells. Alternatively, the TAR vector can contain sequences that would enable selection in mammalian cells enabling direct transfer from yeast.
with one hook that is unique to the gene(s) being isolated and the other hook being a common repeat. The numbers of unique genes isolated per Âľg of total DNA presented to yeast were comparable for the BRCA1 and the HPRT genes. Thus, direct gene isolation is now possible using information derived from only a small portion of a gene. Because only a few weeks are required once the vectors are built, TAR cloning provides new opportunities for investigating genes and chromosomal regions directly from
the BRCA2 gene and these could be easily identified by PCR analysis (see Table 1). Further physical analysis established that several independent copies of the complete gene had indeed been isolated. The utility of TAR cloning for the specific isolation of human genes has now been demonstrated further for the BRCA1 and the HPRT genes (unpublished) and the human ribosomal RNA gene family (Kouprina et al., 1997). As shown in Table 1, specific isolation can be accomplished 611
Resnick et al: TARgeting the human genome to make gene isolation easy individuals. Previously, isolation of specific chromosomal regions would have required the development of a library for each person studied followed by extensive analysis to find the region of interest. These features suggest that TAR cloning can open the way to clinical investigations of whole genes or large chromosomal regions since, for example, only 10 to 20 ml of blood would be needed for the isolation of a specific gene. Another novel utility--referred to as radial TAR cloning--derives from the isolation of the rDNA and the HPRT genes with a vector that has a unique sequence hook and an Alu repeat hook (A and B, respectively, in Figure 1 and T a b l e 1 ). YACs are generated that extend from the unique position to various Alu's. By changing the orientation of the unique hook, a radial series of YACs is developed that surround the unique sequence. There are many applications that include isolating a unique region surrounding a particular STS or EST site. In addition chromosomal changes such as amplifications and translocations in individuals become directly accessible with TAR cloning once a chromosomal sequence is identified. Radial TAR cloning also provides the opportunity to clone a region lacking an ARS-like sequence since the hook with the common repeat enables the isolation of chromosome fragments that are sufficiently large that they are likely to contain such a sequence. The TAR cloning can be used in a cycle that provides for specific human DNA isolation and reintroduction, as described in Figure 2. Once DNA is isolated as a circular molecule it can be modified and even retrofitted with bacterial artificial chromosome sequences and mammalian selectable markers such as neomycin (NEO) or hygromycin resistance (or alternatively the original TAR vector could contain these sequences) using recombination methods standard to yeast (Larionov et al., 1996b, 1997). The YAC/BAC can than be transferred into E. coli in order to obtain large amounts of this DNA and it could subsequently be introduced into human cells. (Large circular molecules may be isolated directly from yeast, so that the step involving transfer to E. coli could be eliminated.) This approach is being applied to the BRCA2, BRCA1 and HPRT genes initially isolated as YACs. The subsequent YAC/BACs are reintroduced into mammalian cells using the NEO marker for selection. Since, as recently shown for HPRT, most of the isolated genes are functional when transferred to mammalian cells (in preparation), the cycle of human DNA isolation and reintroduction can be accomplished with high fidelity. The tremendous success of the human genome project has relied on the development of new approaches. The information generated can be applied to many areas including functional genomics, investigations of genetic diseases, gene manipulation and gene therapy. TAR cloning is one of the new tools that will make our chromosomes more accessible.
Acknowledgment This work was supported in part through a CRADA (Cooperative Research and Development Agreement) between NIEHS and Life Technologies Inc. of Gaithersburg, Maryland.
References Capecchi, M.R. (1 9 8 9 ). Altering the genome by homologous recombination, Science 2 3 3 , 1288-1292. Kouprina, N., Graves, J., Resnick, M.A., Larinov, V. (1 9 9 7 ) Specific isolation of rDNA genes by TAR cloning, Gene 197, 269-276. Larionov, V., Kouprina, N., Eldarov, M., Perkins, E., Porter, G., and Resnick, M. (1 9 9 4 ) Transformation-associated recombination between diverged and homologous DNA repeats is induced by strand breaks, Y e as t 10, 93-104. Larionov, V., Kouprina, N., Graves, J., Chen, X-N., Korenberg, J. R. and Resnick, M.A. (1 9 9 6 a ). Specific cloning of human DNA as YACs by transformationassociated recombination. P r o c . N a t l . A c a d . S c i. 93, 491-496. Larionov, V., Kouprina, N., Graves, J. and Resnick, M.A. (1 9 9 6 b ) Highly selective isolation of human DNAs from rodent-human hybrid cells as circular YACs by TAR cloning. P r o c . Natl Acad. S c i 93, 13925-13930 (1996b). Larionov, V., Kouprina, N., Nikolaishvili, N. and Resnick, M. A., (1 9 9 4 ) Recombination during transformation as a source of chimeric mammalian artificial chromosomes in yeast (YACs), N u c l e i c A c i d s R e s e a r c h 22, 41544162. Larionov, V., Kouprina, N. Solomon, G., Barrett, J. C. and Resnick, M.A. (1 9 9 7 ). Direct isolation of human BRCA2 gene by transformation-associated recombination in yeast. P r o c . N a t l . A c a d . S c i . 94, 7384-7387. Ma, H., Kunes, S., Schatz, P.J., and Botstein, D. (1 9 8 7 ). Plasmid construction by homologous recombination in yeast. Gene 58, 201-216. McCormick M. K.., Campbell, E., Deaven, L., Moyzis, R. (1993) Low-frequency chimeric yeast artificial chromosome libraries from flow-sorted human chromosomes 16 and 21, P r o c . N a t l A c a d . S c i . 90, 1063-1067. Mezard, C., Pompon, D., and Nicolas, A. (1 9 9 2 ). Recombination between similar but not identical DNA sequences during yeast transformation occurs within short stretches of identity, C e l l 70, 659-670. Resnick, M.A. (1 9 7 6 ). The repair of double-strand breaks in DNA: a model involving recombination. J. T h e o r e t i c a l B i o l o g y 59, 97-106. Rudolph, H., Koenig-Rauseo, I., and Hinnen, A. (1 9 8 5 ). One-step gene replacement in yeast by cotransformation, Gene 36, 87-95. Stinchcomb, D. T., Thomas, M., Kelly, J., Selker, E., and Davis, R. W. (1 9 8 0 ). Eukaryotic DNA segments capable of autonomous replication in yeast. P r o c . N a t l A c a d . S c i 77, 4559-4563 (1980).
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Gene Therapy and Molecular Biology Vol 1, page 613 recombination. C e l l 33, 25-35.
Szostak, J.W., Orr-Weaver, T.L., Rothstein, R.J., Stahl, F.W. (1 9 8 3 ), The double-strand break model for
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Control of growth and proliferation by the retinoblastoma protein Robert J. White Institute of Biomedical and Life Sciences, Division of Biochemistry and Molecular Biology, Davidson Building, University of Glasgow, Glasgow, G12 8QQ, U.K. _______________________________________________________________________________________________ Correspondence: Robert J. White, Tel: 0141-330-4628, Fax: 0141-330-4620, E-mail: rwhite@udcf.gla.ac.uk Keywords: Cancer. Retinoblastoma. Tumour suppression. Transcription. Translation
Summary The retinoblastoma susceptibility gene R b is an important tumour suppressor. It will inhibit both growth and proliferation when introduced into many types o f c e l l . Furthermore, i t i s frequently found mutated in a range of human cancers. It is therefore of considerable importance that we should understand fully how this gene operates. The RB gene product is a 110 kDa nuclear phosphoprotein that regulates the activity of a number of key transcription factors. In turn, its activity is controlled through phosphorylation by cyclin-dependent kinases i n response t o the availability of growth factors. It therefore provides a mechanism for coordinating gene expression with growth factor availability. One of the principle targets of RB is a transcription factor called E 2 F . E 2 F c o n t r o l s t h e e x p r e s s i o n o f a p a n e l o f g e n e s t h a t p r o m o t e p r o l i f e r a t i o n . By downregulating these genes through its inhibitory action on E2F, RB provides a restraining influence u p o n c e l l c y c l e p r o g r e s s i o n . I t has been l e s s clear how RB i s able t o suppress the growth (increase in mass) of cells. However, recent studies have suggested that it may achieve this by repressing the production of rRNA and tRNA. Loss of control over the protein synthetic apparatus may constitute an important step in tumour development.
1992). Expression of exogenous Rb was found to inhibit growth, proliferation, soft agar colony formation and tumourigenicity in nude mice (Bookstein et al., 1990; Huang et al., 1988; Qin et al., 1992). Further proof of the importance of Rb in resisting carcinogenesis was provided by the specific mutagenesis of this gene. Although homozygous deletion of Rb is lethal, heterozygous mice survive and display a strong predisposition to cancer (Hu et al., 1994; Jacks et al., 1992; Lee et al., 1992; Maandag et al., 1994; Nikitin and Lee, 1996; Williams et al., 1994). These observations prove unequivocally that Rb is a bona fide tumour suppressor gene. Having established the credentials of RB as an important tumour suppressor, it became a major priority to determine how it achieves this effect. At a cellular level, RB is involved in constraining both growth (increase in cell mass) and proliferation (increase in cell number): without it the ability of cells to shut down these functions is compromised (Weinberg, 1995; Whyte, 1995). Mammalian cells decide between proliferation and quiescence during the first two thirds of G1 phase: if growth factors are plentiful at this time they continue
I. Introduction The retinoblastoma susceptibility gene Rb is essential for life. Its homozygous inactivation causes mouse embryos to die during the fourteenth day of gestation with defective neural and erythroid development (Clarke et al., 1992; Jacks et al., 1992; Lee et al., 1992). The Rb gene encodes a 110 kDa nuclear phosphoprotein that is expressed almost ubiquitously in normal mammalian cells (Weinberg, 1995; Whyte, 1995). It was mapped to chromosome 13q14 by virtue of its association with an inherited predisposition to retinoblastoma, a rare pediatric tumour of the retina (Friend et al., 1986). Inactivating mutations in this gene also occur in many other types of human malignancy, including small-cell lung cancers, several sarcomas and bladder carcinomas (Weinberg, 1995; Whyte, 1995). These observations suggested that Rb is a tumour suppressor and that loss of its function can contribute to oncogenesis. Support for this idea came from experiments in which the wild-type gene was introduced into tumour cells that lacked its function (Bookstein et al., 1990; Huang et al., 1988; Qin et al.,
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White: Control of Growth and Proliferation by RB through the cell cycle, but if conditions are unfavourable they withdraw from cycle and quiesce (Pardee, 1989). Before reaching the end of G1, cells become committed to complete the mitotic cycle regardless of growth conditions (Pardee, 1989). This transition to serum-independence is called the R (restriction) point (Pardee, 1989). RB serves an important function in restraining passage through the R point when growth factors are limiting (Sherr, 1994; Weinberg, 1995; Whyte, 1995). When RB function is lost, the sensitivity of cells to their normal regulatory signals is severely compromised (Sherr, 1994; Weinberg, 1995; Whyte, 1995). This constitutes a major step towards uncontrolled proliferation. Although it is clear that RB regulates passage through the R point, many unanswered questions remain as to how this is achieved in mechanistic terms. To understand fully the complex biological effects of RB, it will be necessary to determine how it operates at the molecular level. Although some aspects of this have been characterised extensively, novel targets for RB are still being discovered (Taya, 1997). The relative contribution of each of these targets in inhibiting growth, proliferation and tumour formation will need to be established. A complete picture of how RB functions will require the careful interlinking of its various activities.
carcinomas. In such cases, the patient inherits two wildtype alleles of Rb, but mutations arise in both copies during tumourigenesis. The most striking examples of this are the small cell lung carcinomas, where Rb changes are found in nearly all cases (Horowitz et al., 1990). Other types of tumour display a lower frequency of Rb mutation. For example, RB was found to be altered or absent in a third of bladder carcinomas that were surveyed (Horowitz et al., 1990). However, many types of tumour express apparently wild-type RB, including melanomas and colon carcinomas (Horowitz et al., 1990). Thus, mutation of Rb is a tumour-specific phenomenon.
B. Inactivation of RB by viral oncoproteins A survey of human cervical carcinoma cell lines found that two out of seven bear small inactivating mutations in RB (Scheffner et al., 1991). Whereas neither of these lines were infected by human papillomavirus (HPV), each of the remaining five that expressed normal RB also contained HPV DNA (Scheffner et al., 1991). HPVs play an etiologic role in most cervical neoplasias (Vousden, 1995). The E7 oncoprotein encoded by HPV can transform established cell lines and has also been shown to bind to RB (Dyson et al., 1989; Munger et al., 1989). Some HPVs, such as HPV-16 and -18, are associated with potentially pre-cancerous genital tract lesions and a large percentage of anogenital cancers, whereas others, such as HPV-6 and -11, are associated with benign proliferative tumours with a low risk of malignant progression (e.g. condyloma acuminata) (Vousden, 1995). E7 proteins from the high risk viruses HPV-16 and -18 have higher binding affinity for RB than E7 from the lower risk types HPV-6 and -11 (Heck et al., 1992; Munger et al., 1989). Single residue substitutions in HPV-6 E7 that cause a substantial increase in affinity for RB also produce a concomitant gain in transforming activity (Heck et al., 1992; Sang and Barbosa, 1992). It is therefore likely that the ability of E7 to bind RB contributes significantly to the oncogenic capacity of HPVs. Therefore, RB function may be lost in most if not all cervical cancers; this occurs by gene mutation in the minority of HPV-negative cases and by complex formation with E7 protein in the remaining instances (Scheffner et al., 1991). The transforming proteins of several other DNA tumour viruses can also bind RB and neutralize its function (Vousden, 1995). This property is shown by the large T antigen of simian virus 40 (SV40) (DeCaprio et al., 1988; Ewen et al., 1989; Ludlow et al., 1989; Moran, 1988) and the E1A protein of adenovirus (Whyte et al., 1988, 1989). Mutagenesis studies have shown that the regions of these oncoproteins that are necessary for binding RB are also required for their transforming properties (DeCaprio et al., 1988; Ewen et al., 1989; Moran, 1988; Whyte et al., 1989). Furthermore, the parts of RB that are needed for association with E1A and T antigen are also common sites for mutations (Hu et al., 1990). By binding to RB, these viral proteins can interfere with its normal
II. RB and cancer A. Mutational inactivation of RB People who inherit a nonfunctional allele of the Rb gene have an approximately 90% chance of developing retinoblastoma at an early age (Whyte, 1995). Inactivation of the remaining allele by somatic mutation seems to be a universal feature of this cancer and is probably the ratelimiting step in its initiation (Horowitz et al., 1990). Individuals who survive hereditary retinoblastoma show a strong predisposition to osteosarcomas and soft tissue sarcomas later in life: this again is associated with loss of the second Rb allele (Whyte, 1995). These osteosarcomas and mesenchymal tumours are less frequent than retinoblastoma in Rb heterozygotes, and loss of the functional copy of Rb may not be rate-limiting for such tumours (Whyte, 1995). Unlike humans, Rb +/- mice do not develop retinoblastoma: instead over 95% die 300-400 days after birth with melanotroph tumours of the intermediate pituitary lobe (Hu et al., 1994; Maandag et al., 1994; Williams et al., 1994). Sequential analyses of the initial stages of spontaneous melanotroph carcinogenesis in heterozygous mice suggest that mutation of the Rb gene is the initiating event of malignant transformation (Nikitin and Lee, 1996). It is not understood why murine and human Rb heterozygotes suffer different types of cancer. Neither is it known why melanotrophs or retinoblasts are particularly sensitive to the inactivation of RB. Many other types of human tumour display somatic mutation of Rb, including osteosarcomas, small cell lung carcinomas, breast cancers, prostate and bladder 614
Gene Therapy and Molecular Biology Vol 1, page 615 cellular functions and thereby mimick the effects of the Rb mutations that occur in many tumours.
C. Inactivation of RB by phosphorylation RB can be switched off through phosphorylation (Hunter and Pines, 1994; Pines, 1995; Sherr, 1994; Weinberg, 1995). This constitutes a normal control mechanism that is used to regulate progress through the cell cycle (Hunter and Pines, 1994; Pines, 1995; Sherr, 1994; Weinberg, 1995). Thus, RB is underphosphorylated during the first two thirds of G1 phase and whilst in this condition it helps prevent cells from passing through the R point (Hunter and Pines, 1994; Pines, 1995; Sherr, 1994; Weinberg, 1995). Near the end of G1, if conditions are propitious, RB becomes phosphorylated at multiple sites and loses its ability to inhibit passage into S phase (Hunter and Pines, 1994; Pines, 1995; Sherr, 1994; Weinberg, 1995). Its affinity for the nuclear compartment is also diminished (Mittnacht and Weinberg, 1991). The cyclin D- and cyclin E-dependent kinases are responsible for controlling RB in this way (Hunter and Pines, 1994; Pines, 1995; Sherr, 1994; Weinberg, 1995). The activity of cyclin D-dependent kinases is abnormally elevated in a variety of cancers and this provides another mechanism whereby RB function is lost (Bates and Peters, 1995; Hunter and Pines, 1994; Pines, 1995; Weinberg, 1995). The gene for cyclin D1 is amplified in at least 15% of primary breast cancers and an even greater proportion of squamous cell carcinomas of the neck, head, oesophagus and lung (Bates and Peters, 1995; Hunter and Pines, 1994). Furthermore, cyclin D1 RNA and protein is overexpressed in 30-40% of primary breast tumours, suggesting that gene amplification is not the only mechanism contributing to increased levels of the product (Bates and Peters, 1995). In some parathyroid adenomas and B cell lymphomas, chromosomal translocations cause overproduction of cyclin D1 (Bates and Peters, 1995; Hunter and Pines, 1994). When EpsteinBarr virus immortalizes B-lymphocytes, cyclin D2 becomes activated (Sinclair et al., 1994). The gene for cyclin-dependent kinase 4 is amplified in many glioblastomas and some gliomas (Weinberg, 1995). In addition to these diverse situations in which cyclins or their associated kinases are activated directly, many other cancers lose the function of p16 and/or p15, which are important repressors of the cyclin D-dependent kinases (Hirama and Koeffler, 1995; Hunter and Pines, 1994; Weinberg, 1995). For example, the genes for p16 and p15 are deleted in many glioblastomas, oesophageal, bladder, lung and pancreatic carcinomas, and are sometimes mutated in familial melanomas (Hirama and Koeffler, 1995; Weinberg, 1995). Thus, the cyclin D-dependent kinases become abnormally active in a broad spectrum of cancers through a variety of mechanisms. This has the effect of switching off RB. It is therefore certain that RB function is lost in a high proportion of tumours. Indeed, it has been suggested that the control pathway involving RB may become deregulated 615
in all human malignancies (Weinberg, 1995). This can be achieved in a variety of different ways - gene mutation, association with viral oncoproteins, or hyperphosphorylation. A good illustration of the importance of inactivating RB during tumour progression was provided by a survey of small cell lung carcinomas (Otterson et al., 1994). This study tested 55 small cell lung cancers and found that 48 lacked normal RB expression but contained wild-type p16; six out of the remaining seven lacked functional p16 (Otterson et al., 1994).
III. RB targets A. E2F As explained above, RB acts as a signal transducer which controls gene expression in response to the availability of growth factors. It does this by targetting a number of key transcription factors and regulating their functions. Perhaps the best characterised of these is E2F (Adams and Kaelin, 1995; La Thangue, 1994; Lam and La Thangue, 1994; Weinberg, 1996). E2F is a heterodimeric transcription factor composed of an E2F polypeptide and a DP polypeptide. In vertebrates, five E2F genes and three DP genes have been identified (Adams and Kaelin, 1995). Heterodimerization results in a synergistic increase in both the DNA-binding and transcriptional activation functions of these proteins (Bandara et al., 1993; Helin et al., 1993; Krek et al., 1993). It also enhances the ability to recognize RB (Helin et al., 1993; Krek et al., 1993). Not only does RB mask the transactivation domain of E2F, but it can exert a dominant silencing activity that represses promoters with E2F-binding sites (Weintraub et al., 1992). When growth factors are limiting RB is underphosphorylated and active; it binds to E2F and inhibits it (F i g u r e 1 ). Following serum stimulation, RB becomes phosphorylated at multiple sites by the cyclin D-dependent kinases; this inactivates it and causes it to dissociate from E2F, thereby allowing the expression of E2F-responsive genes (Adams and Kaelin, 1995). T a b l e 1 lists some of the genes that contain E2F sites in their promoters. Many of these have been shown to be regulated by E2F, but it has not been proven in every case. These potential target genes can be divided into five categories. One group consists of genes encoding subunits of E2F, which suggests that autoregulation may occur. A second category contains Rb and the related gene p107, which implies further opportunities for feedback control. The next class consists of the oncogenes B-myb , N-myc and c-myc. Another group contains several genes that are directly involved in driving the cell cycle, including components of the cyclin-dependent kinases and the cdc25C phosphatase that activates these. The fifth and largest group consists of many genes that encode components of the DNA replication apparatus, including DNA polymerase !, the origin recognition factor HsOrc1, and several enzymes involved in nucleotide biosynthesis. A striking feature of this list is that many of the genes with E2F sites would be predicted to contribute to cellular
White: Control of Growth and Proliferation by RB proliferation. This is the case for the oncogenes and for cdc2 and the cyclins, which have a positive effect on cell
of this is to look for changes in expression following the specific deletion of the Rb gene. Very few of the genes listed in Table 1 pass this test. One study of primary mouse embryonic fibroblasts (MEFs) examined ten genes with E2F sites and found that only cyclin E and p107 synthesis were changed following homozygous inactivation of Rb (Hurford et al., 1997). During G0 and G1 phases, cyclin E and p107 mRNA levels were twofold higher in Rb -/- MEFs compared to wildtype controls (Hurford et al., 1997). The expression of B-myb, cdc2, E2F-1, TS, RRM2, cyclin A2, DHFR, TK, DNA polymerase , and Cdc25C genes were unaffected by the RB-knockout (Hurford et al., 1997). Another study found a ten-fold increase in cyclin E protein and a two- to fourfold increase in cyclin D1 when Rb -/- MEFs were compared to the corresponding Rb +/+cells (Herrera et al., 1996). The surprising lack of effect that deleting Rb has upon most E2F target genes is probably due to redundancy in the RB family. RB has two close relatives called p107 and p130 (Figure 2). These three proteins show substantial similarity in primary sequence and are thought to perform overlapping functions (Whyte, 1995). They are most highly related in a bipartite domain called the pocket, which is responsible for binding E1A and E2F (Whyte, 1995). As a consequence, they are sometimes referred to as the pocket proteins. It may be that when Rb is deleted, its relatives can assume many of its functions. Indeed, a double knockout of Rb and p107 has a more severe phenotype than single knockouts of either (Lee et al., 1996). This is certainly consistent with a functional overlap. However, p107 and p130 are much more similar to each other than they are to RB. Indeed, whereas RB specifically targets E2F-1, -2 and -3, p107 and p130 appear to bind only E2F-4 and E2F-5 (Weinberg, 1995). A p107/-/p130-/- double knockout strongly derepresses B-myb but has no effect on cyclin E (Hurford et al., 1997). It therefore seems that p107 and p130 can only assume some of the functions that are performed by RB. The most striking difference between the pocket proteins is that p107 and p130 have never been found to be mutated in cancers. Even if many of the genes listed in Table 1 are not subject to control by RB, repressing the synthesis of cyclins E and D1 through its action on E2F should in itself be sufficient to provide a brake upon cell cycle progression and hence proliferation. Indeed, under certain circumstances dominant-negative mutants that abolish E2F activity can block the cell cycle (Dobrowliski et al., 1994;Wu et al., 1996). However, this is by no means the
F i g u r e 1 . When growth factors are limiting, RB binds to E2F and represses its ability to activate transcription. Following serum stimulation, RB becomes hyperphosphorylated at multiple sites through the action of cyclin-dependent kinases. This inactivates RB and causes it to dissociate from E2F, which allows expression of E2Fresponsive genes.
Table 1. Genes regulated by E2F 1. E2F Components - E2F-1, -4, -5 - DP-1 2. Pocket proteins - RB - p107 3. Oncogenes - B-myb - c-myc - N-myc 4. Genes that drive the cell cycle - cdc2 - cyclin A - cyclin D - cyclin E - cdc25C 5. Genes required for DNA replication - DNA polymerase ! - HsOrc1 - PCNA - topoisomerase I - thymidylate synthase - thymidine kinase - ribonucleotide reductase
cycle progression. Furthermore, DNA replication is clearly a prerequisite of productive cell division. One would therefore predict that by inhibiting the expression of the batteries of genes listed in Table 1, through its repressive effect on E2F, RB would be able to achieve a very potent block upon proliferation. As yet it is unclear how many of the genes with E2F sites are actually regulated by RB. The most stringent test 616
Gene Therapy and Molecular Biology Vol 1, page 617
B. UBF
F i g u r e 2 . Regions of homology between all three pocket proteins are shown as black blocks. Regions that are homologous between p107 and p130, but are not shared by RB, are shaded.
whole story. In molar terms, RB is two orders of magnitude more abundant than E2F within the cell (Weinberg, 1995). This suggests that RB regulates additional targets besides E2F. Indeed, one study found that the proliferation rate of epithelial cells is not affected when endogenous E2F is inactivated using dominantnegative mutants (Bargou et al., 1996). It is therefore highly likely that E2F-independent pathways contribute to the physiological effects of RB. A diverse array of cellular proteins have been shown to bind RB (Taya, 1997; Whyte, 1995). Some of these are listed in Table 2. They include the tyrosine kinase c-Abl (Welch and Wang, 1993) and the factors BRM and BRG1 that are involved in controlling nucleosome structure (Dunaief et al., 1994; Singh et al., 1995). Several others are transcription factors. I shall concentrate on two of these, UBF and TFIIIB, which may have key roles in controlling cell growth.
Table 2. Some of the cellular proteins that interact with RB PROTEIN
FUNCTION
E2F UBF TFIIIB Elf-1 PU.1 Brm BRG1 MyoD ATF-2 MDM2 c-Abl D-type cyclins PP1
Transcription factor Transcription factor Transcription factor Transcription factor Transcription factor Reorganising chromatin structure Reorganising chromatin structure Transcription factor Transcription factor Oncoprotein Tyrosine kinase Cdk activators Protein phosphatase
A nucleolar transcription factor called UBF was identified as an RB-binding protein by screening a cDNA expression library with purified RB as probe (Shan et al., 1992). Subsequent studies have confirmed the ability of recombinant RB to bind specifically to UBF (Cavanaugh et al., 1995; Voit et al., 1997). Furthermore, immunoprecipitation assays with cellular extracts demonstrated that endogenous RB and UBF associate when present at physiological ratios (Cavanaugh et al., 1995). The identification of UBF as a target for RB was somewhat unexpected. All previous studies on RB had concentrated on genes that are transcribed by RNA polymerase II (pol II), which synthesizes the messenger RNA (mRNA) in cells. However, UBF is only involved in regulating transcription by pol I, the polymerase responsible for synthesizing large ribosomal RNA (rRNA). UBF binds to the promoters of rRNA genes and stimulates transcription in several ways; it helps fold the DNA and it recruits pol I and an essential factor called SL1 or TIF-IB (Reeder et al., 1995). In vitro experiments demonstrated that recombinant RB can indeed inhibit the synthesis of large rRNA by pol I (Cavanaugh et al., 1995; Voit et al., 1997). Whereas RB represses rRNA production in the presence of UBF, it does not affect the low level of basal transcription that occurs in a UBFdepleted system (Cavanaugh et al., 1995). RB diminishes specifically the ability of UBF to bind to DNA (Voit et al., 1997). The physiological relevance of these results was confirmed by immunofluorescence analyses of intact cells (Rogalsky et al., 1993; Cavanaugh et al., 1995). This approach allows one to visualise the nucleolus, which is the site of synthesis of large rRNA. It was found that RB accumulates in the nucleolus when cells stop growing, in parallel with a decrease in pol I activity (Rogalsky et al., 1993; Cavanaugh et al., 1995). Furthermore, immunoprecipitation experiments showed that the interaction between RB and UBF increases when cells down-regulate pol I transcription as their rate of growth decreases (Cavanaugh et al., 1995). Thus, there is a clear in vivo correlation between growth arrest, the association of RB with UBF, and the repression of rRNA synthesis.
C. TFIIIB Although 5.8S, 18S and 28S rRNAs are made by pol I as a single precursor transcript, the 5S rRNA is made separately by pol III, the largest and most complex of the eukaryotic RNA polymerases (White, 1994). 5S rRNA synthesis is independent of E2F and UBF, but is nevertheless repressed by RB (White et al., 1996; Larminie et al., 1997). Indeed, RB appears to be capable of inhibiting the production of all pol III products, including transfer RNA (tRNA), the U6 small nuclear RNA (snRNA) that is required for splicing, and the adenoviral VA RNAs that are involved in subverting the host cell's
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White: Control of Growth and Proliferation by RB
F i g u r e 3 . RB can regulate transcription by all three nuclear RNA polymerases. Pol I synthesizes large rRNA; pol II synthesizes mRNA; and pol III synthesizes tRNA and 5S rRNA. A simplified basal transcription complex is drawn for each polymerase; additional basal factors are required that are not shown in the figure. RB represses pol I via the factor UBF. RB represses a subset of pol II templates via gene-specific regulatory factors such as E2F. RB represses pol III via the general factor TFIIIB.
function of endogenous RB, we made use of knockout mice in which the the Rb gene had been inactivated by site-directed mutagenesis. Nuclear run-on assays were used to measure directly the transcription of endogenous genes in intact nuclei from primary MEFs of these RB-knockout mice. We found that tRNA and 5S rRNA synthesis by pol III is 5-fold more active in the Rb -/- cells than in equivalent fibroblasts from wild-type mice (White et al., 1996). In contrast, the total level of pol II transcription is not increased when the Rb gene is deleted (White et al., 1996). In vitro assays with extracted factors again established that the increased production of tRNA and 5S rRNA in the RB-negative MEFs is due to a more active pol III transcription apparatus (White et al., 1996). Since the only genetic difference between the Rb +/+ and the Rb /- fibroblasts is the presence of the Rb gene, these results established that endogenous RB plays a very major role in suppressing the level of pol III transcription in vivo. RB appears to regulate tRNA and rRNA synthesis by targetting a factor called TFIIIB (Larminie et al., 1997). TFIIIB is a multisubunit complex that contains the TATA-binding protein (TBP) and at least two additional polypeptides (Rigby, 1993; White, 1994). Its function is to recruit pol III to the appropriate promoters and position it at the transcription start site (Kassavetis et al., 1990; White, 1994). We found that recombinant RB interacts with TFIIIB and represses it specifically (Larminie et al.,
translational apparatus (White et al., 1996; Larminie et al., 1997). Initial evidence that this is the case came from biochemical assays which tested whether RB can regulate pol III in a cell-free system. We found that adding recombinant RB to a system reconstituted using fractionated factors repressed expression of every pol III template tested, whereas control protein had little or no effect (White et al., 1996). Support for the in vivo relevance of these observations came from transient transfection experiments, which showed that overexpressing RB can repress pol III transcription without affecting a control promoter (White et al., 1996). These results demonstrated for the first time that high levels of RB can inhibit pol III activity. Overexpressing proteins at abnormally elevated levels can sometimes force proteins into artifactual interactions. It was therefore important to determine whether RB plays a significant role in controlling pol III when present at physiological concentrations within a cell. To begin to address this, we compared two human osteosarcoma cell lines; SAOS2, which expresses only a truncated nonfunctional form of RB, and U2OS, which contains wild-type RB. SAOS2 cells were shown to express a transfected pol III template 5-fold more actively than U2OS cells (White et al., 1996). Transcription assays carried out using extracted proteins confirmed the higher activity of the pol III factors from the RB-negative SAOS2 cells (White et al., 1996). As a more rigorous test of the 618
Gene Therapy and Molecular Biology Vol 1, page 619 1997). Furthermore, immunoprecipitation and cofractionation experiments indicated that a population of endogenous RB molecules associates with TFIIIB at physiological concentrations (Larminie et al., 1997). This interaction is diminished or abolished in SAOS2 osteosarcoma cells, which contain only a truncated mutant form of RB (Larminie et al., 1997). The activity of TFIIIB is elevated specifically in primary fibroblasts from RBdeficient mice (Larminie et al., 1997). These results established that TFIIIB is a target for repression by RB (Figure 3). This conclusion fits well with previous data indicating that TFIIIB activity rises as cells progress from G1 into S phase, the time when RB is silenced through hyperphosphorylation (White et al., 1995). A subsequent investigation by Chu et al. (1997) provided independent support for these analyses. This study confirmed that overexpressing RB represses pol III transcription in transfected cells and in vitro (Chu et al., 1997). Consistent with the earlier investigations, RB was shown to bind to TFIIIB (Chu et al., 1997). Furthermore, clustered substitutions in RB that disrupt the interaction with TFIIIB also prevent repression (Chu et al., 1997). In addition, Chu et al. (1997) reported an interaction between overexpressed RB and another pol III factor called TFIIIC2. A model was proposed in which RB utilises distinct domains to bind either TFIIIB or TFIIIC2 (Chu et al., 1997). However, there was little correlation between TFIIIC2 binding and the ability of RB mutants to repress pol III transcription (Chu et al., 1997). Furthermore, there is no evidence that TFIIIC2 and RB interact when present at physiological ratios. Chu et al. (1997) concluded that TFIIIB is the principal target for RB-mediated repression of pol III, but that a subsidiary interaction with TFIIIC2 may also contribute to the effect. Although it has been shown that RB binds to one or more of the general pol III factors, it remains to be determined how this leads to transcriptional repression. One possibility is that RB blocks interactions with promoter DNA. Precedent for this is provided by the pol I system, where RB interferes with the DNA-binding properties of UBF (Voit et al., 1997). An alternative is that RB disrupts the structure of TFIIIB in some way. A growth suppressor called Dr1 has been shown to use this mechanism (White et al., 1994). Dr1 inhibits tRNA synthesis both in vitro and in vivo (White et al., 1994; Kim et al., 1997). It achieves this by displacing one of the essential subunits of TFIIIB from its interaction with TBP (White et al., 1994). Other possible mechanisms might involve RB disrupting the protein-protein interactions between TFIIIB and TFIIIC or pol III. Order of addition experiments showed that the pol III factors remain susceptible to RB even after they have been assembled into a stable preinitiation complex on the VAI promoter (Larminie et al., 1997). TFIIIB is required for all pol III transcription (White, 1994; Willis, 1993). Therefore by repressing TFIIIB, RB can provide blanket repression of all pol III templates.
619
This contrasts strongly with the situation for pol II, where only a small proportion of promoters, such as those with E2F sites, are controlled by RB. The majority of genes that are transcribed by pol II are not affected directly by the presence of RB (White et al., 1996). Therefore, RB is a gene-specific regulator of pol II but a general regulator of pol III. This distinction is meaningless in the pol I system, since pol I only transcribes a single highly reiterated template that encodes the large rRNA. The number of genes that are controlled by E2F is relatively small and very few of these become activated in Rb -/- knockouts (Herrera et al., 1996; Hurford et al., 1997). UBF probably regulates a larger number of genes, since there are ~400 copies of the large rRNA template in diploid human cells (Long and Dawid, 1980). It remains to be determined whether these are affected by knocking out RB. The number of promoters that require TFIIIB exceeds this by over three orders of magnitude. Thus, a diploid human cell contains around a million Alu genes, 2600 tRNA genes, 600 5S rRNA genes, 200 U6 snRNA genes and a range of other less abundant pol III templates (White, 1994). All of these need TFIIIB to be expressed (White, 1994). The tRNA and 5S genes have been shown to be activated in Rb -/- knockouts, whereas the other classes have yet to be tested in this way (White et al., 1996). Since deleting Rb results in a five-fold increase in tRNA and 5S rRNA production (White et al., 1996), it is highly likely that the majority of these genes are subject to repression by RB. These observations suggest that the pol III templates constitute by far the largest category of genes that are controlled directly by RB.
IV. Control of growth and proliferation by RB Both the growth (increase in mass) and proliferation (increase in number) of cells are suppressed by RB. It is essential that these two processes are coordinated, because a significant imbalance can trigger apoptosis (Kung et al., 1993; Qin et al., 1994; Rueckert and Mueller, 1960; Shan and Lee, 1994). In order to maintain a constant size, a cell must ensure that all its components are duplicated at a similar rate. Thus, DNA content and protein levels generally increase in parallel (Stanners et al., 1979) and attempts to dissociate them with specific inhibitors can have lethal consequences (Kung et al., 1993). The control of proliferation by RB can be largely explained by its ability to regulate E2F. As described above, E2F regulates a range of pol II-transcribed genes that promote cell cycle progression (Adams and Kaelin, 1995; La Thangue, 1994; Lam and La Thangue, 1994; Weinberg, 1996). These include several genes that are required for DNA replication, such as those encoding DNA polymerase ! and the replication origin-binding protein HsOrc1, as well as genes that drive the cell cycle, such as cyclin A and cdc2 (Adams and Kaelin, 1995; Weinberg, 1996). By repressing some of these through its inhibitory effect on E2F, RB can often
White: Control of Growth and Proliferation by RB
Figure 4. Mechanisms that may enable RB to restrict cell growth and proliferation. E2F promotes cell cycle progression. It appears to do this by activating the synthesis of proteins required for DNA replication, such as thymidine kinase (TK), dihydrofolate reductase (DHFR) and DNA polymerase !, as well as proteins that drive the cell cycle, such as cdc2 and cyclins D and E. By repressing E2F, RB may limit the production of these products and therefore provide a brake on proliferation. UBF and TFIIIB are required for the synthesis of rRNA and tRNA, essential raw materials for protein synthesis. By repressing these transcription factors, RB may be able to limit the rate of translation and therefore provide a brake on cellular growth.
One possibility is that this is achieved by regulating the production of tRNA and rRNA, which are major determinants of biosynthetic capacity (Nasmyth, 1996; White, 1997). (Figure 4). A substantial weight of evidence shows that the regulation of protein synthesis is an important aspect of growth control. When cells quiesce, tRNA and rRNA levels decrease, polysomes disperse into free ribosomes, and the overall rate of protein accumulation is reduced. Following mitogenic stimulation, the production of tRNA, rRNA, ribosomal proteins and translation factors accelerates and protein synthesis increases before cells reach S phase (Clarke et al., 1996; Johnson et al., 1974; Kief and Warner, 1981; Mauck and Green, 1974; Redpath and Proud, 1994; Rosenwald, 1996a,b; Stanners et al., 1979; Tatsuka et al., 1992). Ribosome content is proportional to the rate of growth (Kief and Warner, 1981). Indeed, careful measurements in animal cells have demonstrated that growth rate is directly proportional to the rate of protein accumulation (Baxter and Stanners, 1978). The main determinant of protein accumulation is translation, although turnover also makes a significant contribution (Baxter and Stanners, 1978). A 50% reduction in the rate of protein synthesis is sufficient to cause proliferating cells to withdraw from cycle and quiesce (Brooks, 1977; Ronning et al., 1981). Translation is clearly dependent on the availability of tRNA and rRNA. By limiting the production of these, RB may be able to suppress the level of protein synthesis, which could in turn provide a brake on cellular growth. As yet, this
provide a brake on DNA replication and passage through the cell cycle. However, the genes regulated by E2F are primarily involved in controlling proliferation and provide few obvious links to the control of growth. Control of E2F on its own may therefore be insufficient to achieve a balanced regulation of both growth and proliferation. One could imagine that growth is somehow tied to the cell cycle, so that regulating the latter is sufficient to achieve indirect control of the former. However, most of the available evidence argues against this (Nasmyth, 1996). In fact, in bacteria and in yeast the dependence works primarily the other way round, with cell cycle progression requiring attainment of a critical mass (Nasmyth, 1996). The basic principles observed in microrganisms are likely to be conserved in higher orders. For example, murine fibroblasts must reach a certain mass before they can initiate DNA synthesis (Killander and Zetterberg, 1965). Furthermore, a survey of mammalian cell types found that in most cases size continues to increase when DNA synthesis is inhibited using aphidicolin (Kung et al., 1993). HeLa cells provided a striking exception, and in this line biosynthesis and growth decrease in response to the cell cycle block (Kung et al., 1993). HeLa cells are highly abnormal and it is likely that their anomalous behaviour does not provide a reliable indicator of the control mechanisms that operate in most animal cells. In general, the growth of mammalian cells appears not to depend on chromosome duplication, at least in the short term. Thus, the control of proliferation seems insufficient to explain fully the ability of RB to inhibit cell growth. 620
Gene Therapy and Molecular Biology Vol 1, page 621 should still be regarded as speculation, although it must be true to some extent. However, it remains to be determined to what degree the activities of pols I and III are ever limiting for growth under physiological conditions. A good indication that they may be came from a study carried out in S. cerevisiae. It was found that in this yeast a twofold reduction in the level of initiator tRNA results in a three-fold increase in doubling time (Francis and Rajbhandary, 1990). If the same were true of a mammalian cell, then the 5-fold depression in tRNA synthesis that is imposed by RB must surely have a substantial impact upon the rate of growth. During tumour development, when RB function is compromised, the release of pol III from this major constraint may be an important step towards neoplasia.
V. RNA polymerase III and cancer If RB plays an important physiological role in restraining pol III transcription, then one would expect to find that pol III activity is elevated in a broad range of cancers, where RB function is compromised. This is indeed the case. Many studies have observed that the abundance of pol III transcripts is abnormally elevated in transformed and tumour cells. This was first discovered by Kramerov et al. (1982), who examined carcinoma and plasmacytoma lines. Subsequent work extended the observation to include cells that have been transformed by DNA tumour viruses, RNA tumour viruses, or chemical carcinogens (Brickell et al., 1983; Carey and Singh, 1988; Carey et al., 1986; Kramerov et al., 1990; Lania et al., 1987; Majello et al., 1985; Ryskov et al., 1985; Scott et al., 1983; Singh et al., 1985; White et al., 1990). This activation is very general, but not universal, there being a few examples of transformed lines that do not display the characteristic increase in pol III transcript levels (Ryskov et al., 1985; Scott et al., 1983). A tight causal link between pol III activation and transformation is suggested by the fact that two fibroblast lines transformed by temperature-sensitive mutants of the SV40 large T antigen down-regulate pol III transcription at the non-permissive temperature whilst reverting to normal morphology and phenotype (Scott et al., 1983). The abundance of pol III transcripts varies substantially between different SV40transformed lines and the highest levels correlate with progression to a more tumorigenic phenotype (Scott et al., 1983; White et al., 1990). A recent study provided convincing evidence that a pol III product is induced in rodent tumours. This investigation examined a pol III transcript called BC1, which is unusual because it is normally only expressed in neurons (DeChiara and Brosius, 1987). The function of BC1 has yet to be determined. Northern analysis showed BC1 expression in breast carcinomas, colonic adenocarcinomas and skin fibrosarcomas, but not in the corresponding untransformed tissues (Chen et al., 1997). In situ hybridisation studies of theses tumours confirmed the presence of BC1 RNA in the neoplastic cells, whereas it was absent from the surrounding tissues (Chen et al., 621
1997). Although the fibrosarcomas and adenocarcinomas were induced by local inoculation with cells that had been treated with chemical carcinogens, the breast carcinoma analysed was a primary tumour induced by ras (Chen et al., 1997). Similar studies have shown that BC200 RNA, the primate analogue of BC1, is expressed in many, but not all, primary human tumours (Chen et al., 1997). Like BC1, BC200 RNA is found exclusively in the malignant cells and not in the adjacent normal tissue (Chen et al., 1997). Thus, abnormal activation of pol III expression is a frequent feature of tumours in vivo. As already explained, RB function is lost in many human cancers through a variety of mechanisms. It will be important to determine to what extent this is responsible for activating pol III. Deletion and substitution analyses have demonstrated that the RB sequences which control pol III correspond to the domains that are mutated frequently in tumours (White et al., 1996; Chu et al., 1997). Indeed, the minimal region of RB that is necessary to regulate cell growth and proliferation is also sufficient to repress transcription by pol III (White et al., 1996). Several examples have been characterised of highly localised mutations that inactivate RB in human cancers. For example, in one small cell lung carcinoma a single base change in a splice acceptor site gave rise to an RB polypeptide that lacked the 35 amino acids encoded by exon 21 (Horowitz et al., 1990). In another small cell lung carcinoma, a point mutation created a stop codon and a novel splice donor site within exon 22, thereby eliminating 38 residues from the product (Horowitz et al., 1990). A third inactivating mutation from a small cell lung cancer resulted in a single amino acid substitution at codon 706 (Kaye et al., 1990). We tested the ability of each of these three naturally occurring mutants to regulate pol III transcription and found that repression was lost in every case (White et al., 1996). Although this is clearly a limited survey, it nevertheless demonstrates a correlation between the function of RB as a tumour suppressor and its ability to control pol III. As described above, the E1A oncoprotein of adenovirus and the large T antigen of SV40 bind and neutralize RB, a property which is important for their transforming capabilities (DeCaprio et al., 1988; Ewen et al., 1989; Ludlow et al., 1989; Moran, 1988; Whyte et al., 1988; Whyte et al., 1989). Both E1A and T antigen can also stimulate the rate of pol III transcription (Loeken et al., 1988; Patel and Jones, 1990; White et al., 1996). We found that E1A and T antigen can release pol III from repression by RB (White et al., 1996). These viral oncoproteins are believed to regulate gene expression through multiple mechanisms, but one way in which they can stimulate pol III involves overcoming the physiological constraint that is normally provided by RB. In cells transformed by E1A or T antigen, the loss of RB function is likely to contribute substantially to an activation of pol III transcription.
White: Control of Growth and Proliferation by RB 1987). As such, it is of key importance in controlling the rate of translation. Overexpression of eIF-4E in various types of fibroblast stimulates growth and proliferation and induces morphological transformation (Lazaris-Karatzas et al., 1990; Lazaris-Karatzas and Sonenberg, 1992). Cells with abnormally high eIF-4E levels also induce tumours in nude mice (Lazaris-Karatzas et al., 1990; LazarisKaratzas and Sonenberg, 1992). Similarly, overexpression of eIF-4E in HeLa cells accelerates growth and results in the formation of overcrowded multilayered foci (De Benedetti and Rhoads, 1990). Conversely, reducing the level of eIF-4E with antisense RNA inhibits the growth of HeLa cells (De Benedetti et al., 1991) and the tumorigenicity of ras-transformed fibroblasts (RinkerrSchaffer et al., 1993). In serum-starved cells, the recycling of eIF-2 is inhibited by phosphorylation of its ! subunit, thereby impairing translational initiation (Redpath and Proud, 1994). Mutation of eIF-2! so that it can no longer be phosphorylated causes malignant transformation of NIH 3T3 cells (Donze et al., 1995). Malignancy can also be induced by dominant negative forms of the eIF-2! kinase, whereas the wild-type kinase inhibits growth when overexpressed in mammalian fibroblasts or yeast (Chong et al., 1992; Koromilas et al., 1992; Meurs et al., 1993). The eIF-2! kinase (which is also referred to as PKR) is inducible by interferon and is likely to contribute to the action of interferons as growth inhibitors and anti-tumour agents (Clemens, 1992; Lengyel, 1993). The cellular activity of eIF-2! and eIF-4E increases in response to various oncogenes (Rosenwald, 1996b). The levels of eIF-2! and eIF-4E mRNA and protein are elevated in fibroblasts that overexpress c-myc (Rosenwald et al., 1993a,b; Rosenwald, 1995; Jones et al., 1996; Rosenwald, 1996b). This is associated with accelerated rates of protein accumulation and cell growth (Rosenwald, 1996b). v-src and v-abl have similar effects on eIF-2! and eIF-4E, but this may reflect the ability of these oncoproteins to stimulate c-myc production (Rosenwald et al., 1993a,b; Rosenwald, 1995, 1996b). v-src and ras also increase the phosphorylation of eIF-4E, which can activate its function (Frederickson et al., 1991; Rinker-Schaeffer et al., 1992). In addition, ras can deregulate eIF-2 by inducing an inhibitor of eIF-2! kinase (Mundschau and Faller, 1992). These many examples provide abundant evidence that abnormal stimulation of the translation apparatus is a frequent characteristic of transformed cells. This supports the idea that elevated rates of protein synthesis are necessary to sustain the development of many tumours.
VI. Components of the translation apparatus are often deregulated in cancer cells There is a multitude of documented examples in which the translation machinery has become deregulated following transformation (Rosenwald, 1996a). This fact provides strong support for the contention that the control of protein synthesis is an important aspect of growth regulation. For example, fibroblasts transformed by polyoma virus were found to synthesize protein more rapidly than normal parental cells and have lost control over ribosome production (Stanners et al., 1979). In untransformed revertants of these fibroblasts, correct regulation is recovered (Stanners et al., 1979). One study compared levels of expression of ribosomal proteins in colorectal tumours from eight different individuals with normal colonic mucosa from the same patients (PogueGeile et al., 1991). In every case, the adenocarcinomas overexpressed all six ribosomal protein transcripts that were tested (Pogue-Geile et al., 1991). The levels of these mRNAs were also generally elevated in adenomatous polyps, the presumed precursors of the carcinomas (PogueGeile et al., 1991). This implies that increased ribosomal protein production occurs early during the development of these tumours, perhaps concomitant with the onset of neoplasia. Colorectal tumours and tumour-derived cell lines were also reported to produce higher levels of rRNAs than normal colonic mucosa, consistent with a general increase in ribosomal components (Pogue-Geile et al., 1991). Constitutive expression of EF-1!, a translation factor that catalyses the attachment of aminoacyl-tRNAs to the ribosome, makes fibroblasts highly susceptible to transformation by 3-methylcholanthrene or ultraviolet light (Tatsuka et al., 1992). These observations suggest that deregulation of the protein synthesis machinery can predispose cells to malignant transformation. Perhaps the most striking demonstration that the translation apparatus becomes activated during tumourigenesis came from a recent study that used serial analysis of gene expression (SAGE) to document the expression profiles of 45,000 genes in gastrointestinal tumours (Zhang et al., 1997). Only 108 pol II transcripts were found to be expressed at higher levels in primary colon cancers relative to normal colonic epithelium (Zhang et al., 1997). Of these, 48 encode ribosomal proteins and 5 encode translation elongation factors (Zhang et al., 1997). Similar results were obtained with pancreatic cancers (Zhang et al., 1997). These observations provide compelling evidence that deregulation of protein synthesis is intimately linked with tumour formation. Several studies have shown that the abnormal activation of translation factors is actually sufficient to trigger neoplastic transformation (Rosenwald, 1996a). eIF4E, the mRNA cap-binding protein, and eIF-2, which brings initiator methionine tRNA to the 40S ribosomal subunit, appear particularly important in this regard. eIF4E is the least abundant of the translation initiation factors and is rate limiting for protein synthesis (Duncan et al.,
VII. c-Myc: a foot in both camps? The oncogene c-myc may have a foot in both the growth and proliferation camps. The c-myc promoter contains an E2F binding site and is subject to repression by RB (Hiebert et al., 1989; Zou et al., 1997). If c-myc expression is prevented using antisense technology, cells stop growing and arrest in G1 phase (Heikkila et al., 1987; Prochownik et al., 1988). Myc has been shown to 622
Gene Therapy and Molecular Biology Vol 1, page 623 promote cell cycle progression by stimulating production of cdc25 and the activation of cyclin D and E-dependent kinases (Galaktionov et al., 1996; Steiner et al., 1995). In
addition, the activation of c-myc in rodent fibroblasts results in an increase in the abundance of the translation
F i g u r e 5 . Control of c-myc production may provide an additional mechanism for RB to influence both growth and proliferation. Myc can stimulate cell cycle progression through the activation of cyclin-dependent kinases (cdks). Myc may promote protein synthesis and cellular growth by increasing the production of the translation initiation factors eIF-2! and eIF-4E. eIF-4E can stimulate cyclin D1 production. This, in turn, might be expected to switch off RB and thereby activate UBF and TFIIIB. The c-myc promoter contains a binding site for E2F and may therefore be subject to repression by RB. Inhibiting the production of myc may provide a mechanism for RB to control both growth and proliferation.
initiation factors eIF-2! and eIF-4E (Jones et al., 1996; Rosenwald, 1996a,b; Rosenwald et al., 1993a,b). The elevated concentrations of eIF-2! and eIF-4E that accompany activation of c-myc correlate with a rise in the net rate of protein synthesis and accelerated growth (Rosenwald, 1996b). Furthermore, overexpression of eIF4E results in a selective increase in cyclin D1 production (Rosenwald et al., 1993a). This, in turn, might be expected to switch off RB and thereby activate UBF and TFIIIB. By silencing the c-myc promoter, RB may be able to suppress both proliferation and growth. (Figure 5).
VIII. Discussion There is substantial evidence that the deregulation of translation is an important aspect of neoplastic transformation. Rapid growth undoubtedly requires elevated rates of protein accumulation; without it, a tumour would be unable to maintain its increase in mass. For rapidly dividing cells to sustain a high rate of translation will 623
require efficient production of tRNA and rRNA. In addition to the clear correlation between protein accumulation and growth, constitutively elevated translation might drive a population to proliferate. This could work as follows: unbalanced growth in the absence of cell replication is likely to trigger apoptosis; such conditions may select for cells that have acquired the ability to bypass the apoptotic pathway and multiply continuously. In this review, I have drawn a clear distinction between growth and proliferation. I have also argued that separate mechanisms appear to be involved in controlling these processes. However, the point must be emphasised that growth and proliferation are intimately linked and there is undoubtedly substantial cross-talk between the two. Many potential examples of this can be envisaged. For example, E2F is involved in regulating the genes for cyclins A, D and E. Since these cyclins control kinases that can inactivate RB, there is obvious potential for a feedback loop. Moreover, through its action on cyclins and hence RB, E2F might be expected to influence UBF and TFIIIB
White: Control of Growth and Proliferation by RB activity, and hence translation and growth. As explained above, c-myc may also have direct impact upon both the cell cycle machinery and the translation apparatus. It is clearly of benefit to the cell to have growth and proliferation coordinated by unifying control mechanisms. RB may provide such a regulatory switch. RB is a tumour suppressor of major importance, with a key role in controlling cell growth and proliferation. By regulating E2F, RB has the potential to inhibit the synthesis of gene products that are necessary for DNA synthesis, chromosomal replication and cell cycle progression. By repressing UBF and TFIIIB, RB can reduce the production of tRNA and rRNA. This may allow it to limit the rate of protein accumulation, which will provide a brake on cellular growth. Coregulating these essential processes may allow RB to achieve the necessary balance between growth and proliferation (White, 1997). Many other molecular targets have been identified for RB and these provide additional controls over cellular activity (Taya, 1997; Whyte, 1995). Regulating a range of key components may enable RB to coordinate a number of disparate processes. The loss of these controls will undoubtedly constitute a major step towards tumour development.
Acknowledgements
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I apologise to any colleagues whose contributions may not have been mentioned due to limitations of space. I thank S. Mittnacht, N. La Thangue, T. Hunt and K. Nasmyth for helpful and stimulating discussions concerning these ideas. Thanks also to C. Larminie and J. Sutcliffe for comments on the manuscript. Research in my laboratory is supported by the Cancer Research Campaign, the Medical Research Council, the Biotechnology and Biological Sciences Research Council and the Nuffield Foundation. I am a Jenner Research Fellow of the Lister Institute of Preventive Medicine.
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Gene Therapy and Molecular Biology Vol 1, page 629 Gene Ther Mol Biol Vol 1, 629-639. March, 1998.
Transcriptional regulation of the H-ras1 protooncogene by DNA binding proteins: mechanisms and implications in human tumorigenesis G. Zachos1 , 2 and D. A. Spandidos1 , 2 1 Institute of Biological Research and Biotechnology, National Hellenic Research Foundation, 48 Vas. Constantinou Ave., Athens 11635; 2 Medical School, University of Crete, Heraklion, Greece. _______________________________________________________________________________________________ Correspondence to: Professor D.A. Spandidos, Tel/Fax: +(301)-722 6469
Summary Altered expression of ras genes is a common event in human tumors. Transcriptional regulation of the H-ras1 proto-oncogene occurs through nuclear factors that recognize elements in the promoter region of the gene, in the first and fourth intron and in the VTR unit and involves alternative splicing and specific methylation patterns, as well. Aberrant levels of the Ras p21 protein are detected i n a variety o f human tumors and are often correlated with clinical and prognostic parameters. Thus, understanding the regulation of the expression of ras g e n e s p r o v i d e s a u s e f u l target for gene therapy treatments.
I. Introduction B. Ras proteins: structural characteristics and function
ras genes are a ubiquitous eukaryotic gene family. They have been identified in mammals, birds, insects, mollusks, plants, fungi and yeasts. Their sequence is highly conserved, thus revealing the fundamental role they play in cellular proliferation (Spandidos, 1991).
The H-Ras, N-Ras and K-RasA proteins are 189 amino acids long, whereas K-RasB is shorter by one amino acid. They all have a molecular weight of 21kDa and are termed p21 proteins. The p21 proteins are identical at the 86 N-terminal amino acid residues, they possess an 85% homology in the next 80 amino acid residues and diverge highly at the rest of the protein molecule, with the exception of the four C-terminal amino acids which share the common motif CAAX-COOH (C, Cysteine 186; A, Aliphatic amino acid-Leucine, Isoleucine or Valine; X, Methionine or Serine) (Lowy and Willumsen, 1991). The Ras protein is synthesized as pro-p21, undergoes a series of post-translational modifications at the C-terminus increasing the hydrophobicity of the protein and associates with the inner face of the plasma membrane. Sequences at the C-terminus are essential for membrane association and the conserved Cys 186 is required to initiate the posttranslational modifications of pro-p21 (Willumsen and Christensen, 1984). The superfamily of Ras proteins comprises a group of small GTPases, regulating an astonishing diversity of cellular functions (Makara et al, 1996). They are located at the heart of a signal transduction pathway that links cellsurface receptors through a protein kinase cascade to
A. The structure of ras genes Three functional ras genes have been identified and characterized in the mammalian genome, H-ras1, K-ras2 and N-ras, as well as two pseudogenes, H-ras2 and K-ras1 (Barbacid, 1987). All three ras genes have a common structure with a 5' non-coding exon (exon -I) and four coding exons (exons I-IV). The introns of the genes differ widely in size and sequence, with the coding sequences of human K-ras spanning more than 35 kb, while those of Nras and H-ras span approximately 7 and 3 kb, respectively. The K-ras gene has two alternative IV coding exons, thus encoding two proteins, K-RasA and K-RasB (McGrath et al, 1983), with the K-RasB form being more abundant. The H-ras gene also has an alternative exon in the fourth intron (Cohen et al, 1989). In addition, H-ras has a variable tandem repeat sequence (VTR), located downstream of the polyadenylation signal, which exhibits enhancer activity (Spandidos and Holmes, 1987, Cohen et al, 1987).
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Zachos and Spandidos: Transcriptional regulation of the H-ras1 proto-oncogene changes in gene expression and cell morphology and to cell division mechanisms. The Ras p21 protein interacts directly with the Raf oncoprotein to recruit the MAP kinases and their subordinates, thus converting a mitogenic signal initiated by membrane receptors with tyrosine kinase activity to a cascade of Serine/ Threonine kinases with multiple targets, including cytoskeleton, transcription factors, inflammatory mediators and other kinases (Avruch et al, 1994, Marshall, 1995).
boxes (Berg, 1992). The transcription factor AP-1 is the nuclear factor required to mediate transcription induced by phorbol ester tumor promoters and recognizes a short TGACTCA sequence (Lewin, 1991). Both c-Jun, encoded by members from the jun family (jun, junB, junD), and cFos proteins are active components and contribute to the activity of AP-1 by forming c-Jun homodimers as well as c-Jun-c-Fos heterodimers. In addition, there appears to be a mutual antagonism between activation by AP-1 and glucocorticoid receptors at target genes that contain recognition sites for both factors, via protein-protein interactions (Yang-Yen et al, 1990). Finally, the NF-I (CTF) nuclear factor binds the CCAAT element (CAAT box) and is involved in both gene transcription and DNA replication. The NF-I C-terminal region is proline rich and activates transcription through interference with the transcription machinery (Mermod et al, 1989). Ishii et al (1986), identified six GC boxes that bind the Sp-1 transcription factor as the essential regulatory elements within the H-ras promoter. Using deletion analysis of the H-ras promoter region by focus formation assay in NIH 3T3 cells, Honkawa et al (1987) reported a minimum promoter region of 51 bp length, which was GC rich (78%) and contained a GC box. Lowndes et al (1989) located a 47 bp element, distinct of the one reported by Honkawa et al, that upregulated the transcriptional activity of the promoter region by 20- to 40-fold and contained a GC box and a CCAAT box, binding the NF-1 (CTF) factor. Transient expression assays in which a series of mutants spanning the promoter region of H-ras were ligated to a promoterless chloramphenicol acetyl transferase (CAT) vector, were used in this analysis. Jones et al (1987), also identified two NF-I binding sites, one strong, also noted by Honkawa et al, and one weak. Trimble and Hozumi (1987), using CAT transfection experiments in CV-1 cells, identified a 100 nucleotide region, encompassing the consensus CCAAT box and two Sp-1 sites. However, Nagase et al (1990), using deletion mutants in CAT assays in CV-1 and A-431 cells, suggested that the presence of Sp-1 binding sites at specific positions may not be essential for promoter activity, but a number of Sp-1 binding sites in the region could be required. Lee and Keller (1991), transfected recombinant plasmids encompassing internal deletions and point mutations of the promoter region in HeLa cells and performed CAT assays. They reported a GC box, an unidentified element and a new element CCGGAA directly upstream the GC box, as the most important regulatory elements. Spandidos et al (1988), using recombinant plasmids in CAT activity experiments showed that AP-1-like proteins participate in control of H-ras transcription and identified four TPA responsive-AP-1 binding elements in the H-ras promoter. A great variety of the transcription initiation sites was also identified (Lowndes et al, 1989, Nagase et al, 1990,
C. ras oncogenes: mechanisms of activation The ras family of proto-oncogenes, is a frequently detected family of transformation-inducing genes in human tumors. Implication of ras genes in human tumorigenesis occurs by four different mechanisms: point mutations (Kiaris and Spandidos, 1995), gene amplification (Pulciani et al, 1985), insertion of retroviral sequences (Westaway et al, 1986) and alterations in regulation of transcription (Zachos and Spandidos, 1997). With the exception of mutations, all other mechanisms result in activation of the transforming properties of ras genes by quantitative mechanisms. The c-H-ras1 gene is the best studied member of the family and provides a good example for understanding the mechanisms of gene regulation. The H-ras proto-oncogene expression is regulated by elements located in the promoter region, in intronic sequences and in the 3' end of the gene. In addition, H-ras gene expression is regulated by alternative mechanisms such as DNA methylation and alternative splicing (reviewed by Zachos and Spandidos, 1997). Alterations in the H-ras expression levels are a common mechanism of human tumorigenesis.
II. Transcriptional regulation of the Hras gene from promoter-like sequences The H-ras gene promoter contains multiple RNA start sites, multiple GC boxes and has no characteristic TATA box (Ishii et al, 1985). These features are characteristic of housekeeping genes. Most promoter region studies have focused on the region upstream of the 5' splice site of the first intron of the gene (nucleotides 1-577), although others consider the SstI fragment (nucleotides 1-1054) that encompasses a part of the first intron as well, to be the gene promoter (Spandidos et al, 1988). Regulation of gene expression depends on a variety of nuclear factors (Boulikas, 1994). A great number of regulatory elements in the H-ras promoter has been reported, but the results were often controversial, depending on the followed experimental procedure. Transcription factors that interplay on the regulatory regions of the H-ras gene promoter include Sp-1, NF-1, AP-1 and some unknown factors as well. The Sp-1 is a mammalian DNA binding protein activating transcription by interacting through zinc finger domains with guanine-rich DNA sequences called GC 630
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F i g u r e 1 . A synopsis of the reported nuclear factors participating in c-H-ras1 transcriptional regulation. Factors recognize sequences in the H-ras promoter, in intronic sequences and in the VTR region. Exons, rectangles; coding sequences, filled rectangles; VTR, cross-hatched box; B, BamHI cleavage site; S, SstI cleavage site; arrows, major transcriptional initiation sites; ?, unknown regulatory factors; IDX, intron D exon.
B. The Hormone response elements (HREs)
Lee et al, 1991) using S1 nuclease analysis. A synopsis of the reported nuclear factors that participate on the promoter activity of H-ras, and of the major RNA start sites, are shown in F i g . 1 .
Steroid hormone receptors produce an enormous number of biological effects in different tissues as hormone activated transcriptional regulators (Beato, 1989, Beato et al, 1995). Such a process requires the coordinate expression of multiple genes and likely candidates are signal transductors, capable of secondarily controlling the transcription of sets of genes that lack steroid hormone response elements. Proto-oncogenes are theoretically well suited for this role, as they exhibit precise temporal patterns of expression during proliferation and differentiation, they participate in signal transduction and regulate the expression of multiple genes in a cascade fashion (Bishop, 1991). Thus, they may integrate signals from steroids and other regulatory factors and amplify the cellular response to the hormone. Evidence for steroid hormone regulation of proto-oncogenes encoding for nuclear transcription factors has already been provided for c-fos, c-jun and c-myc (Hyder et al, 1994).
III. Regulation of the H-ras gene expression from intronic sequences Intronic sequences play an important role in H-ras regulation. The nuclear factor Sp-1, steroid hormone receptors and the P53 onco-suppressor protein recognize sequences in the first and fourth introns of the H-ras gene.
A. The Sp-1 box There is evidence that the mutant T24 ras 0.8 kb SstI DNA fragment is a more potent activator of gene expression, compared to the corresponding normal H-ras fragment (Spandidos and Pintzas, 1988). A structural basis for this difference was shown to be a 6 bp element in the mutant H-ras fragment, that was absent in the normal H-ras, in the first intron of the gene. This element was proved to contain an Sp-1 binding site (Pintzas and Spandidos, 1991) (F i g . 1 ).
1. Regulation of c-H-ras by steroid hormone receptors Zachos et al (1995), identified sequences in the 3' end of the first intron and in the fourth intron of the H-ras
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Zachos and Spandidos: Transcriptional regulation of the H-ras1 proto-oncogene gene, with high similarity with the glucocorticoid response element (GRE) and estrogen response element (ERE) consensus oligonucleotides, respectively (F i g . 1 ). Using nuclear extracts from human and murine cell lines in gel retardation assays, it was shown that both glucocorticoid receptor (GR) and estrogen receptor (ER) specifically recognize the corresponding H-ras elements (Zachos et al, 1995). H-ras p21 protein, is a G-protein involved in signal transduction beginning from transmembrane growth factor and peptide receptors with tyrosine kinase activity (Avruch et al, 1994, Marshall et al, 1995). Steroid receptors, participate in dinstinct signalling mechanisms, involving transcriptional regulation of genes by ligand activated receptors (Beato, 1989, Beato et al, 1995). Thus, hormone regulation of H-ras provides evidence for a direct interaction of these two pathways, allowing the cell to have an additional regulatory "switch" by hormonally modulating the levels of G-proteins at the transcriptional level (Zachos et al, 1996a). Moreover, the first intron of the gene contains well conserved regions between human and rodents (HashimotoGotoh et al, 1988) and encompasses positive and negative elements influencing H-ras expression, possibly at posttranscriptional level (Hashimoto-Gotoh et al, 1992). It is noteworthy that the GRE, as well as the p53 element that will be discussed later, are located in the conserved region of the intron, thus providing evidence for an essential role in regulating H-ras expression. Moreover, the H-ras GRE is located within the first positive element.
Thus, endometrial and ovarian epithelial cells may have a predisposition to develop neoplastic abnormalities in addition to a second tumorigenic event, e.g. viral infection, loss of an onco-suppressor gene, or mutational activation of a proto-oncogene (Zachos et al, 1996a).
C. The p53 element One of the major roles of wild-type P53 oncosuppressor protein is to trigger cell cycle arrest or apoptosis in response to DNA damage by acting as a sequence specific transcription factor that binds to DNA and activates genes involved in the control of the cell cycle, including p21, gadd45, bax, mdm2 and PCNA (Zambetti et al, 1993, Hainaut, 1995). The mutant forms of P53 promote tumorigenesis by dominant-negative inhibition of wild-type P53 through cross-oligomerization. Moreover, P53 mutants were proved to exert oncogenic functions of their own (Dittmer et al, 1993). 1 . Regulation o f the c-H-ras by the P53 tumor-suppressor protein H-ras contains within its first intron sequences that partially match the p53 consensus binding site (F i g . 1 ). Using gel retardation assays it was shown that wild-type P53, as well as the "hot spot" mutant His 273 recognize the H-ras element with high affinity (Spandidos et al, 1995, Zoumpourlis et al, 1995). Furthermore, the H-ras element functioned as a P53-dependent transcriptional enhancer in the context of a reporter plasmid, thus suggesting that P53 is a physiological regulator of H-ras expression (Spandidos et al, 1995). Activation of H-ras expression by P53 may seem a paradox, since p53 is a tumor suppressor and H-ras a proto-oncogene. However, precedence has already been established for activation of proto-oncogenes by P53. Wild-type P53 induces expression of mdm-2, whose protein product inhibits the tumor suppressor activities of P53 and Rb (Xiao et al, 1995). Interestingly, there are certain similarities in the organization of the p53 elements of the H-ras and mdm-2 genes, which are not shared with the p53 elements of other genes targeted by P53. In H-ras there are three half sites: two of them are contiguous, while the third is 8 nucleotides upstream. In mdm-2 there are again three half-sites: two are contiguous, while the third one is located 28 nucleotides upstream (Wu et al, 1993). In contrast to H-ras and mdm-2, the elements of the other genes regulated by P53 are contiguous. The organization of the half-sites affects the ability of the P53 protein to recognize these elements. Wild-type P53 reversibly switches between two conformations: the "inactive" T state, with dihedral symmetry, which can recognize only non-contiguous half-sites and the "active" R state, which can recognize even contiguous half-sites (Waterman et al, 1995). Thus, it is suggested that H-ras and mdm-2 genes allow regulation by even the "inactive" T state of P53 protein.
2. Interaction of the H-ras and steroid hormone receptors in gynecological cancer The human endometrium and ovary are major targets for action of glucocorticoids. Sex hormones and steroids act as tumor promoters. The level of receptor binding in H-ras hormone response elements was examined in gel retardation assays, using nuclear extracts from human endometrial and ovarian lesions and from adjacent normal tissue (Zachos et al, 1996b). Increased binding of the glucocorticoid receptor in H-ras GRE was observed in more than 90% of endometrial and in all ovarian tumors tested, compared to the adjacent normal tissue. Moreover, elevated binding of the estrogen receptor in H-ras ERE was found in all pairs of ovarian tumor/ normal tissue examined (Zachos et al, 1996b). Thus, it was proposed that H-ras is implicated in human gynecological lesions through elevated steroid receptor binding. In addition, previous data showed cooperation of the Hras with steroids in cell transformation (Kumar et al, 1990) and overexpression of the Ras p21 in ovarian tumors, compared to normal or benign tumor tissue (Katsaros et al, 1995). By combining these data, it was proposed that the high levels of steroid and sex hormones in human genital tract may result in increased amounts of ligand activated steroid receptors. Furthermore, receptors bind to the H-ras DNA and induce elevated transcription of the H-ras oncogene, resulting in an increased oncogenic potential. 632
Gene Therapy and Molecular Biology Vol 1, page 633 The H-ras p53 element is located within the first intron. Interestingly, the p53 element of the mdm-2 is also located in the first intron of the gene (Wu et al, 1993). The significance of this is not understood at this time. The mdm-2 has an internal promoter in the first intron. Transcripts initiating at both promoters contain the entire protein sequence, however they differ in the efficiency with which translation is initiated in codon 1. Thus, transcripts that include the first exon mostly express an N-terminally truncated Mdm-2 protein, whereas transcripts from the internal promoter express a full-length protein. P53 induces expression only from the internal promoter and only the full-length form can associate with P53, closing the autoregulatory feedback loop (Barak et al, 1994). It remains to be determined whether P53 induces expression of transcripts initiating at the first intron of Hras, as well as the biological significance of such transcripts. The p53 tumor suppressor may therefore exert its cellular effects by coordinate activation of genes that suppress and induce cell proliferation. 2. Altered binding of p53 protein to the Hras element in human tumors Mutation and overexpression of the p53 tumor suppressor is a common event in human endometrial and ovarian cancer (Berchuck et al, 1994) and is associated with poor prognosis (Levesque et al, 1995, Kihana et al, 1995). Moreover, aberrant regulation of the H-ras gene expression also participates in the development of human gynecological lesions (Zachos and Spandidos, 1997). Using nuclear extracts from human endometrial and ovarian tumors and from the adjacent normal tissue in gel retardation assays, we examined the binding levels of the P53 protein to the H-ras element (our unpublished results). Elevated P53 binding in the tumor tissue was found in 5/11 (45%) of endometrial and in 2/5 (40%) of ovarian cases. Loss of P53 DNA binding activity was observed in 3/11 (27%) of endometrial and in 1/5 (20%) of ovarian tumors. In the remaining 3/11 (27%) of endometrial and in 2/5 (40%) of ovarian pairs tested, no alteration in the P53 binding levels was observed. In order to interpret the results, all pairs were subsequently tested for mutations in exons 4-9 of the p53 gene using PCR-SSCP analysis. No mutation was observed in any case showing elevated DNA binding activity, thus implying for overexpression of the wild-type p53 gene in these tumors. In addition, no p53 mutational alteration was observed in the cases showing similar DNA binding levels in tumor versus the adjacent normal tissue. However, a mutated allele was detected in all four endometrial and ovarian cases showing loss of P53 DNA binding activity. We therefore suggest that P53 could directly modulate the H-ras oncogenic potential in human endometrial and ovarian lesions, depending on the expressed levels of P53 and the status of the protein (wild-
type or mutated forms), thus providing additional evidence for the role of H-ras in human carcinogenesis.
IV. The role of the VTR Variable tandem repeats (VTRs, minisatellites) are highly polymorphic structures characterized by the tandem repetition of short (up to 100 bp) sequence motifs. Several observations on tandemly-repetitive elements within viral genomes (Yates et al, 1984) have led to the speculation that some human minisatellites might serve as regulatory regions for cellular transcription or replication.
A. The H-ras minisatellite sequence as transcriptional enhancer The human H-ras gene contains a VTR region located 1 kb upstream the polyadenylation signal (F i g . 1). It consists of 30 to 100 copies of a 28 bp consensus repeat. Four common alleles and more than 25 rare alleles have been described (Krontiris et al, 1993). It was shown that the H-ras VTR sequences possess endogenous enhancer activity, independently from orientation, however this activity is promoter specific (Spandidos and Holmes, 1987, Cohen et al, 1987). The 28 bp repeat unit of the minisatellite binds four proteins (p45, p50, p72 and p85) which are members of the rel/ NF-!B family of transcriptional regulatory factors (Trepicchio and Krontiris, 1992).
B. VTR rare alleles of the H-ras and ovarian cancer risk Women who carry a mutation in the BRCA1 gene have an 80% risk of breast cancer and a 40% risk of ovarian cancer by the age of 70 (Easton et al, 1995). The varying penetrance of BRCA1 suggests a role for other genetic and epigenetic factors in tumorigenesis of these individuals. H-ras was the first example of a modifying gene on the penetrance of an inherited cancer syndrome. Rare alleles of the H-ras VTR locus duplicate the magnitude of ovarian cancer risk for BRCA1 carriers, but not the risk for developing breast cancer (Phelan et al, 1996). It was suggested that H-ras VTR alleles show differences in modulating gene transcription, that H-ras VTR alleles are in linkage disequilibrium with other genes important in tumorigenesis, or that rare alleles provide a marker for genomic instability (Phelan et al, 1996).
V. The role of the DNA methylation status DNA methylation is essential for embryonic development and alterations in the DNA methylation status are common in cancer cells. CpG sites in vertebrates are either clustered in 0.5-2 kb regions called
Table I. ras gene overexpression in human tumors and correlation with clinical parameters. 633
Zachos and Spandidos: Transcriptional regulation of the H-ras1 proto-oncogene
Tumor type Neuroblastoma Head and neck Esophagus Larynx Thyroid Lung Liver Small intestine Stomach Pancreas Colon Breast Bladder Endometrium Ovary Leukemias
Frequency (%) 50-80 54 40 57-86 85 64-85 60 70 35 42 31 65-70 39-58 18-95 45 39-67
ras gene
Stage in tumorigenesis early early unknown unknown early late unknown unknown late unknown early unknown early late late unknown
H-, ras H-, KHH-, K-, Nras ras ras ras K-, ras ras H-, K-, ras ras H-, K-, Nras ras H-, K-, N-
Prognosis of the disease favourable favourable unknown unknown unknown poor unknown unknown poor unknown poor unknown poor unknown poor unknown
processing. The predicted product of the alternate transcript (p19) lacks transforming potential, since the C terminal sequence of p21 that is necessary for attachment of the protein to the inner site of the cellular membrane is absent. It is suggested that alternative splicing patterns operate to suppress the H-ras p21 expression. This negative control is abolished by mutations that interfere with this process.
CpG islands, or are dispersed, in which case they are mostly methylated and constitute mutational hotspots (Jones, 1996). The CpG islands are associated with gene promoters (e.g. H-ras) or coding regions (e.g. p16) and are unmethylated in autosomal genes. 5' Methyl-cytosine can affect transcription by altering the DNA binding activities of transcription factors. This could be done either directly, for example binding of trans-acting proteins at AP-2 sites is inhibited (Comb and Goodman, 1990), or indirectly, by enhanced binding of methylated DNA binding protein (MDBP) which stereochemically inhibits DNA binding of transcription factors (Boyes and Bird, 1991). The promoter region of the H-ras gene is hypomethylated in human tumors compared to the corresponding normal tissue (Feinberg and Vogelstein, 1983). Furthermore, methylation of cis-elements decreases H-ras promoter activity in vitro (Rachal et al, 1989) and inhibits the transforming activity of the oncogene (Borello et al, 1987). It is therefore suggested that epigenetic and reversible mechanisms, like DNA methylation, can regulate the expression of proto-oncogenes and silence genetically activated human oncogenes.
VII. Overexpression of ras genes in human tumors Overexpression of ras genes is a common event in human tumors (reviewed by Zachos and Spandidos, 1997). Table I summarizes the experimental results by indicating the tumor type where elevated expression of ras genes was observed, the frequency of the overexpression, the activated member of the ras gene family, the stage in tumorigenesis and correlation of altered ras gene expression with prognosis of the disease. Where no particular ras gene is mentioned (referred as: ras), no discrimination between the ras family members was performed, nor was their status (mutated or wild-type alleles) defined. Elevated ras gene expression was observed in human neuroblastomas (Spandidos et al, 1992), head and neck tumors (Field, 1991), esophageal (Abdelatif et al, 1991), laryngeal (Kiaris et al, 1995), thyroid (Papadimitriou et al, 1988), lung (Miyamoto et al, 1991), liver (Tiniakos et al, 1989), small intestine (Spandidos et al, 1993), stomach (Motojima et al, 1994), pancreatic (Song et al, 1996), colorectal (Spandidos and Kerr, 1994), breast (Dati et al, 1991), bladder (Ting-jie et al, 1991), endometrial
VI. Differential expression of the H-ras gene is controlled by alternative splicing A proportion of H-ras pre-mRNA is spliced to incorporate an alternative exon, termed IDX (intron D exon), which contains an in-frame translational termination codon that prevents expression of the genetic information specified by the exon IV as shown in F i g . 1 (Cohen et al, 1989). The abundance of these transcripts is low, apparently due to message instability or defective
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Gene Therapy and Molecular Biology Vol 1, page 635
F i g u r e 2 . A synopsis of molecular therapeutic strategies developed against activated ras oncogenes. Strategies include antisense oligonucleotides, ribozymes, farnesyltransferase inhibitors and activation of T-cell response after mutant Ras peptide vaccination of patients.
(Long et al, 1988) and ovarian tumors (Scambia et al, 1993) and in leukemias (Gougopoulou et al, 1996). The frequency of elevated expression of the ras family of genes varies from 30% in endometrial and colorectal tumors, to 85-90% of cases in endometrial, lung and laryngeal tumors. In a number of tumors including neuroblastomas, head and neck, thyroid, colorectal and bladder cancers, ras overexpression is considered to be an early genetic event. However, in lung, stomach, endometrial and ovarian lesions, overexpression of ras genes appears in a later stage in tumorigenesis. Elevated expression of the Ras p21 protein is correlated with poor prognosis in lung, stomach, colorectal, bladder and ovarian lesions, whereas it is a favourable marker for neuroblastomas and head and neck tumors.
VIII. Molecular therapeutic strategies The development of effective molecular strategies for therapy is the aim of tumor biology. Current therapeutic strategies include ribozymes against mutant ras gene products, antisense strategies, inhibitors of Ras protein post-translational modifications and Ras peptide vaccination (F i g . 2 ).
635
A. Ribozymes Molecular biology applies the site-specific RNAse properties of ribozymes to gene therapy for cancer. The anti-ras ribozymes are designed to cleave only activated ras RNA (F i g . 2 ). To develop this strategy into practical means, methods must be developed to accomplish high efficiency delivery of the ribozyme to target neoplastic tissue. An adenoviral-mediated delivery was designed (Feng et al, 1995). Using anti-Ras ribozymes, it was possible to reverse the neoplastic phenotype in mutant Hras expressing tumor cells with high efficiency (KashaniSabet et al, 1994).
B. Antisense strategies The antisense strategy involves reduction of a particular gene expression by introduction of a cDNA segment in antisense orientation, in order to bind the target mRNA and prevent its translation (Stein and Cheng, 1993) (F i g . 2 ). Critical to the success of such an antisense agent is its ability to enter living cells, to specifically bind the target mRNA and induce RNAse-H cleavage of the target RNA. Activated ras genes, by mutation or overexpression, are a common target of these
Zachos and Spandidos: Transcriptional regulation of the H-ras1 proto-oncogene therapeutic trials in cell-free and in vitro systems (Monia et al, 1992, Schwab et al, 1994).
Avruch J, Zhang X and Kyriakis JM (1 9 9 4 ) Raf meets Ras: completing the framework of a signal transduction pathway. TIBS 19, 279-283.
C. Inhibitors of Ras post-translational modifications
Barak Y, Gottlieb E, Juven-Gershon T and Oren M (1 9 9 4 ) Regulation of mdm2 expression by p53: alternative promoters produce transcripts with nonidentical translation potential. G e n e s D e v 8, 1739-1749.
Farnesylation of the CAAX motif of Ras protein is essential for the subcellular localization of Ras to the plasma membrane and is critical to Ras cell-transforming activity. Inhibitors of farnesyltransferase have been developed as potential cancer therapeutic agents (Gibbs et al, 1994) (F i g . 2 ). Requirements for Ras farnesylation inhibitors include specificity for farnesyl protein transferase, ability to inhibit post-translational modifications of the mutant ras specifically, high potency, activity in vivo and lack of toxicity (Kelloff et al, 1997).
Barbacid M (1 9 8 7 ) ras genes. A n n R e v B i o c h e m 56, 779-827. Beato M, Herrlich P and Schutz G (1 9 9 5 ) Steroid hormone receptors: many actors in search of a plot. C e l l 83, 851857. Beato M (1 9 8 9 ) Gene regulation by steroid hormones. C e l l 56, 335-344. Berchuck A, Kohler MF, Marks JR, Wiseman R, Boyd J and Bast RC (1 9 9 4 ) The p53 tumor suppressor gene is frequently altered in gynecologic cancers. A m J O b s t e t G y n e c o l 170, 246-252.
D. Ras peptide vaccination
Berg JM (1 9 9 2 ) Sp1 and the subfamily of zinc finger proteins with guanine-rich binding sites. Proc Natl Acad Sci USA 89, 11109-11110.
Ras peptide vaccination is a recent, developing molecular strategy for cancer therapy. Mutant Ras peptides are candidate vaccines for specific immunotherapy in cancer patients. An amount of mutant Ras p21 is degraded in the cytoplasm and fragments are attached with class I MHC glycoproteins, in the outer surface of the cell membrane (F i g . 2 ). When vaccinated with a synthetic Ras peptide representing the ras mutation in tumor cells, a transient Ras-specific T-cell response is induced, towards the fragments of mutant Ras protein associated with MHC molecules. Ras peptide vaccination was proved to be effective in 40% of patients with pancreatic cancer (Gjertsen et al, 1995, 1996). However, peptide vaccination of patients, like all other gene therapy strategies previously mentioned, requires considerable development before useful anti-cancer agents can emerge.
Bishop JM (1 9 9 1 ) Molecular themes in oncogenesis. C e l l 64, 235-248. Borello MG, Pierotti MA, Bongarzone I, Donghi R, Modellini P and Della Porta G (1 9 8 7 ) DNA methylation affecting the transforming activity of the human Ha-ras oncogene. Cancer Res 47 , 75-79. Boyes J and Bird A (1 9 9 1 ) DNA methylation inhibits transcription indirectly via a methyl-CpG binding protein. C e l l 64, 1123-1134. Boulikas T (1 9 9 4 ) A compilation and classification of DNA binding sites for protein transcription factors from vertebrates. C r i t R e v E u k a r y o t G e n e E x p 4, 117321. Cohen JB, Broz SD and Levinson AD (1 9 8 9 ) Expression of the H-ras proto-oncogene is controlled by alternative splicing. C e l l 58, 461-472.
IX. Concluding remarks
Cohen JB, Maureen VW and Levinson AD (1 9 8 7 ) A repetitive sequence element 3' of the human c-Ha-ras1 gene has enhancer activity. J C e l l P h y s i o l (Suppl) 5: 75-81.
Regulation of the c-H-ras1 gene expression is a complicated procedure, including regulation by a variety of regulatory proteins (transcription factors, hormone receptors, tumor-suppressor proteins), alternative mechanisms (methylation, splicing) and by sequences located in the promoter region, in introns and downstream of the coding sequence. Understanding the molecular mechanisms of the expression of ras genes is of great significance for studying human tumorigenic events and developing effective strategies for gene therapy.
Comb M and Goodman HM (1 9 9 0 ) CpG methylation inhibits proenkephalin gene expression and binding of the transcription factor AP-2. N u c l e i c A c i d s R e s 18, 3975-3982. Dati C, Muraca R, Tazartes O, Antoniotti S, Perrotean I, Giai M, Cortese P, Sismondi P, Saglio G and DeBortoli M (1 9 9 1 ) c-ErbB-2 and Ras expression levels in breast cancer are correlated and show a cooperative association with unfavorable clinical outcome. I n t J C a n c e r 47: 833-838.
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Pintzas A and Spandidos DA (1 9 9 1 ) Sp1 specific binding sites within the human H-ras promoter: potential role of the 6 bp deletion sequence in the T24 H-ras1 gene. Anticancer Res 11: 2067-2070.
Ting-jie M, Ze W and Nianli S (1 9 9 1 ) Correlation between the expression of the p21 ras oncogene product and the biological behavior of bladder tumors. Eur Urol 20, 307-310.
Pulciani S, Santos E, Long LK, Sorrentino V and Barbacid M (1 9 8 5 )ras gene amplification and malignant transformation. M o l C e l l B i o l 5, 2836-2841.
Tiniakos D, Spandidos DA, Kakkanas A, Pintzas A, Pollice L and Tiniakos G (1 9 8 9 ) Expression of ras and myc oncogenes in human hepatocellular carcinoma and nonneoplastic liver tissues. Anticancer Res 9, 715-722.
Rachal MJ, Yoo H, Becker FF and Lapeyre J-N (1 9 8 9 ) In vitro DNA cytosine methylation of cis-regulatory elements modulates c-Ha-ras promoter activity in vivo. N u c l e i c A c i d s R e s 17, 5135-5147.
Trepicchio WL and Krontiris TG (1 9 9 2 ) Members of the rel/ NF-!B family of transcriptional regulatory proteins bind the HRAS1 minisatellite DNA sequence. N u c l e i c A c i d s R e s 20, 2427-2434.
Scambia G, Catozzi L, Panici PB, Ferrandina G, Coronetta F, Barazzi R, Baiocchi G, Uccelli L, Piffanelli A and Mancuso S (1 9 9 3 ) Expression of ras oncogene p21 protein in normal and neoplastic ovarian tissues: correlation with histopathologic features and receptors for estrogen, progesterone and epidermal growth factor. Am J Obstet Gynecol 168, 71-78.
Trimble WS and Hozumi N (1 9 8 7 ) Deletion analysis of the cHa-ras oncogene promoter. FEBS Lett 219, 70-74. Yang-Yen H-F, Chambard J-C, Sun Y-L, Smeal T, Schmidt TJ, Drouin J and Karin M (1 9 9 0 ) Transcriptional interference between c-Jun and the glucocorticoid receptor: mutual inhibition of DNA binding due to direct protein-protein interaction. C e l l 62, 1205-1215.
Schwab G, Chavany C, Duroux I, Goubin G, Lebeau J, Helene C and Saison-Bechmoaras T (1 9 9 4 ) Antisense oligonucleotides adsorbed to polyalkylcyanoacrylate nanoparticles specifically inhibit mutated Ha-rasmediated cell proliferation and tumorigenicity in nude mice. Proc Natl Acad Sci USA 91, 10460-10464.
Yates J, Warren, N, Reisman D and Sugden B (1 9 8 4 ) A cisacting element from the Epstein-Barr viral genome that permits stable replication at recombinant plasmids in latently infected cells. P r o c N a t l A c a d S c i U S A 81, 3806-3810.
Song MM, Nio Y, Sato Y, Tamura K and Furuse K (1 9 9 6 ) Clinicopathological significance of Ki-ras point mutation and p21 expression in benign and malignant exocrine tumors of the human pancreas. Int J Pancreatol 20, 8593.
Waterman JLF, Shenk JL and Halazonetis TD (1 9 9 5 ) The dihedral symmetry of the p53 tetramerization domain
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Gene Therapy and Molecular Biology Vol 1, page 639 mandates a conformational switch upon DNA binding. EMBO J 14, 512-519. Westaway D, Parkoff J, Moscovici C and Varmus HE (1 9 8 6 ) Identification of a provirally activated c-Ha-ras oncogene in an avian nephroblastoma via a novel procedure: cDNA cloning of a chimaeric viral-host transcript. EMBO J 5, 301-309. Willumsen BM and Christensen A (1 9 8 4 ) The p21 ras Cterminus is required for transformation and membrane association. Nature 310, 583-586. Wu X, Bayle JH, Olson D and Levine AJ (1 9 9 3 ) The p53mdm2 autoregulatory feedback loop. Genes D e v 7, 1126-1132. Zachos G and Spandidos DA (1 9 9 7 ) Expression of ras protooncogenes: regulation and implications in the development of human tumors. Crit R e v O n c o l Hematol 26, 65-75. Zachos G, Zoumpourlis V, Sekeris CE and Spandidos DA (1 9 9 5 ) Binding of the glucocorticoid and estrogen receptors to the human H-ras oncogene sequences. I n t J O n c o l 6, 595-600. Zachos G, Varras M, Koffa M, Ergazaki M and Spandidos DA (1 9 9 6 a ) The association of the H-ras oncogene and steroid hormone receptors in gynecological cancer. J Exp Ther Oncol 1, 335-341. Zachos G, Varras M, Koffa M, Ergazaki M and Spandidos DA (1 9 9 6 b ) Glucocorticoid and estrogen receptors have elevated activity in human endometrial and ovarian tumors as compared to the adjacent normal tissues and recognize sequence elements of the H-ras proto-oncogene. Jpn J Cancer Res 87, 916-922. Zambetti GP and Levine AJ (1 9 9 3 ) A comparison of the biological activities of wild-type and mutant p53. FASEB J 7, 855-865. Zoumpourlis V, Zachos G, Halazonetis TD, Ergazaki M and Spandidos DA (1 9 9 5 ) Binding of wild-type and mutant forms of P53 protein from human tumors to a specific DNA sequence of the first intron of the H-ras oncogene. Int J Oncol 7, 1035-1041.
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Periodicity of DNA bend sites in eukaryotic genomes Ryoiti Kiyama Institute of Molecular and Cellular Biosciences, University of Tokyo, Yayoi, Bunkyo-ku, Tokyo 113, and National Institute of Bioscience and Human-Technology, Tsukuba-shi, Ibaraki 305, Japan __________________________________________________________________________________________________ Correspondence to: Ryoiti Kiyama, Tel: 81-3-3812-2111, ext. 7835, Fax: 81-3-3818-9437, E-mail: kiyamar@hgc.ims.u-tokyo.ac.jp
Summary We found that DNA bend sites are distributed regularly and periodically in the genomic DNA of eukaryotes. Their locations were conserved during molecular evolution in otherwise unstable intergenic regions of genomic DNA at intervals of approximately 700 bp, which corresponds to a length of four nucleosomes, suggesting their active role in chromatin organization. By further examination of these sites with respect to chromatin structure, we obtained evidence that these sites may act as signals for nucleosome phasing. Here, we summarize our results regarding periodic bent DNA in the human -globin, c-myc, and immunoglobulin heavy chain loci and discuss their biological functions.
formed by the intrinsic nature of the DNA or by protein binding, as well as sequence information, would be a good signal for structural recognition. Note that the non-B DNA structures themselves are the result of nucleotide sequence information, although the sequence-structure relationship is not simple.
I. Bent DNA in biological reactions Genomic DNA is a source of genetic and functional information in the form of nucleotide sequences. DNA has a relatively simple composition of purine or pyrimidine bases attached to a common phosphate backbone which would not give rise much local structural variation. However, recent studies have revealed that non-B DNA or "unusual" DNA structures are actively involved in biologically important reactions as functional elements (Crothers et al., 1990). For example, Z-DNA is known to activate transcription and recombination presumably by exposing bases on the outside of the phosphate backbone, thereby increasing the chance of interaction with proteins or other bases (Rich et al., 1984; Blaho and Wells, 1989). Other structures such as triplex DNA and unisomorphic DNA have been discussed in studies of transcriptional activation and recombination mechanisms (Crothers et al. , 1990; Soyfer and Potaman, 1996). Although their mechanisms of action are quite different, these non-B DNA structures seem to act as signals for recognition by protein factors in a way different from searching nucleotide bases. Bent DNA was first discovered as anomalous migration of DNA fragments in gels, and has been extensively studied due to its potential involvement as a transcriptional modulator (Travers, 1989; Hagerman, 1990; Crothers et al., 1990; Trifonov, 1991; Ioshikhes et al., 1996; Werner et al., 1996). Such structures also play important roles in activation of recombination (Nash, 1990). Binding of proteins to DNA can cause DNA bending, which would further enhance the recognition by other proteins of the site of the protein-DNA complex (Khan and Crothers, 1992). Therefore, DNA bending
II. Assays for DNA bend sites The presence and the location of DNA bend sites can be analyzed by several assays. Among these, the circular permutation assay (Wu and Crothers, 1984) has been commonly utilized for mapping the bend sites in DNA fragments of several hundred bp to 1 kb in length. The assay procedure is schematically illustrated in Figure 1. Plasmids containing the tandem dimers of the fragment of interest are cloned, and after digestion of the plasmid DNA with the restriction enzymes that cut the fragment once the plasmid DNA samples are resolved by electrophoresis. We routinely use 8% polyacrylamide (mono: bis = 29: 1) gels, which can resolve bands up to 1 kb in size (Wada-Kiyama and Kiyama, 1994). Electrophoresis should be performed at 4˚C twice or three times overnight to obtain better resolution of the bands. Cloning of the tandem dimers can be performed by direct cloning of two identical fragments into the multiple cloning site of the vector, or cloning them into two different sites one after another. Most of the clones could be obtained by the former method under conditions where the fragment (0.1 to 1 µg) is present in excess over the amount of vector DNA (ten times or more) in a smallvolume reaction mixture (5 to 10 µl). After transformation of E. coli, only direct repeats of the fragments, but not
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Kiyama: Periodicity of DNA bend sites in eukaryotic genomes complicate calculations. However, this method does have some technical limitations. Firstly, the assay is totally dependent upon the availability of suitable restriction sites. If there are no appropriate restriction sites, the bend sites cannot be localized to a small region of DNA. Secondly, the DNA structure of the other part of the same fragment could influence the mobility. As a result of this effect, mapping a bend site to a very small region by this method would not always give a precise location. Although rare, we observed a slight difference in the location of a site in the !-globin gene region between clones of different sizes used for mapping. Therefore, the lower limit of resolution would be 50 to 100 bp. The more precise location of the bend sites could be achieved by several other methods. For a relatively large region, sites can be examined with deletion constructs. When the bend center is completely deleted from the construct, all restricted fragments have the same mobility. Meanwhile, mapping the site in regions of approximately 100 bp or less would be achieved by using concatenated oligonucleotides of 20 or 30 bp (Wada-Kiyama and Kiyama, 1995). When the oligonucleotide contains a bend site, the concatemers exhibit retardation on polyacrylamide gel electrophoresis. The effect of bending is greater as the length of the oligonucleotide increases. The bend angle can be estimated by comigration of standards such as A3N7 (0.63Ë&#x161;/ base) (Calladine et al., 1988). The bend angle could also be determined by the assay based on ring closure of concatenated oligonucleotides (Zahn and Blattner, 1987).
III. The human -globin locus
Fig. 1. Assay for bent DNA.
inverted repeats, can be obtained in dimeric form. The circular permutation assay is a very simple and reliable method to identify and roughly map DNA bend sites. The results of mapping are reproducible under identical electrophoresis conditions and generally reproducible among different subclones containing the same site, and the patterns can be interpreted without 642
Using the circular permutation assay, we mapped the DNA bend sites in the human "-globin locus which is located on chromosome 11 and contains five (!-, G#-, A#-, $- and "-) active genes and one (%"-) pseudogene (Figure 2). This locus is ideal for mapping the sites because the nucleotide sequence of over 70 kb has been reported. Furthermore, since most of the locus is intergenic, the influence of the coding region could be excluded. The similarity of the exon-intron structure and the sequences of the flanking region would be ideal for evolutional study of the sites. The chromatin structure in this locus has been extensively characterized in that switching of globin gene expression is paralleled by alterations of chromatin structure as revealed by DNase I-hypersensitivity (reviewed by Stamatoyannopoulos and Nienhuis, 1993; Evans et al., 1990). The periodicity of the bend sites at intervals of 680 bp on average was first identified in the !-globin region (Wada-Kiyama and Kiyama, 1994). Further studies of the sites in the regions of other globin genes revealed that relative locations of the sites to their cap sites were conserved among most of the members of this family which were separated as much as 200 million years ago.
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Fig. 2. Periodic bent DNA in the human "-globin locus. Mapped DNA bend sites are shown as shadowed boxes. Hatched boxes indicate putative 150 bp sites aligned at 680 bp intervals as a reference for periodicity.
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Kiyama: Periodicity of DNA bend sites in eukaryotic genomes
Fig. 3. Conservation of periodic bent DNA in the translocation of the c-myc and IgÂľ loci. DNA bend sites in the c-myc (bottom) and IgÂľ (top) loci are aligned to highlight the conservation of the periodicity of the hypothetical sites (shadowed columns) based on their universal periodicity, after the rearrangements observed in Manca (A), BL22 (B) and Ramos (C) cell lines. Only three hypothetical sites near the junctions are shadowed but they were matched throughout the loci. Reproduced from Ohki et al. (1997).
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Gene Therapy and Molecular Biology Vol 1, page 645 Table 1. Average intervals of periodic bent DNA. ________________________________________________________________________________________ Locus Mapped No. of Average S. D. a Ref. region (kb) sites (bp) (bp) ________________________________________________________________________________________ "-globin 66 98 679.2 229.6 b c-myc 8 12 694.2 281.4 Ohki et al. 1997 IgÂľ 7 11 654.5 222.7 Ohki et al. 1997 Erythropoietin receptor 9 13 651.2 221.0 b c Estrogen receptor 3 5 688.1 210.9 Kuwabara et al. 1997 ________________________________________________________________________________________ a Standard deviation. b Unpublished results. c 5'-region containing the alternative cap site, P0.
IV. The human c-myc and the immunoglobulin heavy chain loci
The duplication of the two #-globin genes, which occurred most recently, was immediately followed by diversification of the non-coding region by as much as 70%, while all of the bend sites were conserved (Slightom et al., 1980). Insertion of an Alu sequence might have disturbed the periodicity, although as observed in the region upstream of the !-globin gene, the interval seemed to have returned to the average after a long period of molecular evolution. The positions of the bend sites were conserved even between the human "- and mouse "majglobin genes (Wada-Kiyama and Kiyama, 1996b). Mapping of over 90 bend sites in the locus revealed that the periodicity of the bend sites exists throughout the locus with an average interval of 680 bp (Wada-Kiyama and Kiyama, 1994, 1995, 1996b; unpublished results). However, we observed disturbance of the periodicity at several locations. Interestingly, all of the locations of the disturbed periodicity located upstream of the !-globin genes that caused the distances of the adjacent bend sites to be longer than average were found in or close to the DNase I-hypersensitive sites, which constitute the locus control region ("-LCR). The "-LCR is composed of four or five developmentally-regulated DNase I-hypersensitive sites (Crossley and Orkin, 1993; Evans et al., 1990; Felsenfeld, 1993). These sites are designated as open chromatin regions and act as the sites of interaction of transcription factors and the enhancer-binding protein NFE2. This region interacts with the promoter region of each member of the "-like globin gene family and controls their expression during development. One of the DNase Ihypersensitive sites, HS2 located 11 kb upstream of the cap site of the !-globin gene, was located in the center of two adjacent bend sites separated by a distance of 860 bp, which is longer than average (unpublished results). It seemed as if HS2 was placed far from the bend sites to minimize the influence of the sites. This is discussed again below.
The human c-myc gene has three exons and occupies a region of approximately 5.5 kb on chromosome 8. As observed in the "-globin locus, this locus contained periodic bent DNA. DNA bend sites were mapped at an average interval of 694.2 bp and were present in the 5'and 3'- non-coding regions, introns and the non-coding exon (exon 1), but not present in the coding region (Ohki et al., 1997). Interestingly, one of the bend sites corresponded to the location of TATA box of the P2 promoter, suggesting that prebending of the promoter region can facilitate transcriptional enhancement. The c-myc gene is involved in the progression of Burkitt's lymphoma by translocation of the locus into one of the immunoglobulin genes located on chromosomes 2, 14 or 22. These translocation events often result in reshuffling the location of regulatory elements. Deregulation of the expression by juxtaposition of the Âľ enhancer to the c-myc promoter is one of the mechanisms of tumor progression caused by this oncogene. The mechanism of these translocation events has not been well documented except that immunoglobulin-specific recombination is somehow involved (Specer and Groudine, 1991). Translocation junctions were formed at various locations in the locus yet no specific sequences were commonly found in their immediate proximity. However, when the periodic bent DNA was mapped in the c-myc and the IgÂľ loci, at least three stable cell lines containing the translocation junctions within these regions showed conservation of the periodicity before and after the rearrangements (Figure 3). This would be readily explained if we assume that the periodic bent DNA is a key element for chromatin structure. It would be necessary for a stable cell line to maintain a similar chromatin structure as to that before the rearrangement. Otherwise, a secondary rearrangement could alter the sequence until a stable structure is eventually formed.
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V. Other loci in eukaryotic genomes We have already determined that periodic bent DNA is present in the human erythropoietin receptor and the human estrogen receptor loci (Kuwabara et al., 1997; unpublished results). The intervals of the sites in these loci were 651.2 or 688.1 bp, respectively, which are close to the values for other loci (Table 1). In both cases, its periodicity was disturbed by exons. For the estrogen receptor gene, the alternative cap site located approximately 2 kb upstream of the canonical site caused a shift of the nearby sites. For the erythropoietin receptor gene, the 700 bp periodicity of bend sites was conserved even within the long introns (1st and 6th introns) although the sites were shifted when the length of introns was not sufficient to accommodate two sites. One of the sites in the estrogen receptor gene contained motifs of the estrogen response element, the binding site for the hormone-responsive trans-activating factors, and had the affinity to the nuclear scaffold. This site might play a role in determining the nuclear localization of this gene as well as a role in transcriptional regulation. For other loci of eukaryotes, we examined the potential bend sites by a computer search (Wada-Kiyama, and Kiyama, 1996a). Based on the observation that physically mapped bend sites often contain A+T-rich sequences including An or T n tracts at intervals of ten or multiples of ten nucleotides, we searched A2N8A2N8A2 and the complementary T 2N8T2N8T2 for a periodicity. There was a statistically significant sequence periodicity at an interval of roughly 700 bp in eukaryotic genomic DNA. This tendency was absent in prokaryotes and in eukaryotic cDNA, suggesting that the periodicity is universal among eukaryotic genomes, especially in intergenic regions.
VI. Biological significance of the bend sites The observations that periodic bent DNA is conserved during molecular evolution and its intervals are maintained precisely in otherwise unstable intergenic regions suggested that these sites are biologically relevant. Despite the systematic and organized patterns of chromatin folding, no specific signals have been determined as key elements for the folding mechanism. A computer search further revealed the non-random distribution of nucleotide sequences on the genomic DNA, while it failed to deduce any specific sequences in common, suggesting the presence of unidentified codes which are not apparent from sequence information alone. Therefore, judging from the periodicity and the length of their intervals, periodic bent DNA may be closely associated with chromatin structure, presumably with the formation of nucleosomes. We reported that some of the sites were indeed involved in the formation of nucleosomes by having high affinity to histone core particles (Wada-Kiyama and Kiyama, 1996b). Chromatin structure seems to be extensively stabilized when the overall periodicity is maintained before and after the rearrangement. Meanwhile, open chromatin regions, 646
revealed by DNase I-hypersensitivity (Gross and Garrard, 1988), could be at least partly due to disturbance of the periodicity. While the nucleosome phasing activity of these sites might be effective when the distances of the bend sites are less than or equal to the length of four nucleosomes, open chromatin regions would be more efficiently formed when their distances are more than the length of four nucleosomes. Some of the sites seem to be used as multiple sites for chromatin organization, as observed in the estrogen receptor gene. We are currently investigating chromatin structure at the replication origin based on the alignment of bend sites to examine the relationship of these sites with DNA replication. Our results indicated that periodic bent DNA is a key element of chromatin structure and plays a role in various biological reactions.
References Blaho, J. A. and Wells, R. D. (1989) Left-handed Z-DNA and genetic recombination. Prog. Nucl. Acid Res. Mol. Biol. 37, 107-126. Calladine, C. R., Drew, H. R. and McCall, M. J. (1988) The intrinsic curvature of DNA in solution. J. Mol. Biol. 201, 127-137. Crossley, M. and Orkin, S. (1993) Regulation of the "-globin locus. Curr. Opin. Genet. Dev. 3, 232-237. Crothers, D. M., Haran, T. E. and Nadeau, J. G. (1990). Intrinsically bent DNA. J. Biol. Chem. 265, 7093-7096. Evans, T., Felsenfeld, G. and Reitman, M. (1990) Control of globin gene transcription. Ann. Rev. Cell Biol. 6, 95-124. Felsenfeld, G. (1993) Chromatin structure and the expression of globin-encoding genes. Gene 135, 119-124. Gross, D. S. and Garrard, W. T. ( 1988) Nuclease hypersensitive sites in chromatin. Ann. Rev. Biochem. 57, 159-197. Hagerman, P. J. (1990). Sequence-directed curvature of DNA. Ann. Rev. Biochem. 59, 755-781. Ioshikhes, I., Bolshoy, A., Derenshteyn, K., Borodovsky, M. and Trifonov, E. N. (1996) Nucleosome DNA sequence pattern revealed by multiple alignment of experimentally mapped sequences. J. Mol. Biol. 262, 129-139. Khan, J. D. and Crothers, D. M. (1992). Protein-induced bending and DNA cyclization. Proc. Natl. Acad. Sci. USA 89, 63436347. Kuwabara, K., Wada-Kiyama, Y., Sakuma, Y. and Kiyama, R. (1997) Multiple interactions of periodic bent DNA in the promoter region of the human estrogen receptor gene with the nuclear scaffold, core histones and nuclear factors. submitted. Nash, H. A. (1990) Bending and supercoiling of DNA at the attachment site of bacteriophage lambda. Trends Biochem. Sci. 15, 222-227 Ohki, R., Hirota, M., Oishi, M. and Kiyama, M. (1997) Conservation and continuity of periodic bent DNA in genomic rearrangements between the c-myc and immunoglobulin heavy chain Âľ loci. submitted.
Gene Therapy and Molecular Biology Vol 1, page 647 Rich, A., Nordheim, A. and Wang, A. H.-J. (1984) The chemistry and biology of left-handed Z-DNA. Ann. Rev. Biochem. 53, 791-846. Slightom, J. L., Blechl, A. E. and Smithies, O. (1980). Human fetal G#- and A#-globin genes: Complete nucleotide sequences suggest that DNA can be exchanged between these duplicated genes. Cell 21, 627-638. Soyfer, V. N. and Potaman, V. N. (1996) In Triple-Helical Nucleic Acids. Springer Verlag, New York. Specer, C. A. and Groudine, M. (1991). Control of c-myc regulation in normal and neoplastic cells. Adv. Cancer Res. 56, 1-48. Stamatoyannopoulos, G. and Nienhuis, A. W. (1993) Hemoglobin switching. In The molecular basis of blood diseases, Stamatoyannopoulos, G., Nienhuis, A. W., Majerus, P. and Varmus, H. (eds). W. B. Saunders, Philadelphia. pp107-155. Travers, A. A. (1989) DNA conformation and protein binding. Ann. Rev. Biochem. 58, 427-452. Trifonov, E. D. (1991) DNA in profile. Trends Biochem. Sci. 16, 467-470. Wada-Kiyama, Y. and Kiyama, R. (1994). Periodicity of DNA bend sites in the human !-globin gene region: Possibility of sequence-directed nucleosome phasing. J. Biol. Chem. 269, 22238-22244. Wada-Kiyama, Y. and Kiyama, R. (1995). Conservation and periodicity of DNA bend sites in the human "-globin gene locus. J. Biol. Chem. 270, 12439-12445. Wada-Kiyama, Y. and Kiyama, R. ( 1996a) Conservation and periodicity of DNA bend sites in eukaryotic genomes. DNA Res. 3, 25-30. Wada-Kiyama, Y. and Kiyama, R. (1996b) An intrachromosomal repeating unit based on DNA bending. Mol. Cell. Biol. 16, 5664-5673. Werner, M. H., Gronenborn, A. M. and Clore, G. M. (1996) Intercalation, DNA kinking, and the control of transcription. Science 271, 778-784. Wu, H.-M. and Crothers, D. M. (1984). The locus of sequencedirected and protein-induced DNA bending. Nature 308, 509-513. Zahn, K. and Blattner, F. R. (1987). Direct evidence for DNA bending at the lambda replication origin. Science 236, 416422.
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Gene Therapy and Molecular Biology Vol 1, page 649 Gene Ther Mol Biol Vol 1, 649-660. March, 1998.
DNA methyltransferase: a downstream effector of oncogenic programs; implications for therapy. Moshe Szyf Department of Pharmacology and Therapeutics, McGill University, 3655 Drummond street, Montreal PQ H3G 1Y6, Canada. __________________________________________________________________________________________________ Correspondence: Moshe Szyf, Tel: 514-398-7107, Fax: 514-398-6690, E-mail: mcms @musica.mcgill.ca
Summary DNA MeTase is an attractive anticancer target. A molecular analysis of its regulation suggests that it is a downstream effector of many oncogenic pathways and that its down modulation can i n h i b i t t u m o r g r o w t h . I t i s p o s s i b l e t h a t D N A M e T a s e i n h i b i t o r w i l l b e e f f e c t i v e i n a broad spectrum o f cancers because i t l i e s downstream t o nodal cellular checkpoints that could be activated by multiple ways. An important challenge is to design novel inhibitors of DNA MeTase that are highly specific. Such inhibitors are potentially important pharmacological agents with wide therapeutic applications.
I. Introduction A. What is DNA methylation?
A. Working hypothesis: DNA MeTase is an anticancer target
DNA methylation is a postreplicative covalent modification of DNA that is catalyzed by the DNA methyltransferase enzyme (DNA MeTase) ( Razin and Szyf, 1984; Bestor et al., 1988). The main concept in DNA methylation is the idea of a pattern of DNA methylation and its correlation with the state of activity of genes (Yisraeli and Szyf, 1984). In vertebrates, the cytosine moiety at a fraction of the CpG sequences is methylated (60-80%); the non methylated CpGs are distributed in a nonrandom manner generating a pattern of methylation that is gene and tissue specific (Yisraeli and Szyf, 1984). Plant DNA is also methylated at CG as well as CXG sequences (Gruenbaum et al., 1984). DNA methylation plays an important role in development of plant cells and might be a critical element involved in silencing transgene expression in plants (Meyer, 1995).
This review summarizes recent findings demonstrating that the cytosine DNA Methyltransferase (DNA MeTase) is a downstream effector of cellular pathways leading either to oncogenesis or a change in the state of differentiation of vertebrate cells (Rouleau et al., 1992; Rouleau et al., 1995; MacLeod et al., 1995; MacLeod and Szyf, 1995; Szyf et al., 1992). It is becoming clear that control of gene expression by pharmacological means is one of the great challenges and hopes of current therapeutics. DNA MeTase is a master regulator of gene expression programs. This review suggests that genomic programs could be specifically modulated by pharmacological inhibition of DNA MeTase. Since DNA MeTase is believed to be involved in similar processes in a broad group of animals and plants, my hypothesis is that agents that inhibit DNA MeTase specifically will have broad pharmacological applications (Szyf 1994; Szyf 1996). Recent data from our laboratory using different classes of DNA MeTase inhibitors supports this hypothesis.
B. DNA Methylation patterns encode epigenetic information 1. Correlation of gene expression and DNA methylation patterns
II. Background
Does the pattern encode epigenetic information? A large number of papers published in the last two decades have shown a correlation between the pattern of methylation and the state of activity of genes (Yisraeli and Szyf, 1984). That is, some or all of the CpG sites in
The goal of this section is to illustrate the logical progression of concepts and data leading to our working hypothesis and to the proposal that DNA MeTase inhibitors could serve as important pharmacological agents. 649
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Figure 1. DNA methylation patterns fix gene expression programs, DNA MeTase inhibitors alter gene expression programs . The genome of a vertebrate (first line) bears many potential sites for modification by methylation (open circles). However, a subset of these sites is methylated (indicated by an M in the circle) in different tissues. When one looks at the methylation pattern of different genes (a to e) in different tissues (for example tissues A and B), one observes that they bear a different pattern of methylation. One also observes that in general inactive genes are modified whereas active genes bear sites of methylation that are not modified. It is proposed that both binding of transcription factors to regulatory sites of the genes (indicated by the triangles and ovals-activators in green and methylated-DNA binding factors-repressors) as well as the pattern of methylation (methylated sites attract methylated-DNA dependent repressors) define the state of activity of vertebrate genes. The pattern of methylation is maintained because of limiting level of DNA MeTase, thus the level of DNA MeTase locks the gene expression program of a tissue. Inhibition of DNA MeTase by DNA MeTase inhibitors results in transient demethylation and unlocking of the gene expression program. Demethylation enables reorganization of the interactions of transcription factors with DNA and resetting of a new program of gene expression. The direction that this reorganization will take is limited by the repertoire of transcription factors in the cell.
introduced into cells can suppress their activity (Stein et al., 1982, for a review of this question see Szyf, 1996). One possible solution to this dilemma is the suggestion that there is a dynamic interrelationship between DNA methylation and gene expression (Szyf, 1996). DNA methylation can play both a primary and secondary role in gene expression. That is, methylation of certain sites precipitates gene repression whereas in other instances the chromatin structure of a repressed gene can trigger DNA methylation. The combination of these processes should result in formation of a stable state of gene repression by a covalent modification of the DNA structure itself. Thus, the pattern of methylation in a cell will stabilize a gene expression program for this cell (F i g . 1 ).
regulatory regions of a specific gene will be methylated in all tissues where the gene is silenced but the same sites will be nonmethylated in tissues that express the gene (Yisraeli and Szyf, 1984) (F i g . 1 ).
2. DNA methylation and gene expression: cause or effect? There is a longstanding and unresolved discussion whether the state of methylation of genes is a cause or effect of their state of expression. Whereas a series of experiments demonstrated that inactivation of a gene precedes its repression (Lock et al., 1987), other studies have shown that methylation of genes before they are
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to transcription factors and is not transcribed. Figure 2. mechanisms for repression methylation.
Two gene by
DNA methylation can inhibit gene expression by two different mechanisms. The model gene described in this figure is activated (horizontal arrow indicates mRNA transcript) by interaction of transcription factors (triangle) with a cis acting sequence (shaded box) located in front of the transcription initiation site. The gene has a number of methylatable sites (open circles), one of which is located at the transcription factor recognition sequence. Mechanism A describes a case where the methylatable site located at the transcription recognition site is methylated (M). This methylation inhibits the recognition of the cis acting sequence by the transcription factor. Mechanism B describes a case where a regional methylation occurred in the body of the gene. This methylation results in binding of methylated-DNA binding protein(s) to the methylated region (open oval). The binding of this protein precipitates the spreading of an inactive chromatin structure, the gene becomes inaccessible
involved in the precipitation of an inactive chromatin structure on methylated DNA or whether other mechanisms are involved in building of inactive chromatin around methylated DNA (F i g . 2 ).
3. What is the mechanism of gene repression by methylation? A series of publications suggest that DNA methylation can repress gene expression directly, by inhibiting binding of transcription factors to regulatory sequences (Becker et al., 1987), or indirectly, by signaling the binding of methylated-DNA binding factors that repress gene activity or by precipitating an inactive chromatin structure (Razin and Cedar, 1977; Keshet et al., 1986). Two methylated DNA binding proteins that can repress transcription in a methylation dependent manner have been recently characterized MeCP2 and MeCP1(Cross, et al., 1997; Nan et al., 1997). The carboxy terminal half of MeCP2 contains a repressor domain which can interact with the transcriptional machinery. MeCP2 is also suggested to precipitate or stabilize an inactive chromatin structure. Kass et al., have shown that methylated DNA is assembled into an inactive chromatin structure (Kass et al., 1997). It is not clear yet whether MeCP2 is generally
4. Summary: methylation plays an important role in control of genomic functions. A long list of data supports the hypothesis that DNA methylation plays an important role in the control of genomic functions. It is well established that regulated changes in the pattern of DNA methylation occur during development (Brandeis et al., 1993) , parental imprinting (Peterson and Sapienza, 1993) and cellular differentiation (Razin et al., 1985) and that aberrant changes in the pattern of methylation occur in cellular transformation (Feinberg et al., 1983; de Bustros et al., 1988; Baylin et al., 1991). A targeted mutation, by homologous recombination in ES cells, of the DNA MeTase gene results in embryonic
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Szyf: DNA Methyltransferase in cancer therapy lethality (Li et al., 1992) and inhibition of DNA MeTase by expression of an antisense to the DNA MeTase results in a change in the identity of a cell from a fibroblast to a cell of myogenic lineage (Szyf et al., 1992). Whereas the most studied biological process regulated by DNA methylation is gene expression, it is possible that DNA methylation directly regulates other genome functions such as replication and recombination. These processes might be as important as gene expression in the events leading to oncogenesis (Szyf, 1996) . If a DNA methylation pattern locks certain gene expression programs, then modulating the pattern should unlock these programs and play important therapeutic roles (Szyf, 1994; Szyf, 1996) (F i g . 1 ). To be able to design therapeutic strategies to unlock a pattern of methylation and gene expression, one has to understand the mechanisms that control DNA methylation patterns.
C. The level of DNA MeTase is an important determinant of DNA methylation patterns. 1. The semiconservative model of methylation inheritance The accepted model in the field has been that patterns of methylation are maintained because the DNA MeTase is very efficient in methylating hemimethylated DNA generated in the process of replication (maintenance methylation) but very inefficient in methylating nonmethylated DNA (de novo methylation) (Razin and Riggs, 1980). However in spite of the simplicity of this model, both the cloned DNA MeTase (Tollefsbol and Hutchinson, 1995) and a putative new enzyme (Lei et al., 1996) have been shown to bear de novo methylation activity.
2. The role of cis acting signals If de novo methylation is possible, what determines Figure 3. What determines DNA methylation patterns? A model. DNA methylation patterns are determined by an interplay between: Transacting factors (triangle-factors enhancing methylation, oval-factors enhancing demethylation), Signals in DNA (red-enhancing methylation, green-enhancing demethylation), Levels of DNA MeTase and DNA demethylase activities. The pattern of methylation could be altered by modulation of any of these factors. The ideal targets for pharmacological intervention are the enzymatic activities.
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the specificity of DNA methylation? One important factor is cis signals contained in the sequence and putative cellular factors recognizing these signals (Szyf et al., 1989; Szyf, 1991) (F i g . 3). These signals possibly direct the general DNA methylation machinery to specific regions. Several experiments have shown that the presence of certain cis-acting sequences protect adjacent sequences from methylation (Szyf et al., 1990) while other sequences target adjacent sequences to become methylated (Szyf et al., 1989).
3. DNA methylation patterns are regulated by an interplay between local signals and the level of DNA MeTase activity. The role of cell legacy Could the pattern of DNA methylation be determined also by central cellular signals? Very limited attention has been given to the role that the cellular level of the enzyme catalyzing DNA methylation might play in determining and controlling DNA methylation patterns. One obvious reason why this level of regulation was not considered is because it had been difficult to explain how a general change in the level of the enzyme could lead to discrete changes in DNA methylation. To address that question I have previously suggested that the pattern of methylation is determined by an interplay between local signals, as suggested for example by the de novo methylation of the C21 gene in Y1 cells (Szyf et al., 1989), and the level of DNA MeTase (Szyf et al., 1984) and demethylase activities (Szyf 1994; Szyf et al., 1995) in the cell (F i g . 3). The affinity of each CpG site to DNA MeTase is determined by either the properties of the sequence or the DNA binding proteins interacting with it in specific cell types. Thus, the final pattern of methylation will reflect the legacy of the cell, its specific repertoire of DNA binding factors and resulting chromatin structure. According to this hypothesis we predict that a general change in DNA MeTase will result in a predictable change in DNA methylation pattern that is specific per cell type. In accordance with this hypothesis it has been shown that a limited inhibition of DNA MeTase results in specific
Gene Therapy and Molecular Biology Vol 1, page 653 Figure 4: Regulation of the DNA MeTase, a model. The DNA MeTase promoter and 5â&#x20AC;&#x2122; upstream region: the filled boxes indicate the first exons. Two translation initiation sites were identified, potentially resulting in a long and a short form of DNA MeTase (indicated by ATG) which might exhibit different transforming capabilities. Each of these initiation sites is regulated by a different promoter. The lower translation initiation site is a product of a transcript (transcription initiation sites indicated by horizontal arrows) initiating downstream of a promoter that is regulated by the Ras signaling pathway. The AP-1 recognition sequences located at (~1.7) upstream of the lower transcription initiation sites are indicated as ovals. The expression of DNA MeTase is regulated at multiple levels: First, by choice of promoter resulting in two different DNA MeTase forms. Second, the basal promoter which maintains limiting levels of DNA MeTase expression and is possibly regulated by tumor suppressors. Third, the mRNA is destabilized at G0 phase of the cell cycle. This is regulated by the retinoblastoma protein. T antigen can remove this regulation and stabilize the DNA MeTase mRNA. Fourth, a cluster of AP-1 sites can mediate transactivation of DNA MeTase by signal transduction pathways and by oncogenic signals. The activation by AP-1 could be repressed by glucocorticoid receptor.
alterations in DNA methylation patterns and differentiation of the cell to the next stage in differentiation rather than a chaotic loss of identity (Szyf et al., 1992). Animal experiments and clinical trials performed two decades ago have shown that general inhibition of DNA MeTase by the DNA MeTase inhibitor 5-Aza-CdR resulted in specific activation and demethylation of ! globin gene in animals and patients (Ley et al., 1982; DeSimone et al., 1982).
During the last five years, we have shown that the DNA MeTase is regulated at both the transcriptional and posttranscriptional level by nodal cellular signaling pathways (Szyf, 1994; Szyf, 1996). Cloning and characterizing the promoter of the DNA MeTase enabled us to determine that it bears AP-1 sites which are transactivated by Jun, a downstream effector of the nodal cellular and oncogenic Ras signaling pathway (Rouleau et al., 1992; Rouleau et al., 1995) (F i g . 4). Down regulation of the Ras-Jun pathway in the mouse adrenocarcinoma cell line Y1 leads to inhibition of DNA MeTase expression, inhibition of DNA methylation, alteration of DNA methylation patterns and reversal of oncogenesis (MacLeod and Szyf, 1995; MacLeod et al.,
4. DNA MeTase is regulated by central cellular signals at the transcriptional and posttranscriptional level a. Transcriptional regulation: 653
Szyf: DNA Methyltransferase in cancer therapy 1995). Recently, an additional start site upstream to the one identified by us which encodes a new translation initiation site resulting in a larger protein has been reported (Tucker et al., 1996). This start site is located in a CG island which is characteristic of housekeeping genes. My hypothesis is that differential utilization of these two promoters, an AP-1-regulated activity versus a housekeeping basal regulation plays an important role in determining the cellular level of DNA MeTase and its substrate specificity. Our recent data demonstrates that the human DNA MeTase promoter region bears as well a large number of AP-1 sites and is also upregulated by the Ras signaling pathway (Ramchandani et al., unpublished data). b. Posttranscriptional regulation: The DNA MeTase is also regulated with the phase of the cell cycle (Szyf et al., 1991). Whereas transcription of the message continues throughout the cell cycle, DNA MeTase mRNA is absent in Go cells which is consistent with posttranscriptional control of DNA MeTase mRNA. It has been recently shown that posttranscriptional regulation of DNA MeTase is also associated with muscle differentiation (Liu et al., 1996). Our recent data has linked the posttranscriptional regulation of DNA MeTase to basic cellular pathways that are known to play a critical role in cellular transformation (F i g . 4 ). We have recently shown that ectopic expression of SV40 T antigen in nontransformed 3T3 cells results in induction of DNA MeTase mRNA, DNA MeTase protein levels and genomic DNA methylation. A T antigen mutant which has lost the ability to bind pRb does not induce DNA MeTase. Surprizingly, this upregulation of DNA MeTase by T antigen occurs mainly at the posttranscriptional level by altering mRNA stability. Inhibition of DNA MeTase by 5-Aza-CdR reverses T antigen induced transformation suggesting a causal role for increased DNA MeTase activity in T antigen triggered transformation (Pinard et al., unpublished observations). This data links the Rb tumor suppressor pathway to the posttranscriptional regulation of DNA MeTase. It is interesting to note that Ras and T antigen can only transform primary cells when they are jointly expressed. It is tempting to speculate that their cooperative role in cellular transformation reflects the fact that they regulate DNA MeTase at two different levels. An alternative mechanism that might be responsible for the specificity of DNA MeTase is regulated alternative splicing of exons encoded by the DNA MeTase gene. Recent data from my laboratory suggests that the human DNA MeTase is encoded by 40 exons and that there is a potential for in -frame alternative splicing that will result in different forms of DNA MeTase (Deng et al., unpublished results). Regulation of this process with different developmental stages might play an important role in specifying specific classes of sites for methylation. In summary, our observations do not only establish the regulation of DNA MeTase by central cellular signaling pathways, but also suggest a potential molecular 654
link between DNA methylation and oncogenic pathways (Szyf, 1994) (F i g u r e s 4 , 5). The concept that DNA methylation patterns could be controlled by altering the level of the DNA MeTase leads to the idea that partial inhibition of DNA MeTase by pharmacological agents could result in altering gene expression programs (F i g . 4). One example of many possible applications of my hypothesis is the use of inhibition of DNA methylation to inhibit cellular transformation (F i g . 5 ). Inhibitors of DNA methylation could possibly be used to alter and control genetic programs in humans, plants and animals and might have broad application in clinical medicine, veterinary medicine and agriculture (Meyer, 1995).
III. DNA MeTase is an important therapeutic target. A. DNA methylation and cellular transformation 1. Is methylation involved in oncogenesis? The findings that the level of DNA MeTase as well as the pattern of DNA methylation might be controlled by oncogenic pathways leads to the question of whether DNA methylation plays an important role in cellular transformation? An activity that has a widespread impact on the genome such as DNA MeTase is a good candidate to play a critical role in cellular transformation. This hypothesis is supported by many lines of evidence that have demonstrated aberrations in the pattern of methylation in transformed cells.
2. Induction of dMTase activity in cancer cells explains the hypomethylation observed in these cells. Although it is clear that methylation patterns are altered in cancer cells, the direction that these changes take is perplexing. While many reports show hypomethylation of both total genomic DNA (Feinberg et al., 1988) and individual genes in cancer cells (Feinberg et al., 1983), other reports have indicated that hypermethylation of specific loci such as "tumor suppressor" genes is an important characteristic of cancer cells (Makos et al., 1992; Baylin et al., 1988; Baylin et al., 1991). How can one resolve this contradiction? We have recently suggested (Szyf, 1994) that the hypomethylation observed in cancer cells is a consequence of increased DNA demethylation activity induced by oncogenic pathways such as Ras7 which acts on a different subset of sites than those that are hypermethylated (Szyf et al, 1995). We have recently characterized a bona fide demethylase activity that is especially abundant in all cancer cells (Bhattacharya and Szyf unpublished results). We suggest that the dMTase recognizes CpG sites with different specificity than the DNA MeTase resulting in concomitant hypomethylation of some sites and hypermethylation of other sites (Szyf, 1994).
Gene Therapy and Molecular Biology Vol 1, page 655
Figure 5: DNA MeTase hyperactivation and tumorigenesis; reversal by DNA MeTase antagonists, a model. Regulated expression of DNA MeTase (standing rectangle indicating MeTase level is partly filled) is critical for maintaining the pattern of methylation (open circles indicate methylatable sites and M indicates methylation) and locking a somatic cell in in its program. Two facets of genome functions are regulated by DNA methylation. First, the profile of gene expression (the maintenance of the cognate program is indicated by a lock, expressed genes are indicated by horizontal arrows, transcription factors are indicated by ovals and triangles, methylated-DNA binding repressors are indicated by red ovals). Second, control of DNA replication is regulated by methylation (indicated by a stop sign). The replication control sequences (indicated by the open square) are not methylated in a resting somatic cell, signaling arrest of DNA replication. Oncogenic signaling pathways can induce the DNA MeTase resulting in hypermethylation of certain sequences, both genes and replication control regions. This results in loss of the original gene expression profile of the cell (open lock) and loss of the control over replication. Inhibitors of DNA MeTase can reduce the level of DNA MeTase, resulting in hypomethylation and activation of the replication control regions as well as restoration of some of the original gene expression program.
lation is that it is a consequence of the limited increase in DNA MeTase activity observed in many tumor cells (Kautiainen and Jones, 1986; el-Deiry et al., 1991). Recently Belinsky et al., have shown that increased DNA MeTase activity is an early event in carcinogen induced lung cancer in mice (Belinsky et al., 1996). Forced expression of exogenous DNA MeTase cDNA causes transformation of NIH 3T3 cells supporting the hypothesis that overexpression of DNA MeTase can cause cellular transformation (Wu et al., 1993). Our data demonstrating that the increase in DNA methylation activity in cancer cells is an effect of activation of either the oncogenic Ras-
3. Induction of DNA MeTase by oncogenic pathways is a critical component of oncogenic programs The critical remaining question is whether the "hypermethylation" observed in cancer cells is a programmed or random event? Random events are more difficult to control pharmacologically. However, if hypermethylation is a consequence of a programmed increase in DNA MeTase activity, the probability of reversing this state by inhibitors of DNA MeTase is high. A possible explanation for this observed hypermethy655
Szyf: DNA Methyltransferase in cancer therapy Jun signaling pathway (Szyf, 1994; MacLeod et al., 1995) or the oncogenic pathway induced by T antigen (Pinard and Szyf, unpublished data) supports the hypothesis that increased DNA MeTase is a critical component of diverse oncogenic programs. Several lines of evidence obtained by us support a causal role for increased DNA MeTase in oncogenesis. First, treatment of Y1 adrenocortical carcinoma cells with the DNA MeTase inhibitor 5-azaCCdR or stably expressing an antisense to DNA MeTase in these cells results in inhibition of tumorigenesis in vitro (MacLeod and Szyf, 1995). Second, when the DNA MeTase antisense transfected Y1 cells are injected into a syngeneic mouse, tumor formation in vivo is significantly inhibited (MacLeod and Szyf, 1995). Third, 5-Aza-CdR treatment of T antigen transformed 3T3 cells results in inhibition of cellular transformation in vitro . Fourth, intra peritoneal administration of phosphorothioate modified DNA MeTase antisense oligonucleotides inhibits tumorigenesis in vivo in LAF/1 mice (syngeneic strain) bearing Y1 tumors (Ramchandani et al., 1997). Similarly, Laird et al., (1995) have shown that treating mice bearing the Min mutation with 5-Aza-CdR significantly reduces the appearance of intestinal polyps. Based on these data, our working hypothesis is that induction of the enzymatic machinery controlling DNA methylation is a critical component of oncogenic programs and that oncogenesis could be reversed by inhibiting this induction.
B. What is the mechanism by which overexpression of the DNA MeTase induces tumorigenesis?
increases the rate of mutagenesis when DNA MeTase is present in the cell. This data strongly suggests that the mechanism by which the DNA MeTase inhibitor inhibits polyp formation in mice does not involve inhibition of mutagenesis.
2. Inactivation of tumor suppressors The second proposed mechanism discussed above is that hypermethylation results in silencing of "tumor suppressor" genes (Pokora and Schneider, 1992; OhtaniFujita et al., 1993; Merlo et al., 1995; Royer- Merlo et al., 1995;Herman et al., 1995). Although there is solid evidence that DNA methylation is an important mechanism involved in silencing "tumor suppressors", it is not clear whether methylation of tumor suppressor genes is a consequence of a programmed change in the level of DNA MeTase, such as that occurring in the Y1 system. An inherent problem in the "tumor suppressor" model is how can a general increase in DNA methylation result in site-specific methylation of specific genes. One possible model is that additional factors such as the"imprintors", proposed to function in parental imprinting of genes, might be involved in translating the increase in DNA methylation into site specific methylation events (Szyf, 1991). Alternatively, induction of DNA MeTase might directly activate cellular regulatory pathways that result in inactivation of tumor suppressor genes. Following inactivation, the tumor suppressors undergo methylation.
3. Direct control of cell growth
If induction of DNA MeTase is an important component of an oncogenic program, what is the mechanism? Based on what is known about the functions of DNA methylation in diverse biological systems, three alternative possible modes of actions emerge. Hypermethylation can result in stable mutations, can repress tumor suppressor genes or possibly directly control DNA replication.
1. DNA MeTase induces C to T transitions The first mechanism proposed by Peter Jones is that hypermethylation can increase the probability of reversion of 5mC to T by deamination, resulting in mutagenesis (Jones et al., 1992). However, this is an irreversible mechanism which is inconsistent with recent data. Laird et al., have previously shown that treatment of mice bearing the Min allele of APC with the DNA MeTase inhibitor 5-Aza-CdR reduces the frequency of polyp formations in these mice suggesting that DNA MeTase is critical for tumor formation in Min mice. If the mechanism by which DNA MeTase induces tumorigenesis is an increase in mutation rate, then treatment with 5azaCdR should have resulted in reduced mutagenesis. However, recent data by Jackson-Grusby et al., (1997) suggests that incorporation of 5-Aza-CdR into DNA 656
A third hypothesis is that DNA methylation directly controls the progression of the cell cycle (Szyf, 1996) . Recent evidence suggests that hypermethylated CG clusters is a marker of active origins (Rein et al., 1997), and that methylation of origins of replication occurs concurrently with replication (Araujo et al. unpublished). I have therefore proposed (Szyf, 1996) that hypermethylation causes firing of normally silent origins, explaining the chromosomal abnormalities observed in cancer cells. One interesting question is what is the kinetics of transformation induced by methylation. One hypothesis is that the increased MeTase is required to maintain random events of de novo methylation of tumor suppressor genes. Cells that have acquired these methylations are selected. If this model is true, transformation induced by methylation should be slow and the number of transformed cells should increase with time. On the other hand if increased MeTase levels target central controls of cell growth , then transformation by methylation should be rapid. Future experiments will most probably resolve this question. In summary, I will like to suggest this unifying hypothesis explaining the involvement of DNA methylation in cancer. The basic oncogenic programs in the cell trigger an induction of both DNA MeTase and dMTase activities resulting in hypermethylation of certain
Gene Therapy and Molecular Biology Vol 1, page 657 sites and hypomethylation of others. The specificity of this process is determined by the different affinities of the different sites to these respective enzymes. I suggest that the milieu of DNA binding factors in the cell directs the DNA MeTase to growth suppressor sites and the dMTase to growth stimulating sites. Thus, the coordinate induction of DNA MeTase and dMTase results in a simultaneous repression of all growth suppression functions and induction of growth activation functions (F i g . 5 ). If a general induction in DNA MeTase activity is indeed responsible for launching this program, then inhibition of the enzyme by pharmacological means should direct the cell towards the original program of a nontransformed cell.
Inhibitors of SAH hydrolysis such as periodate-oxidized adenosine or 3-deazaadenosine analogs (Chiang et al., 1992) were used before as inhibitors of DNA methylation. However, SAH, its analogues and inducers will inhibit a large number of different methylation reactions in the cell and must have nonspecific side effects (Papadopoulos et al., 1987) (F i g . 6 ).
3. Antisense oligonucleotides We have recently shown that a DNA MeTase antisense mRNA that is expressed in Y1 tumor cells inhibits tumorigenesis ex vivo and in vitro (MacLeod and Szyf, 1995). The advent of antisense oligodeoxynucleotides as specific inhibitors of protein expression in vivo offers new opportunities to test the therapeutic value of inhibition of DNA MeTase as well as to use these as novel therapeutic agents. We have recently shown that a phosphorothioate-modified antisense oligodeoxynucleotide directed against the DNA MeTase inhibits DNA MeTase as well as inhibits the growth of tumors in syngeneic mice in vivo (Ramchandani et al., 1997). These results have now been extended to xenografts of human cancer lines in nude mice. Active DNA MeTase antisense compounds that can inhibit human DNA MeTase mRNA as well as growth of human tumor cell lines in vivo have been identified (MacLeod et al., unpublished). DNA MeTase antisense oligonucleotides are potential candidates for anticancer agents in humans (F i g . 6).
IV. Therapeutic applications of DNA MeTase inhibitors A. DNA MeTase inhibitors An essential step in the developing of new pharmacological concepts is identifying novel targets. DNA MeTase was not considered a pharmacological target of importance because of the prevalent conception that inhibitors of DNA methylation might be carcinogenic (Platt, 1995). Because of this prevalent conception, no inhibitors to the cytosine DNA MeTase were developed since the introduction of 5-azacytidine. 5-azacytidine was originally synthesized as a nucleoside analog and was later found to inhibit DNA methylation after its incorporation into cellular DNA by covalently trapping the DNA MeTase (Wu and Santi, 1985; Jones, 1985). Our basic research of the mechanisms involved in regulating DNA MeTase activity and DNA methylation reviewed in the previous sections introduces the DNA MeTase as an important and very broad pharmacological target.
4. Direct inhibitors of DNA MeTase Antisense oligonucleotides only inhibit de novo synthesis but not the existing DNA MeTase protein. There might be certain situations where the turn-over rate of the enzyme will be too slow. In addition, antisense compounds are species specific and can not be used in animal models as well as nonanimal models such as plants which will most probably be of significant commercial potential. It is clear that new inhibitors are required to fully realize the research and applied potential of DNA methylation. Other approaches that are now tested in my laboratory is to use analogs of the CG substrate as direct inhibitors of DNA MeTase. DNA MeTase is an attractive candidate for DNA based antagonists since in distinction from other DNA binding protein it forms a covalent transition state intermediate with the DNA substrate (Wu and Santi, 1985). An ideal DNA based antagonists would therefore bind the MeTase but would not be an acceptor for methyl transfer. Thus, a stable complex would be formed between the enzyme and the substrate (F i g . 6). Recent unpublished data from our laboratory suggests that some analogs inhibit DNA MeTase activity at the nanomolar range and inhibit DNA MeTase and tumor growth in living cells (Bigey et al., unpublished).
1. 5-Aza-CdR a DNA MeTase inhibitor with serious side effects The only specific inhibitor of DNA MeTase that is currently available is 5-Aza-CdR which is phosphorylated by cellular kinases, incorporated into DNA and traps DNA MeTase molecules by forming a covalent bond with the catalytic site of the protein (Wu and Santi, 1985). This mechanism of action results in potential toxicities and side effects that limit the utility of 5-azadC as a therapeutic agent as well as a research tool (see review in Szyf, 1996). Recent data suggests that the mutagenicity induced by 5azaCdR is a consequence of the interaction of DNA MeTase with 5-azaCdR incorporated into the DNA (Juttermann et al., 1994; Jackson-Grusby et al., 1997). It is clear that new DNA MeTase inhibitors that are not incorporated into DNA should be developed (F i g . 6 ).
2. SAM analogs S-adenosyl- homocysteine, an analogue of SAM and one of the products of the methylation reaction is an inhibitor of DNA methylation (Mixon and Dev, 1983).
B. Potential side effects of DNA MeTase inhibitors 657
Szyf: DNA Methyltransferase in cancer therapy One important issue that might challenge the utility of DNA MeTase inhibitors as therapeutics is potential side effects resulting from activation of unwanted genes. It is obviously impossible to assess the full systemic effect of DNA MeTase inhibitors at this stage. However, previous data as well as our understanding of the mechanisms of action of DNA methylation suggest that these effects will not be a major issue. First, demethylation is insufficient per se to activate genes, the presence of the proper transcription machinery is required. Demethylation will only activate those genes in the cell that have the appropriate transcription factors available. Second, the new pattern of methylation generated after demethylation will be dictated by the legacy of the cell. This is probably why extensive demethylation of cell lines results in
activation of the next stage in differentiation rather than chaotic activation of many possible programs (Szyf et al., 1992). Third, DNA MeTase inhibitors will only have an effect on dividing cells since they passively inhibit methylation during replication but do not remove methyl groups from DNA. Fourth, chronic treatment of mice with 5-Aza-CdR (Laird et al., 1995) or DNA MeTase antisense (Ramchandani et al., 1997) does not result in apparent systemic toxicity.
Acknowledgements The work discussed in this review was supported by the MRC, NCIC and contracts with MethylGene Inc. and Hybridon Inc.
Figure 6: Inhibitors of DNA MeTase DNA MeTase could be inhibited at different levels. The first line illustrates a scheme (not to scale) of the first exons and introns of the DNA MeTase gene. The second and third lines are schemes of mRNAs encoded by the DNA MeTase. A n t i s e n s e o l i g o n u c l e o t i d e s : Antisense oligonucleotides (line under the mRNA) directed against some of the splice junctions, reduce DNA MeTase mRNA level (MacLeod et al., unpublished). Two different messages are transcribed from the DNA MeTase gene. Antisense oligonucleotides could be directed against the sequence encoding the ATG translation initiation site of each protein specifically. Once the protein is synthesized, it could be inhibited by either of three different ways. Hairpin i n h i b i t o r s : First, a modified hairpin oligonucleotide substrate (left) bearing a hemimethylated CG sequence will bind the DNA MeTase and form a stable complex with the substrate. As the modification of the hairpin inhibits the transfer of a methyl group from SAM, the enzyme remains bound to the substrate and is unavailable for methylating genomic DNA. S A M a n a l o g s : SAH and its analogs bind the SAM binding pocket of the DNA MeTase and inhibit methylation. The first line describes the DNA methylation reaction. A double stranded DNA bearing a methylated C in a CG dinucleotide on the parental strand and a nonmethylated C in the CG dinucleotide on the nascent strand is reacted with S-adenosyl-methionine (SAM) in a reaction catalyzed by the DNA methyltransferase (DNA MeTase). The resulting products of the reactions are a double stranded methylated DNA and S-adenosyl homocysteine (SAH).
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Li E, Bestor TH and Jaenisch R ( 1 9 9 2 ) Targeted mutation of the DNA methyltransferase gene results in embryonic lethality. C e l l 69, 915-926.
el-Deiry, W. S., Nelkin, B. D., Celano, P., Yen, R. W., Falco, J. P., Hamilton, S. R. and Baylin, S. B. ( 1 9 9 1 ) High expression of the DNA methyltransferase gene characterizes humanneoplastic cells and progression stages of colon cancer. P r o c . N a t l . A c a d . S c i . U S A 88, 3470-3474.
Liu Y, Sun L and Jost JP. ( 1 9 9 6 ) In differentiating mouse myoblasts DNA methyltransferase is posttranscriptionally and posttranslationally regulated. N u c l . A c i d s R e s . 24, 2718-2722. Lock LF, Takagi N and Martin GR ( 1 9 8 7 ) Methylation of the Hprt gene on the inactive X occurs after chromosome inactivation. C e l l 48, 39-46.
Feinberg AP and Vogelstein B ( 1 9 8 3 ) Hypomethylation distinguishes genes of some human cancers from theirnormal counterparts. Nature 301, 89-92. Feinberg AP, Gehrke CW, Kuo KC and Ehrlich M ( 1 9 8 8 ) Reduced genomic 5-methylcytosine content in human colonic neoplasia. Cancer Res. 48, 1159-1161.
MacLeod AR and Szyf M ( 1 9 9 5 ) Expression of an antisense to the DNAmethyltransferase mRNA induces DNA demethylation and inhibits tumorigenesis. J . B i o l . C h e m . 270, 8037-8043.
Gruenbaum Y, Naveh-Many T, Cedar H and Razin A ( 1 9 8 4 ) Sequence specificity of methylation in higher plant DNA. Nature 292, 860-862.
MacLeod AR, Rouleau J and Szyf M ( 1 9 9 5 ) Regulation of DNA methylation by the Ras signaling pathway. J . B i o l . C h e m . 270, 11327-11337.
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Szyf: DNA Methyltransferase in cancer therapy Makos M, Nelkin BD, Lerman MI, Latif F, Zbar B and Baylin SB ( 1 9 9 2 ) Distinct hypermethylation patterns occur at altered chromosome loci in human lung and colon cancer. P r o c . N a t l . A c a d . S c i . U S A 89, 1929-1933. Merlo A, Herman JG, Mao L, Lee DJ, Gabrielson E, Burger P, Baylin SB and Sidransky D (1995) 5â&#x20AC;&#x2122; CpG island methylation is associated with transcriptional silencing of the tumor suppressor p16/CDKN2/MTS1 in human cancers. Nature Med. 1, 686-692. Meyer P. ( 1 9 9 5 ) DNA methylation and transgene silencing in Petunia hybrida. Curr. T o p i c s M i c r o b i o l . I m m u n o l . 197, 15-28. Mixon JC and Dev VG ( 1 9 8 3 ) Fragile X expression is decreased by 5-azacytidine and S-adenosylhomocysteine. Am J Hum Genet. 35, 1270-1275. Nan X, Campoy FJ, Bird A ( 1 9 9 7 ) MeCP2 is a transcriptional repressor with abundant binding sites in genomic chromatin. C e l l 88, 471-481. Peterson K and Sapienza C ( 1 9 9 3 ) Imprinting the genome: imprinted genes, imprinting genes, and a hypothesis. A n n u . R e v . G e n e t . 27, 7-31. Ohtani-Fujita N, Fujita T, Aoike A, Osifchin NE, Robbins PD and Sakai T ( 1 9 9 3 ) CpG methylation inactivates the promoter activity of the human retinoblastoma tumorsuppressor gene. O n c o g e n e 8, 1063-1067.
specific methylation pattern in 11p13 detected by PFGE. Genes Chromosom. Cancer 5, 132-140. Stein R, Razin A and Cedar H ( 1 9 8 2 ) In vitro methylation of the hamster adenine phosphoribosyltransferase gene inhibits its expression in mouse L cells. Proc Natl Acad Sci USA 79,3418-3422. Szyf M, Avraham-Haetzni K, Reifman A, Shlomai J, Kaplan F, Oppenheim A and Razin A (1 9 8 4 ) DNA methylation pattern is determined by the intracellular level of the methylase. P r o c N a t l A c a d S c i USA 81, 32783282. Szyf M, Schimmer BP and Seidman JG ( 1 9 8 9 ) Nucleotidesequence-specific de novo methylation in a somatic murine cell line. P r o c . N a t l . A c a d . S c i . U S A 86, 6853-6857. Szyf M Tanigawa G and McCarthy PL Jr. ( 1 9 9 0 ) A DNA signal from the Thy-1 gene defines de novo methylation patterns in embryonic stem cells. M o l . C e l l . B i o l . 10, 4396-4340. Szyf M, Bozovic V and Tanigawa G ( 1 9 9 1 ) Growth regulation of mouse DNA methyltransferase gene expression. J . B i o l . C h e m . 266,10027-10030. Szyf M ( 1 9 9 1 ) DNA methylation patterns: an additional level of information?. B i o c h e m . C e l l B i o l . 69, 764767.
Papadopoulos V, Kamtchouing P, Drosdowsky MA and Carreau S ( 1 9 8 7 ) Effects of the transmethylation inhibitor S-adenosyl-homocysteine and of the methyl donor S-adenosyl-methionine on rat Leydig cell function in vitro. J . S t e r o i d B i o c h e m . 26, 93-98.
Szyf M, Rouleau J, Theberge J and Bozovic V ( 1 9 9 2 ) Induction of myogenic differentiation by an expression vector encoding the DNA methyltransferase cDNA sequence in the antisense orientation. J B i o l C h e m . 267, 12831-12836.
Platt OS ( 1 9 9 5 ) Sickle cell paths converge on hydroxyurea. Nature Med. 1, 307-308.
Szyf M ( 1 9 9 4 ) DNA methylation properties: consequences for pharmacology. T r e n d s P h a r m a c o l S c i . 15, 233238.
Ramchandani S, MacLeod AR, Pinard M, von Hofe E and Szyf M ( 1 9 9 7 ) Inhibition of tumorigenesis by a cytosineDNA methyltransferase antisense oligodeoxynucleotide. P r o c . N a t l . A c a d . S c i . U S A 94, 684-689 . Razin A and Cedar H ( 1 9 7 7 ) Distribution of 5methylcytosine in chromatin. P r o c . N a t . A c a d . S c i USA 74, 2725-2728. Razin A and Riggs AD ( 1 9 8 0 ) DNA methylation and gene function. S c i e n c e 210, 604-610. Razin A and Szyf M. ( 1 9 8 4 ) DNA methylation patterns. Formation and function. B i o c h i m . B i o p h y s . A c t a . 782: 331-342. Razin A, Feldmesser E, Kafri T and Szyf M ( 1 9 8 5 ) Cell specific DNA methylation patterns; formation and a nucleosome locking model for their function. P r o g . C l i n i c . B i o l . R e s . 198, 239-253. Rein T, Zorbas H and DePamphilis M ( 1 9 9 7 ) Active mammalian replication origins are associated with a highdensity cluster of mCpG dinucleotides. M o l . C e l l . B i o l . 17, 416-426. Rouleau J, Tanigawa G and Szyf M. ( 1 9 9 2 ) The mouse DNA methyltransferase 5'-region. A unique housekeeping gene promoter. J . B i o l . C h e m . 267, 7368-7377. Rouleau J, MacLeod AR and Szyf M ( 1 9 9 5 ) Regulation of the DNA methyltransferase by the Ras-AP-1 signaling pathway. J . B i o l . C h e m . 270,1595-1601. Royer-Pokora B and Schneider S ( 1 9 9 2 ) Wilms' tumor-
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Szyf M Theberge J and Bozovic V ( 1 9 9 5 ) Ras induces a general DNA demethylation activity in mouse embryonal P19 cells. J . B i o l . C h e m . 270, 12690-12696 Szyf M ( 1 9 9 6 ) The DNA methylation machinery as a target for anticancer therapy. Pharmacol.Ther. 70, 1-37. Tollefsbol TO and Hutchinson CAIII ( 1 9 9 5 ) Mammalian DNA (Cytosine-5-)-methyltransferase expressed in Escherichia coli, purified and characterized J . B i o l . Chem. 270, 18543-18550. Tucker KL, Talbot D, Lee MA, Leonhardt H and Jaenisch R ( 1 9 9 6 ) Complementation of methylation deficiency in embryonic stem cells by a DNA methyltransferase minigene. P r o c . N a t l A c a d . S c i . U S A 93, 1292012925. Wu JC and Santi DV ( 1 9 8 5 ) On the mechanism and inhibition of DNA cytosine methyltransferases. P r o g . C l i n i c . B i o l . R e s . 198, 119-129. Wu J, Issa JP, Herman J, Bassett DE Jr, Nelkin BD and Baylin SB ( 1 9 9 3 ) Expression of an exogenous eukaryotic DNA methyltransferase gene induces transformation of NIH 3T3 cells. P r o c . N a t l . Acad. S c i . U S A 90, 8891-8895 Yisraeli, J., and Szyf, M. ( 1 9 8 4 ) The pattern of methylation of eukaryotic genes. in DNA M e t h y l a t i o n : Biochemistry & Biological Significance, (Razin, A. Cedar, H. & Riggs, A. D., eds) pp 353-378, Springer-Verlag, New York.
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Gene Therapy and Molecular Biology Vol 1, page 661 Gene Ther Mol Biol Vol 1, 661-679. March, 1998.
Correlation between DNA methylation and poly(ADP-ribosyl)ation processes Giuseppe Zardo°, Stefania Marenzi* and Paola Caiafa°# ° Departments of Biomedical Sciences and Technologies, University of L'Aquila and of *Biochemical Sciences “A.Rossi Fanelli” University of Rome “La Sapienza", #C.N.R. Centre for Molecular Biology, Rome, Italy.
___________________________________________________________________________________________________ Corresponding author: Dr. Paola Caiafa, Dipartimento di Scienze e Tecnologie Biomediche, Università dell’Aquila; Via Vetoio, Loc. Coppito, I-67100 L’Aquila, Italy. Tel: (+39) 862-433431; Fax: (+39) 862-433433; E-mail: caiafa@axscaq.aquila.infn.it
Summary The aim of this article is to show the close relationship between DNA methylation and poly(ADPribosyl)ation which are two important nuclear enzymatic mechanisms. An open question i s to explain how some CpG dinucleotides, in particular those present into GpG islands, can maintain their unmethylated state in spite of the presence of active DNA methyltransferase in chromatin. This paper illustrates some data indicating that H1 histone i s a possible trans-acting factor involved in protecting genomic DNA from full methylation and proposes that the somatic variant H1e, in its poly(ADP-ribosyl)ated isoform, is the protein capable of undertaking this role.
methylation pattern results from the combination of maintenance and de novo methylation and of demethylation processes, Figure 2. The maintenance methylase recognizes and modifies hemimethylated sites generated during DNA replication thus preserving the tissue-specific methylation pattern (Razin and Riggs, 1980). In higher eukaryotes this enzymatic process takes place within a minute or two after replication (Leonhardt et al., 1992).
I. Introduction A. Inhibitory effect of DNA methylation on gene expression. DNA methylation is a specific post-synthetic modification of DNA that, in eukaryotic cells, appears to play an important role in the epigenetic modulation of gene expression. It is the major enzymatic DNA modification that transfers methyl groups from S-adenosyl methionine (S-AdoMet) to cytosine (C) and converts these residues into 5-methylcytosine (5mC) (Bestor and Ingram, 1983). Although in vertebrates the presence of 5mC has occasionally been reported to be found in dinucleotide sequences CpC, CpA and CpT (Woodcock et al., 1987, 1988; Toth et al., 1990; Tasheva and Roufa, 1994; Clark et al., 1995), the best substrate for DNA methyltransferase is cytosine located in the CpG dinucleotide (Gruembaun et al., 1981).
The final "correct" methylation pattern is reportedly obtained, in somatic cells, during the early stages of embryonic development, through a combination of demethylation and de novo methylation steps (Brandeis et al., 1993). Demethylation occurs by an active reaction (Frank et al., 1991; Brandeis et al., 1993; Jost, 1993; Weiss et al., 1996) where a 5-methyldeoxycytidine excision repair system cleaves the DNA strand at 5mCpG sites, removes the methylcytosine from DNA and replaces it with cytosine. Subsequently, a burst of de novo methylation starts the differentiation process leading to a bimodal pattern of methylation in which the "CpG islands" at the 5' end of the housekeeping genes remain constitutively unmethylated, while other genomic sequences undergo a massive wave of de novo methylation. Demethylation of individual genes occurs also during tissue-specific differentiation (Razin et al.,
As for the distribution of 5mCs, evidence already existed (Yisraeli and Szyf, 1984) that they are distributed in a non-random fashion in genomic DNA. Successive studies have shown that the methylated cytosines are present in bulk DNA (Bloch and Cedar, 1976) while the unmethylated ones are essentially located within some particular DNA regions termed "CpG islands" (Bird et al., 1985; Bird, 1986,1987), F i g u r e 1 . The specific DNA 661
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Maintenance DNA methylation 5' XXXC mGXXXC mGXXX 3' XXXGC XXXGC XXX 5' XXXC mGXXXC mGXXX 3' XXXGmC XXXGmC XXX
Replication !!!!!"
5' XXXC mGXXXC mGXXX 3' XXXGmC XXXGmC XXX Maintenance Methylation !!!!!!!!!!!!" 5' XXXC mGXXXC mGXXX 3' XXXGmC XXXGmC XXX
5' XXXCG XXXCG XXX 3' XXXGmC XXXGmC XXX Substrate: hemimethylated DNA Role: to preserve the tissue specific methylation pattern When : 1-2 min. after replication. !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!
De novo DNA methylation 5' XXXCG XXXCG XXX 3' XXXGC XXXGC XXX Substrate: Role: When :
5' XXXC mGXXXCG XXX 3' XXXGmC XXXGC XXX
De novo methylation !!!!!!!!!"
unmethylated CpG sequences. to define the final correct tissue specific methylation pattern involved in the differentiation process, or repress the active genes in somatic cells. during the early stages of embryonic development, or during carcinogenesis.
!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!
Active DNA demethylation 5' XXXC mGXXXC mGXXX Demethylation 5' XXXC mGXXXC mGXXX Demethylation 5' XXXCG XXXCG XXX 3' XXXGmC XXXGmC XXX !!!!!!" 3' XXXGC XXXGC XXX !!!!!!" 3' XXXGC XXXGC XXX Substrate: Role: When :
fully methylated DNA emimethylated DNA . to define the final correct methylation pattern, or gene activation in somatic cells. after replication, during the early stages of embryonic development (blastula stage), or during tissue specific differentiation. !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! F i g u r e 1 . Processes involved in defining the DNA methylation pattern.
Distribution of CpG and 5'meCpG dinucleotides in eukaryotic DNA. Bulk
CpG island G+C content
40%
60% CpG level (CpG/GpC)
0.2
> 0.6 Methylation level
High
Unmethylated
F i g u r e 2 . Non-random distribution of CpG and 5mCpG dinucleotides in genomic DNA.
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Gene Therapy and Molecular Biology Vol 1, page 663 difficult to demonstrate up to now. Research on the identification of factors able to link methylated DNA has met with greater success. The first protein identified as able to bind methylated DNA sequences is the methylated DNA binding protein (MDBP) a ubiquitous family of closely related proteins in vertebrates (Huang et al., 1984; Zhang et al., 1993). Its consensus sequence is composed of 14 bp and has a substantial degree of degeneration. MDBP sites can have up to 3 CpG's and generally the degree of binding increases when more of these are methylated and when they are methylated on both strands. In spite of the high level of degeneracy of the consensus sequence, MDBP can be considered sequence-specific in its binding because mutations in inopportune positions cause this protein to lose its ability to link the sequence. This protein can also link the consensus sequence independently of its methylated level provided the C is substituted by T and T is present in TpG or TpA dinucleotides (Zhang et al., 1986; Khan et al., 1988). It is possible that the consensus methylation independent sequences could derive from the spontaneous deamination of 5mC in T. From transfection experiments a role of down-regulation of gene expression has been proposed for this protein (Asiedu et al., 1994; Zhang et al., 1995). The MDBP-2-H1 is another protein that binds itself preferentially to certain DNA sequences containing a simple mCpG pair (Pawlak et al., 1991). Although the protein is not sequence specific, its affinity for the consensus sequence is highest in the promoter region (+2 +32) of vitellogenin II gene where it plays an down-regulatory role of gene expression (Pawlak et al., 1991). Further investigations have shown (Jost and Hofsteenge, 1992) that this protein - identified as H1 histone-like - must undergo phosphorylation before to its interaction with the methylated DNA sequence (Bruhat and Jost, 1995). Two other proteins named MeCP1 and MeCP2, that have the ability to link DNA regions in which the CpG dinucleotides are methylated to higher or lower levels, have been proposed as proteins involved in the silencing of gene expression (Meehan et al., 1989; Boyes and Bird, 1991; Lewis et al., 1992). In particular MeCP1, whose molecular weight is of about 800 KDa, is suggested to be involved in a mechanism through which its association with methylated DNA could prevent the linkage of transcription factors in these DNA regions. This protein binds sequences containing about 12 or more methylated CpGs and the enrichment in CpG dinucleotides argues that these DNA regions are "CpG island-like" (Meehan et al., 1989).
1986; Brandeis et al., 1993; Jost and Jost, 1994), this process being probably required for gene activation. To explain the demethylation process two different mechanisms have been described. The first one involves a proteic factor 5methylcytosine endonuclease activity that is able to remove the 5-methylcytosine and to substitute it with cytosine (Jost, 1993; Jost and Jost, 1994; Jost et al., 1995). The second one involves the presence of a ribozyme or maybe a ribozyme associated with a proteic factor that is able to remove the mCpG dinucleotide and to substitute it with CpG dinucleotide (Weiss et al., 1996). The fact that CpG dinucleotides are present in an unmethylated state in "CpG islands" is of interest since their frequency in them is five times more than in bulk DNA, Figure 1. As far as the correlation between DNA methylation and gene expression is concerned, the "CpG islands", that go from 500-2000 base pairs in size, are usually found in the 5' promoter region of housekeeping genes and overlap genes to variable extents (Bird, 1986). There is evidence that transcription of genes, correlated with "CpG islands", is inhibited when these regions are methylated (Keshet et al., 1985). That the "CpG islands" are not by themselves unmethylable is demonstrated by in vitro experiments (Carotti et al., 1989; Bestor et al., 1992). A great deal of investigation has been and is performed in order to clarify why the "CpG islands" remain untouched by the action of DNA methyltransferase (Ysraeli and Szyf, 1984) in spite of their localization on promoter region of housekeeping genes which are, in decondensed chromatin, permanently accessible to the transcriptional factors. A question yet to be solved is to identify different cisacting signals and trans-acting protein factors that may play a key role in defining the bimodal pattern of methylation involved in cell differentiation and gene expression. It has been suggested that the density of CpG dinucleotide inside "CpG-islands" could be per se a signal involved in protecting the unmethylated state of these DNA regions (Frank et al., 1991) but further experiments suggest that there are some sequence motifs that are intrinsically protected against de novo methylation (Szyf et al., 1990; Christman et al., 1995; Tollefsbol and Hutchinson, 1997) and/or that there are some cis-acting "centers of methylation" capable of preventing the methylation pattern of flanking DNA sequences (Szyf et al., 1990; Szyf, 1991; Mummaneni et al., 1993; Brandeis et al., 1994; Hasse and Schultz, 1994; MaCleod et al., 1994; Magewu and Jones, 1994; Mummaneni et al., 1995). The simple possible explanation that there are trans-acting protein factors associated with "CpG islands" which prevent access to those DNA regions, has been
The strength of promoter and the density of mCpGs (Boyes and Bird, 1992) are two factors which regulate the association of MeCP1 with DNA. It is clear that low levels of methylation can repress transcription of a weak promoter but not of a strong promoter. In fact, sparsely methylated genes bind MeCP1 weakly and the
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Gene Therapy and Molecular Biology Vol 1, page 664 transcription is partially repressed if the gene promoter is weak while if the promoter is strong the gene is expressed (Boyes and Bird, 1992). The MeCP2 factor is able to bind DNA that contains a single mCpG pair (Lewis et al., 1992; Meehan et al., 1992). MeCP2, for which a transcriptional repressor role has been described, is very abundant in bulk vertebrate genomic DNA - 100 times more abundant than MeCP1 - where it is in competition with H1 histone. This result supports the hypothesis that MeCP2 is involved in condensing chromatin structure (Nan et al., 1997). Although these proteins play an important role in mediating the methylation-dependent repression of genes, an open question to answer is how the CpG moieties of the "CpG islands", become vulnerable or resistant to the action of DNA methyltransferase and can thus lose or maintain their characteristic pattern of methylation. This is the goal of our research: our aim is to identify and pinpoint a nuclear protein trans-acting factor directly involved in maintaining the unmethylated state of "CpG islands".
II. H1 histone and DNA methylation. A. Methylation-dependent binding of H1 histone to DNA. An attractive hypothesis to explain the repressive effects of DNA methylation on gene expression is that H1 histone binds itself preferentially to DNA sequences containing mCpG dinucleotides. Although H1 histone is mainly present in highly methylated condensed chromatin there is ample disagreement in the scientific literature - at variance from the other above mentioned proteins - as to whether or not its presence is dependent on the methylated state of DNA. A preference of H1 histone for double-stranded DNA with a relatively high abundance of methylated CpGs has however been recently shown by McArthur and Thomas (1996), who have suggested that the condensing ability of H1 histone could thus be favored by the higher level of DNA methylation existing in transcriptionally inactive chromatin. Parallel experiments (Caiafa et al., 1995; Reale et al., 1996) have been performed in order to examine whether in oligonucleosomal DNA, purified from inactive chromatin fraction, an increased methylation of CpG residues would interfere with the formation of the appropriate H1-H1 interactions critical for attainment of folded chromatin structures. Conflicting results respect to those of McArthur and Thomas (1996) were obtained since the introduction of new methyl groups into oligonucleosomal DNA was surprisingly found to decrease its ability to allow these H1-H1 interactions (Figure 3),
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suggesting that, in vivo, the presence of some unmethylated CpGs in linker DNA is likely to be an important prerequisite for chromatin compaction. These differences could be explained by differences in the DNAs selected for the two experiments as, despite the common aim of avoiding sequence-specific effects in H1-DNA binding, there are indeed considerable differences in terms of CpG frequency and of the overall methylation level of the DNAs. The DNA sequences used by McArthur and Thomas (1996), chosen as representative of a large region of the sea urchin genome, are essentially obtained from unmethylated CpG-rich DNA regions, while our oligonucleosomal DNA was extracted from human placenta inactive chromatin fraction whose relatively scarce CpG moieties have a rather high basal methylation level. The band shift assays did not solve the problem of methylation dependent binding of H1 histone to DNA. In fact experiments carried out using DNA fragments with different amounts of CpGs dinucleotides, failed to show any effect of CpG methylation on H1 histone binding since H1 histone has shown an identical affinity for either methylated or non-methylated DNA (Campoy et al., 1995). It may be recalled that while Higurashi and Cole (1991) have also found that the interaction of H1 histone with CCGG is independent of the methylation level, Levine et al. (1993) have shown a preferential binding of total H1 histone to plasmid methylated DNA.
B. Inhibitory effect of H1 histone on in vitro DNA methylation. In our research on a nuclear proteic factor involved in DNA methylation process, we focused our attention on histone proteins since previous papers have reported a possible inhibitory role played by histones on DNA methylation (Kautiainen and Jones, 1985; Davis et al., 1986). Our experiments (Caiafa et al., 1991) have shown that the ability of total histones to affect in vitro enzymatic DNA methylation was essentially due to a single H1 histone that, in the "physiological" range (0.3:1, w/w) histone:DNA ratio, was the only one able of exerting a consistent (90%) inhibition on methylation of double stranded DNA, catalyzed by human placenta DNA methyltransferase. Neither H1-depleted preparations of "core" histones nor, separately, any other single histone (H2a, H2b, H3) were able to affect the methylation process, Figure 4. Since H1 is known to be preferentially associated to linker DNA (van Holde, 1988) its ability to suppress in vitro DNA methylation is consistent with previous
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F i g u r e 3 A : SDS-PAGE patterns of H1 histone after treatment, in the presence of native (lanes 1, 2, 3) or of artificially overmethylated oligonucleosomal DNA (lanes 4, 5, 6), with dithiobis-(succinimidylpropionate) at different H1:DNA ratios -- 0.1, 0.3, 0.5 (w/w) -- in 40 mM NaCl. In lane 7, H1 histone treated with DSP in the absence of DNA; in lane 8, untreated H1 histone. (B ): Electrophoretic patterns, in 1% agarose stained with ethidium bromide, of glutaraldehyde-fixed H1-DNA complexes, formed in 40 mM NaCl at H1:DNA ratios ranging from 0.1 to 0.9 (w/w), using native oligonucleosomal DNA (left panel) or artificially overmethylated DNA (right panel). DNA molecular marker III from Boehringer is in lane III. Naked DNA controls (native in the left panel, artificially overmethylated in the right one) are in lanes C and C1 . "Reprinted from B i o c h e m . B i o p h y s . R e s . C o m m . 2 2 7 , Reale et al.. H1-H1 Cross-linking efficiency depends on genomic DNA methylation, 768-774, (1996) with kind permission of Academic Press, Inc." F i g u r e 4 . Effect, on the in vitro activity of human placenta DNA methyltransferase, of histones H1, H2a, H2b and H3 (from calf thymus) renaturated by progressive dialysis at decreasing urea and NaCl concentrations in the presence (closed triangles) or absence (closed circles) of 5 mM EDTA. Each point represents the mean result of at least five different experiments in triplicate, S.D. "Reprinted from B i o c h i m . B i o p h y s . A c t a 1 0 9 0 , Caiafa et al. Histones and DNA methylation in mammalian chromatin. I째 Differential inhibition by histone H1, 38-42, (1991) with kind permission of Elsevier Science Publishers - NL Sara Burgerhartstraat 25, 1055 KV Amsterdam, The Netherlands".
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nucleosome sliding may have significantly altered the regions of chromatin DNA accessible to methylation. In the first set of experiments we measured the proportion of labelled methyl groups remaining in the "100 bp minicore particles" upon extensive staphylococcal nuclease digestion of in vitro methylated H1-depleted oligonucleosomes. As shown in Figure 5a,b - where H1 had been taken away, respectively from oligonucleosomes and from nuclei by two alternative procedures - nuclease treatment removed the majority (85% in one case, 75% in the other) of the labelled 5methylcytosine residues. By contrast, nuclease digestion removed from native oligonucleosomes (H1-containing) only a relatively small portion of the 5-methylcytosine residues which had been inserted by in vitro enzymatic DNA methylation, Figure 5c.
findings of higher 5mC levels in nucleosomal core DNA as compared to linker DNA (Razin et al., 1977; Solage and Cedar, 1978; Adams et al., 1984; Caiafa et al., 1986). Some experiments were carried out to assess whether the observed hypomethylation of linker DNA sequences reflect an intrinsic deficiency in CpG dinucleotides or whether the well-documented association between DNA and H1 histone causes a local inhibition of enzymatic DNA methylation process (D’Erme et al., 1993). The net level of methyl-accepting ability of CpG dinucleotides in linker DNA - defined as the DNA region which can be hydrolyzed by staphylococcal nuclease digestion of H1-depleted oligonucleosomes - was evaluated by making use of a number of distinct experimental strategies in order to minimize possible artefacts. Since the removal of H1 histone by two alternative procedures yielded quite similar results, it is unlikely that artefactual
a- R e s i d u a l m e t h y l g r o u p s i n 1 0 0 b p “ m i n i c o r e ” p a r t i c l e s a f t e r n u c l e a s e d i g e s t i o n o f m e t h y l a t e d H1-depleted oligonucleosomes (treated with 0.6 M NaCl): H1 - depleted oligonucleosome s
in vitro !!" methylation # methyl - 3 H incorporated (2480 dpm/10µg DNA = 100%) !!"
staphylococcal nuclease digestion
“minicore” particles (100 bp) # methyl - 3 H incorporated (390 dpm/10µg DNA = 15.6%)
!!"
b - Residual methyl groups in the nuclease-resistant fraction from methylated H1-depleted nuclei (treated at low pH) : H1 - depleted nuclei
in vitro methylation
staphylococcal nuclease digestion
methyl - 3 H incorporated (717 dpm/10µg DNA = 100%)
nuclease-resistant fraction methyl - 3 H incorporated (174 dpm/10µg DNA = 24.3%)
c - Residual methyl groups in 145 bp “core” particles after nuclease digestion of methylated native oligonucleosomes: native oligonucleosomes
in vitro methylation
staphylococcal nuclease digestion
methyl - 3 H incorporated (1215 dpm/10µg DNA = 100%)
“core” particles (145 bp) methyl - 3 H incorporated (716 dpm/10µg DNA = 58.9%)
F i g u r e 5 . Evaluation, by three distinct experimental strategies involving nuclease digestion after in vitro methylation, of the distribution of methyl-accepting CpGs in the nuclease-sensitive fraction. The data obtained refer to a similar set of experiments run in parallel, so as to obtain comparable values. Five other similar experiments gave slightly different results in terms of absolute incorporation of methyl groups, but almost identical as percent radioactivity values remaining in the nucleaseresistant fractions. "Reprinted from B i o c h i m . B i o p h y s . A c t a 1 1 7 3 , D'Erme et al.. Inhibition of CpG methylation in linker
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Zardo et al: DNA methylation and poly(ADP-ribosyl)ation DNA by H1 histone, 209-216, (1993) with kind permission of Elsevier Science Publishers - NL Sara Burgerhartstraat 25, 1055 KV Amsterdam, The Netherlands". a - Direct methylation of 100 bp “minicore” particles vs H1 - depleted oligonucleosomes: H1 - depleted oligonucleosomes
staphylococcal nuclease digestion
“minicore” particles (100 bp)
in vitro methylation
in vitro methylation
methyl - 3 H incorporated (3380 dpm/10µg DNA = 100%)
methyl -3 H incorporated (340 dpm/10µg DNA = 10.1%)
b - Methylation of purified DNA’s from 145 bp “core” particles and from 100 bp “minicore” particles vs oligonucleosomal DNA: native oligonucleosomes
staphylococcal nuclease digestion
H1 - depleted oligonucleosomes
staphylococcal nuclease digestion
“core” particles (145 bp) “minicore” particles (100 bp) purified DNA from “minicore” particles
purified DNA from oligonucleosomes
in vitro methylation
in vitro methylation
purified DNA from “core” particles (145 bp) in vitro methylation methyl - 3 H incorporated (1067 dpm/10µg DNA = 25.9%)
methyl - 3 H incorporated (1010 dpm/10µg DNA = 24.3%)
3
methyl - H incorporated (4175 dpm/10µg DNA = 100%)
F i g u r e 6 . Evaluation, by two distinct experimental strategies involving nuclease digestion before in vitro methylation, of the distribution of methyl-accepting CpGs in the nuclease-sensitive fraction. "Reprinted from B i o c h i m . B i o p h y s . A c t a 1 1 7 3 , D'Erme et al.. Inhibition of CpG methylation in linker DNA by H1 histone, 209-216, (1993) with kind permission of Elsevier Science Publishers - NL Sara Burgerhartstraat 25, 1055 KV Amsterdam, The Netherlands".
Histone proteins added none
H1 (0.3 mg/mg DNA
“core” histones (1.0 mg/mg DNA
H2a (1.0 mg/mg DNA
Number of experiments:
n=6
n=6
n=3
n=3
Native oligonucleosomes
48.4±0.7
-
-
-
H1-depleted oligonucleosomes
100.0
58.0±1.3
101.4±3.5
102.6±2.7
Purified DNA from oligonucleosomes
155.0±2.1
41.6±0.8
153.8±5.8
-
T a b l e 1 . Inhibition by H1 of the methyl-accepting ability of oligonucleosomal DNA. The incorporation of labeled methyl groups in the DNA of H1-depleted oligonucleosomes is made equal to 100 and all the other results obtained in a same set of experiments are referred to this value.
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Gene Therapy and Molecular Biology Vol 1, page 668 of the H1 to enzyme ratio. It seems therefore unlikely, at least in our experimental conditions, that competition between the histone and enzyme for some common DNA binding site(s) is the main mechanism regulating the incorporation of methyl groups in the CpG sequences of chromatin. The methyl-accepting ability of intact oligonucleosomes was, on the other hand, far from negligible. Although it underwent a two-fold increase upon H1 histone depletion (with a further 50% increment if also all other proteins were removed), it went back, indeed, to the same level as in native chromatin when excess H1 histone was added to H1-depleted oligonucleosome preparations (Table 1).
In a complementary approach, when the "100 bp minicore particles" obtained by digestion with staphylococcal nuclease of H1-depleted oligonucleosomes were used as substrates for subsequent in vitro methylation, their methyl-accepting ability was found to be, on a DNA basis, only one-tenth of that of the original H1-depleted oligonucleosomes, Figure 6a. By assaying the susceptibility to methylation of the purified DNAs from the same particles, the methyl-accepting ability of oligonucleosomal DNA was four times larger than that of either the "145 bp core particles" or of the "100 bp minicore particles" Figure 6b. These data (Dâ&#x20AC;&#x2122;Erme et al., 1993) have shown that the lower level of DNA methylation in linker regions than in "core" particles (Razin and Cedar, 1977; Solage and Cedar, 1978; Adams et al., 1984; Caiafa et al., 1986) was not due to an intrinsic CpG deficiency of linker DNA, which was, in H1-depleted oligonucleosomes, susceptible to extensive in vitro methylation, but can rather be ascribed to the inhibition exerted by H1 histone on the process of enzymatic DNA methylation (Caiafa et al., 1991), which would occur in these linker DNA regions because of their preferential association with H1 histone.
Other two hypotheses can account for these results: the presence of some particular variant(s) more or less capable of inhibiting enzymatic DNA methylation and/or the presence of DNA regions escaping the negative control of H1 histone.
The ability and the specificity of H1 histone to inhibit CpG methylation in linker DNA were assayed by readding purified H1 to H1-depleted oligonucleosomes or to the DNA purified from them, Table 1. H1-depletion doubled the methyl-accepting ability of oligonucleosomes, with a further 50% increase as the remaining proteins were also removed. Addition of H1, in a protein-to-DNA (w/w) ratio of 0.3, reduced the incorporation of labelled methyl groups in H1-depleted oligonucleosomes and in the purified oligonucleosomal DNA to the same level occurring in native oligonucleosomal particles. This inhibition was paralleled by a re-condensing effect occurring upon addition of H1 to H1-depleted oligonucleosomes, as shown in Figure 7. Both phenomena are apparently specific to H1 histone, since they could not be obtained by addition of other histones or of serum albumin up to a 1:1 protein/DNA (w/w) ratio.
Figure 7 . CD spectra, in the region of DNA chromophores, of native (__) and H1-depleted oligonucleosomes (---) and re-condensing effect occurring upon addition to the H1-depleted oligonucleosomes of "core" histones (protein/DNA ratio, w/w=1) or of H1 histone (protein/DNA ratio, w/w = 0.1: -.-.- ; w/w = 0.2:-o-o-o-). Oligonucleosomes were suspended, at a DNA concentration of 60 mg/ml, in a 60 mM NaCl, 5 mM Tris-HCl buffer (pH 7.4). "Reprinted from B i o c h i m . B i o p h y s . Acta 1 1 7 3 , D'Erme et al.. Inhibition of CpG methylation in linker DNA by H1 histone, 209-216, (1993) with kind permission of Elsevier Science Publishers - NL Sara Burgerhartstraat 25, 1055 KV Amsterdam, The Netherlands".
These experiments and others previously performed by Davis et al. (1986) have shown that enzymatic DNA methylation is not entirely suppressed by the intrinsic presence of H1 histone. The hypothesis of a competition (Santoro et al., 1993) between the enzyme and histone H1 for some common DNA negative control of H1 histone was investigated and disproved by performing experiments in which increasing amounts of purified DNA methyltransferase were added to Micrococcus luteus ds-DNA in the presence of a constant amount of H1 histone, the H1/DNA ratio being fixed to its "physiological" value of 0.3. As shown in Figure 8, the enzymatic DNA methylation in vitro was independent 668
Zardo et al: DNA methylation and poly(ADP-ribosyl)ation
III. H1 histone somatic variants. The hypothesis that some particular variant could be specifically involved in the in vitro inhibition of enzymatic DNA methylation stems from the fact that H1 histone is composed of a family of different somatic variants termed H1a, H1b, H1c, H1d and H1e (Cole, 1987). They all have a three domain structure, with a highly conserved central globular domain (98% identity in 80 aa sequence). The differences between the variants are located in the N-terminal and C-terminal tails, which consist of about 40 and 100 amino acids respectively (Cole, 1987), with the overall variation in molecular mass being approx 1.0-1.4 kDa.
A. Tight correlation between H1e variant and the inhibition of DNA methylation. Some experiments were performed to verify whether or not these variants could differ from each other in their ability to exert a negative control on DNA methylation. Calf thymus H1 histone somatic variants were purified by reverse phase HPLC, the protein components in effluent composition being characterized by SDS/slab gel electrophoresis in 15% (w/v) polyacrylamide. As shown in Figure 9, only a restricted number of fractions, eluting as a single peak ("p3") was able to cause over 80% inhibition while the other fractions, namely "p1" and "p2" were totally ineffective (Santoro et al., 1995). The SDS/PAGE characterization of the various fractions indicated, according to Lennox et al. (1984) and to Lindner et al. (1990) the presence in "p1" of H1a, in "p2" of H1d and in "p3" of H1e and H1c. Having not yet achieved a satisfactory separation of H1e and H1c, we managed to purify H1c and H1e (Zardo et al., 1996) in order to individuate which variant is really involved in the inhibition of DNA methylation process. A good separation in four peaks was obtained when H1 histone from L929 mouse fibroblasts was purified. The HPLC retention time of each peak, combined with the electrophoretic mobility of various bands, allowed us to identify the H1a, H1b, H1e and H1c variants. When the H1e vs H1c variants were assayed for their effect on in vitro DNA methyltransferase activity, only H1e was effective in causing a marked inhibition, at H1:DNA "physiological" ratio, Figure 10, so that it can be concluded that H1e is the unique variant involved in the inhibition of the DNA methylation process.
F i g u r e 8 . Variations in the extent of Micrococcus luteus ds DNA methylation in vitro, as a function of added DNA methyltransferase, in absence (open circles) or presence (open triangles) of H1 histone at a constant histone-to-DNA ratio equal to 0.3 (w/w). "Reprinted from B i o c h e m . B i o p h y s R e s . C o m m . 1 9 0 , Santoro et al.. Effect of H1 histone isoforms on the methylation of single- or doublestranded DNA, 86-91, (1993) with kind permission of Academic Press, Inc."
The number and relative amounts of these variants differ in various tissues and species throughout the development stages of the organism and in neoplastic systems (Liao and Cole, 1981a,b; Pehrson and Cole, 1982; Lennox and Cohen, 1983; Huang and Cole, 1984; Lennox, 1984; Cole, 1987; Davie and Delcuve, 1991; Baubichon-Cortay et al., 1992; Giancotti et al., 1993; Schulze et al., 1993; De Lucia et al., 1994), so that they may play different roles in chromatin organization, with a non-random distribution.
669
B. H1e: the only one variant able to bind the "CpG-rich" sequences. Gel retardation assays were carried out in order to test the affinity of the different H1 variants for various synthetic oligonucleotides which varied in terms of their sequence and of the relative abundance in methylated or unmethylated CpGs with respect to NpGs (i.e. to all dinucleotide sequences having G as their second moiety). As a representative of genomic DNA we also used a 145 bp DNA prepared by digestion of human placenta chromatin with Staphylococcus aureus nuclease. Experiments have shown (Santoro et al., 1995) that among H1 histone somatic variants, the H1a variant was able to bind a 145 bp genomic DNA fragment but was unable to bind 44 bp ds-oligonucleotides containing two or more CpG dinucleotides. The other variants were capable of binding sequences containing up to three CpGs, while the fraction H1e-c was unique in binding CpG rich
Gene Therapy and Molecular Biology Vol 1, page 670 DNA sequences. Later, using H1e and H1c purified variants, we assessed that H1e variant binds itself better than H1c to the 6CpG oligonucleotide, Figure 11. Our experimental data underline two important characteristics of H1e variant: this is the only variant which suppresses enzymatic DNA methylation and it is the only variant able to bind itself to CpG-rich sequences.
F i g u r e 1 0 . Separation and characterization of H1e and H1c variants from L929 fibroblasts and their effect on in vitro DNA methylation: a) HPLC separation of H1 histone variants and electrophoretic pattern, in 12% SDSpolyacrylamide gel of the eluted fractions (upon visualization by silver staining). b ) Inhibition of DNA methyltransferase activity by H1e (open circles) or H1c (closed circles), at different protein-to-DNA ratios. "Reprinted from B i o c h e m . B i o p h y s R e s . C o m m . 2 0 , Zardo et al.. Inhibitory effect of H1e histone somatic variant on in vitro DNA methylation process, 102-107, (1996) with kind permission of Academic Press, Inc."
Figure 9 . Separation and characterization of calf thymus H1 histone variants and their effect on in vitro DNA methylation: a) elution profile from the RP-HPLC column; b ) SDS gel electrophoresis of all protein fractions, evidenced by Coomassie Brilliant Blue; c ) effect of total H1 histone ("t") and of the various fractions eluted from the RP-HPLC column, at a protein/DNA ratio equal to 0.2 (w/w), on the in vitro activity of human placenta DNA methyltransferase. Each point represents the average results of ten different separations by RP-HPLC. "Reprinted from B i o c h e m . J . 3 0 5 , Santoro et al.. Binding of histone H1e-c variants to CpG-rich DNA correlates with the inhibitory effect on enzymatic DNA methylation, 739-744, (1995) with kind permission of Portland Press"
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Zardo et al: DNA methylation and poly(ADP-ribosyl)ation
F i g u r e 1 1 . Binding of H1e (open circles) and H1c (closed circles) to 44 bp synthetic 6CpG duplex oligonucleotide with the cytosines in the CpG moieties in unmethylated state. The binding was evaluated by gel retardation after incubation of the H1e and the H1c variants with the appropriate oligonucleotide, the relative amount of free DNA being measured by densitometric scanning of the autoradiograms. "Reprinted from B i o c h e m . B i o p h y s R e s . C o m m . 2 0 , Zardo et al.. Inhibitory effect of H1e histone somatic variant on in vitro DNA methylation process, 102-107, (1996), with kind permission of Academic Press, Inc."
IV. Why poly(ADP-ribosyl)ation was selected out of all H1 histone postsynthetic modifications. To explain how H1 histone could be involved in playing many multiplex very important structural and functional roles in chromatin it is important to remember that everyone of the genetic somatic variants can be dynamically modified by different post-synthetic enzymatic reactions (Wu et al., 1986; Davie, 1995) and sometimes the same protein can be substrate for more than one modification. Only in this way can we consider H1 histone as a protein characterized by a big macroheterogeneity that allows different possible interactions with DNA or with other proteins. Some experimental data have led us to focus our attention on the poly(ADP-ribosyl)ation process. A starting point derived from our results showing that when H1e is poly(ADP-ribosyl)ated it loses its condensing effect on chromatin structure even though it remains associated with linker DNA (D’Erme et al., 1996), Figure 12. Taking into account polyADP-ribose dependent chromatin decondensation (Poirier et al., 1982;
671
Aubin et al., 1983; D’Erme et al., 1996), we considered the possibility that this modification may alter the interaction of H1 histone with linker DNA, causing a change in the methyl-accepting ability of CpG dinucleotides present essentially in their unmethylated form on linker DNA. Our aim was, therefore, to compare the methyl-accepting ability of native nuclei with that of nuclei in which chromatin decondensation was induced by poly(ADP-ribosyl)ation. Figure 13A shows the incorporation of ADP-ribose polymers into H1 histone during the experimental time and that at the same time the methyl-accepting ability was not increased in the decondensed chromatin structure induced by the poly(ADPribosyl)ation process, Figure 13B. These data suggest that the poly(ADP-ribosyl)ated H1 histone has not been removed from linker DNA, despite possible alterations in the H1-DNA interactions and that, even if poly(ADPribosyl)ation decrease the H1e-H1e interactions that are essential for the formation of the higher levels of chromatin structure, the poly(ADP-ribosyl)ated isoform of H1e could be present in decondensed chromatin structure where the housekeeping genes are located. The second starting point was the observation that the demethylation process utilizes an excision-repair mechanism to remove 5-methylcytosine. Since it is known that the poly(ADP-ribosyl)ation of H1 histone plays a relevant role in the repair mechanism (Boulikas, 1989; Realini and Althaus, 1992; Malanga and Althaus, 1994) H1 histone in its poly(ADP-ribosyl)ated isoform could indeed, following the demethylation process, remain bound to demethylated regions and regulate thede novo remethylation process that defines the methylation pattern where the "CpG islands" are in an unmethylated state.
V. Correlation between DNA methylation and poly(ADP-ribosyl)ation processes. A. Poly(ADP-ribosyl)ation process. Poly(ADP-ribose) polymerase (EC 2.4.2.30) is a nuclear enzyme that has been implicated in a number of important biological processes (Jacobson and Jacobson, 1989; de Murcia et al., 1995). Although poly(ADPribose) polymerase is able to bind undamaged DNA, it needs DNA strand breaks for its activation. Each monomer of this enzyme, which is a dimer in its catalytic form (Mendoza-Alvarez and Alvarez-Gonzales, 1993), has three domains which play specific roles in the poly(ADPribosyl)ation process. The zinc finger motifs in the Nterminal domain are responsible for the DNA recognition site, taking advantage of DNA strand breaks rather than of specific polynucleotide sequences (Ménissier de Murcia et al., 1989; Gradwohl et al., 1990; Ikejma et al., 1990; de
Gene Therapy and Molecular Biology Vol 1, page 672
F i g u r e 1 2 . A ) Cross-linking analysis to investigate the role played by each H1 histone variant on the formation of H1-H1 polymers: SDS-PAGE patterns of H1 histone variants, at 30% (w/w) H1:DNA ratio, incubated with 1.2 kb oligonucleosomal DNA in 40 mM NaCl for 1 hour at room temperature and then treated with dithiobis(succinimidyl)propionate (DSP 0.2 mg/ml) for 20 min: H1a, H1b, H1e, H1c (lane1-4). In lanes 5 and 6, untreated histone H1 and histone H1 treated with DSP were run as controls in the absence of DNA and B ) the effect of the "enriched" poly-ADP-ribosylation of H1e variant, vs the native one, on the formation of H1-H1 polymers: SDS-PAGE patterns of the product of cross-linking of the H1e histone isoforms at different (w/w) H1:DNA ratio, incubated with 1.2 kb oligonucleosomal DNA, in 40 mM NaCl for 1 hour at room temperature and then treated with dithiobis(succinimidyl)propionate (DSP 0.2 mg/ml) for 20 min: 30%, 20% and 10% (w/w) of H1e:DNA (lane1-3); 30%, 20% and 10% (w/w) of "enriched" poly(ADP-ribosyl)ated H1e:DNA (lane 4-6). "Reprinted from B i o c h e m . J . 3 1 6 , D'Erme et al.. Cooperative interactions of oligonucleosomal DNA with the H1e histone variant and its poly(ADP-ribosyl)ated isoform, 475-480, (1996) with kind permission of Portland Press".
F i g u r e 1 3 . Methyl-accepting ability as assay to study the interactions of H1 histone to linker DNA in native nuclei vs poly(ADP-ribosyl)ated ones. A): time course of incorporation of [32 P] ADPribose polymers associated to H1 histone extracted by 10% PCA (w/v) from nuclei incubated with 50 ÂľM [ 32 P]-NAD; B ): methyl-accepting ability of native nuclei (open circles) vs poly(ADP-ribosyl)ated ones (closed circles). "Reprinted from B i o c h e m . J . 3 1 6 , D'Erme et al.. Co-operative interactions of
672
Zardo et al: DNA methylation and poly(ADP-ribosyl)ation oligonucleosomal DNA with the H1e histone variant and its poly(ADP-ribosyl)ated isoform, 475-480, (1996) with kind permission of Portland Press".
Murcia and Ménissier de Murcia, 1994). The C-terminal domain contains the catalytic site (de Murcia et al., 1995). As for the central domain, it undergoes automodification upon binding of the enzyme on the damaged DNA by introducing ribose polymers -- up to 200 residues according to Alvarez-Gonzalez and Jacobson (1987) -- on 28 automodification sites (Kawaichi et al., 1981; Desmarais et al., 1991) which are essentially localized in this domain.
µCi/ml 3H-SAM, have shown that the poly(ADP-ribose)free isoform of H1 histone failed to inhibit in vitro DNA methylation when added up to a protein/DNA ratio of 0.25 (w/w) while the poly(ADP-ribosyl)ated one was, instead, highly inhibitory under the same condition, Figure 14B.
The active enzyme can then start a series of heteromodification reactions that modulate the functions of chromatin proteins (Ferro et al., 1983; Yoshihara et al., 1985; Boulikas, 1989; Scovassi et al., 1993).
Other experiments were carried out in order to verify whether ADP-ribose polymers by themselves could play a direct role in the modulation of DNA methyltransferase activity. ADP-ribose polymers, isolated from L929 fibroblasts incubated with 50 µM 32P-NAD were fractionated on Sephadex G-50. These protein-free polymers caused a clear-cut inhibition of in vitro methylation of dsDNA but not of ssDNA. The extent of this inhibition is directly dependent on the size of the polymers, as compared to a control assay in absence of polymers considered as 100%, Figure 15. Since a high ADP-ribose polymers/DNA ratio did not affect methylation of ssDNA the polymers can hardly be visualized as directly interacting with DNA methyltransferase.
In vitro experiments have shown that this poly(ADPribosyl)ation mechanism can involve H1 histone binding polymers both in a covalent and in a non-covalent manner. The covalent modification introduces in the C and N-terminal tails of this histone short polymers (8-10 units), whose sizes are specifically defined by the histone itself (Naegeli and Althaus, 1991), while long branched polymers of ADP-ribose are able to form non-covalent interactions with this chromatin protein (Panzeter et al., 1992).
B. Effect of poly(ADP-ribosyl)ated H1 histone on in vitro DNA methylation. The aim of these experiments was to examine, in vitro the possible correlation between DNA methylation and poly(ADP-ribosyl)ation processes and, in particular, whether or not the inhibitory effect exerted by H1 histone on in vitro enzymatic DNA methylation (Caiafa et al., 1991) could be essentially due to the poly(ADPribosyl)ated isoform of this protein. In order to verify this hypothesis the poly(ADPribosyl)ated and the poly(ADP-ribose)-free H1 histone isoforms were purified. The modified protein was purified by affinity chromatography on an aminophenylboronate column of H1 histone obtained from permeabilized L929 mouse fibroblasts (Zardo et al., 1997) incubated for 10 min with 500 µM NAD, Figure 14A, while the unmodified one was obtained from mouse fibroblasts preincubated for 24 hours with 8 mM 3-aminobenzamide, a well-known inhibitor of the poly(ADP-ribosyl)ation process (Griffin et al., 1995). In both preparations the entire H1 histone fraction was isolated by overnight extraction in 0.2 M H2SO4 followed by a second extraction in 10% (w/v) PCA (Johns, 1977). DNA methyltransferase assays, performed in presence of 5 units DNA methyltransferase purified from human placenta nuclei and using as methyl donor 16 µM SAM plus 50
673
C. Effect of ADP-ribose polymers on in vitro DNA methylation.
In the close relationship existing between poly(ADPribosyl)ation and DNA methylation processes, the poly(ADP-ribosyl)ation of H1 histone appears to play a key role. Since the association of H1 histone with ADPribose polymers can be either covalent (Naegeli and Althaus, 1991) or non-covalent (Panzeter et al., 1992), further investigations are needed to ascertain whether also the latter adduct is effective in maintaining CpG dinucleotides in their unmethylated state. To go into this question some in vivo experiments were performed in which the correlation between DNA methylation and poly(ADP-ribosyl)ation processes was investigated by using the methyl-accepting ability assay on isolated nuclei and/or purified DNA from L929 mouse fibroblasts. The results shown in Figure 16, support the working hypothesis of an in vivo relationship between the two nuclear processes suggesting a role of poly(ADPribosyl)ation in preserving a number of CpG dinucleotides from endogenous methylation, maintaining them in an unmethylated state. By gel retardation assay we could also show that poly(ADP-ribosyl)ated H1 histone has a high capacity of linking CpG-rich ds-oligonucleotide, so that it is possible to suppose that it has a preferential location on genomic DNA in regions rich in these nucleotides. Since, on the other hand, only relatively short poly-ADPribose chain(s) are bound to H1 histone (D'Erme et al., 1996), it is unlikely that they can be responsible by themselves for
Gene Therapy and Molecular Biology Vol 1, page 674 the intense inhibitory effect exerted on the methylation of ds DNA by the poly(ADP-ribosyl)ated isoform of H1
histone. In conclusion our hypothesis is that after DNA packaging into nucleosomes, the access to the DNA of a
Figure 1 4 . A) Purification of poly(ADPribosyl)ated H1 histone isoform on an aminophenylboronate column chromatography, monitoring the absorbance at 230 nm (closed circles), or the radioactivity (open circles). B ) Comparison between poly(ADP-ribose)-free H1 histone (closed squares) and the purified poly(ADP-ribosyl)ated isoform (closed circles) for their inhibitory effect on in vitro DNA methylation. Each value is the average value of three different experiments. "Reprinted from B i o c h e m i s t r y 3 6 , Zardo et al.. Does poly(ADP-ribosyl)ation regulate the DNA methylation pattern?, 7937-7943, (1997) with kind permission of the American Chemical Society".
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Zardo et al: DNA methylation and poly(ADP-ribosyl)ation F i g u r e 1 5 . Effect of ADP-ribose polymers of different size (A: striped bars, n>40; B : white bars, n <6n<40; C: horizontally striped bars, n<20) on in vitro DNA methylation. Control assay, taken as 100%, was performed in absence of polymers. Different polymers/DNA ratios, ranging from 0.25 to 1.00, are indicated in the abscissa. The assay was carried out for 1 h at 37°C in the presence of 50 units/ml DNA methyltransferase purified from human placenta nuclei, using 30 µg/ml Micrococcus luteus dsDNA (left panel) and ssDNA (right panel) as substrates and 30 µCi/ml 3 H-SAM as donor of methyl groups. The incorporation of 3 H-SAM in control dsDNA was 4.1 ± 0.1 picomoles and in control ssDNA 4.6 ± 0.3 picomoles. Histograms, in which error bars have been included, represent the average value of three different experiments. "Reprinted from B i o c h e m i s t r y 3 6 , Zardo et al.. Does poly(ADP-ribosyl)ation regulate the DNA methylation pattern?, 7937-7943, (1997) with kind permission of the American Chemical Society".
F i g u r e 1 6 . - Methyl-accepting ability experiments. In panel A the endogenous methyl accepting ability of native nuclei, obtained from 6.5x10 6 L929 fibroblasts preincubated for 24 hrs without (control) and with 8 mM 3ABA, was performed in the presence of 16 µM 3 H-SAM. The level of methyl groups has been evaluated on the total DNA purified from cells. Control DNA, whose incorporation was 2.8 ± 0.1 picomoles of 3 H SAM, was considered as 100%. In panels B and C, DNA samples (3 mg each) purified from the nuclei -- obtained from 6.5x106 L929 fibroblasts preincubated for 24 hrs without (control) and with 8 mM 3ABA and where the endogenous methyl accepting ability had previously been saturated with 16 µM “cold” SAM -- were used as substrates for evaluating their residual methyl accepting ability in the presence either of 50 units/ml human DNA methyltransferase or of 50 units/ml bacterial SssI methylase. The incorporation of 3 H SAM in control DNA was 0.3 ± 0.02 picomoles in panel B and 6 ± 0.2 picomoles in panel C. Histograms, in which error bars have been included, represent the average value of three different experiments. "Reprinted from B i o c h e m i s t r y 3 6 , Zardo et al.. Does poly(ADP-ribosyl)ation regulate the DNA methylation pattern?, 7937-7943, (1997) with kind permission of the American Chemical Society".
moving methyltransferase would then be limited by the presence of poly(ADP-ribosyl)ated H1 and/or by preferentially long and branched polymers linked in a noncovalent way to the histone, so as to afford protection of the unmethylated state of those CpG-rich DNA regions (Zardo et al., 1997).
Acknowledgment. This work was supported by the Italian Ministry of University and Scientific and Technological Research 675
(60% Progetti di Ateneo and 40% Progetti di Interesse Nazionale) and by Fondazione "Istituto PasteurFondazione Cenci Bolognetti".
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Gene Therapy and Molecular Biology Vol 1, page 681 Gene Ther Mol Biol Vol 1, 681-699. March, 1998.
Poly (ADP-ribosyl)ation as one of the molecular events that accompany mammalian spermatogenesis. Piera Quesada Department of Organic and Biological Chemistry, University Federico II of Naples; via Mezzocannone 16, 8O134 Napoli, Italy. __________________________________________________________________________________________________ Correspondence: Piera Quesada: Phone +39-81-7041235, Fax: +39-81-5521217, E-mail quesada@unina.it
Summary It is known that mammalian spermatogenesis is a synchronous process of cellular differentiation during which morphological changes occur, concomitantly with alterations in the complement of constituent proteins, that reflect differences in the mRNA populations coding for stage-specific proteins. Moreover, the most dramatic changes in chromatin structure observed in eukaryotes, take place during spermiogenesis, and the main nuclear processes occur in well-defined cell stages. Rat testis has been used as experimental model in a research project carried out at various l e v e l s , represented by rat germinal c e l l s (primary and secondary spermatocytes, round spermatids), chromatin fractions (transcriptionally active chromatin, nuclear matrix, MARs) and purified nuclear proteins (histone and non-histone proteins). Specific experiments have been carried out in order t o determine the poly(ADPR)polymerase content at different stages o f germ-cell differentiation, the poly(ADP-ribose) amount, length and complexity inside the nucleus, and the poly(ADP-ribose) acceptors among tissue- and stage-specific nuclear proteins. The results indicate that regulation o f the poly(ADPribosyl)ation system accompanies the earlier phases o f the germinal cell differentiation. Indeed, poly(ADPribose)polymerase is particularly active in primary spermatocytes, being possibly implicated i n the recombination events that characterize the p a c h y t e n e p h a s e o f t h e m e i o t i c d i v i s i o n . D i f f e r e n t c l a s s e s o f p o l y ( A D P r i b o s e ) modify different chromatin fractions (DNase I-sensitive, DNase I-resistant chromatin, and nuclear matrix) implicated in DNA replication, repair and transcription. Moreover, the H1 variant H1t, specifically expressed in pachytene spermatocytes, represents the main poly(ADPribose) acceptor, together with poly(ADPR)polymerase itself, in rat germ-cells. Its modification can amplify the role of histone H1 variants as modulators of chromatin structure.
As a result of years of work by many expert biologists a detailed histological description of the process is available; the endocrine and paracrine hormonal control has been assessed. However, very little is still known about the molecular and cellular events that underlie spermatogenesis. Such evidences can now be achieved applying the powerful tools of molecular genetics to this important area of biology (Meiestrich, 1993).
I. Introduction A. Overview of mammalian spermatogenesis Mammalian spermatogenesis is a highly specialized process of differentiation and one of the most dramatic events that any single cell manifests. It is a continuous developmental process that occurs in the adult, and it offers unique opportunity to study the regulation and execution of cell differentiation programs.
Spermatogenesis is composed of three major phases: (i ) mitotic proliferation of spermatogonia, (i i ) two reductional divisions of meiosis that produce the haploid 681
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Figure 1. Schematic representation of the different populations of germinal cells which characterize the different stages of spermatogenesis. The nucleoproteins present during various stages of spermatogenesis are correlated with cell morphology.
process occurs during the adult life and can be more easily studied.
spermatids, and (i i i ) extensive remodelling of these cells during spermiogenesis to form the mature spermatozoa (Figure 1).
The unique events of meiosis include the pairing and recombination of chromosomes during prophase of meiosis I, and the segregation of homologous chromosomes during anaphase of meiosis I. In meiotic prophase chromosomes must find their homologues, establish and maintain pairing, and they must carry out the steps of recombination. Later on, chromosomes undergo condensation, and recombination events are resolved as crossover, that in the metaphase, stabilize the alignment of homologues at the spindle equator, thus ensuring regular segregation of homologous chromosomes from one another during the ensuing anaphase (Hawley, 1988).
Because cytokinesis is incomplete at each of the mitotic and meiotic cell divisions, descendants of a single stem cell develop within a syncytium in which cells are connected by intracellular bridges (Braun et al., 1995). It has been proposed that the cytoplasmic bridges allow the passage of various macromolecules between cells, thus ensuring synchronous development of all cells within a clone, and gametic equivalence between haploid spermatids. The modification of the genome that occurs during spermatogenesis establishes the pattern of paternal expression that is essential for successful embryonic development and the normal phenotype of the adult (Meiestrich, 1993).
All these events are essential for production of chromosomally balanced gametes; errors in pairing or recombination in meiotic prophase lead to errors in anaphase I segregation, giving rise to aneuploid offspring by the production of chromosomally unbalanced gametes. Mutagens, in particular aneuploidogens and clastogens, can induce such errors. To understand the etiology of chromosomally abnormal gametes, the knowledge of the normal mechanisms of chromosome behaviour during meiosis is required.
B. Meiosis and recombination Of the three phases of the spermatogenesis (mitotic proliferation, meiosis and post-meiotic differentiation) meiosis most closely defines gametogenesis since no mammalian cell other than germ cells undergo meiosis. Moreover, in female gametocytes the meiosis occurs during the fetal life, whereas in male gametocytes this
Cell-cycle check-points describe the mechanism by which cells assess the completion of events required for
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Quesada: Poly (ADP-ribosyl)ation in mammalian spermatogenesis successful progress through the cell cycle and production of euploid cell products (Hartwell and Weinert, 1989). There may be a number of spermatogenesis meiosis checkpoints and these may assess both completion of recombination and accumulation of precursors for spermiogenesis.
and Cook, 1986 ); then it has been suggested that the nuclear matrix may play a key role in genome organization and gene potentiation. For instance, the common structural features of replication origins in all life forms have been assessed (Boulikas, 1996); this knowledge is of importance for the understanding of the mechanisms underlying the differential expression of genes that colocalize with matrix associated regions.
For instance, it is not known what happens during pachytene and why it is so lengthy. The supposition might previously have been that the process takes days because this much time is needed to carry out the genetic events of recombination. However it has been demonstrated that the recombination events have reached the point when chiasmata can be formed (Collins and Newton, 1994). Thus, it may be not the genetic events that account for the length of time that the spermatocyte spends in this phase of its differentiation. Instead, the length of pachytene may be determined by the need to synthesize precursors that will be used for post-meiotic spermiogenesis. Different kinds of pairing occur during meiosis, which involve base-pairing (DNA-DNA) interactions. Recently an alternative model has been presented based upon protein-DNA interactions involved in transcription (Cook, 1997). Interestingly, the condensation of chromatin into discernible chromosomes usually inhibits transcription. In contrast the "aligned" chromosomes during meiotic prophase I are transcriptionally active. Indeed, it is remarkable the observation that chromosomes only pair when they are transcriptionally active. A general model has been described for pairing based upon promoter-polymerase interactions on the light of the observation that each chromosome in the haploid set has a unique array of transcription units. Indeed a correct pairing would be nucleated when a promoter binds productively to a homologous site in another transcription factory and is then the consequence of transcription of partially condensed chromosomes. A concept is emerging that multiple components of nuclear organization contribute to competence for gene expression. Chromatin structure, nucleosome organization and gene-nuclear matrix interactions provide a basis for rendering sequences accessible to transcription factors, supporting integration of activities at independent promoter elements of cell cycle and tissue specific genes.
As in the somatic nucleus, chromatin within the male gametes is organized in discrete loops; however, these loops differ from their somatic counterparts with respect to the packaging of their DNA and their average size. Loops within the sperm nucleus are approx. 27Kb in size (compared to 60Kb in somatic cell nuclei). The sperm nuclear matrix attachment regions (SMARs) show a somatic-like organization; furthermore, it has been demonstrated that a specific subset of haploid specific and constitutively expressed genes are associated with the sperm nuclear matrix (Kramer and Krawets, 1996); thus the mature sperm genome is organized in a specific nonrandom manner.
C. Chromatin structure and function in differentiating germinal cells The most dramatic changes in chromatin structure and function observed in eukaryotes take place during spermatogenesis: (i ) DNA replication occurs in spermatogonia and prior meiosis in preleptotene spermatocytes. Spermatocytes undergo meiosis producing spermatids which no longer divide but differentiate into mature spermatozoa; (i i ) spermatogonia, spermatocytes, and early spermatids are active in nuclear transcription, whereas spermatids undergoing differentiation as well as spermatozoa are totally inactive; (i i i ) an interval of regulated DNA nicking followed by repair synthesis occurs in meiotic cells at pachytene phase (DNA recombination); (i v ) late spermatids are genetically inactive in DNA replication and transcription; however, these cells are still able to repair their genetic damage before the final nuclear condensation of chromatin occurs; (v) the chromatin of spermatids undergoing differentiation to spermatozoa becomes relaxed in late spermatids, exposing binding sites on DNA at regions of nucleosome disassembly. Cell type-specific expression of genes encoding these germ-line nuclear proteins may be regulated at the transcriptional level and/or at the post-transcriptional level.
The structure of the eukaryotic nucleus is still an area of interest in cell biology; it is well established that both interphase chromatin and mitotic chromosomes are organized into loops, anchored to a nuclear matrix by specific DNA sequence landmarks named MARs (matrix associated regions) or SARs (scaffold associated regions) (Boulikas, 1993a). Such DNA sequences have been demonstrated to contain origins of replication and transcription enhancers (Jackson and Cook, 1985; Jackson
Differences in post-transcriptional processing have been observed during spermatogenesis. Stabilization of mRNA via polyadenylation and the increased efficiency of translational reinitiation associated with this process may be critical. In this context, the replication-independent histone mRNAs that are restricted to specific cell types (e.g. those encoding H5, the H1 variant in nucleated red 683
Gene Therapy and Molecular Biology Vol 1, page 684 blood cells) are polyadenylated, while the short-lived replication dependent histone transcripts do not contain poly(A) tracts. The polyadenylated histone mRNA lacks the conserved terminal hairpin structure seen in nonpolyadenylated transcripts and instead contains the (polyA) addition sequence (AAUAAA) upstream of the poly(A) tract.
organelles. During this differentiation process a flagellum is constructed the acrosomal vesicle is formed and the nucleus is compacted to approximately one-tenth of its volume (Hecht, 1986). Spermiogenesis is characterized by chromatin condensation and the replacement of histones typical of earlier spermatogenic cells by the highly basic protamines. During this period the nucleosomal pattern of somatic chromatin is lost in round spermatids and replaced by an highly compacted structure in mature sperm. The replacement of histones involves the elimination of somatic and germ cell specific histones. Variants for histone H1, H2a, H2b and H3 that appear during the mitotic and meiotic phases of spermatogenesis have been described in both mouse and rat germinal cells (Meiestrich, 1989).
The rat testis-specific TH2b histone gene assumes a hypomethylated chromatin structure at all stages of spermatogenesis. The H1t mRNA level rises sharply in meiotic pachytene spermatocytes, being very low in premeiotic spermatogenic cells as a result of transcriptional repression of the gene by a pre-meiotic cell-specific protein. A temporal correlation has been observed between the appearance of testis specific DNA binding proteins and the onset of transcription of the testis-specific histone H1t gene (Grimes et al., 1992).
Several reports suggested that histone synthesis ceases during spermiogenesis as these proteins are replaced by a set of transitional proteins (TPs) in both rat and mouse. Later in spermiogenesis the TPs are replaced by protamines which persist in mature sperm. While the organization of sperm chromatin is unknown, the appearance of germ cell-specific histone variants, TPs and protamines suggest that multiple proteins are involved in chromatin remodelling.
The appearance of stage-specific mRNA during spermatogenesis has been demonstrated (Thomas et al., 1989). Experiments involving in vitro translation of mRNA from isolated germ cells have suggested that transcription of the phosphoglycerate kinase-2 (PGK-2) and lactate dehydrogenase (LDH-C) genes first occurs in pachytene spermatocytes and continues in round spermatids. The expression of two DNA repair related enzymes poly(ADPR)polymerase and DNA polymerase !, also varies in germinal cells. While the spermatocytes were shown to contain both enzymes as well as their transcripts, in other cell types this has not been observed (Menegazzi et al., 1991).
The detailed mechanisms by which nucleosomes are disassembled and the DNA finally compacted during spermiogenesis remain unknown. Several factors affect this mechanism including post-translational modification of histones. A correlation exists between the occurrence of extensive H4 acetylation in spermatids and the presence of protamines in spermatozoa (Meiestrich, et al. 1992). Acetylation of specific lysines, as well as other kind of post-translational modification, can reduce protein positive charge which is believed to modify their interaction with DNA (Oliva et al., 1987)
Screening of testis cDNA libraries have identified cDNA clones that are transcribed specifically by round spermatids, such as the protamines (Thomas et al., 1989). Developmentally-regulated patterns of gene expression have been explored and the data suggest the following: (i ) stage-specific patterns of transcription occur coincidentally with the appearance and accumulation of distinct germ cell types within the seminiferous epithelium; (i i ) transcription of a variety of genes occurs exclusively within haploid spermatids, while specific transcripts accumulate in spermatogonia and meiotic germ cells but not in spermatids; (i i i ) several transcripts are detected initially during meiotic prophase and continue to accumulate in round spermatids; (i v ) several multigene families express germ cell-specific isotypes, including "tubulin, !-actin, LDH and PGK; (v) various oncogenes are expressed in germ cells.
Besides acetylation, phosphorylation and methylation, ADPribosylation also plays a fundamental role in gene regulation as it can dramatically influence DNA/histone interactions, particularly during cell differentiation (Lautier et al., 1993). In recent years the elucidation of the molecular mechanisms of the poly(ADPribosyl)ation reactions has advanced rapidly. The nuclear enzyme poly(ADPR)polymerase (PARP) participates in several nuclear events (DNA replication, transcription and repair) by catalyzing the post-translational modification of chromosomal proteins. After its activation by DNA strand breaks, this enzyme catalyzes its self-modification and the modification of other DNA binding proteins with variably sized ADPribose polymers (pADPR) consuming the nuclear pool of !-NAD (Althaus and Richter, 1987).
Collectively, these observations suggest that precise controls exist for determining stage specific gene expression during spermiogenesis (Erikson, 1990). The differentiation of round spermatids into mature spermatozoa requires the synthesis of hundreds of new proteins and the assembly of a unique collection of 684
Quesada: Poly (ADP-ribosyl)ation in mammalian spermatogenesis pADPR is a homopolymer made up of adenosine diphosphate ribose units. The nature of this polymer is now well characterized as structurally similar to a nucleic acid. Branching points up to a proportion of 3% can be found within the polymer, contrary to DNA or RNA. ADPribose polymers vary in complexity and may reach a length of more than 200 residues. The half-life of pADPR varies in relation to the length of the polymer chain as well as to the nature of the acceptor protein: after stimulation of pADPR synthesis with alkylating agents, the half-life of pADPR is less than 1 min. The very short life of pADPR suggests the presence of very high polymer turnover in intact cells.
poly(ADPribose); (i i i ) the poly(ADPribose) acceptors among tissue- and stage-specific nuclear proteins. 1. Cellular level There is a great deal of evidence advocating for a role of PARP in the propagation of epigenetic information. PARP activity correlates with the species-specific life span among mammalian species (Grube and Burkle, 1992). Malignant transformation has been correlated to changes in the regulation and expression of genes caused by poly(ADPribosyl)ation dysfunction (Borek and Cleaver, 1986). The expression level of the PARP gene varies during differentiation in several cell lines (Smulson et al., 1995) and, in general, PARP seems to be more active in the S and G2 phases of the cell cycle (Leduc et al., 1988).
Indeed, many enzymes are involved in pADPR metabolism. The poly(ADPR)polymerase, responsible for the synthesis of the pADPR, catalyzes the initiation, elongation and branching steps. PARP has also an abortive NADase activity, with a Km four times that of the polymerase activity. The elongation mode is probably distal and is accomplished in a distributive fashion with respect to the acceptor (Mendoza-Alvarez and AlvarezGonzales, 1993).
Moreover, it has been inferred that pADPR metabolism is involved mainly in base excision repair (Mathis and Althaus, 1990; Satoh and Lindahl, 1992; Malanga and Althaus, 1994; Lindahl et al., 1995). Differences in PARP activity can be interpreted in many ways: evidence has been obtained for changes in the pADPR synthesis pattern (Satoh et al., 1994), and in the nature of the poly(ADPribosyl)ated proteins (Boulikas, 1990), as a function of particular chromatin structure activities.
pADPR catabolism in cells is achieved by three different enzymes the poly(ADPR)glycohydrolase (Hatakeiama, et al. 1986 ) the ADPribosyl protein lyase (Okayama et al. 1978) and a phoshodiesterase that breaks the pyrophosphate moieties of pADPR (Futai and Mizuno, 1967). The action of poly(ADPR)glycohydrolase (PARG) is the most important and catalyzes polymer degradation by exoglycosidic hydrolysis following an endoglycosidic incision. Two different forms have been found in many tissues: the existence of a nuclear and a cytoplasmic pADPR glycohydrolase has been suggested. Various factors modulate pADPR glycohydrolase activity including the nature of the acceptors and the length of the polymer (Hatakeiama, et al. 1986 )
A functional correlation was observed between early phases of spermatogenesis and poly(ADPribosyl)ation in the rat (Quesada et al., 1996). 2. Nuclear level It is now possible to have a better insight into the internal structure of the interphase nucleus. The nuclear framework is now characterized as a non-histone protein scaffold supporting the attachment points of DNA loops (Jack and Eggert, 1992), this structure seems to influence gene replication and transcriptional activity. It is widely accepted that DNA replication and transcription (Jackson and Cook, 1985; Jackson and Cook, 1986) as well as DNA repair (Mc Cready and Coock, 1984) are actively coordinated by the nuclear matrix.
D. Poly(ADPribosyl)ation reactions during rat germinal cell differentiation
It is thought that PARP plays a role in the maintenance of genetic integrity. As an example, PARP is defective in the cells from patients suffering from xeroderma pigmentosum, who are unable to excise pyrimidine dimers induced by ultraviolet radiation (Wood et al. 1988).
In order to define the role of the poly(ADPribosyl)ation system during germinal cell differentiation, we have used the rat testis as an experimental model. Our investigations have been carried out at various levels: (i ) at the cellular level using different rat germinal cells (primary and secondary spermatocytes, haploid spermatids); (ii) at the nuclear level using isolated chromatin fractions (transcriptionally active chromatin, nuclear matrix, MARs); (iii) at the protein level using purified nuclear proteins (histone and non-histone proteins).
A possible mechanism for the involvement of the poly(ADPribosyl)ation reaction, in different chromatin functions has been proposed (Realini and Althaus, 1992) that takes into account all the described features of the poly(ADPribosyl)ation reaction. According to this mechanism, histone proteins are reversibly detached from the chromatin by the concerted action of poly(ADPR)polymerase and poly(ADPR)glycohydrolase;
In addition, we have carried out specific experiments in order to determine: (i ) the content and enzymatic activity of poly(ADPR)polymerase at different stages of germ-cell differentiation; (i i ) the amount, length and complexity of 685
Gene Therapy and Molecular Biology Vol 1, page 686 the long ADPribose chains linked to the PARP enzyme would be responsible for the dissociation of the chromatin structure, and would then be degraded by the deADPribosylating enzyme to allow reassociation of histones to DNA.
3. Protein level In all cellular events involving poly(ADPribosyl)ation, the state of chromatin represents a signal. It has been shown that poly(ADPribosyl)ation could affect chromatin structure by direct covalent modification of chromosomal proteins and by non-covalent interaction with histones (Lautier et al., 1993; Panzeter et al., 1992 ) due to the high number of long and branched pADPR chains linked to the PARP itself. A modulation in chromatin superstructure, induced by synthesis and degradation of pADPR, has been visualized by de Murcia et al. (1988) using electron microscopy.
Boulikas (1993b) also proposed a model showing how pADPR chains, linked to the automodified form of PARP might be involved in removing histones from matrix associated regions of chromatin (MARs). Indeed, poly(ADPribosyl)ated histone H1 might contribute to the destabilization of nucleosome structure, unfolding the DNA around the core histone octamer. The complete removal of histones from short sequences of DNA (1-4 nucleosomes) seems to be required for repair as well as for initiation of DNA replication and transcription (Boulikas, 1993b)
Moreover, several nuclear proteins have been identified as ADPribose acceptors in vivo in different systems (Althaus and Richter, 1987): the list includes both structural chromosomal proteins (histones, HMGs, nuclear matrix proteins, etc.) and nuclear enzymes (DNA polymerase ", DNA ligase, Topoisomerase I and II, etc.).
In rat testis, a functional form of PARP has been identified which did not seem to be an intrinsic component of the nuclear matrix; this form of PARP was rather indirectly associated to the matrix structure (Quesada et al., 1994).
It has been show that the modification of the linker histone H1 by pADPR alters drastically chromatin conformation (de Murcia et al., 1988; Boulikas, 1990). It has been demonstrated that the stage- and testis-specific histone H1 variant H1t are the preferential ADP-ribose acceptors among acid-soluble chromosomal proteins in rat testis (Quesada et al., 1990). Poly(ADPR)polymerase has been found preferentially associated to transcriptionally active chromatin domains (Hough and Smulson, 1984). The microheterogeneity of H1 is known to play a role in the compaction of DNA into the nucleosome fiber. Among the H1 variants, H1t exerts the lowest condensing effect (De Lucia et al., 1994) and is mostly associated with transcriptionally active chromatin regions (De Lucia et al., 1996). Moreover, the same variants appeared to be ADPribosylated to different extents. Thus, taken together these findings indicate that poly(ADPribosyl)ation of histone proteins could contribute to the structural dynamics characteristic of the transcriptionally competent chromatin. Our studies have explored the possible role of ADPribosylation reactions during spermatogenesis; the spermatogenesis offers a good model to investigate the relationship between poly(ADPribosyl)ation and structural and functional changes that chromatin undergoes during cellular differentiation.
F i g u r e 2 . Temporal appearance of germinal cells during development of rat seminiferous epithelium.
II. Results The temporal appearance of germinal cells in the rat seminiferous epithelium has been confirmed by cytofluorimetric analysis of total germinal cells isolated by collagenase digestion from testes of rats of different age. Figure 2 shows that the seminiferous epithelium
Data are expressed as a percentage of total cells, solubilized by collagenase digestion from seminiferous tubules, characterized as haploid spermatids, diploid and tetraploid spermatocytes, on the basis of their DNA content determined by cytofluorimetric analysis.
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Quesada: Poly (ADP-ribosyl)ation in mammalian spermatogenesis contains different numbers of three cell types, a function of animal age. On the basis of their DNA content rat germinal cells can be discerned as haploid cells (spermatids), diploid cells (mainly secondary spermatocytes), and tetraploid cells (primary spermatocytes). These three cellular populations are indicative of different stages of germinal cell differentiation. Indeed, the pachytene stage occurs in the interval between 25-35 days of age which is the longest portion of the prophase, characterized by the formation of the synaptonemal complexes and by a high level of genetic recombination. A difference is evident in the percentages of diploid/haploid cells in an interval from 28-32 days of
Fi gure 3. Poly(ADPR)polymerase activity in testis of rats of different ages. The specific activity values of the enzyme are reported, as determined in isolated nuclei, with the enzymatic assays described in Materials and Methods. Each value represents the average of four experiments done in duplicate. S . T .: seminiferous tubules isolated by collagenase digestion. Crypt.: artificial cryptorchid testes obtained by elevating the temperature
postnatal age; the amount of tetraploid cells is always less than 25% and does not change significantly until 60 days of age. F i g u r e 3 shows that PARP activity varies in rat testis in animals of different ages; the major level of PARP activity was detected in testes of 30 day-old animals coincident with a crucial phase of the spermatogenesis (meiosis), when the germ cell content (diploid/haploid ratio) changes drastically in the seminiferous tubules. Moreover, the PARP enzyme is specifically present in the seminiferous epithelium and its activity can be
to 37째C and placing them in the body cavity.
Figure 4 : Levels of PARP mRNA in testis and prostate of differently aged rats. Northern-blot analysis of total RNA extracted from testis and prostate of 30-days and 60-days old rats. Aliquots of 20 mg (A) and 50 mg (B) of RNA were hybridized with [32 P]-labelled pRat cDNA probe. The migration of 3,6 Kb of PARP mRNA is reported on the right, and that of 28S and 18S ribosomal RNA on the left
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Figure 5. Cytofluorimetric analysis of isolated rat germ cell populations. Fractions of rat germ cells isolated by centrifugal elutriation, were analyzed after propidium staining, by cytofluorimetry. (A) total germinal cells isolated by collagenase digestion from testes of 45 days old rats; (B ) Fraction IV containing haploid spermatids; (C) Fraction VI containing diploid secondary spermatocytes; (D) Fraction VII containing tetraploid primary spermatocytes.
elutriation. Round spermatids were identified by optic microscope mainly in fractions V, whereas fraction VI contains mainly secondary spermatocytes, and fraction VII primary spermatocytes (data not shown). The enrichment rate obtained in these fractions was determined by cytofluorimetric analysis. Figure 5 shows that on fraction IV, VI and VII approx. 85% enrichment was obtained for haploid, diploid and tetraploid cells respectively. Among these fractions, fraction VII represents a particularly interesting sample since it contains the highest percentage of tetraploid spermatocytes, the cells implicated in the pachytene phase of the meiotic division characterized by a high rate of RNA synthesis and the expression of stage-specific chromosomal proteins (histones TH2B and H1t).
functionally related to the ongoing process of spermatogenesis, since it is drastically reduced in testes in which artificial cryptorchidism is induced. Preliminary results obtained by Northern blot analysis of total RNA extracted from testes of rats of different ages showed that the expression level of PARP varies specifically in testis (not in other tissues) in relation with the animal age (Figure 4). The higher PARP expression level was observed in testes from 60-day old rats and could be correlated to the post-meiotic gene expression observed in mammals for instance to that for oncogenes (Erikson, 1990). Using the procedure illustrated on T a b l e 1 the three cellular fractions have been isolated by centrifugal 688
Quesada: Poly (ADP-ribosyl)ation in mammalian spermatogenesis The three germinal cell populations were analyzed for poly(ADPR)polymerase and poly(ADPR)glycohydrolase content. The results of the enzymatic activity assays are reported on Figure 7. A different value of PARP specific activity was detected; the sample enriched on tetraploid spermatocytes shows an enzymatic activity of 1.5 mU/mg of DNA higher than the 0.88 mU/mg of DNA detected in diploid cells, and three times as much as the 0.48 mU/mg of DNA of haploid spermatids. On the contrary, the PARG specific activity did not change significantly (0.8-1.0 mU/mg of DNA) in the same three samples. These findings have been confirmed by Western-blot analysis of the same samples, using polyclonal antibodies directed against PARP. Figure 8 shows that, using immunodetection, the amount of PARP in primary spermatocytes cellular extract is higher than that in secondary spermatocytes and haploid spermatids. Figure 6: Uridine and Thymidine uptake in rat germ cell populations.
These results are in agreement with a previous report by Corominas and Mezquita (1985) showing different pADPR levels in successive stages of rooster spermatogenesis, and support the functional relationship between spermatogenesis and poly(ADPribosyl)ation we have postulated (Quesada et al., 1990).
The values indicate the [3 H] incorporation observed in the acid-insoluble material of isolated germ cell fractions.
A potential role for PARP in cellular differentiation has been inferred from studies on enzymatic activity and protein amounts during differentiation in rat astrocytes (Chambert et al., 1992) and 3T3LI preadipocytes (Smulson et al., 1995). Changes in PARP expression accompany also the differentiation of HL60 cells induced by retinoic acid (Bhatia et al., 1990) and the proliferation of lymphocytes with PHA (McKerney et al., 1989). Wein et al. (1993) reported a cell cycle-related expression of PARP in proliferating rat thymocytes. In this case, a translational regulation of PARP occurs in the G1 phase, whereas a translational as well as transcriptional activation of PARP occurs in the S phase of the cell cycle. It seems that different mechanisms of regulation of PARP also accompany germinal cell differentiation. Variations in the enzymatic activity have been observed which can be interpreted as changes in the number of PARP molecules, their enzymatic stimulation, modulation of PARP synthesis at the transcriptional and posttranscriptional levels, as well as in terms of differences in pADPR turnover rates.
Figure 7. Poly(ADPR)polymerase and poly(ADPR)glycohydrolase activity in rat germ cell populations. The specific activity values of the two enzymes are reported, as determined in total cellular fractions, with the enzymatic assays described in Materials and Methods. Each value represents the average of four determinations done in duplicate.
Germinal cell viability was assessed measuring [3H]thymidine and [3H]uridine uptake. Figure 6 shows that the isolated cellular populations are able to incorporate the two precursors into the TCA precipitable material, and that the highest incorporation value is associated with primary spermatocytes.
Our evidence indicates that an active poly(ADPribosyl)ation system is associated mainly with tetraploid spermatocytes raising the possibility that PARP
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Figure 8: Poly(ADPR)polymerase levels in rat germ cells. Western-blot analysis of PARP levels in crude extracts of (1) haploid spermatids, (2) secondary spermatocytes and (3) primary spermatocytes. (A) Coomassie-Blue staining; (B ) immunodetection with polyclonal antibody directed against human PARP.
enzyme acts as a modulator of the nuclear processes that occur in these cells by its automodification or heteromodification (via covalent bond or non-covalent interaction) of specific nuclear proteins. As in other experimental systems we can presume that poly(ADPribosyl)ation acts by inducing structural changes of chromatin, thereby facilitating DNA metabolism, which involves various protein-protein and DNA-protein interactions. The high rate of replicational activity of spermatocytes is well known and is accompanied by DNA recombination, a finely controlled crucial event during spermatogenesis ensuring integrity of the genetic material. In order to determine the distribution and activities of the poly(ADPribosyl)ation molecules inside the nucleus, we have isolated and characterized different chromatin fractions, according to the procedure illustrated in Figure 9. Moreover, these fractions have been analyzed for their ability to incorporate [32P]CTP in newly synthesized RNA. A high level of transcriptional activity is present in the DNase I-sensitive chromatin and also in the nuclear matrix in agreement with findings already reported in other systems. We have also examined the level of ADPribosylation in the same fractions, taking into account both the extent of the modification and the size of the ADPribose oligomers. Figure 10 shows DNA, proteins, PARP and pADPR content of the three fractions. In particular, the percentage of pADPR which was found associated with nuclear matrices prepared from nuclei incubated with 200 ÂľM [ 32P]NAD is approximately 10% of the total nuclear
Figure 9: Isolation procedure of rat testis nuclear matrix. 32
The distribution of DNA, proteins and newly synthesized P-RNA in different chromatin fractions is also reported.
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Quesada: Poly (ADP-ribosyl)ation in mammalian spermatogenesis
F i g u r e 1 0 : DNA, Proteins, PARP and pADPR content (%) of different chromatin fractions isolated after incubation of intact nuclei with 32 P NAD. 1. DNase I-sensitive chromatin; 2. DNase I-resistant/ 2M NaCl extractable chromatin; 3. Nuclear matrix.
activity. It is noticeable that the pADPR content was not directly related to the amount of both DNA or protein. These results seem to indicate that the nuclear matrix is enriched in ADPribosylated proteins. To characterize the kind of pADPR synthesized in nuclei, aliquots of nuclear fractions were subjected to alkaline hydrolysis (pH 12), thus detaching the intact polymer, which was then processed for electrophoresis on sequencing polyacrylamide gels and autoradiography. The typical ladder of pADPR indicates that a polymer population is synthesized in isolated nuclear matrix different from that synthesized in DNase I-sensitive and resistant chromatin fractions; the matrix polymer is enriched in ADPR chains longer than 20 moieties, and in a fraction to the top of the gel which is known to be enriched in branched polymers (Figure 11). Thus, nuclear proteins appear to be modified by three classes of pADPR differing in length and complexity.
F i g u r e 1 1 : Autoradiography of 32 P-pADPR analyzed on a 20% polyacrylamide sequencing slab-gel.
To identify ADPribose acceptors, protein components extracted by 2M NaCl from nuclei were separated by means of 20% polyacrylamide gel electrophoresis on acetic-acid-urea (1st dimension) and SDS 15% polyacrylamide gel electrophoresis (2nd dimension) and processed for autoradiographic analysis. Autoradiography of [32P]ADP-ribosylated nuclear proteins revealed that
1. Nuclear matrix; 2. DNase I sensitive chromatin; 3. DNase I resistant/ 2M NaCl extractable chromatin.
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Gene Therapy and Molecular Biology Vol 1, page 692 amounts of PARP in nuclear matrices isolated from different sources (Wesierska-Gadek and Sauerman, 1985; Cardenas-Corona et al., 1987; Alvarez-Gonzales and Ringer, 1988). According to Kaufmann et al. (1991) the recovery of PARP in rat liver nuclear matrix varies with the extraction conditions, since the association of the enzyme to this structure is mediated by the formation of intermolecular disulfide bonds. Thus, the amount of pADPR (10%) associated with the rat testis nuclear matrix is far from being negligible. An interesting hypothesis can be drawn concerning the well known automodification mechanism of PARP: the presence of a portion of ADPR tightly associated to the nuclear matrix raises the possibility of the occurrence of an auto-ADPribosylated form of the enzyme anchored to the nuclear matrix. In order to identify the ADPribose acceptors among the rat testis nuclear proteins their ADPribosylation has been induced in intact nuclei, by incubation with 32P-NAD. Acid-soluble proteins have been than extracted with 0.2 M H2SO4 and separated by reverse-phase HPLC as reported in Materials and Methods. In a one step procedure, several histone and non-histone proteins have been purified, which appeared to be ADPribosylated in different extent. The histogram in Figure 12 shows the specific radioactivity calculated for each of the purified acid-soluble proteins. This analysis indicates that it is histone H1 which is mainly ADPribosylated, compared to core histones and HMG like chromosomal proteins. Moreover, among the different histone H1 variants, H1t represents a better ADPribose acceptor. F i g u r e s 1 3 a n d 1 4 show the reverse phase (RP)HPLC pattern of H1 variants. Indeed, the autoradiography of purified proteins determined by 15% polyacrylamide gel electrophoresis in the presence of SDS, confirmed the preferential ADPribosylation of the tissue- and stagespecific variant of the histone H1, H1t. Moreover, the H1t histone H1 variant appeared to be modified by ADPribose oligomers ranging from 4 to 20 residues compared with no longer than 12 ADPR residues on H1a (data not shown).
Figure 1 2 : Electrophoretic pattern of loosely-bound chromosomal proteins.
The modification of the linker histone H1 by ADPribosylation is well documented in different systems. The ADPribosylation sites have been also identified as the # COOH group of the two glutamic residues in the Nterminal and C-terminal domain of the protein, besides the COOH-terminal group itself. All these residues are conserved in the histone H1 variants and this means that the different extents of their ADPribosylation can not be explained on the account of unavailability of ADPribosylation sites.
2D-gel analysis (see Materials and Methods) of 2M NaCl extractable chromosomal proteins isolated from intact nuclei incubated with 32 P NAD.
radioactivity was associated to histone-like proteins and to component(s) with low electrophoretic mobility and high molecular weight which can be identified as the automodified form(s) of PARP. Our results are in line with recent reports on the presence, within the nucleus, of multiple classes of PARP molecules, representing different functional forms of the enzyme. Others have reported the presence of variable
The existence of a non-covalent interaction of pADPR molecules with histone proteins has been described. This represents a different ADPribosylation mechanism, due to 692
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III. Conclusions Our data have some important implications with regard to the role and versatility of the poly(ADPribosyl)ation reactions in mammalian spermatogenesis. (i ) Regulation of the poly(ADPribose) turnover, variations in the number of PARP molecules synthesized, as well as changes in PARP transcription level, all seem to accompany the earlier phases of the germinal cell differentiation. (i i ) Three classes of poly(ADPribose) molecules of different length and complexity modify chromosomal proteins and PARP in the three chromatin fractions which were isolated (DNase I-sensitive DNase I-resistant chromatin, and nuclear matrix) is implicated in DNA replication, repair and transcription. (i i i ). The testis-specific histone H1t is the main poly(ADPribose) acceptor, together with PARP itself, in rat germ-cells. Short oligomers modify covalently histone components whereas long and branched polymers are bound to non-histone proteins (PARP, nuclear matrix proteins, etc.). Two general conclusions can be drawn: (i ) The observation of a poly(ADPribosyl)ation system particularly active in pachytene spermatocytes is in agreement with the proposed role of PARP as a guardian of the genome in preventing aberrant recombination. Such a role is particularly important in primary spermatocytes undergoing the pachytene phase of the meiotic division, characterized by DNA recombination events.
F i g u r e 1 3 : Purification procedure of nuclear proteins.
32
(i i ) The observation of the preferential ADPribosylation of the histone H1 variant H1t, among the chromosomal proteins, can be related to the different roles attributed to the H1 variants in the compaction of DNA into the nucleosome fiber. Among the H1 variants H1t exerts the lower condensing effect and is mostly associated with transcriptionally active chromatin regions. This can explain why H1t is specifically expressed in pachytene spermatocytes. The different ADPribosylation patterns of histone H1 variants can amplify their role as modulators of chromatin structure, in relation to the various nuclear events that require chromatin remodelling.
P ADPribosylated
The histogram reports the specific radioactivity value that has been calculated for single component purified by reversephase HPLC.
the long and branched polymers linked to the PARP, able to compete with DNA for histone binding (Panzeter et al., 1992). Both the covalent and non-covalent ADPribosylation of histone H1 variants has to be taken into account to understand how PARP molecules modulate chromatin structure.
How to study the molecular and cellular events of the spermatogenesis using poly(ADPribosylation) as a marker.
The differential activation of gene expression between hetero- and euchromatin is sustained by a non-uniform distribution of somatic and tissue-specific H1 variants. However, the specific involvement of different H1 variants in transcriptional processes needs to be further investigated; in this scenario, the ADPribosylation could play an important role.
As already stated, to understand the etiology of chromosomally abnormal gametes, the knowledge of the normal mechanisms of chromosome behaviour is required. To obtain this goal it would be advantageous to understand all the events of meiosis carried out by purified spermatocytes in culture. Procedures for separation of
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F i g u r e 1 4 : Reverse-Phase HPLC of 32 P ADPribosylated histone H1 variants. Chromatographic pattern of histone H1 variants separated by reverse-phase HPLC on a Vydac C4 silica column (see Materials and Methods). The Figure shows also the electrophoretic pattern on SDS 15% polyacrylamide gel and the corresponding autoradiography of proteins contained in each peak.
germ-cells, such as the centrifugal elutriation, allow the isolation of cell populations at defined stages of differentiation. This method provides large amounts of meiotic cells and round spermatids suitable for biochemical studies (Quesada et al., 1996). The purity of the fractions is about 90% with a cell survival of 99%. Thus gene expression can be analyzed during the different stages of spermatogenesis with this approach.
To study changes in nuclear and chromatin composition and genomic activity during spermatogenesis several agents can be used to alter sex-organ development. Cryptorchidism can be artificially-induced in animals maintained on a retinol-depleted diet since vitamin A has long be known to be essential for normal male reproductive function (La Borde et al., 1995). Spermatogenesis can be restored by the oral administration or intratesticular injection of retinoids which have been demonstrated to activate cell division in type A spermatogonia and induce their differentiation in vitro.
Recently, short-term cultures of pachytene spermatocytes have been assessed that will be useful for investigating the defining events of pachytene substage of meiotic prophase, namely maintenance of chromosome pairing and recombination (Handel et al., 1995).
Cis-DDP is a drug frequently used in the treatment of testicular neoplasia; Cis-DDP determines changes in Sertoli cells function and is responsible for spermatid killing (Chu, 1994). Cis-DDP can be administrated acutely or chronically in combination with a gonadotrophin releasing hormone analogue to evaluate the effects of this agent on spermatogenesis, with the aim to study the damage induced by chemotherapeutic drugs, and whether pretreatment with LRA is able to prevent this damage (Scott et al., 1996) .
Rats of different ages can also be used to study the progression of germinal cell differentiation; since the spermatogenesis is a synchronous process, it is possible to correlate the presence in the seminiferous epithelium of different amount of each kind of germinal cells to the animal age. Synaptonemal complex analysis is a novel approach for identifying substances hazardous to the germ cells. This sensitive cytological procedure reveals induction of structural damages and pairing abnormalities in SCs of meiotic prophase chromosomes, together with other germline toxic effects, and can allow an estimate of chemical mutagenicity (Allen et al., 1987).
Undifferentiated germ cells can offer a rapid, sensitive and inexpensive alternative to screening for teratogenicity and genotoxicity of diverse chemicals on live animals. As an example methyl-methanesulphate (MMS) can be used which is known to affect mouse sperm chromatin structure and testicular cell kinetics. MMS is a potent electrophilic, 694
Quesada: Poly (ADP-ribosyl)ation in mammalian spermatogenesis monofunctional alkylating agent which induces clastogenic damage including dominant lethal mutations. It has been demonstrated that MMS alkylates cysteine -SH groups in sperm protamines, thereby destabilizing sperm chromatin structure and leading to chromosomal breakage and mutations (Evenson et al., 1993).
IV. Materials and methods Materials. Wistar rats (28-130 days of age) were used in all the experiments. [U14 C]NAD+, nicotinamide [U14 Cadenine dinucleotide ammonium salt (248 mCi/mmol), [32 P]NAD+, nicotinamide adenine dinucleotide di(triethylammonium) salt (adenylate 32 P), 1000 Ci/mmol, [32 P]CTP (3000 Ci/mmol), [3 H] methylthymidine (40 Ci/mmol) and [3 H]Uridine (25-30 Ci/mmol) were supplied by Amersham International plc. DNase I (EC 3.1.21.1) (2,000 U/mg), collagenase type XI, phenyl-methyl-sulphonyl-fluoride (PMSF), leupeptin, spermine, and spermidine were obtained from SIGMA Chemical Company; guanidine thiocyanate was from FLUKA, pure grade. Electrophoretic molecular weight markers were purchased from Pharmacia, X-Omat RP films from Kodak, and nitro-cellulose filters (0.45 µm pore size, type HA) from Millipore. Reverse-Phase HPLC silica 300-C4 columns were from Vydac(ODS 5 mm particles, 0.5 x 25 cm)
Alternatively, physical agents can be used to alter cell progression. The DNA damage induced after exposure to ionizing radiation can be measured at different cellular stages of spermatogenesis as well as the DNA repair rate, as well as in relation to the apoptotic index (Van Loom et al., 1991). The comet assay represents a sensitive a rapid method for DNA damage detection on individual cells, able to size highly fragmented DNA and DNA repair efficiency (Fairbain et al., 1995) Treatment with okadaic acid (OA) seemingly abrogates a checkpoint that holds mid-pachytene spermatocytes in prophase for several days. Since OA is a polyether monocarboxylic acid able to inhibit several protein phosphatases, the phosphorylation state of critical proteins may be an important element of spermatogenic meiosis cell-cycle control (Wiltshire et al., 1995).
Isolation of germinal c e l l s by centrifugal e l u t r i a t i o n . Testes from two rats were decapsulated, resuspended in 10 ml of Dulbecco's minimal essential medium (DMEM) and seminiferous tubules free of the interstitial tissues were obtained by collagenase treatment (0.25 mg/ml). The seminiferous tubules were then incubated at 30°C for 60 min in DMEM containing 0.25 mg/ml collagenase, 0.075 mg/ml DNase I, and 0.5% bovine serum albumin (BSA). After incubation the cell suspension was centrifuged for 10 min at 1,200g. Aliquots of the pellet were complexed with propidium and subjected to cytofluorimetric analysis in a BectonDickinson cytofluorimeter. Total germinal cells resuspended in DMEM, in the presence of 0.1 mg/ml DNase I and 0.5% BSA, were separated into fractions enriched in various cell types by sedimentation at unit gravity (centrifugal elutriation), as described by (Quesada et al., 1996).
Going from genes to their product, the cellular protein pattern is also representative of the functional state of the cell. Moreover post-translational modification of proteins represents also a signal of the normal or abnormal progression of the cell cycle. Several post-translational protein modifications can be directly related to the nuclear metabolism. The role of protein modification, could be studied in vitro and/or in vivo. Reconstitution experiments, involving addition of specific proteins to DNA in vitro could help determine how these proteins affect chromatin structure. A direct but very difficult method would be to specifically prevent their synthesis in vivo and then determine the resulting modification on chromatin structure. Two dimensional gel electrophoresis of proteins can be used to detect and purify hundreds of proteins from a single sample simultaneously. The protein map of rat spermatocytes and round spermatids has been obtained with this method, and several proteins were successfully identified in the SWISS-PROT protein database, in order to highlight changes in protein expression during meiosis (Cossio et al., 1995). On the basis of these considerations and in the light of the experimental evidence already available, it is now possible to better understand the functional correlation between poly(ADPribosyl)ation and spermatogenesis. Alterations in the poly(ADPribosyl)ation system might be useful markers in gene therapy of germ-cell line.
A cell suspension of 10 ml (180-220x10 6 cells obtained from two testes of 40-45 day aged rats) was loaded into a JE-6 Beckman elutriator rotor and separation was performed with speeds of 3,000-2,000 rpm and flow rates of 13-40 ml/min (Table 1 ). The buffer employed was phosphate buffered saline (PBS) containing 0.5% BSA; several fractions of 50 ml were collected. Aliquots of the pellet of single fractions were complexed with propidium and subjected to cytofluorimetric analysis in a Becton-Dickinson cytofluorimeter. Isolation of sub-cellular fractions was carried out essentially as described by (Quesada et al., 1996). Testes were homogenized in a medium containing 0.32 M sucrose, 2 mM MgCl 2 , 5 mM !-mercaptoethanol, centrifuged at 2,000g for 10 min and the nuclei recovered in the sediment. The supernatant was centrifuged at 20,000 g for 60 min in a SW40 rotor, to obtain in the pellet the microsomal fraction separate from the soluble fraction. [ 3 H] Thymidine and [ 3 H ] U r i d i n e i n c o r p o r a t i o n . Seminiferous tubules were labelled for 1 hr at 37°C in DMEM in the presence of 30 mCi/ml of [3 H] methylthymidine or [3 H]Uridine. The tissue was washed three times with the medium and the cells isolated as described above. The cellular
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Gene Therapy and Molecular Biology Vol 1, page 696 fractions were washed with 5% trichloracetic acid, ethanol, ethanol/ether and ether to remove unbound radioactivity. The residue was suspended in 0.2 M HClO 4 and portions of the solution were used for the determination of radioactivity on a Beckman LS8100 liquid scintillation spectrometer.
enzyme source an aliquot of cells or sub-cellular fractions corresponding to 30 µg of proteins in a total volume of 100 µl. After 10 minutes of incubation at 37°C, the reaction was stopped with ice-cold 20% trichloracetic acid and the radioactivity present in the acid-soluble material determined on a Beckman LS8100 liquid scintillation spectrometer. One enzymatic unit was defined as that liberating 1 µmole of ADPribose, per minute at 37°C.
[ 32 P ] R N A s y n t h e s i s . RNA synthesis was followed by incubation of nuclei in the presence of [32 P]CTP according to (Greengerg and Zif, 1984) and [32 P]RNA was measured as 25% thricloroacetic acid-insoluble radioactivity.
B l o t t i n g e x p e r i m e n t s . Activity-blots and immunoblots were performed as described by (Simonin et al., 1991). Aliquots of 10 7 cells were suspended in 100 µl of 50 mM glucose, 25 mM Tris-HCl pH 8, 10 mM EDTA, 1 mM PMSF and sonicated with 60-s pulses at 180V. The crude extract, after incubation at 45°C for 15 min in 50 µl of 50 mM TrisHCl pH 6.8, 6 M urea, 6% ß-mercaptoethanol, 3% SDS, was separated on 10% polyacrylamide slab-gel in the presence of 1% SDS and electrotransferred onto nitro-cellulose sheets at 4°C for 2 hrs at 200 mA. For activity-blot experiments proteins transferred onto nitrocellulose sheets were incubated for 1 hr at room temperature in renaturation buffer (50 mM Tris-HCl pH 8, 100 mM NaCl, 1 mM DTT, 0.3% (v/v) Tween 20, 20 µM Zn(II)acetate, 2 mM MgCl 2 ) containing DNase I activated DNA (2 µg/ml) and, for 2 hr more, in the same buffer containing [ 32 P]NAD 1 µCi/ml. The blots were then washed several times with the renaturation buffer, dried and analyzed by autoradiography. For immuno-blot experiments nitrocellulose sheets were treated for 1 hr with the blocking solution (50 mM Tris-HCl pH 8, 150 mM NaCl, 0.5% (v/v) Tween 20 and 3% (w/v) gelatin). Incubation with anti-human PARP antibodies was performed, for 2 hrs at room temperature in the same solution supplemented with 0.3% gelatin. The blots were then washed twice with TBS-Tween and antibody binding was detected by using alkaline phosphatase conjugated goat anti-rabbit IgG second antibody with BCIP/NBT (SIGMA) tablets as substrate for the color reaction.
I s o l a t i o n o f c h r o m a t i n f r a c t i o n s . Rat testis nuclei were isolated by homogenization and differential centrifugation, as previously described (Quesada et al., 1990). Proteases were irreversibly inhibited by 1 mM PMSF and 0.1 mM leupeptin. The nuclear matrix isolation procedure was essentially as described by (Tubo and Berezney, 1987) and consisted in the endogenous digestion of isolated nuclei 45 mins at 37°C, followed by a three times repeated extraction with an high salt buffer (2 M NaCl, 0.2 mM MgCl 2 , 1 mM PMSF in 10 mM Tris-HCl pH 7.4) followed by tree washes with low-salt buffer (0.2 mM MgCl2 , 1 mM PMSF in 10 mM Tris-HCl pH 7.4). The nuclear matrix, was then re-suspended in one third volume of low-salt buffer. Alternatively, nuclei were suspended in 10 mM TRIS-HCl pH 7.5, 1 mM EDTA, 1 mM PMSF, digested with DNase I (600U/mg of DNA) and lysed for 1 hr at 0°C. Lysed nuclei were centrifuged for 20 min at 12,000 g, the supernatant collected and the pellet re-extracted twice as above (DNase I resistant chromatin). The insoluble fraction was digested with DNase I (1,000 U/mg DNA) in 60 mM TRIS-HCl pH 7,5, 60 mM NaCl, 20 mM MgCl2 at 37°C for 1 hr, to obtain after centrifugation 12,000g 20 min, the nuclear scaffold. N u c l e a r p r o t e i n e x t r a c t i o n . Acid-soluble proteins contained in nuclei, and nuclear fractions were extracted three times by 1 h stirring at 4°C in 0.2 M H2 SO4 , and centrifuged at 10,000g for 15 min. The extracts were pooled and proteins precipitated with 6 vols. of ice-cold acetone at -20°C overnight. Precipitates were collected by centrifugation at 18,000g for 20 min at -10°C
For Northern blot experiments total cellular RNA (20-50 µg per sample) was first separated by electrophoresis on a 1% agarose gel containing 6% formaldehyde, then blotted onto a Hybond N membrane and hybridized with a fragment of rat PARP cDNA (pRatC) [32 P]-labelled using a Multiprime DNA labelling kit (Amersham International plc).
P o l y ( A D P r i b o s e ) p o l y m e r a s e and p o l y ( A D P R ) g l y c o h y d r o l a s e a c t i v i t y a s s a y . In a typical PARP activity assay, the reaction mixture (final volume 250 µl) contained: 100 mM Tris-HCl pH 8, 14 mM !mercaptoethanol, 10 mM MgCl 2 , 4 mM NaF, 200 µM [14 C]NAD (10,000 cpm/nmol), 12 µg DNase I, and, as enzyme source, an amount of cells or sub-cellular fractions corresponding to 30 µg of proteins. After 10 min incubation at 20°C, the reaction was stopped with ice-cold trichloracetic acid and the radioactivity present in the acid-insoluble material, collected on a HAWP (0.45µm) filter, determined on a Beckman LS8100 liquid scintillation spectrometer. One enzymatic unit was defined as the enzyme activity catalyzing the incorporation, per minute at 20°C, of one µmole of ADPribose into acid-insoluble material.
P r o t e i n a n d D N A a s s a y . Protein concentration was determined following the method described by (Burton, 1968) using bovine serum albumin as standard. DNA content was determined on the basis of the absorbance at 260 nm (1.0 OD260nm = 50µg /ml DNA) or by the diphenylamine method described by Burton (1968). E l e c t r o p h o r e t i c a n a l y s i s . Acid-soluble nuclear proteins were analysed by electrophoresis on urea-acetic acid 20% polyacrylamide slab-gels (pH 2.9) and, SDS 7-15% polyacrylamide slab-gels, as described by (Quesada et al., 1996). Each labelled sample was run in duplicate and either stained with Coomassie Brilliant Blue R-250 or autoradiographed.
The PARG activity assay was performed in a standard reaction mixture containing 50 mM potassium phosphate (pH 7.2), 10 mM !-mercaptoethanol, 100 µg/ml BSA, 10 mM [14 C] poly(ADPribose) (20 residues long on average) and, as
Analysis of reaction products. Intact [32 P]poly(ADP-ribose) moieties incorporated into the proteins were detached by incubation at 60°C for 3 h with 10 mM Tris, NaOH pH 12, 1 mM EDTA. Samples were extracted with
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Quesada: Poly (ADP-ribosyl)ation in mammalian spermatogenesis phenol/CHCl 3 /isoamyl alcohol (49:49:2), dried in a SpeedVac and dissolved in 50% urea, 25 mM NaCl and 4 mM EDTA, pH 7.5, to be analyzed on 20% polyacrylamide slab-gel (Panzeter and Althaus, 1990)
Boulikas T., ( 1 9 9 3 b ) "Relation between carcinogenesis chromatin structure and poly(ADPribosylation)" Anticancer Res ., 14, 885-894. Boulikas, T., ( 1 9 9 6 ) "Common structural features of replication origins in all life forms" J . C e l l . B i o c h e m ., 60, 297-316.
Reverse-Phase HPLC analysis. Acid soluble nuclear proteins were purified by reverse-phase HPLC using a 300-5 C4 silica column and a gradient elution system formed by 0.1% trifluoracetic acid (solvent A) and 95% CH 3 CN in 0.1% trifluoracetic acid (solvent B). The gradient elution was from 15 to 65% of solvent B in 70 mins (slope 0.65%/min).
Braun, R.E., Lee, K., Schumaker, J.M., and Fajardo, M.A., ( 1 9 9 5 ) “Molecular genetic analysis of mammalian spermatids differentiation” in “Recent P r o g r e s s i n H o r m o n e R e s e a r c h” 50, 275-286, Acad. Press New York. Burton, K., (1 9 6 8 ) "A study of the conditions and mechanism of diphenylammine reaction for the colorimetric estimation of DNA" C e l l , 23, 585-593.
Acknowledgements I thank all my colleagues Prof. Benedetta Farina, Dr. M.Rosaria Faraone-Mennella, Dr. Maria Malanga, Dr. Filomena De Lucia, Dr. Luigia Atorino and Dr. Filomena Tramontano for their contribution to the work summarized in this review. I thank also Dr. Roy Jones, Principal Scientific Officer at the Babrahan Institute of the BBSRC in Cambridge, U.K., for his constant support and for hosting me in his laboratory. This work was supported by funding (40%) and 60% M.U.R.S.T..
Cardenas-Corona, M.E., Jacobson, E.L., Jacobson, M.K., ( 1 9 8 7 ) "Endogeous polymers of ADPribose are associated with the nuclear matrix" J . B i o l . C h e m . 262, 14863-14866. Chambert, M.G., Niedergang, C.P., Hog, F., Partisani, M., Mandel, P., ( 1 9 9 2 ) "Poly(ADPR)polymerase expression and activity during proliferation and differentiation of rat astrocytes and neuronal cultures" B i o c h i m . B i o p h y s . Acta, 1136, 196-202.
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Jackson, D.A., and Cook, P.R., ( 1 9 8 6 ) "Transcription occurs at a nuclear skeleton" EMBO J., 5, 1403-1410. Kaufmann, S.H., Brunet, G., Talbot, B., Lamarre, D., Dumas, C., Shaper, J..H., Poirier, G.G., ( 1 9 9 1 ) "Association of poly(ADPribose)polymerase with the nuclear matriix: the
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Quesada: Poly (ADP-ribosyl)ation in mammalian spermatogenesis Okayama, H., Honda, M., Hayaishi, O., (1 9 7 8 ) "Novel enzyme from rat liver that cleaves an ADP-ribosyl histone linkage" P r o c . N a t l . Acad. S c i . USA 75, 22542257.
Tubo, R.A., and Berezney, R., ( 1 9 8 7 ) "Identification of 100 and 150S DNA polymerase " megacomplexes solubilized from the nuclear matrix of regenerating rat liver" J . B i o l . C h e m . 262, 5857-5865.
Oliva, R., Bazett-Jones, D., Mezquita, P., Dixon, G.H., ( 1 9 8 7 ) "Factors affecting nucleosome disassembly by protamines in vitro" J . B i o l . Chem., 35, 1701617025.
Van Loom, A.A.W.M., Den Boer, P.J., Van der Schans, Mackenbach, P., Grootegoed J.A., Baan, R.A., Lohman, P.H.M., ( 1 9 9 1 ) "Immunochemical detection of DNA damage induction and repair at different cellular stages of spermatogenesis of the hamster after in vitro or in vivo exposure to ionizing radiation" E x p . C e l l R e s ., 193, 303-309.
Panzeter, P.L., and Althaus, F.R., ( 1 9 9 0 ) "High resolution size analysis of ADPribose polymers using modified DNA sequencing gels" Nucl. Acid Res .,18, 2194.
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Haucke: Membrane biogenesis: from mechanism to disease Gene Ther Mol Biol Vol 1, 701-706. March, 1998.
Membrane biogenesis: from mechanism to disease Volker Haucke Department of Cell Biology & Howard Hughes Medical Institute, Yale University School of Medicine, 295 Congress Avenue, New Haven, CT 06510, USA __________________________________________________________________________________________________ Correspondence to: Volker Haucke, phone: +1-203-737 4469, fax: +1-203-737 1762, E-mail: volker.haucke@yale.edu Keywords: membrane biogenesis-peroxisomal disorders-nerve terminal-endocytosis-autoimmune diseases-cancer
Summary The biogenesis o f membranes i n v o l v e s the continous f l o w o f proteins and lipids which are selectively targeted to or retrieved from specific compartments within eukaryotic cells. While some diseases are caused by the impairment of particular protein transport pathways or mislocalization of a certain protein others may be related to altered signal transduction cascades resulting from defective endocytosis of plasma membrane receptors or other membrane trafficking defects. The implications of this hypothesis for our understanding of the proper functioning of a eukaryotic cell and for the treatment of human diseases are being discussed.
I. Overview Unlike bacteria, eukaryotic cells are elaborately subdivided into membrane-bounded, structurally and functionally distinct compartments. Each of these organelles contains a specific set of proteins, lipids and other molecules which enables them to fulfill characteristic functions within the cell. On average, the membranebounded compartments together occupy nearly half the volume of a cell, and about one third of all proteins within a eukaryotic cell are membrane proteins. Thus, membrane biogenesis and organelle maintenance are major tasks which are essential for all eukaryotic cells (Palade, 1975). During the past two decades it has become clear that a number of inherited metabolic and neurological disorders result from the mistargeting of particular proteins to an incorrect destination within the cell. As an example for this class of diseases I will describe a number of disorders resulting from defective peroxisome biogenesis. Other pathological states pertaining to membrane trafficking however may arise from autoimmune impairment of cells expressing a particular antigen or from genetic defects resulting in the generation of an abnormal protein as exemplified by the deposition of !-amyloid protein in brains of patients suffering from Alzheimer's disease. In the second part of this chapter I will, therefore, focus my discussion on the trafficking of membranes at the nerve terminal in normal and certain pathological states.
II. Peroxisome biogenesis & dysfunction A. How peroxisomes are formed 701
Peroxisomes are ubiquitous eukaryotic organelles which are involved in a variety of metabolic processes such as the scavenging and destruction of peroxides, the !-oxidation of fatty acids and the biosynthesis of ether lipids. However, unlike mitochondria and chloroplasts they do not contain their own DNA and like most other intracellular membranes cannot be formed de novo. Biologists and physicians alike have become increasingly interested in the biogenesis of these organelles since Goldfischer reported in 1973 that patients with the cerebro-hepato-renal syndrome Zellweger's disease lacked demonstrable peroxisomes. Until now the number of peroxisomal biogenesis disorders (PBD) has grown to sixteen which fall into eleven different complementation groups. In order to learn more about the molecular basis of these diseases investigators have studied the way by which peroxisomes import their constituent proteins from the cytosol using both mammalian cell cultures and yeast as model systems. Protein targeting to the peroxisomal matrix is mediated by evolutionary conserved peroxisomal targeting signals (PTSs) which bind to specific PTS receptors as depicted in F i g u r e 1 . The majority of peroxisomal matrix proteins carries a C-terminal tripeptide (SKL or closely similar) termed PTS1. PTS2 is a conserved N-terminal nonapeptide (R/K) (L/V/I) (X5) (H/Q) (L/A) and is used by a smaller subset of matrix proteins. Other internally located PTSs have been identified but, as with the targeting signals of peroxisomal membrane proteins, no consensus sequence has been found (Rachubinski & Subramani, 1995).
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Figure 1: Hypothetical model for how proteins get imported into peroxisomes. (a) Import of proteins containing the PTS1 signal for targeting to the peroxisomal matrix. PTS1R, receptor for PTS1 containing proteins. (b ) Import of proteins containing the PTS2 signal for targeting to the peroxisomal matrix. PTS2R, receptor for PTS2 containing proteins. Components of a putative import channel across the peroxisomal membrane (gray) are indicated by purple rectangles.
Yeast and human cells selectively deficient in the PTS1 or PTS2 import pathway have been used to identify the PTS receptors (PTSR). PTS1 signals are recognized by the PTS1R now collectively referred to as Pex5p, a tetratricopeptide repeat (TPR) protein of 64-69 kDa. It is still unclear whether this protein is localized to the cytoplasm, the outer face of the peroxisomal membrane, or the peroxisomal matrix (van der Leij et al., 1993; Dodt et al., 1995; Szilard et al., 1995; Wiemer et al., 1995). Independent reports from three different laboratories now suggest that the src-homology domain 3 (SH3 domain) of the peroxisomal membrane protein Pex13p functions as a docking site for the mobile cytosolic PTS1R Pex5p to facilitate the delivery of PTS1 containing proteins (Elgersma et al., 1996; Erdmann & Blobel, 1996). PTS2 signals are recognized by the PTS2R termed Pex7p. Again it is unclear whether this protein resides in the cytosol or the peroxisomal matrix (Zhang & Lazarow, 1995). The clinical documentation of a series of similar human peroxisomal disorders (i.e. Zellweger syndrome, neonatal adrenoleukodystrohy, rhizomelic chondrodysplasia punctata (RCDP) etc.) has led to the identification of the human homolog of PTS1R (Dodt et al., 1995; Wiemer et al., 1995). The PTS1 and PTS2 pathways may be linked through a direct interaction between the tetratricopeptide repeat (TPR) region (a recently identified protein-protein 702
interaction motif) of PTS1R and the WD40 repeats (another distinct protein-protein interaction motif) of PTS2R although rigorous biochemical evidence for such an interaction has not yet been reported (Rachubinski & Subramani, 1995). Unlike most other protein translocation systems peroxisomes are capable of importing stably folded (Walton et al., 1995) or even oligomeric proteins (Glover et al., 1994; McNew & Goodman, 1994). How these proteins actually cross the membrane is unknown. One possibility is that peroxisomes contain very large pores, but no experimental evidence for the existence of such pores has been reported. Alternatively, some form of pino- or endocytosis at the peroxisomal membrane might be involved in the protein transport process. It is also possible that most peroxisomal proteins are imported into as yet unidentified peroxisomal precursors and that peroxisomes are derived from these precursors by maturation. We also know very little about the energetics of protein transport into peroxisomes although ATP hydrolysis is required for the import of proteins into the matrix (Subramani, 1996). Thus, protein translocation into peroxisomes turns out to obey somewhat different rules than the protein translocation systems of mitochondria (Haucke & Schatz, 1997) or the endoplasmic reticulum (Rapoport et al., 1996).
Haucke: Membrane biogenesis: from mechanism to disease
B. Human peroxisomal disorders Human peroxisomal biogenesis disorders occur with a relatively high frequency of about 1/50 000 live births and are a genetically heterogenous group of autosomal, recessive, lethal diseases that fall into at least eleven distinct complementation groups as identified by cell fusion complementation analysis. Twelve out of the sixteen PBDs known to date are associated with severe neurological disability while even patients suffering from the remaining PBDs show some sort of neurological defect. These disorders can be grouped into three different classes, A,B, and C, according to the molecular defect leading to the disease. In group C the subcellular localization or activity of a single peroxisomal protein or enzyme is compromised. These disorders usually show the least severe phenotype. Patients with group A or B disorders often exhibit the presence of non-functional peroxisome ghosts which miss a few or many peroxisomal matrix proteins due to deficiencies in either the PTS1 or PTS2 or both protein import pathways (Rachubinski and Subramani, 1995). We will now turn to a more detailed analysis of the defects associated with these classes of disorders.
1. Zellweger Syndrome Zellweger syndrome (ZS), a rare fatal disorder in newborn infants was originally described by Goldfischer et al. in 1973. It is an inherited metabolic disease associated with a number of cerebral, hepatic and renal defects and belongs to group A of the peroxisomal biogenesis disorders. Cells isolated from ZS patients have peroxisome ghosts lacking many peroxisomal matrix proteins and these patients show elevated levels of very long-chain fatty acids and are deficient in plasmalogens (ether lipids). ZS is the most severe PBD known to date and is invariably fatal. A number of similar diseases such as neonatal adrenoleukodystrophy and infantile refsum disease have been described all of which show a related but less severe phenotype compared to ZS. It appears that a number of mutations can lead to ZS and cells belonging to these various complementation groups show differences in their capability of importing proteins via either the PTS1 or PTS2 pathways. Cells from patients in complementation group2 with ZS have mutations in their PTS1 receptor gene. Elegant studies in vitro have shown that the human PTS1 receptor can complement the protein import defect in these cells suggesting that the mutated PTS1 receptor is indeed the cause for the disease (Dodt et al., 1995; Wiemer et al., 1995). Thus, gene therapeutic approaches may soon provide means of treating this horrible disease.
2. Rhizomelic chondrodysplasia punctata Rhizomelic chondrodysplasia punctata (RCDP) is a rare autosomal recessive phenotype associated with complementation group 11 of the peroxisome biogenesis disorders and is characterized by severe growth failure,
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profound developmental delay, cataracts, rhizomelia, and a severe deficiency in plasmalogens (Braverman et al., 1997). Cells from RCDP patients are unable to import peroxisomal thiolase, an enzyme targeted to peroxisomes via the PTS2 pathway. Recently, the molecular defects leading to RCDP have been elucidated. Analysis of cells from RCDP patients have revealed a number of mutations within a single gene with homology to the yeast PTS2 receptor (Baverman et al., 1997; Motley et al., 1997; Purdue et al., 1997). Subsequent cloning identified this gene as the human PTS2R, Pex7 (Braverman et al., 1997; Motley et al., 1997; Purdue et al., 1997). Expression of human Pex7 in RCDP cells rescues PTS2 targeting and restores the activity of dihydroxyacetone-phosphate acyltransferase, a peroxisomal enzyme of plasmalogen synthesis (Purdue et al., 1997). The two pathways of protein import into peroxisomes may however not be completely separate since several ZS patients with defective PTS1 receptors also show reduced amounts of PTS2 targeted enzymes. Moreover, an isoform of the human PTS1 receptor Pex5 is required for the efficient import of PTS2 targeted proteins and the tetratricopeptide repeats (TPR) of Pex5 directly interact with the WD40 domain of Pex7 in the two-hybrid system (Braverman et al., 1997). It is therefore possible that the molecular defects associated with some complementation groups of PBDs may result from a mutant receptor which not only is unable to bind its import substrates but may additionally fail to associate or cooperate with the other PTS receptor protein resulting in multiple import defects.
3. Peroxisome-to-mitochondrion mistargeting An interesting example of a group C peroxisome biogenesis disorder which is caused by the mistargeting of a single peroxisomal protein is represented by primary hyperoxaluria type 1 (PH1). PH1 is an autosomal recessive disease associated with a normally occuring P11L polymorphism and a PH1-specific G170R mutation in the gene encoding for the homodimeric enzyme alanine:glyoxylate aminotransferase 1 (AGT) (Leiper et al., 1996). The P11L substitution creates an amino-terminal mitochondrial targeting signal which competes with its carboxy-terminal peroxisomal import signal and in vitro is sufficient to direct the protein into mitochondria. This mutation alone does not interfere with the peroxisomal targeting of AGT in living cells. AGT containing both mutations, however, is mistargeted to mitochondria both in vitro and in vivo. Recent work has now shed light on this phenomenon: the G170R mutation abolishes the ability of the protein to form homodimers in the cytosol and thereby prevent its mistargeting to mitochondria which are unable to import fully folded or dimeric proteins (Haucke and Schatz, 1997). Thus, mistargeting is due to the unlikely occuring polymorphism that generates a functionally weak mitochondrial targeting signal and a disease-specific mutation which, in combination with the polymorphism, inhibits AGT dimerization and therefore allows the protein to cross the mitochondrial membranes.
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Figure 2: Components involved in the formation of a clathrin-coated bud that mediates synaptic vesicle endocytosis. The individual proteins are indicated by differentially colored symbols.
III. Membrane trafficking at the nerve terminal & disease: a putative link between synaptic vesicle endocytosis and the biology of cancer I will now turn to the description of the mechanism by which synaptic vesicles are retrieved and recycled at the plasma membrane and discuss a number of recent observations which suggest that endocytosis, and by extrapolation also other membrane trafficking events may play an important role in regulating signal transduction pathways which in turn are intimately linked to the biology of cancer, Alzheimer's disease and other major human diseases.
A. The synaptic vesicle cycle Synaptic vesicles (SV) are specialized secretory organelles involved in synaptic transmission in the nervous system. Upon stimulation SVs dock and fuse with the plasma membrane and release their content into the synaptic cleft. Membrane fusion occurs by a closely similar mechanism from yeast to neurons, and is mediated by specific pairing of SNARE proteins on the two membranes undergoing fusion (Ferro-Novick and Jahn, 1994). Following exocytosis, SV membranes are retrieved and reused for the generation of new SVs. This entire cycle occurs with high specificity and can be very rapid (less than one minute) (Ryan, 1996). The most widely accepted model for how SVs are being regenerated proposes that SVs are retrieved through clathrinmediated endocytosis (Cremona and De Camilli, 1997) involving a coat complex consisting of the heavy and light
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chains of clathrin, the plasma membrane-specific adaptor complex AP2 (a heterotetramer composed of ",!, Âľ and # subunits) and the accessory protein AP180. The importance of clathrin coats in SV endocytosis has recently been corroborated by genetic studies in Drosophila and C. elegans. It is still unclear what recruits this complex to the membrane, but one possibility is that synaptotagmin (Zhang et al., 1994), an abundant protein of SVs may facilitate this process by interacting with the AP2 adaptor. However, both AP2 and AP180 have been found to interact directly with membrane phosphoinositides (PIs) indicating that both lipid and protein may participate in anchoring the coat to membranes. Vesicle fission of the mature coated bud is then effected by the recruitment and oligomerization of the GTPase dynamin to the stalk of endocytic pits. Upon hydrolysis of GTP dynamin disassembles and the clathrin-coated vesicle pinches off to eventually re-enter the pool of SVs awaiting a stimulus for another round of exocytosis. This step may be aided or regulated by the inositol 5-phosphatase synaptojanin (Mc Pherson et al, 1996), which is selectively concentrated in nerve terminals in association with endocytic intermediates of SV membranes. Recent evidence suggests that the SH3 domain of the nerve terminal phosphoprotein amphiphysin I (Bauerfeind et al, 1997), together with its partner protein amphiphysin II plays an important role in recruiting dynamin to the invaginated endocytic pit (Shupliakov et al., 1997; Wigge et al., 1997). Through its affinity for both, dynamin and the adaptor AP2, amphiphysin may link the assembly of the clathrin coat to the formation of dynamin rings, thereby coordinating these two events leading to the generation of calthrin coated vesicles (David et al., 1996; Ramjaun et al., 1997; Wigge et al., 1997).
Haucke: Membrane biogenesis: from mechanism to disease
B. A putative link between endocytosis and the biology of cancer Amphiphysin I is a neuron-specific protein which was originally found as a component associated with SVs (Lichte et al., 1992) and as the autoantigen in a subgroup of patients suffering from Stiff-man syndrome (SMS) (De Camilli et al., 1993). Stiff-man syndrome is a rare disease of the central nervous system characterized by painful spasms of limbs, trunk and abdominal muscles (Layzer, 1988). Group II patients are characterized by the presence of autoantibodies against amphiphysin I and all suffer from breast cancer (Folli et al., 1993). In an effort to elucidate the connection between amphiphysin I autoimmunity and cancer Floyd et al. (submitted for publication) have analyzed the expression of amphiphysin I in breast cancer tissues. Amphiphysin I was present as an alternatively spliced, overexpressed 108 kDa isoform in several breast cancer tissues and as two 128 and 108 kDa forms in the breast cancer of a SMS patient. Although it is not yet clear whether the high amphiphysin expression level is directly linked to the enhanced proliferation of the malignant cells, the observation that amphiphysin I is overexpressed in some forms of cancer supports the idea that amphiphysin family members play a role in the biology of cancer cells. It is well conceivable that overexpression of a mutant protein involved in endocytosis could alter signaling cascades initiated by endocytosed plasma membrane receptors and could thereby lead to tumorigenesis as described below. Another link between endocytosis and signal-dependent cell proliferation has recently emerged from studies in transfected mammalian cells. First, inactivation of the clathrin- and dynamin-dependent uptake of the receptor for epidermal growth factor (EGFR) by overexpressing a mutant form of dynamin leads to enhanced proliferation of these endocytosis-defective cells (Vieira et al., 1996). The altered proliferative response is presumably due to the hyperphosphorylation of a subset of EGF-dependent signal transducing molecules suggesting an important role for EGFR signaling in establishing and controlling specific signaling pathways. Second, Grb2, an SH3-SH2-SH3 domain containing protein involved in transducing signals from growth factor receptors (i.e. EGFR) to the Ras pathway upon stimulation with EGF transiently associates with dynamin, a GTPase involved in vesicle fission from the plasma membrane (as described above) (Wang and Moran, 1996). The transient interaction between dynamin and Grb2 is required for the internalization of the EGFR as microinjection of a peptide corresponding to the Grb2 SH3 domain blocks endocytosis. Thus, activation and termination of EGF signaling appear to be regulated by the diverse interactions of Grb2 with either signal transducing or endocytic components providing another link between endocytosis and the attenuation of signal transduction events from the plasma membrane.
IV. Perspectives 705
The examples described in this article are just some out of a growing number of studies on how mislocalization of certain proteins due to genetic alterations either in the protein itself or in its targeting machinery or perturbation of membrane trafficking pathways may lead to disease. Although many of the described connections between membrane traffic, complex inherited disorders, signalmediated growth control, and pathogenesis remain mechanistically poorly understood accumulating evidence suggests that the biogenesis of membranes and the trafficking of organelles and molecules within the cell may be intimately linked to the regulatory and signal transduction networks governing the physiological state of a cell. A better understanding of this crosstalk might eventually lead to improved treatments for today's diseases including cancer, Alzheimer's disease, diabetes and others.
Acknowledgements The author was supported by a long-term fellowship from the European Molecular Biology Organization (EMBO) and currently holds a long-term fellowship from the Human Frontier Science Program (HFSP).
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McNew, J. A., and Goodman, J. M. (1 9 9 4 ). An oligomeric protein is imported into peroxisomes in vivo. J . C e l l B i o l . 127, 1245-1257. McPherson, P. S., Garcia, E. P., Slepnev, V. I., David, C., Zhang, X., Grabs, D., Sossin, W., Bauerfeind, R., Nemoto, Y., and De Camilli, P. (1 9 9 6 ). A presynaptic inositol-5phosphatase. Nature 379, 353-357. Motley, A. M., Hettema, E. H., Hogenhout, E. M., Brites, P., A.L.M.A., t. A., F.A., W., Baas, F., Heijmans, H. S., Tabak, H. F., Wanders, R. J. A., and Distel, B. (1 9 9 7 ). Rhizomelic chondrodysplasia punctata is a peroxisomal protein targeting disease caused by a non-functional PTS2 receptor. Nature Genet. 15, 377-380. Palade, G. E. (1 9 7 5 ). Intracellular aspects of the process of protein synthesis. S c i e n c e 189, 347-358. Purdue, P. E., Zhang, J. W., Skoneczny, M., and Lazarow, P. B. (1 9 9 7 ). Rhizomelic chondrodysplasia punctata is caused by deficiency of human PEX7, a homologue of the yeast PTS2 receptor. Nature Genet. 15, 381-384. Rachubinski, R. A., and Subramani, S. (1 9 9 5 ). How proteins penetrate peroxisomes. C e l l 83, 525-528. Ramjaun, A. R., Micheva, K. D., Bouchelet, I., and McPherson, P. S. ( 1 9 9 7 ). Identification and characterization of a nerve terminal-enriched amphiphysin isoform. J . B i o l . C h e m . 272, 16700-16706. Rapoport, T. A., Rolls, M. M., and Jungnickel, B. (1 9 9 6 ). Approaching the mechanism of protein transport across the ER membrane. Curr. Op. Cell B i o l . 8, 499-504. Ryan, T. A., Smith, S. J., and Reuter, H. (1 9 9 6 ). The timing of synaptic vesicle endocytosis. P r o c . N a t l . A c a d . S c i . USA 93, 5567-5571.
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Whelan, J., Omahony, P., and Harmey, M. A. (1 9 9 0 ). Processing of Precursor Proteins by Plant Mitochondria. A r c h i v B i o c h e m B i o p h y s 279, 281-285. Wigge, P., Kohler, K., Vallis, Y., Doyle, C. a., Owen, D., Hunt, S. P., and McMahon, H. T. (1 9 9 7 ). Amphiphysin heterodimers:potential role in clathrin-mediated endocytosis. M o l . B i o l . C e l l 8, 2003-2015. Wiemer, E.A., Nuttley, W.M., Bertolaet, B.L., Li, X., Francke, U., Wheelock, M.J., Anne, U.K., Johnson, K.R. and Subramani, S. (1 9 9 5 ) Human peroxisomal targeting signal-1 receptor restores peroxisomal protein import in cells from patients with fatal peroxisomal disorders. J . C e l l B i o l ., 130, 51-65. Zhang, J. Z., Davletov, B. A., S端dhof, T. C., and Anderson, R. G. W. (1 9 9 4 ). Synaptotagmin I is a high-affinity receptor for clathrin AP-2: implications for membrane recycling. C e l l 78, 751-760. Zhang, J.W. and Lazarow, P.B. (1 9 9 5 ) PEB1 (PAS7) in Saccharomyces cerevisiae encodes a hydrophilic, intraperoxisomal protein that is a member of the WD repeat family and is essential for the import of thiolase into peroxisomes. J . C e l l B i o l ., 129, 65-80.
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Cdc25 protein phosphatase: regulation and its role in cancer Jens W. Eckstein Mitotix, Inc., One Kendall Square, Cambridge, MA 02139 USA ________________________________________________________________________________________________ Correspondence: Jens W. Eckstein, Tel: (617) 225-0001 x255, Fax: (617) 225-0005, E-mail: eckstein@mitotix.com
Summary The family o f the Cdc25 dual-specific protein tyrosine/threonine phosphatases i s critically involved in cell cycle control. The substrates of Cdc25 are cyclin-dependent kinases, which are regulated by the phosphorylation of threonine and tyrosine residues. Cdc25 regulation and activity reveals a complex network o f counter-balancing mechanisms and puts i t on the crossroads of fundamental cellular events like cell proliferation, cell cycle arrest and apoptosis. Our present knowledge of the biology and biochemistry of Cdc25 phosphatases makes them attractive targets for drug discovery efforts: (a) they phase critical, non-redundant cell cycle regulatory functions; (b) they are bona fide checkpoint genes; (c) they have tight substrate specificities and a well-defined mechanism of catalysis; (d) they are potential targets of at least two oncogenes (Raf1 and c-Myc) that are frequently altered in human cancers; (e) they co-operate with other oncogenes i n cell transformation and thus are bona fide proto-oncogenes; and lastly (f) their expression is altered in tumors.
complexes (for a recent review on Cdc25 cell biology and biochemistry, see Draetta and Eckstein, 1997). Recent investigations into the regulation of Cdc25 itself are beginning to shed light on an intruiging and complex network of players (please refer to Figure 2 throughout the text). They place Cdc25 squarely on the crossroads between cell proliferation, apoptosis, mitogenic signal transduction, and cancer. This chapter reviews briefly the emerging understanding of Cdc25 regulation and its implications for human cancer.
I. Introduction: Cdc25 and the cell cycle The role of Cdc25 as an inducer of mitosis first emerged from studies of yeast genetics that linked the phosphorylation state of the Cdc2 cyclin-dependent kinase to the activity of a protein phosphatase (Russell and Nurse, 1986). Later, the gene product of the cdc25 gene was identified to be a dual-specificity phosphatase that removes inhibitory phosphorylations of Cdc2, both from a highly conserved tyrosine residue (Tyr15) and a less conserved threonine residue (Thr14, Figure 1). Homologs of the yeast gene were identified in a wide variety of organisms. This functional conservation of Cdc25 throughout evolution illustrates its fundamental role in controlling the cell cycle. The regulation of proteins of the cyclin-dependent kinase (Cdk) family has been studied in great detail, and several cdc25 genes have been identified in mammals. In humans the three homologs that were isolated are Cdc25A, B and C. Cdc25 C is the mitotic inducer; its substrate is the hyperphosphorylated complex of Cdc2/cyclin B. The functions of Cdc25A and B are less clear, with their possible substrates ranging from Cdk4/cyclin D (Terada et al., 1995), Cdk2/cyclin E and cyclin A complexes (Hoffmann et al., 1994) to Cdc2/cyclin A and cyclin B
II. Cdc25 is a phosphoprotein The Cdc25 protein undergoes phosphorylation during the cell cycle (Izumi et al., 1992), a step that triggers its phosphatase activity. The phosphorylation of all three human versions of Cdc25 is essential for cell cycle progression. Several phosphorylation sites have been mapped, suggesting the possibility that more than one kinase is involved in this regulation of Cdc25 by posttranslational modification. Cdc25 can be phosphorylated by its own substrate, cyclin-dependent kinases (Cdks). Cdc25C is phosphorylated and activated by Cdc2/cyclin B in vitro. In vivo, this activation occurs at the G2/M transition, which initiates mitosis (Hoffmann et al., 1993; Izumi and Maller, 1993; Strausfeld et al., 1994). Cdc25A was later 707
Eckstein: Cdc25 protein phosphatase in cancer
F i g u r e 1 . Cdc25 is a dualspecificity protein phosphatase activating cyclindependent kinases (Cdks) Cdks bind to cyclins and are phosphorylated on three residues. Thr160 phosphorylation (Pa, shown in green) activates the kinase. The phosphorylations on Thr14 and Tyr15 (Pi, shown in red) are inhibitory and removed by Cdc25, resulting in an active kinase. The kinases Wee1 and Myt1 are counteracting Cdc25 and phosphorylate Tyr15 and Thr14, respectively.
Figure 2 Cdc25 and the cell cycle. This scheme summarizes the regulation and function of human Cdc25A, B and C in the cell cyle, as described in the text. Green arrows indicate induction, (de)phosphorylation or activation, red lines inhibition. Proteins with a Ubi tag are degraded via the ubiquitin-dependent pathway. Approximate timing of events and activity of proteins is indicated by the brown dashed lines subdividing the cell cycle into G0/G1, S, and G2/M phases.
Cdc25A occurs during the S-phase (Jinno et al., 1994). These results suggest regulation of Cdc25 via a self-
shown to be phosphorylated by Cdk2/cyclin E in vitro (Hoffmann et al., 1994). In vivo, hyperphosphorylation of 708
Gene Therapy and Molecular Biology Vol 1, page 709 amplifying feedback loop. Such a cooperative phenomenon has been cited to explain the sharp rise of Cdc2 kinase activity at the G2/M transition (Hoffmann et al., 1993; Izumi and Maller, 1993; Strausfeld et al., 1994). In addition to Cdks, other kinases are implicated in Cdc25 phosphorylation, as well. For example, the Raf1 kinase turned out to associate with Cdc25. Using double immunofluorescence microscopy, Cdc25A and B were found to co-localize with Raf1 and Ras at the cell membrane (Galaktionov et al., 1995), a process that is dependent on serum stimulation. Raf1 kinase phosphorylates Cdc25A and B in vitro, leading to an increase in phosphatase activity (Galaktionov et al., 1995). In a two-hybrid screen experiment, Raf1 also was found to be associated with members of the 14-3-3 protein family, which in turn associated with Cdc25A and B (Conklin et al., 1995). 14-3-3 proteins have been implicated in a number of mitogenic signaling pathways, including the kinase cascade that contains Raf1 (Fantl et al., 1994; Freed et al., 1994). Moreover, a role for 14-3-3 proteins in the regulation of Cdc25 was reported in connection with DNA damage sensing in cells. The response of cells to UV-induced DNA damage is multifaceted. It involves induction of cyclindependent kinase inhibitors, such as p21Cip1/Waf1, as well as hyperphosphorylation of the Cdks, and ultimately leads to cell cycle arrest (Poon et al., 1996). Recently, a new pathway has been proposed that links the gene sensing DNA damage in the yeast S. pombe—Rad3—to Cdc25 activity (Furnari et al., 1997; Sanchez et al., 1997). Rad3 is related to the human ATM protein that is defective in ataxia telangiectasia patients, a rare genetic disorder whose varied symptoms include possibly a high risk of developing tumors (Xu and Baltimore, 1996). DNA damage induces increased phosphorylation of the Chk1 kinase by a Rad3-dependent process. Cdc25 is potentially a direct target of Chk1, and Chk1's phosphorylation of a specific serine residue (Ser216 in human Cdc25C) results in binding of Cdc25 to 14-3-3 protein (Peng et al., 1997). It was proposed that 14-3-3 binding sequesters Cdc25C from functionally interacting with Cdc2, leading to a G2 arrest in the cell cycle. Regulation of Cdc25 by spatial sequestering rather than inhibition of the phosphatase activity seems to be the main effect of the phosphorylation of Cdc25 via the Chk1 kinase. The Chk1 phosphorylation site is conserved in Cdc25A and B, as well, suggesting that a similar regulatory mechanism is involved in other DNA damage checkpoints earlier in the cell cycle. The role of the 14-3-3 proteins in connection with the Raf1 kinase is still unclear. One can speculate that 14-3-3 proteins act as docking sites—or adaptors—for both Cdc25 and Raf1, and that subsequent phosphorylation of Cdc25 by Raf1 leads to the release and activation of Cdc25. This example nicely illustrates the fine balance of counteracting processes in cell cycle regulation. Furthermore, it
identifies Cdc25C, and possibly Cdc25A and B, as bona fide checkpoint genes. Yet other kinases have been reported to phosphorylate Cdc25 protein, suggesting that there are additional mechanisms for coordinating the regulation of cyclindependent kinases with various mitotic processes, such as chromosome segregation (Kumagai and Dunphy, 1996).
III. Other regulatory mechanisms The level of Cdc25 protein is tightly regulated by both transcriptional and post-translational mechanisms (Ducommun et al., 1990; Moreno et al., 1990). In humans, Cdc25A is expressed early in the G1 phase of the cell cycle following serum stimulation of quiescent fibroblasts (Jinno et al., 1994). Cdc25 B is expressed closer to the G1/S transition, and Cdc25C is activated in G2 (Sadhu et al., 1990). Recently, Galaktionov and colleagues observed that Cdc25 mRNA became more abundant following activation of the Myc proto-oncogene. They were able to show that Cdc25A, and possibly Cdc25B, are physiologically relevant and direct targets of c-Myc (Galaktionov et al., 1996). Their studies suggest furthermore that Cdc25 is a general mediator of Myc function. Therefore, Cdc25 is not only essential to normal cell proliferation but also for inducing Myc-dependent apoptosis. Downregulation of Cdc25 was reported to be achieved by at least two different mechanisms in the cell: repression and ubiquitin-dependent degradation. As an example for repression, consider TGF-ß. Its effect on cyclin-dependent kinase activity has been extensively studied as a model anti-mitogenic response, in particular in connection with cyclin-dependent kinase inhibitors (CKIs). In a recent report, Iavarone et al. conclude that induction of the cyclin-dependent kinase inhibitor p15Ink4B and downregulation of Cdc25A by TGF-ß constitute two complementary mechanisms of inhibition of the cyclin D-dependent kinase (Iavarone and Massague, 1997). Their experiments indicate that Cdc25A downregulation by TGF-ß occurs at transcription; it remains to be determined whether Myc participates in this process. Ubiquitin-dependent degradation of proteins is an important regulatory mechanism for all sorts of cellular processes (reviewed in Ciechanover, 1994) and has been found to play a key role in the degradation of the mitotic cyclins (Glotzer et al., 1991). In a study on Cdc25 degradation in S.pombe, Nefsky and Beach isolated a gene named Pub1, which encodes an E6-AP like protein (Nefsky and Beach, 1996). E6-AP belongs to a family of ubiquitin ligases, or E3s, which assist in transferring a ubiquitin molecule or a polyubiquitin chain to a target protein. Once the target protein is tagged with ubiquitin, it is rapidly degraded by the 26S proteasome. Cdc25 was ubiquitinated in a Pub1-dependent fashion, and loss of
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Eckstein: Cdc25 protein phosphatase in cancer Pub1 function lead to elevated levels of Cdc25 protein and increased Cdc25 activity in vivo.
I thank my wife, Gabrielle Strobel, for editorial assistance.
IV. Cooperation of Ras and Myc
References
The regulation of Cdk activity involves inhibitory small proteins (cyclin-dependent kinase inhibitors, or CKIs) from the Ink and the Waf1/Kip1/Cip1 families. Recent findings suggest, firstly, that the regulation of Cdc25 and the CKI proteins through Ras and Myc is tightly interconnected and, secondly, that the cooperation of active Ras and Myc leads to accumulation of G1 Cdk activity (Leone et al., 1997). Expression of Myc and Ras results in a loss of p27 Kip1 protein (probably through ubiquitin-dependent proteolysis) and leads to increased Cdk2/cyclin E activity. At the same time, Cdc25A is induced by c-Myc and activated by Raf1, a downstream target of Ras. This leads to a synergistic effect in removing an inhibitory protein and inhibitory phosphorylations on Cdk2/cyclin E, culminating in induction of S-phase. Interestingly, the competition between p21 and Cdc25 can be demonstrated directly in binding experiments. Saha et al. identified a consensus sequence in p21 and Cdc25 that is important for their binding to Cdk complexes (Saha et al., 1997). p21 protein directly competes with Cdc25A and vice versa, suggesting that the two proteins utilise similar docking sites on the Cdk/cyclin complexes.
Ciechanover, A. (1 9 9 4 ). The ubiquitin-proteasome proteolytic pathway. C e l l 79, 13-21. Conklin, D. S., Galaktionov, K., and Beach, D. (1 9 9 5 ). 143-3 proteins associate with cdc25 phosphatases. Proc Natl Acad Sci U S A 92, 7892-7896. Draetta, G., and Eckstein, J. (1 9 9 7 ). Cdc25 protein phosphatases in cell proliferation. Biochim. B i o p h y s . Acta 1332, M53-M63. Ducommun, B., Draetta, G., Young, P., and Beach, D. (1 9 9 0 ). Fission yeast cdc25 is a cell-cycle regulated protein. B i o c h e m B i o p h y s R e s C o m m u n 167, 301309. Fantl, W. J., Muslin, A. J., Kikuchi, A., Martin, J. A., MacNicol, A. M., Gross, R. W., and Williams, L. T. (1 9 9 4 ). Activation of Raf-1 by 14-3-3 proteins. Nature 371, 612-4. Freed, E., Symons, M., Macdonald, S. G., McCormick, F., and Ruggieri, R. (1 9 9 4 ). Binding of 14-3-3 proteins to the protein kinase Raf and effects on its activation. S c i e n c e 265, 1713-6. Furnari, B., Rhind, N., and Russell, P. (1 9 9 7 ). Cdc25 mitotic inducer targeted by chk1 DNA damage checkpoint kinase [In Process Citation]. S c i e n c e 277, 1495-7. Galaktionov, K., Chen, X., and Beach, D. (1 9 9 6 ). Cdc25 cell-cycle phosphatase as a target of c-myc. Nature 382, 511-517.
V. Cdc25 and cancer
Galaktionov, K., Jessus, C., and Beach, D. (1 9 9 5 ). Raf1 interaction with Cdc25 phosphatase ties mitogenic signal transduction to cell cycle activation. G e n e s & D e v 9 , 1046-1058.
Cdc25A and B have oncogenic properties. In rodent cells, human Cdc25A and Cdc25B, but not Cdc25C, phosphatases cooperate with either an activated Ras allele or loss of Rb1 in oncogenic focus formation (Galaktionov et al., 1995). Such transformants are highly aneuploid, grow in soft agar, and form high-grade tumours in nude mice. Based upon these criteria, Cdc25A and B are bona fide cellular proto-oncogenes. Indeed, Cdc25B mRNA is expressed at high levels in 32 percent of human primary breast cancers tested (Galaktionov et al., 1995). Similar findings have come from breast cancer studies on Cdc25 A (M. Loda et al., unpublished). Overexpression of Cdc25A and Cdc25B, but not Cdc25C, has also been reported in more than 50 percent of tested squamous cell carcinomas of the head and the neck (Gasparotto et al., 1997). Given the tight connection between Cdc25 and the well-known oncogenes Ras and Myc, overexpression and activation of Cdc25 might be an important feature in cancer development, making Cdc25 an attractive target for future cancer therapy.
Galaktionov, K., Lee, A. K., Eckstein, J., Draetta, G., Meckler, J., Loda, M., and Beach, D. (1 9 9 5 ). CDC25 phosphatases as potential human oncogenes. S c i e n c e 269, 1575-1577. Gasparotto, D., Maestro, R., Piccinin, S., Vukosavljevic, T., Barzan, L., Sulfaro, S., and Boiocchi, M. (1 9 9 7 ). Overexpression of CDC25A and CDC25B in head and neck cancers. Cancer Res 57, 2366-8. Glotzer, M., Murray, A. W., and Kirschner, M. W. (1 9 9 1 ). Cyclin is degraded by the ubiquitin pathway. Nature 349, 132-138. Hoffmann, I., Clarke, P. R., Marcote, M. J., Karsenti, E., and Draetta, G. (1 9 9 3 ). Phosphorylation and activation of human cdc25-C by cdc2--cyclin B and its involvement in the self-amplification of MPF at mitosis. EMBO J 12, 53-63. Hoffmann, I., Draetta, G., and Karsenti, E. (1 9 9 4 ). Activation of the phosphatase activity of human cdc25A by a cdk2-cyclin E dependent phosphorylation at the G1/S transition. EMBO J 13, 4302-4310. Iavarone, A., and Massague, J. (1 9 9 7 ). Repression of the CDK activator Cdc25A and cell-cycle arrest by cytokine TGF-beta in cells lacking the CDK inhibitor p15. Nature 387, 417-22.
Acknowledgement
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Izumi, T., and Maller, J. L. (1 9 9 3 ). Elimination of cdc2 phosphorylation sites in the cdc25 phosphatase blocks initiation of M-phase. M o l B i o l C e l l 4, 1337-50.
Xu, Y. and Baltimore, D. (1 9 9 6 ) Dual roles of ATM in the cellular response to radiation and in cell growth control. G e n e s D e v 10, 2401-2410.
Izumi, T., Walker, D. H., and Maller, J. L. (1 9 9 2 ). Periodic changes in phosphorylation of the Xenopus cdc25 phosphatase regulate its activity. M o l B i o l C e l l 3, 927-939. Jinno, S., Suto, K., Nagata, A., Igarashi, M., Kanaoka, Y., Nojima, H., and Okayama, H. (1 9 9 4 ). Cdc25A is a novel phosphatase functioning early in the cell cycle. EMBO J 13, 1549-56. Kumagai, A., and Dunphy, W. G. (1 9 9 6 ). Purification and molecular cloning of Plx1, a Cdc25-regulatory kinase from Xenopus egg extracts. S c i e n c e 273, 1377-80. Leone, G., DeGregori, J., Sears, R., Jakoi, L., and Nevins, J. R. (1 9 9 7 ). Myc and Ras collaborate in inducing accumulation of active cyclin E/Cdk2 and E2F. Nature 387, 422-6. Moreno, S., Nurse, P., and Russell, P. (1 9 9 0 ). Regulation of mitosis by cyclic accumulation of p80cdc25 mitotic inducer in fission yeast. Nature 344, 549-52. Nefsky, B., and Beach, D. (1 9 9 6 ). Pub1 acts as an E6-AP-like protein ubiquitiin ligase in the degradation of cdc25. EMBO J. 15, 1301-1312. Peng, C. Y., Graves, P. R., Thoma, R. S., Wu, Z., Shaw, A. S., and Piwnica-Worms, H. (1 9 9 7 ). Mitotic and G2 checkpoint control: regulation of 14-3-3 protein binding by phosphorylation of Cdc25C on serine-216 [In Process Citation]. S c i e n c e 277, 1501-5. Poon, R. Y. C., Jiang, W., Toyoshima, H., and Hunter, T. (1 9 9 6 ). Cyclin-dependent kinases are inactivated by a combination of p21 and Thr-14/Tyr-15 phosphorylation after UV-induced DNA damage. J B i o l Chem 271, 13283-91. Russell, P., and Nurse, P. (1 9 8 6 ). cdc25+ functions as an inducer in the mitotic control of fission yeast. Cell 45, 145-53. Sadhu, K., Reed, S. I., Richardson, H., and Russell, P. (1 9 9 0 ). Human homolog of fission yeast cdc25 mitotic inducer is predominantly expressed in G2. Proc Natl Acad Sci U S A 87, 5139-43. Saha, P., Eichbaum, Q., Silberman, E. D., Mayer, B. J., and Dutta, A. (1 9 9 7 ). p21CIP1 and Cdc25A: competition between an inhibitor and an activator of cyclin-dependent kinases. M o l C e l l B i o l 17, 4338-45. Sanchez, Y., Wong, C., Thoma, R. S., Richman, R., Wu, Z., Piwnica-Worms, H., and Elledge, S. J. (1 9 9 7 ). Conservation of the chk1 checkpoint pathway in mammals: linkage of DNA damage to cdk regulation through cdc25 [In Process Citation]. S c i e n c e 277, 1497501. Strausfeld, U., Fernandez, A., Capony, J. P., Girard, F., Lautredou, N., Derancourt, J., Labbe, J. C., and Lamb, N. J. (1 9 9 4 ). Activation of p34cdc2 protein kinase by microinjection of human cdc25C into mammalian cells. Requirement for prior phosphorylation of cdc25C by p34cdc2 on sites phosphorylated at mitosis. J B i o l Chem 269, 5989-6000. Terada, Y., Tatsuka, M., Jinno, S., and Okayama, H. (1 9 9 5 ). Requirement for tyrosine phosphorylation of Cdk4 in G1
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Gene Ther Mol Biol Vol 1, 713-740. March, 1998.
Nucleocytoplasmic trafficking: implications for the nuclear import of plasmid DNA during gene therapy Teni Boulikas Institute of Molecular Medical Sciences, 460 Page Mill Road, Palo Alto, California 94306 and Regulon Inc., 249 Matadero Avenue, Palo Alto, CA 94306
__________________________________________________________________________________ Correspondence: Teni Boulikas, Regulon Inc., 249 Matadero Avenue, Palo Alto, CA 94306, Tel (650) 813-9264, Fax: (650) 424-9594, E-mail: Boulikas@Worldnet.att.net
Key words: pore complex, nucleoporins, nuclear localization signals, karyopherin, nuclear export signals, mRNA export,
Summary Trafficking of nuclear proteins from the site of their synthesis in the cytoplasm to the sites of function in the nucleus through pore complexes is mediated by nuclear localization signals (NLSs) on proteins t o be imported into nuclei. Protein translocation from the cytoplasm t o the n u c l e o p l a s m i n v o l v e s ( i ) t h e f o r m a t i o n o f a c o m p l e x o f karyopherin with NLS-protein, (ii) subsequent binding of karyopherin , ( i i i ) b i n d i n g o f t h e c o m p l e x t o F X F G p e p t i d e r e p e a t s o n nucleoporins, (iv) docking of Ran-GDP to nucleoporin and to karyopherin heterodimer by p10, (v) a number of association-dissociation reactions on nucleoporins which dock the import substrate toward the nucleoplasmic side with a concomitant GDP-GTP exchange reaction transforming RanGDP into Ran-GTP and catalyzed by karyopherin , and finally (vi) dissociation from karyopherin and release of the karyopherin / N L S - p r o t e i n b y R a n - G T P t o t h e n u c l e o p l a s m . A n u m b e r of processes have been found to be regulated by nuclear import including nuclear translocation of the transcription factors NF- B, rNFIL-6, ISGF3, SRF, c-Fos, GR as well as human cyclins A and B1, casein kinase II, cAMP-dependent protein kinase II, protein kinase C, ERK1 and ERK2. Failure of cells to import specific proteins into nuclei can lead to carcinogenesis. For example, BRCA1 is mainly localized in the cytoplasm in breast and ovarian cancer cells whereas in normal cells the protein is nuclear. mRNA is exported through the same route as a complex with nuclear proteins p o s s e s s i n g n u c l e a r e x p o r t s i g n a l s ( N E S ) . T h e m a j o r i t y o f p r o t e i n s w i t h N E S are RNA-binding p r o t e i n s w h i c h b i n d t o a n d e s c o r t R N A s t o t h e c y t o p l a s m . H o w e v e r , o t h e r proteins with NES function in the export of proteins; CRM1, which binds to the NES sequence on other proteins and interacts with the nuclear pore complex, is an essential mediator of the NES-dependent nuclear export of proteins in eukaryotic cells. Nuclear localization and export signals (NLS and NES) are found on a number of important molecules including p53, v-Rel, the transcription factor NF-ATc, the c-Abl nonreceptor tyrosine kinase, and the fragile X syndrome mental retardation gene product; the deregulation of their normal import/export trafficking has important implications for human disease. Both nuclear import and export processes can be manipulated by conjugation of proteins with NLS or NES peptides. During gene therapy the foreign DNA needs to enter nuclei for its transcription; a pathway is proposed involving the complexation of plasmids and oligonucleotides with nascent nuclear proteins possessing NLSs as a prerequisite for their nuclear import. Covalent linkage of NLS peptides to oligonucleotides and plasmids or formation of complexes of plasmids with proteins possessing multiple NLS peptides is proposed to increase their import rates and the efficiency of gene expression. Cancer cells are predicted to import more efficiently foreign DNA into nuclei compared with terminally differentiated c e l l s because o f their increased rates of proliferation and protein import.
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Gene Therapy and Molecular Biology Vol 1, page 714 of their therapeutic gene loads. In addition, complexation of the plasmid into colloidal particles with proteins possessing multiple NLS peptide motifs is proposed to facilitate nuclear import.
I. Introduction Evolution has effectively secluded nuclear functions from cytoplasmic activities by the nuclear envelope barrier in order to circumvent the increased organizational and regulatory problems associated with the advent of the large genomes in more complex organisms. The nuclear envelope has evolved to allow nuclear and cytoplasmic environments to be developed and to selectively keep certain regulatory proteins, such as replication factors, out, allowing cells to regulate their cell cycle and level of ploidy. This double-membrane structure effectively separates transcription of genes from translation of their mRNA into proteins and allows proteins destined to function in the nucleus to pass selectively through the lumen of the nuclear pores (reviewed by Burke, 1990; Boulikas, 1993, 1994; Laskey et al, 1996).
II. Morphology of the pore complexes The nuclear envelope is often studded across both sides with transcisternal "holes." These hollow cylindrical organelles spanning the two nuclear membranes are called pore complexes. Pore complexes have a width (distance from cytoplasm to nucleoplasm) of ~70 nm and a diameter of 133 nm (Hinshaw et al 1992). Their frequency greatly depends on the cell type ranging from 1 to 60 pores/mm 2 .
The nuclear membrane prevents reinitiation of DNA replication in Xenopus eggs, by excluding a "licensing factor" that is essential for DNA replication; replication licensing may involve the MCM (minichromosome maintenance) complex and ORC, the origin recognition complex (Laskey et al, 1996). The interior of the nuclear envelope is lined by a polymer of lamins intimately attached to chromatin DNA. Phosphorylation of lamins at four serines by S6 kinase II and other kinases, all of which appear to be controlled by cdc2 kinase, specifically occur as cells traverse the G2 to M checkpoint of the cell cycle; this process result in lamin depolymerization and in nuclear envelope breakdown. After completion of mitosis these processes are reversed and new nuclear envelopes are assembled around daughter cell nuclei. Selective transport through pores creates a unique biochemical environment within the nucleus. All proteins are synthesized in the cytoplasm; the selective import of proteins through the pore complexes that straddle the inner and outer nuclear membranes is a sophisticated process dependent on energy and upon the presence of short karyophilic peptides, termed nuclear localization signals (NLS), only on nuclear proteins (Dingwall et al, 1982; Kalderon et al, 1984). The ultimate target of gene therapy is the cell nucleus. The knowledge on nucleocytoplasmic trafficking could be used for enhancing the nuclear import of plasmids or small oligonucleotides designed to act in the nucleus. Since only a small portion (less than 1%) of the plasmid molecules that reach the cytoplasm might ultimately enter the nucleus, and only 15% of water soluble oligonucleotides which reach the cytoplasm might ultimately diffuse through pore complexes (Boutorine and Kostina, 1993), covalent linkage of NLS peptides via random-coil peptide arms to oligonucleotides or to plasmids is expected to increase their import rates and the efficiency of expression
Their refined model structure (Hinshaw et al, 1992) is rather complex (Figures 1-4). They appear as tripartite structures composed of two concave rings and a central granule. The concave rings are called pore annuli (one cytoplasmic and one nucleoplasmic), each containing eight granules arranged in an 8-fold rotational symmetry, lying on top of the pore rims. A three-dimensional electron microscopy analysis coupled with image analysis to calculate 2D and 3D maps of detergent released pore complexes revealed that this highly symmetric framework is built from many distinct and interconnected subunits arranged in such a way so as to construct a large central channel (Hinshaw et al, 1992, Figures 1-4). Electron microscopy of nuclear pore complexes isolated from Xenopus laevis oocytes spread on a carboncoated film has shown that each of the eight spokes seen in en face views of pore complexes is built from four morphological features: the annular, the column, the ring, and the lumenal subunits (Hirshaw et al, 1992). Each spoke holds two copies of each subunit. Furthermore, an intricate network connects these subunits to one another. In addition to the large central channel, the spokes are responsible for the construction of eight peripheral channels of unknown function (Hinshaw et al, 1992). Image analysis of spokes on en face electron micrographs of pore complexes (Figure 2a) show that they are composed of (i ) bilobed regions that form an inner annulus encircling a 42 nm diameter hole; (i i ) a central region of a radius of 41 nm and (i i i ) an outer region of 52.5 nm . The maps obtained by Hinshaw and coworkers (1992) provide compelling evidence for a highly symmetric structure of pore complexes in accordance with previous studies (Unwin and Milligan 1982; Akey, 1989). In the oocytes of the frog Xenopus laevis the pore annuli have an inside diameter close to 80 nm and an outer diameter of 120 nm. The inner diameter of the pore complexes is highly constant within a certain cell type. A total of up to 100 distinct proteins (nucleoporins) have been estimated to participate in the structure of the
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F i g u r e 1 . Electron micrographs of nuclear pore complexes, (NPCs), released from the nuclear envelope by detergent. (a) En face views of NPCs (lower right) and rings (upper left) in the same field of view. (b ) Image of a deep pool of stain; the two pore complexes indicated by arrows represent edge views whereas the arrowheads show oblique views of NPCs (see also the model in Figure 3); a number of en face views are also present in the same field of view. Scale bar = 500 nm. Image by courtesy of Ron Milligan and Jenny Hinshaw, The Scripps Research Institute, La Jolla, California. From Hinshaw JE, Carragher BO, Milligan RA (1 9 9 2 ) Architecture and design of the nuclear pore complex. C e l l 69, 1133-1141. Reproduced with kind permission from Cell Press and the authors.
Figure 2. Projection maps obtained by averaging images of each of the four structures identified in Figure 1. Regions where biological material is concentrated are darker and are enclosed by contours; regions where the negative stain is concentrated are lighter. (a) Average of 168 (n=168) e n f a c e images of detergent-released NPCs. (b ) E d g e v i e w of detergent released NPCs, n=48. (c ) R i n g v i e w s (n=400). (d) Intermediate structures (n=23). Scale barr = 50 nm. Image by courtesy of Ron Milligan and Jenny Hinshaw, The Scripps Research Institute, La Jolla, California. From Hinshaw JE, Carragher BO, Milligan RA (1 9 9 2 ) Architecture and design of the nuclear pore complex. C e l l 69, 1133-1141. Reproduced with kind permission from Cell Press and the authors.
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Gene Therapy and Molecular Biology Vol 1, page 717 pore complex (Iovine et al, 1995). A pore-specific transmembrane glycoprotein gp210 (mw 204 kD) with two transmembrane domains and 13 glycosylation sites is thought to anchor the pore complex to the membrane of the nuclear envelope (Wozniak et al, 1989). The central granule, exclusive route for import of nuclear protein, occupies the pore center. Its diameter varies from 2.5 to 35 nm and its appearance ranges from compact spherical to thin rod shaped (Akey, 1989). Fibrils, which protrude deeply into the nuclear interior forming a central ring of spokes, emanate from the central granule. The fibrils, ~3nm in diameter and extending ~200nm to the interior of the nucleus, are involved in nuclear transport: nucleoplasmin-coated gold particles associate with these tentacles (Richardson et al, 1988) and are docking sites for the karyopherin ! NLS-protein complex (Rexach and Blobel, 1995). Antibodies to the Olinked glycoproteins seem to bind close to the 8-fold axis and away from the central plane of the NPC (Snow et al, 1987); thus, it is unlikely for those glycoproteins, involved in nuclear import, are integral parts of the spoke subunits (Hinshaw et al, 1992).
III. Nuclear localization signals (NLSs) A. Historical background Figure 3 Renderings of the 822-symmetrized nuclear pore complex map. En face (a), oblique (b ), edge (c ), and front half view (d) of the 3D map. A slight ridge indicates the central plane of the 3D map and divides the assembly into two symmetrical halves. Manipulation and display of the maps were done with specific programs. Annular subunits are green, rings are yellow, and lumenal subunits are blue. The remaining tan colored parts of the 3D map enclose the column subunits. Image by courtesy of Ron Milligan and Jenny Hinshaw, The Scripps Research Institute, La Jolla, California. From Hinshaw JE, Carragher BO, Milligan RA (1 9 9 2 ) Architecture and design of the nuclear pore complex. C e l l 69, 1133-1141. Reproduced with kind permission from Cell Press and the authors.
The selective import of proteins that have a function in the nucleus through the pore complexes that straddle the inner and outer nuclear membranes is a sophisticated process dependent on the presence of short karyophilic peptides, termed nuclear localization signals (NLS) (reviewed by Boulikas, 1993, 1994, 1996, 1997b). A very short sequence of seven amino acids (Pro-Lys-Lys-LysArg-Lys-Val or PKKKRKV), first recognized by Kalderon and coworkers (1984) in the SV40 large T antigen, is required for its normal nuclear localization. Yoneda et al. (1988) have raised antibodies against the peptide DDDED supposed to be present in nuclear pore or cytoplasmic receptor (transporter) protein molecules and to be involved in ionic interactions with the NLS (KKKRK) of SV40 large T protein. Indirect immunofluorescence with these antibodies against the acidic peptide has shown punctuate staining at the nuclear rim or the nuclear surface in rat, human, bovine and murine cell lines; in addition, the antibody blocked nuclear import. A single protein may possess more than one signals for nuclear import (Standiford and Richter, 1992). The rate of nuclear import is directly related to the number of NLS it possesses (Dworetzky et al, 1988), as was first suggested by Dingwall and coworkers (1982). A smaller number of nuclear proteins contain "bipartite" NLS hypothesized to be reconstituted by two moieties brought together by protein folding or conformational change as for
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example on the cytoplasmic glucocorticoid receptor by hormone binding (Welsh et al, 1986; Picard and Yamamoto, 1987). Three approaches have been used for NLS identification: (i ) gene fusion experiments between a NLScoding DNA segment and the gene coding for puruvate kinase, "-galactosidase, or other cytoplasmic proteins; (i i ) nuclear import of non-nuclear proteins conjugated to synthetic NLS peptides; (i i i ) site-directed mutagenesis of the NLS of a nuclear protein resulting in its cytoplasmic retention (Boulikas, 1993; Tables 1-4).
B. Rules to predict nuclear localization of an unknown protein Several simple rules have been proposed for the prediction of the nuclear localization of a protein of an unknown function from its amino acid sequence: (i ). An NLS is defined as four arginines (R) plus lysines (K) within an hexapeptide; the presence of one or more histidines (H) in the tetrad of the karyophilic hexapeptide, often found in protein kinases that have a
Figure 4 oblique map of the pore complex (Figure 3b) superimposed over an electron micrograph of nuclear pore complexes showing the central plug of the structures. Image by courtesy of Ron Milligan and Jenny Hinshaw, The Scripps Research Institute, La Jolla, California. From Hinshaw JE, Carragher BO, Milligan RA (1 9 9 2 ) Architecture and design of the nuclear pore complex. C e l l 69, 1133-1141. Reproduced with kind permission from Cell Press and the authors.
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Gene Therapy and Molecular Biology Vol 1, page 718 mechanism might ensure a certain stoichiometric ratio of the two molecules in the nucleus and might be of physiological significance.
cytoplasmic and a nuclear function, may specify a weak NLS whose function might be regulated by phosphorylation or may specify proteins that function in both the cytoplasm and the nucleus (Boulikas, 1996).
(x i ) A number of proteins may be imported via other mechanisms not dependent on classical NLS (se below).
(i i ). The K/R clusters are flanked by the !-helix breakers G and P thus placing the NLS at a helix-turnhelix or end of an !-helix. Negatively-charged amino acids (D, E) are often found at the flank of the NLS and on some occasions may interrupt the positively-charged NLS cluster.
C. NLS on adenovirus proteins The pentapeptide KRPRP of Adenovirus E1a when linked to the C-terminus of E. coli galactokinase, was sufficient to direct its nuclear accumulation after microinjection into Vero monkey cells (Lyons et al., 1987). The synthetic peptide CGGLSSKRPRP from adenovirus type 2/5 E1a crosslinked to chicken bovine albumin and microinjected into HeLa cells caused nuclear localization (Chelsky et al., 1989).
(i i i ). Bulky amino acids (W, F, Y) are not present within the NLS hexapeptide. (i v ). NLS signals may not be flanked by long stretches of hydrophobic amino acids (e.g. five); a mixture of charged and hydrophobic amino acids serves as a mitochondrial targeting signal.
Two NLS, PPKKRMRRRIE and PKKKKKRP were found on adenovirus 5 D B P (DNA-binding protein) which is expressed in nuclei of infected cells and is involved in virus replication and early and late gene expression. Both NLS are needed, and disruption of either site impaired nuclear localization of the 529 amino acid protein (Morin et al., 1989).
(v). The higher the number of NLSs the more readily a molecule is imported to the nucleus (Dworetzky et al, 1988). Even small proteins, for example histones (10-22 kDa), need to be actively imported to increase their import rates compared with the slow rate of diffusion of small molecules through pores. (v i ). Signal peptides are stronger determinants than NLSs for protein trafficking; signal peptides direct proteins to the lumen of the endoplasmic reticulum for their secretion or insertion into cellular membranes (presence of transmembrane domains) (Boulikas, 1994).
The NLS RLPVRRRRRRVP was determined on adenovirus pTP1 and pTP2 (preterminal proteins, 80 kD) between amino acid residues 362-373. The 140 kDa DNA polymerase of adenovirus when it had lost its own NLS could enter the nucleus via its interaction with pTP. This NLS, fused to the N-terminus of E. coli "galactosidase, was functional in nuclear targeting (Zhao and Padmanabhan, 1988).
(v i i ). Signals for the mitochondrial import of proteins (a mixture of hydrophobic and karyophilic amino acids) may antagonize nuclear import signals and proteins possessing both type of signals may be translocated to both mitochondria and nuclei (Beasley and Schatz, 1991; Neupert and Lill, 1995).
A "tripartite" or "doubly bipartite" NLS was found on adenovirus DNA polymerase (AdPol) having the sequences: signal I: AHRARRLH (amino acids 6-13); signal II: PPRRRVRQQPP (amino acids 23-33); and signal III: PARARRRRAP (amino acids 39-48). Signals I and II functioned interdependently as an NLS for the nuclear targeting of AdPol, for which signal III was dispensable. The combined signal II-III was more efficient NLS than signal I-II (Zhao and Padmanabhan, 1991).
(v i i i ). Strong association of a protein with large cytoplasmic structures (membrane proteins, intermediate filaments) make such proteins unavailable for import even though they posses NLS-like peptides (Boulikas, 1994). (i x ). Transcription factors and other nuclear proteins posses a great different number of putative NLS stretches; of the sixteen possible forms of putative NLS structures the most abundant types are the ##x##, ###x#, ####, and ##x#x# where # is R or K, together accounting for about 70% of all karyophilic clusters on transcription factors (Boulikas, 1994).
IV. Nucleoporins A number of nucleoporins (proteins of the pore complex) posses FXFG motifs and display modification of Ser/Thr by single N-acetyl-glucosamine residues; these include Nup98, p62, Nup153, and Nup214 in vertebrates and NUP1, NUP2, and NSP1 in S. cerevisiae. A different subset of pore complex proteins including p270, Nup214, Nup153, and Nup98 contain FXFG and GLFG repetitive peptide motifs and are able to bind specifically to NLScontaining protein models; a single motif may be a low affinity binding site and the affinity of binding could be
(x). A small number of nuclear proteins seem to be void of a typical karyophilic NLS; in this case either non karyophilic peptides function for their nuclear import, such molecules possess bipartite NLSs, or these NLS-less proteins depend absolutely for import on their strong complexation in the cytoplasm with a nuclear protein partner able to be imported (Boulikas, 1994); this 718
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Table 1 Simple NLS Signal oligopeptide
Protein and features
PKKKRKV
Wild-type SV40 large T protein A point mutation converting lysine-128 (double underlined) to threonine results in the retention of large T in the cytoplasm. Transfer of this peptide to the N-terminus of "-galactosidase or pyruvate kinase at the gene level and microinjection of plasmids into Vero cells showed nuclear location of chimeric proteins.
PKKKRMV
SV40 large T with a K$M change. Site-directed mutagenesis only slightly impaired nuclear import of large T.
PKKKRKVEDP
Synthetic NLS peptide from SV40 large T antigen crosslinked to BSA or IgG mediated their nuclear localization after microinjection in Xenopus oocytes. The PKKGSKKA from Xenopus H2B was ineffective and PKTKRKV was less effective.
CGYGPKKKRKVGG
Synthetic peptide from SV40 large T antigen conjugated to various proteins and microinjected into the cytoplasm of TC-7 cells. Specified nuclear localization up to protein sizes of 465 kD (ferritin). IgM of 970 kD and with an estimated radius of 25-40 nm was retained in the cytoplasm.
CYDDEA TAD SQH STPPKKKRKVEDPK DFESELLS
SV40 large T protein long NLS. The long NLS but not the short NLS, was able to localize the bulky IgM (970 kD) into the nucleus. Mutagenesis at the four possible sites of phosphorylation (double underlined) impaired nuclear import.
CGGPKKKRKVG
SV40 large T protein. This synthetic peptide crosslinked to chicken serum albumin and microinjected into HeLa cells caused nuclear localization.
PKKKIKV
A mutated (R$I) version of SV40 large T NLS. Effective NLS.
MKx11CRLKKLKCSKEKPKCAKCLKx5 Rx3KTKR 74 N-terminal amino acid
Yeast GAL4 (99 kD). Fusions of the GAL4 gene portion encoding the 74 N-terminal amino acid with E. Coli "-galactosidase introduced into yeast cells specify nuclear localization.
MKx11CRLKKLKCSKEKPKCA 29 N-terminal amino acid
Yeast GAL4. Acted as an efficient nuclear localization sequence when fused to invertase but not to "galactosidase introduced by transformation into yeast cells.
PKKARED VSRKRPR
Polyoma large T protein. Identified by fusion with puruvate kinase cDNA and microinjection of Vero African green monkey cells. Mutually independent NLS. Can exert cooperative effects.
CGYGVSRKRPRPG
Polyoma virus large T protein. This synthetic peptide crosslinked to chicken serum albumin and microinjected into HeLa cells caused nuclear localization.
APTKRKGS
SV40 VP1 capsid polypeptide (46 kD). NLS (N terminus) determined by infection of monkey kidney cells with a fusion construct containing the 5' terminal portion of SV40 VP1 gene and the complete cDNA sequence of poliovirus capsid VP1 replacing the VP1 gene of SV40.
APKRKSGVSKC (1-11)
Polyoma virus major capsid protein VP1 (11 N-terminal amino acid). Yeast expression vectors coding for 17 N-terminal amino acid of VP1 fused to "-galactosidase gave a protein that was transported to the nucleus in yeast cells. Subtractive constructs of VP1 lacking A1 to C 11 were cytoplasmic. This, FITC-labeled, synthetic peptide crosslinked to BSA or IgG, caused nuclear import after microinjection into 3T6 cells. Replacement of K3 with T did not.
PNKKKRK (amino acid position 317-323)
SV40 VP2 capsid protein (39 kD). The 3' end of the SV40 VP2-VP3 genes containing this peptide when fused to poliovirus VP1 capsid protein at the gene level resulted in nuclear import of the hybrid VP1 in simian cells infected with the hybrid SV40.
EEDGPQKKKRRL (307-318)
Polyoma virus capsid protein VP2. A construct having truncated VP2 lacking the 307-318 peptide transfected into COS-7 cells showed cytoplasmic retention of VP2. The 307-318 peptide crosslinked to BSA or IgG specified nuclear import following their microinjection into NIH 3T6 cells.
GKKRSKA
Yeast histone H2B. This peptide specified nuclear import when fused to "-galactosidase.
KRPRP
Adenovirus E1a. This pentapeptide, when linked to the C-terminus of E. coli galactokinase, was sufficient to direct its nuclear accumulation after microinjection in Vero monkey cells.
CGGLSSKRPRP
Adenovirus type 2/5 E1a. This synthetic peptide crosslinked to chicken bovine albumin and microinjected into HeLa cells caused nuclear localization.
LVRKKRKTE3SP (NLS 1) LKDKDAKKSKQE (NLS2)
Xenopus N1 (590 amino acid). Abundant in X. laevis oocytes, forming complexes with histones H3, H4 via two acidic domains each containing 21 and 9 (D+E), respectively. The NLS1 is required but not sufficient for nuclear accumulation of protein N1. NLS 1 and 2 are contiguous at the C-terminus.
GNKAKRQRST
v-Rel or p59 v-rel the transforming protein, product of the v-rel oncogene of the avian reticuloendotheliosis retrovirus strain T (Rev-T). v-Rel NLS added to the normally cytoplasmic "galactosidase directed that protein to the nucleus.
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PFLDRLRRDQK PKQKRKMAR
NS1 protein of influenza A virus, that accumulates in nuclei of virus-infected cells. Determined to be an NLS by deletion mutagenesis of NS1 in recombinant SV40. The 1st NLS is conserved among all NS1 proteins of influenza A viruses.
SVTKKRKLE
Human lamin A. Dimerization of lamin A was proposed to give a complex with two NLSs that was transported more efficiently.
SASKRRRLE
Xenopus lamin A. NLS inferred from its similarity to human lamin A NLS.
TKGKRKRID
Xenopus lamin LI . NLS inferred from its sequence similarity to human lamin A NLS.
CVRTTKGKRKRIDV
Xenopus lamin LI. This synthetic peptide crosslinked to chicken bovine albumin and microinjected into HeLa cells caused nuclear localization.
CGGAMINO ACIDKRVKLD
Human c-myc oncoprotein. This synthetic peptide crosslinked to chicken bovine albumin and microinjected into HeLa cells caused nuclear localization.
PAMINO ACID KRVKLD (M1, fully potent NLS)
Human c-myc oncoprotein. Conjugation of the M1 peptide to human serum albumin and microinjection of Vero cells gives complete nuclear accumulation. M2 gave slower and only partial nuclear localization.
RQRRNELKRSP (M2, medium potency NLS) SALIKKKKKMAP
Murine c-abl (IV) gene product. The p160gag/v-abl has a cytoplasmic and plasma membrane localization, whereas the mouse type IV c-abl protein is largely nuclear.
PPKKRMRRRIE PKKKKKRP
Adenovirus 5 DBP (DNA-binding protein) found in nuclei of infected cells and involved in virus replication and early and late gene expression. Both NLS are needed, and disruption of either site impaired nuclear localization of the 529 amino acid protein.
YRKCLQAGMNLEARKTKKKIKGIQQ ATA (497-524 amino acid)
Rat GR, glucocorticoid receptor (795 amino acid) NLS1 determined by fusion with "-galactosidase (116 kD). NLS1 is 100% conserved between human, mouse and rat GR. Whereas the 407-615 amino acid fragment of GR specifies nuclear location, the 407-740 amino acid fragment was cytoplasmic in the absence of hormone, indicating that sequence 615-740 may inhibit the nuclear location activity. A second (NLS2) is localized in an extensive 256 amino acid C-terminal domain. NLS 2 requires hormone binding for activity.
RKDRRGGRMLKHKRQRDDGEGRGE VGSAGDMRAMINO ACIDNLWPSPLMIKRSKK. (amino acid 256-303)
Human ER (estrogen receptor, 595 amino acid) NLS. NLS is between the hormone-binding and DNAbinding regions; ER, in contrast with GR, lacks a second NLS. Can direct a fusion product with "galactosidase to the nucleus.
RKFKKFNK
Rabbit PG (progesterone receptor). 100% homology in humans; F$L change in chickens. When this sequence was deleted, the receptor became cytoplasmic but could be shifted into the nucleus by addition of hormone; in this case the hormone mediated the dimerization of a mutant PG with a wild type PG molecule.
GKRKNKPK
Chicken Ets1 core NLS. Within a 77 amino acid C-terminal segment 90% homologous to Ets2. When deleted by deletion mutagenesis at the gene level the mutant Ets1 became cytoplasmic.
PLLKKIKQ
c-myb gene product; directs puruvate kinase to the nucleus.
PPQKKIKS
N-myc gene product; directs puruvate kinase to the nucleus.
PQPKKKP
p53; directs puruvate kinase to the nucleus.
SKRVAKRKL
c-erb-A gene product; directs puruvate kinase to the nucleus.
CGGLSSKRPRP
Adenovirus type2/5 E1a. This synthetic peptide conjugated with a bifunctional crosslinker to chicken serum albumin (CSA) and microinjected into HeLa cells directed CSA to the nucleus.
MTGSKTRKHRGSGA MTGSKHRKHPGSGA
Yeast ribosomal protein L29. Double-stranded oligonucleotides encoding the 7 amino acid peptides (underlined) and inserted at the N-terminus of the "-galactosidase gene resulted in nuclear import.
RHRKHP KRRKHP KYRKHP KHRRHP KHKKHP RHLKHP KHRKYP KHRQHP
Mutated peptides derived from yeast L29 ribosomal protein NLS, found to be efficient NLS. The last two are less effective NLS, resulting in both nuclear and cytoplasmic location of "-galactosidase fusion protein.
PETTVVRRR GRSPRRRTPSPRRRR SPR RRRSQS (One sequence, C-terminus)
Double NLS of hepatitis B virus core antigen. The two underlined arginine clusters represent distinct and independent NLS. Mutagenesis showed that the antigen fails to accumulate in the nucleus only when both NLS are simultaneously deleted or mutated.
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Gene Therapy and Molecular Biology Vol 1, page 721 ASKSRKRKL
Viral Jun, a transcription factor of the AP-1 complex. Accumulates in nuclei most rapidly during G2 and slowly during G1 and S. The cell cycle dependence of viral but not of cellular Jun is due to a C$S mutation in NLS of viral Jun. This NLS conjugated to rabbit IgG can mediate cell cycle-dependent translocation.
GGLCSARLHRHALLAT
Human T-cell leukemia virus Tax trans-activator protein. The most basic region within the 48 Nterminal segment. Missense mutations in this domain result in its cytoplasmic retention.
DTREKKKFLKRRLLRLDE (604-620)
Mouse nuclear Mx1 protein (72 kD), Induced by interferons (among 20 other proteins) . Selectively inhibits influenza virus mRNA synthesis in the nucleus and virus multiplication. The cytoplasmic Mx2 has R$S and R$E changes in this region.
CGYGPKKKRKV (SV40 large T) CGYGDRNKKKKE (human retinoic acid receptor) CGYGARKTKKKIK (human glucocorticoid receptor) CGYGIRKDRRGGR (human estrogen receptor) CGYGARKLKKLGN (human androgen receptor)
Synthetic peptides crosslinked to bovine serum albumin (BSA) and introduced into MCF 7 or HeLa S3 cells with viral co-internalization method using adenovirus serotype 3B induced nuclear import of BSA.
RKRQRALMLRQAR 30-42
Human XPAC (xeroderma pigmentosum group A complementing protein) involved in DNA excision repair. By site-directed mutagenesis and immunofluorescence. NLS is encoded by exon 1 which is not essential for DNA repair function.
EYLSRKGKLEL (at the N-terminus)
T-DNA -linked VirD2 endonuclease of the Agrobacterium tumefaciens tumor-inducing (Ti) plasmid. A fusion protein with "-galactosidase is targeted to the nucleus. The T-plasmid integrates into plant nuclear DNA; VirD2 produces a site-specific nick for T integration. VirD2 also contains a bipartite NLS at its C-terminus (see Table 2).
KKSKKKRC (95-102)
Putative core NLS of yeast TRM1 (63 kD) that encodes the tRNA modification enzyme N 2, N 2dimethylguanosine-specific tRNA methyltransferase. Localizes at the nuclear periphery. The 70-213 amino acid segment of TRM1 causes nuclear localization of "-galactosidase fusion protein in yeast cells. Site-directed mutagenesis of the 95-102 peptide resulted in its cytoplasmic retention. TRM1 is both nuclear and mitochondrial. The 1-48 amino acid segment specifies mitochondrial import.
PQSRKKLR
Max protein; specifically interacts with c-Myc protein. Fusion of 126-151 segment of Max to chicken pyruvate kinase (PK) gene, including this putative NLS, followed by transfection of COS-1 cells and indirect immunofluorescence with anti-PK showed nuclear targeting.
QPQRYGGG RGRRW
Gag protein of human foamy retrovirus; a mutant that completely lacks this box exhibits very little nuclear localization; binds DNA and RNA in vitro.
mediate docking of nuclear proteins across the pore; these multiple docking sites were suggested to extend over a distance of 250 nm from the cytoplasmically exposed fibers to the nucleoplasmic baskets (Radu et al, 1995).
proportional to the number of peptide motifs (see Radu et al, 1995 and the references cited therein). The nucleoporin Nup98, containing 16 perfect and imperfect GLFG repeats and 3 FXFG repeats, is located asymmetrically at the nucleoplasmic site of the pore complex in rat cells, and, along with Nup153, is a constituent of the nuclear pore basket structure and/or nucleoplasmic ring (Radu et al, 1995). Karyopherins !/" bind cooperatively to FXFG but not GLFG repeat regions; binding of the NLS-protein/ karyopherin !/" heterodimer to FXFGs stimulated dissociation of the NLS-protein from the karyopherin !/" (Rexach and Blobel, 1995). Nup98 functions as a docking protein via its N-terminal half which contains all of the peptide repeats forming, with peptide repeats of other nucleoporins, an array of sites to
Nup133 and Nup145 in S. cerevisiae are involved in maintaining the architecture of the nuclear envelope and the position of the pore complex in the nuclear envelope; disruption of their genes leads to clustering of pore complexes. Nup116 in yeast (Nup98 in rat, p97 in Xenopus) interacts with Kap95 (karyopherin " in mammals); Nup116 has a number of GLFG repeats which are required for pore function whereas the repeats in Nup49, Nup57, Nup100, and Nup145 are not. Overexpression of Nup116 blocked export of mRNA (Iovine et al, 1995).
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Table 2 "Bipartite" or "split" NLS Signal oligopeptide
Protein and features
C-terminus
Xenopus nucleoplasmin. Deletion analysis demonstrated the presence of a signal responsible for nuclear location.
AVKRPAMINO ACIDT KKAGQA KKK
Xenopus nucleoplasmin
RPAMINO ACIDT KKAGQA KKKKLD
Xenopus nucleoplasmin. Whereas these 17 amino acids had NLS activity, shorter versions of the 17 amino acid sequences were unable to locate pyruvate kinase to the nucleus.
(AVK)RPAMINO ACIDTKKAGQA KKK(KLD)
Xenopus nucleoplasmin. This 14 amino acid segment was identified as a minimal nuclear location sequence but was unable to locate puruvate kinase to the nucleus; three more amino acids at either end (shown in parenthesis) were needed.
CGQAKKKKLD
Xenopus nucleoplasmin-derived synthetic peptide; crosslinked to chicken serum albumin and microinjected to HeLa cells specified nuclear localization. This suggests that nucleoplasmin may possess a simple NLS.
KRPAMINO ACIDT KKAGQA KKKK
Xenopus nucleoplasmin bipartite NLS. Two clusters of basic amino acids (underlined) separated by 10 amino acid are half NLS components.
HRKYEAPRHx6PRKR
Yeast L3 ribosomal protein (387 amino acid) N-terminal 21 amino acid. Possible bipartite NLS. (Ribosomal proteins are transported to the nucleus to assemble with nascent rRNA). Fusion genes with "-galactosidase were used to transform yeast cells followed by fluorescence staining with b-gal antibody. The 373 amino acid of L3 fused to "-gal failed to localize to the nucleus, unless a 8 amino acid bridge containing a proline was inserted between L3 and "-gal.
NKKKRKLSRGSSQKTKGTSASAKARHK SV40 Vp3 structural protein. (35 amino acid C-terminus). By DEAE-dextran-mediated transfection RRNRSSRS (one sequence) of TC7 cells with mutated constructs. RVTIRTVRVRRPPKGKHRK
Simian sarcoma virus v-sis gene product (p28 sis). The cellular counterpart c-sis gene encodes a precursor of the PDGF B-chain (platelet-derived growth factor). The NLS is 100% conserved between v-sis gene product and PDGF. This protein is normally transported across the ER; introduction of a charged amino acid within the hydrophobic signal peptide results in a mutant protein that is translocated into the nucleus. Puruvate kinase-NLS fusion product is transported less efficiently than cytoplasmic v-sis mutant proteins to the nucleus.
KRKIEEPEPEPKKAK
Putative bipartite NLS of Xenopus laevis protein factor xnf7. Inferred by similarity to the bipartite NLS of nucleoplasmin. During oocyte maturation xnf7 is cytoplasmic until mid-blastulaâ&#x20AC;&#x201D;gastrula stage due to high phosphorylation. Partial dephosphorylation results in nuclear accumulation.
KKYENVVIKRSPRKRGRPRKD
Yeast SWI5 gene product, a transcription factor. Underlined basic amino acid show similarity to bipartite NLS of Xenopus nucleoplasmin. The SWI5 gene is transcribed during S, G2 and M phases, during which the SWI5 protein remains cytoplasmic due to phosphorylation by CDC28-dependent histone H1 kinase at three serine residues two near and one (double underlined) in the NLS. Translocated at the end of anaphase/G1 due to dephosphorylation of NLS. NLS confers cell cycleregulated nuclear import of SWI5â&#x20AC;&#x201D;"-galactosidase fusion protein.
MKRKRNS 735-741 GIESIDNVMGMIGILPDMTPSTEMSMRG VRISKMGVDETSSAEKIV 449-495
Bipartite NLS of influenza virus polymerase basic protein 2 (PB2). Mutational analysis of PB2 and transfection of BHK cells showed that both regions are involved in nuclear import. Deletion of 449495 region gives perinuclear localization to the cytoplasmic side.
AHRARRLH 6-13 (BSI) PPRRRVRQQPP 23-33 (BSII) PARARRRR AP 39-48 (BSIII)
"Tripartite" or "doubly bipartite" NLS of adenovirus DNA polymerase (AdPol). BSI and II functioned interdependently as an NLS for the nuclear targeting of AdPol, for which BSIII was dispensable. BSIIIII was more efficient NLS than BSI-II.
KRKx11KKKSKK 207-226
Human poly(ADP-ribose) polymerase (116 kD). The linear distance between the two basic clusters is not crucial for NLS activity in this bipartite NLS. Lysine 222 (double underlined) is an essential NLS component. DNA binding and poly(ADP-ribosyl)ating active site are independent of NLS.
(GRKRAFHGDDPFGEGPPDKKGD)
Herpes simplex virus ICP8 protein (infected-cell protein). This C-terminal portion of ICP8 introduced into pyruvate kinase (PK) caused nuclear targeting in transfected Vero cells. Inclusion of additional ICP8 regions to PK led to inhibition of nuclear localization.
KRPREDDDGEPSERKRARDDR
Bipartite NLS of VirD2 endonuclease of rhizogenes strains of Agrobacterium tumefaciens. Within the C-terminal 34 amino acid. Each region (underlined) independently directs "-glucuronidase to the nucleus, but both motifs are necessary for maximum efficiency. VirD2 is tightly bound to the 5' end of the single stranded DNA transfer intermediate T-strand transferred from Agrobacterium to the plant cell genome.
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Gene Therapy and Molecular Biology Vol 1, page 723 Table 3. "Nonpositive NLS" lacking clusters of arginines/lysines Signal oligopeptide
Protein and features
QLVWMACNSAMINO ACIDFEDL RVLSFIRGTKVSPRG 327-356
Influenza virus nucleoprotein (NP). The underlined region (327-345) when fused to chimpanzee a 1globin at the cDNA level and microinjected into Xenopus oocytes specifies nuclear localization.
MNKIPIKDLLNPQ (NLS1 at N-terminus) VRILESWFAKNIENPYLDT (NLS2 amino acid 141-159, part of homeodomain)
Yeast MAT a2 repressor protein, containing a homeodomain. The two NLS are distinct, each capable of targeting "-galactosidase to the nucleus. However, deletion of NLS2 results in a2 at accumulation at the pores. NLS1 and 2 may act at different steps in a localization pathway. Part of the the homeodomain mediates nuclear localization in addition to DNA binding. The core pentapeptide containing proline and two other hydrophobic amino acids flanked by lysines or arginines (underlined) was suggested as one type of NLS core.
Rx7Kx15KIPRx3HFYEERLSWYSDNED 152-206 (C-terminal segment).
Drosophila HP1 (206 amino acids) that binds to heterochromatin and is involved in gene silencing. NLS identified by "-galactosidase/HP1 fusion proteins introduced by P-element mediated transformation into Drosophila embryos.
FVx7-20 MxSLxYMx4MF
Adenovirus type 5 E1A internal, developmentally-regulated NLS. This NLS functions in Xenopus oocytes but not in somatic cells. This NLS can be utilized up to the early neurula stage.
Table 4. Nucleolar localization signals (NoLS) Signal oligopeptide
Protein and features
MPKTRRR PRRSQRKRPPTP
Nucleolus localization signal in amino terminus of human p27x-III protein (also called Rex) of T cell leukemia virus type I (HTLV-I). When this peptide is fused to N-terminus of "-galactosidase, directs it to the nucleolus. Deletion of residues 2-8 (underlined), 12-18 (double-underline) or substitution of the central RR (dotted-underlined) with TT abolish nucleolar localization. Other amino acids between positions 20-80 increase nucleolar localization efficiency.
RLPVRRRRRRVP
Adenovirus pTP1 and pTP2 (preterminal proteins, 80 kD) between amino acid residues 362-373. The 140 kD DNA polymerase of adenovirus when it has lost its own NLS can enter the nucleus via its interaction with pTP. The staining was nuclear and nucleolar with some perinuclear staining as well. The NLS fused to the N-terminus of E. coli "-galactosidase was functional in nuclear targeting.
GRKKRRQRRRP
HIV (human immunodeficiency virus) Tat protein; localizes pyruvate kinase to the nucleolus. Tat is constitutively nucleolar.
RKKRRQRRR(AHQ) Nucleolar localization signal
Tat positive trans-activator protein of HIV-1 (human immunodeficiency virus type 1). The 3 amino acids shown in parenthesis are essential for the localization of the "-galactosidase to the nucleolus. The 9 amino acid basic region is able to localize "-gal to the nucleus but not to the nucleolus.
PAMINO ACID KRVKLDQRRRP
Artificial sequence from c-Myc and HIV Tat NLSs that effectively localizes pyruvate kinase to the nucleolus.
FKRKHKKDISQNKRAVRR
Human HSP70 (heat shock protein of 70 kD); localizes pyruvate kinase to the nucleus and nucleolus. HSP70 is physiologically cytoplasmic but with heat-shock HSP70 redistributes to the nucleoli, suggesting that the nucleolar targeting sequence is cryptic at physiological temperature and is revealed under heat-shock.
RQARRNRRRR WRERQR (35-50)
HIV-1 Rev protein (116 amino acid; nucleolar). Mutations in either of the two regions of arginine clusters severely impair nuclear localization. "-galactosidase fused to R 4W was targeted to the nucleus, and fused to the entire 35-50 region, was targeted to the nucleolus.
RQARRNRRRRWRERQRQ (35-51)
HIV-1 Rev protein. A fusion of this Rev peptide with "-galactosidase became nuclear but not nucleolar. The 1-59 amino acid segment of Rev fused to "-galactosidase localized entirely within the nucleolus. Whereas the NRRRRW (bold) is responsible for nuclear targeting, the RR and WRERQRQ (double underlined) specify nucleolar localization. Rev may function to export HIV structural mRNAs from the nucleus to the cytoplasm.
Transport across the pore complex is a two-step process involving binding at a site toward the periphery of the pore, docking of the molecules over the central transporter channels across the lumen of the pore complex, and release to the nucleoplasm (Akey and Goldfarb, 1989;
V. Mechanism of nuclear import and translocation across the pore complex A. Molecular mechanisms
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Boulikas: Nucleocytoplasmic trafficking reviewed by Nigg et al, 1991). The guided diffusion involves docking to and undocking from multiple sites across the pore (Radu et al, 1995); the movement of proteins across the pore is a stochastic process operating by means of repeated association-dissociation reactions of the NLS-protein with FXFG repeats on nucleoporins (Rexach and Blobel, 1995). Only the translocation step requires ATP (Richardson et al, 1988). The mechanism of export involves mRNA complexed with proteins containing NLSs forming a large globular mRNP (Mehlin et al, 1992); export might be specified by the presence of a large polyanion (RNA) in the complex whereas import might be specified by the NLS in the protein.
protein to be imported in the cytoplasm. Karyopherin ! accompanies the proteins to be imported from their site of synthesis through the pores to the sites of their function in the nucleus (Görlich et al, 1995b). Two other cytosolic proteins with molecular weights of 56 and 66 kDa have been identified, along with the 66 and 90 kDa karyopherins, to form with NLS-protein a five protein complex (Imamoto et al, 1995). Karyopherins ! and " cooperate to bind to FXFG but not GLFG repeats on nucleoporins (Rexach and Blobel, 1995). Görlich and coworkers (Görlich et al, 1994, 1995b) have identified the 60 and 90 kDa importin subunits in both Xenopus and human cells corresponding to karyopherins ! and " (Moroianu et al, 1996); together they constitute a cytosolic receptor for NLS binding; both subunits appear bound to the pore complex but only the larger subunit enters the nuclear interior. The 60 kDa subunit shows homology to S. cerevisiae S R P 1 , a pore complex protein that contributes to the maintenance of the nucleolar structure (Yano et al, 1992, 1994). Importin-! mediates nuclear protein import by binding nuclear localization signals and importin-". A role for the ! subunit of importin in RNP export has been considered (Laskey et al, 1996).
A single transport gate is located in the central domain of the transporter located within the pore complex that restricts passive diffusion; this was shown using small gold particles coated with polyethylene glycol (PEG; total particle diameter 40-70 Å) or large PEG-particles (total diameter 110-270 Å) which were microinjected into the cytoplasm or nucleoplasm of Xenopus oocytes; cytoplasmic injections of small gold particles showed that the particles were approximately 11 times more concentrated in the cytoplasmic half of the transporter structure whereas the particles were approximately 7 times more concentrated in the nuclear half after nuclear injections. Larger particles were less mobile and after cytoplasmic injection migrated to the surface of the pore complex, but entered the transporter less frequently (Feldherr and Akin, 1997). Macromolecules are poor electrical charge carriers and this can be exploited to detect their movement along electrolyte-filled pores: translocating macromolecules reduce the net conductivity of the medium inside the pore; lesser values of ion conductance indicate greater macromolecular translocation (in size and/or number). This is the principle used in Coulter counter, an instrument for counting and sizing particles. The principle that ion flow is restricted during translocation of macromolecules containing nuclear targeting signals was demonstrated by Bustamante et al (1995). At least four soluble transport proteins have been identified recently: the ! subunit of karyopherin involved in NLS binding (a number of other candidate NLS-binding proteins are known or might be discovered in the future), the " subunit of karyopherin, the Ran, and p10 (Rexach and Blobel, 1995; Radu et al, 1995; Nehrbass and Blobel, 1996).
SRP1 interacts with the Nup1p and Nup2p nuclear pore proteins, is also implicated in the correct orientation of mitotic tubulin spindles, and has been proposed to anchor structural cytoplasmic components to pores thereby organizing proper nuclear matrix structures (Yano et al, 1994). SRP1 and importin 60 are also homologous to Rch1, a protein that interacts with the immunoglobulin
B. Components of the soluble import machinery 1. The
The second human homolog of yeast SRP1, hSRP1, was identified using the yeast two-hybrid system (Zervos et al, 1993) because of its interaction with a RAG-1 activator of V(D)J recombination in immunoglobulin genes (Cortes et al, 1994); the domain of RAG-1 interacting with hSRP1 was not required for recombination (Cortes et al, 1994). hSRP1 contains eight degenerate repeats of 40-45 amino acids four of which (repeats 4-7 between amino acids 245 and 437 not including the acidic stretches of the molecule) are involved in interaction with RAG-1 (Cortes et al, 1994); these repeats are known as arm motifs, have been found in other proteins, and are involved in specific protein-protein interactions (Peifer et al, 1994). For example, arm motifs participate in the interaction between the tumor suppressor adenomatous polyposis coli and "-catenin (Rubinfeld et al, 1993; Su et al, 1993). The human SRP1, interacting with RAG-1 and perhaps also with RNA polymerases, was proposed to localize recombination and transcription near the pore complex providing the anchoring activity and assembling components essential for these processes (Cortes et al, 1994).
subunit of karyopherin
The ! subunit of karyopherin, equivalent to the 60 kDa importin (Görlich et al, 1994, 1995a,b) and to SRP1 and SRP1a recognizes and binds the NLS peptide of the
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Gene Therapy and Molecular Biology Vol 1, page 725 gene recombinase RAG-1 perhaps via the NLS of RAG-1 (Cuomo et al, 1994). 2. The
4. The p10 protein The p10 protein can associate with Ran-GDP (but not to Ran-GTP) and to karyopherin ". p10 binds to nucleoporins possessing peptide repeats (Nehrbass and Blobel, 1996). Addition of GTP to the p10/nucleoporin/ Ran-GDP/karyopherin !/" complex resulted in formation of Ran-GTP causing dissociation of karyopherin ! leaving the karyopherin " bound to nucleoporin (Nehrbass and Blobel, 1996). Release of karyopherin !/NLS-protein then allows the protein to be imported and karyopherin ! to diffuse into the nucleus across the central plug (Görlich et al, 1995a,b).
subunit of karyopherin
The " subunit of karyopherin, equivalent to the 90 kDa importin (Görlich et al, 1994, 1995a,b) and to Kap95 in yeast binds to the karyopherin !/NLS-protein complex in the cytoplasm and mediates docking of the complex to nucleoporins with repetitive tetrapeptide motifs (Iovine et al, 1995; Radu et al, 1995; Weis et al, 1995; Moroianu et al, 1996). Karyopherin " enhances binding of karyopherin ! to NLS-protein (Rexach and Blobel, 1995). Only the " subunit is able to bind pores and binding of the a subunit to the pore depends on karyopherin "; karyopherin " moves only to a distance of 100 nm from its initial cytoplasmic docking site but remains associated with pores and does not appear in the nucleoplasm (Görlich et al, 1994, 1995a,b). The FXFG repeats on nucleoporins appear to stimulate the dissociation of the NLS-protein from the karyopherin !/" heterodimer (Rexach and Blobel, 1995).
The entrance of karyopherin ! in the nucleus is consistent with the model of a shuttling nuclear import receptor (Adam et al, 1989). Dissociation of the NLSprotein from karyopherin ! in the nucleoplasm might be mediated by a difference in the ionic environment between the nucleoplasm and the cytoplasm (Boulikas, 1994), by association of karyopherin ! with other nuclear factors (Görlich et al, 1995b), by association of karyopherin ! with shuttling proteins during their exit from the nucleus (Schmidt-Zachmann et al, 1993), or by phosphorylation of karyopherin ! by a mammalian homolog of the yeast SRP1 kinase (see Radu et al, 1995; Moroianu et al, 1996).
3. RanGTP Ran (in complex with p10) release the docked complex by displacing karyopherin !/NLS-protein; RanGTP and karyopherin ! bind to overlapping sites on karyopherin "; a cluster of basic residues on karyopherin " are the binding sites for RanGTP and karyopherin ! (Moroianu et al, 1996). The small GTPase Ran executes the energydependent step of translocation across the pore complex, results in accumulation of import substrate and karyopherin ! in the nucleus, and in the retention of karyopherin " in the pore complex on both sides of the nuclear pore; in the absence of Ran or energy, karyopherin ! accumulates in the pore but not in the nucleoplasm in permeabilized HeLa cells (Görlich et al, 1995a,b). Ran causes the dissociation of the NLS-protein/ karyopherin ! from the karyopherin " (Rexach and Blobel, 1995; Paschal and Gerace, 1995); incubation of RanGTP with karyopherin !/" heterodimer led to the dissociation of the ! subunit and to the association of the " subunit with Ran; RanGDP had no effect (Rexach and Blobel, 1995). Ran/TC4 is absolutely required for the efficient transport (Moore and Blobel, 1993; Görlich et al, 1995a,b). Ran requires the p10 protein as an active component for its efficient functioning (Nehrbass and Blobel, 1996).
The entrance of karyopherin ! in the nucleus is consistent with the model of a shuttling nuclear import receptor (Adam et al, 1989). Dissociation of the NLSprotein from karyopherin ! in the nucleoplasm might be mediated by a difference in the ionic environment between the nucleoplasm and the cytoplasm (Boulikas, 1993, 1994), by association of karyopherin ! with other nuclear factors (Görlich et al, 1995a), by association of karyopherin ! with shuttling proteins during their exit from the nucleus (Schmidt-Zachmann et al, 1993), or by phosphorylation of karyopherin ! by a mammalian homolog of the yeast SRP1 kinase (see Radu et al, 1995; Moroianu et al, 1996).
C. A summary on the translocation process In summary, protein translocation from the cytoplasm to the nucleoplasm involves the following steps (Nehrbass and Blobel, 1996; Moroianu et al, 1996) (Figure 5): (i ). A weak complex of karyopherin !/NLS-protein is formed in the cytoplasm.
The binding determinants of karyopherin " for RanGTP are similar to Ran BP1, a cytoplasmic Ran-GTPbinding protein, (Coutavas et al, 1993) and to similar domains on nucleoporin Nup 358 (Yokoyama et al, 1995). Displacement of Ran-GTP from karyopherin " may be a requisite for GTP hydrolysis by Ran-GAP (Floer and Blobel, 1996) and may serve to recycle karyopherin".
(i i ). Karyopherin " interacts with karyopherin ! forming a strong karyopherin "/!/NLS-protein complex. Additional proteins may participate to the formation of a larger cytoplasmic complex (Imamoto et al, 1995). (i i i ). The complex binds to FXFG peptide repeats on nucleoporins at the cytoplasmic side of the pore complex
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Boulikas: Nucleocytoplasmic trafficking via karyopherin ". The FXFG repeats may dissociate the NLS-protein from the karyopherin !/".
internalized by receptor-mediated endocytosis into late endosomes. A conformational change of peptides on hemagglutinin (HA) spikes at the lower pH of the endosome (pH 5.5) causes disruption of the endosomal membrane and release of the virus into the cytoplasm (reviewed by Bui et al, 1996). The fusogenic peptides of HA protein of influenza virus have been exploited in gene therapy for the efficient release of DNA-cationic polymer complexes from endosomes (see Fusogenic peptides in Boulikas, 1998, pages 1-172, this volume).
(i v ). p10 docks Ran-GDP to nucleoporin and to the karyopherin heterodimer. (v). A number of association-dissociation reactions on nucleoporins dock the import substrate toward the nucleoplasmic side; this process requires the GTPase Ran and p10. (v i ). A GDP-GTP exchange reaction takes place transforming Ran-GDP into Ran-GTP catalyzed by karyopherin ! which shares sequence homology with a GDP-GTP exchange factor of Ras (GĂśrlich et al, 1994; Peifer et al, 1994). A cytosolic Ran-GTPase activating protein (Ran-GAP) in yeast has been found keeping Ran primarily in the GDP-bound form. Ran-GTP is a secondary product found locally at the pore.
Since the genome is segmented, eight separate helical viral RNPs are formed containing the antisense viral RNA and numerous copies of the 56 kDa (one every 20 nucleotides); other viral proteins in the complex include the three subunits of the polymerase and the 27 kDa M1 viral matrix protein which is released from the vRNPs, presumably in the acid pH of the endosome (see Bui et al, 1996 and the references cited therein). This dissociation step is essential for nuclear import of the vRNPs; two anti-influenza virus drugs, amantadine and rimantadine, inhibit the dissociation of M1 protein from vRNPs. M1 assumes a master regulatory role for the transport of vRNPs across the cell membrane; however, the association of vRNPs with M1 inhibits their nuclear transportation across the pore complex; acidification of the cytosolic compartment caused dissociation of M1 from vRNPs and eliminated the inhibition in import.
(v i i ). Ran-GTP (but not Ran-GDP) causes dissociation of the heterodimeric !/" complex by binding to karyopherin " thus releasing the karyopherin !/NLSprotein. (v i i i ). A complex of karyopherin !/NLS-protein diffuses into the nucleoplasm whereas karyopherin " remains bound to the pore (because of its affinity to FXFG repeats and p10). Shuttling proteins might contribute to coordinating nucleocytoplasmic import/export; these proteins include nucleolin and NO38 (Borer et al 1989), two hsp70-related proteins in Xenopus oocytes (Mandell and Feldherr 1990), the A1 pre-mRNA binding protein (PiĂąol-Roma and Dreyfuss 1992), Nopp 140 (Meier and Blobel, 1992), progesterone receptor (Guiochon-Mantel et al, 1991), La antigen, and several protein kinases (reviewed in Boulikas 1993, 1996). A cap-binding protein has been identified that might mediate export of RNA polymerase II transcripts (Izaurralde et al, 1992).
M1 appears to prevent reimport of vRNPs into the nucleus of the infected cell and thus commits them to an assembly pathway leading to the budding of the virus particles at the cell membrane (Bui et al, 1996). When M1 was dissociated from vRNPs at late times during infection the vRNPs failed to be reimported into nuclei; cell fusion techniques, however, have shown that vRNPs which were dissociated from M1 in acid pH were import competent in the uninfected nucleus; for some unknown reason, infected nuclei, although capable of general nuclear import were no longer able to import vRNPs (Bui et al, 1996). The Hepatitis B virus that, like influenza virus, replicates in the nucleus can be reimported into the nucleus in infected cells, a fact that may explain the chronic nature of Hepatits B infection.
In conclusion, several independent import/export pathways seem to operate in the same cell. Nonbound nucleolin is exported from nuclei and the rate of export is determined by structural domains involved in interactions in the nucleolus; a fusion construct between the cytoplasmic pyruvate kinase and the NLS of T antigen is able to shuttle between nucleus and cytoplasm; lamin B2, a normally nuclear protein, can be converted into a shuttling protein by introducing mutations on its nuclear signal (Schmidt-Zachmann et al, 1993).
VI. Regulated protein import A number of processes have been found to be regulated by nuclear import. These include: the NF- B translocation; the import of Dorsal protein in dorsal but not ventral compartments of Drosophila embryos, a process playing a decisive role in cell type establishment during morphogenesis; the nuclear import of the factors rNFIL-6 and ISGF3 after their phosphorylation in the cytoplasm; the Xenopus nuclear factor 7 which is retained in the cytoplasm from fertilization through the
D. Import of influenza virus ribonucleoproteins Influenza virus is unusual among RNA viruses in that it replicates in the nucleus. When infecting cells it first binds to receptors containing sialic acid and is then
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Gene Therapy and Molecular Biology Vol 1, page 727 mid-blastula stage (Li et al, 1994); and the import of a number of protein kinases including casein kinase II, the catalytic subunit of cAMP-dependent protein kinase II following stimulation of cells with cAMP, the RSK after its phosphorylation by MAP kinase, protein kinase C after stimulation of cells with the phorbol ester TPA, and the Mitogen-Activated Protein kinases (MAPK) ERK1 and ERK2 following stimulation of cells with growth factors or mitogens, (reviewed by Boulikas, 1995).
cytoplasmic protein such as glucocorticoid receptor (GR) induces a conformational change that exposes the NLS, or reconstitutes a functional NLS from remote half NLSs, and the protein is rapidly transported to the nucleus (Picard and Yamamoto, 1987). The ability of GRs to bind DNA is an important determinant for localization and tight binding of GR to the nucleus; mutant GRs localized to the nucleus were only weakly associated with the nuclear compartment (Sackey et al, 1996). However, the related estrogen receptor and retinoic acid receptor, contrary to the glucocorticoid receptor, are nuclear even in the absence of their receptor hormone/morphogen (Picard et al., 1990). An unusual case has been described by Lutz and collaborators (1992) where prenylation of the C-terminus of prelamin A by addition of a 15-carbon or 20-carbon isoprenoid, a posttranslational modification that functions in the proteolytic processing of prelamin A to lamin A in the nucleus, is also required for its nuclear import.
MAPK is activated in cytoplasm by MAPK kinase (MAPKK) in response to extracellular signals; whereas MAPK then is translocated to nucleus, MAPKK remains cytoplasmic because of a NES in the N-terminal region (residues 32-44) rich in leucine residues; the NES peptide of MAPKK, inhibited the nuclear export of ovalbumin (Fukuda et al, 1996). The NLS of the serum response factor (S R F ) is in close proximity to potential phosphorylation sites for the cAMP-dependent protein kinase (A-kinase) and nuclear transport of SRF proteins requires basal A-kinase activity (Gauthier-Rouviere et al, 1995).
VII. Deregulation in nuclear import and molecular carcinogenesis
The NLS needs to be exposed on the surface of the protein and available for binding to nuclear transporter protein molecules. The SV40 large T protein NLS is nonfunctional when it is located in a region of pyruvate kinase predicted not to be exposed to the surface (Roberts et al., 1987). In addition, nuclear proteins have been identified which are synthesized in the form of precursor molecules which remain in the cytoplasm, presumably because their NLS is hidden. Cleavage of such proteins into mature molecules by specific proteases exposes their NLS, and they are then rapidly transported to the nucleus. As an example, the p50 subunit of the transcription factor NF- B (50 kD) is synthesized in the form of a 110 kD precursor with the NLS buried in the protein; proteolytic cleavage giving the 50 kD transcription factor exposes the NLS (Henkel et al., 1992) and facilitates nuclear localization.
Several studies have provided a link between the deregulation in nuclear import mechanisms of specific proteins and cancer. The BRCA1 breast cancer marker protein was found to be mainly localized in the cytoplasm in 16 of 17 breast and ovarian cancer lines and in 17 of 17 samples of cells from malignant effusions whereas in normal cells the protein was nuclear (Chen et al, 1995). The transforming oncoprotein v-Abl of Abelson murine leukemia virus, a mutated form of the c-Abl nonreceptor tyrosine kinase, is a fusion protein in which portions of the retroviral Gag protein replace the Nterminal SH3 domain of c-Abl; this results in loss of phosphorylation sites in v-Abl that down-regulate its activity and render the tyrosine kinase activity of v-Abl constitutive; in addition, the viral Gag sequence provides a myristoylation site on v-Abl which confers a predominantly inner plasma membrane anchorage whereas c-Abl is predominantly located in the nucleus (Wong et al, 1995). Both of these properties of v-Abl, not found in cAbl, contribute to its ability to transform cells.
Nuclear translocation of protein factors, presumably by exposure of their hidden NLS or by reconstitution of a functional NLS from two remote half NLS, can be triggered by phosphorylation (Shelton and Wasserman, 1993), dephosphorylation (Moll et al., 1991; Nasmyth et al., 1990), subunit association (Levy et al., 1989), or by dissociation of an inhibitory subunit (Baeuerle and Baltimore, 1988a,b). Interferon-! regulates nuclear translocation of the transcription factor ISGF3 (Kessler et al., 1990). Thus, distant peptide regions can either "mask" the NLS or anchor the protein in the cytoplasm; examples of proteins regulated in this manner include protein kinase Ca (James and Olson, 1992), human c y c l i n s A and B1 (Pines and Hunter, 1991), and c-Fos (Roux et al., 1990). In other cases, binding of hormone to a
VIII. RNA and protein export A. An historical perspective The concepts that (i ) transcription occurs preferentially at the nuclear periphery near pores versus (i i ) transcription occurring at any location within nuclei both have been entertained and data to support either model are available. Transcripts from the interior of nuclei have been visualized passing through channels originating at the sites of transcription within the interior of the nucleus and emanating to the pore complex (Huang and Spector,
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Boulikas: Nucleocytoplasmic trafficking 1991). RNA transcripts seem to be exported vectorially toward a single pore or a small subset of nuclear pores (Lawrence et al., 1989). This vectorial export of RNA might contribute to an asymmetric mRNA distribution within the cytoplasm consistent with experimental evidence (Weeks and Melton, 1987). Foci where splicing of pre-mRNA takes place are often seen associated with pore complexes via morphologically distinct channels; U3 snRNA was exclusively detected in the nucleolus and U2, U5 and U6 snRNAs were in discrete nucleoplasmic foci (Carmo-Fonseca et al, 1991).
dependent process requiring the 5' cap structure (Hamm and Mattaj, 1990; Dargemont and Kuhn, 1992). RNP particles become attached to filaments which project into the nucleoplasm and which guide the particles to the pores. The central channel can expand to permit export of large complexes such as ribosomal subunits and mRNPs and import of large nuclear proteins. RNA export comprises (i) initial binding to the tentacles of the central plug (ii) energy-dependent translocation toward the cytoplasmic side of the pore complex (Mehlin et al, 1992). Some asymmetries between the cytoplasmic and nucleoplasmic rings have been described; for example, the cytoplasmic ring appears larger and the nucleoplasmic filaments of the pore are longer, forming a basket structure. One of the nucleoporins is attached to the nucleoplasmic side (Snow et al, 1987). These asymmetries are likely to contribute to the RNP export distinct from protein import into nuclei (Mehlin et al, 1992).
RNA export is facilitated by proteins that shuttle between nucleus and cytoplasm. Assays based on interspecies heterokaryons and microinjection of Xenopus oocytes using nucleolin, mutant lamins, differing in their abilities to be incorporated into the lamina and pyruvate kinase-NLS artificial reporter protein, have shown that proteins unable to interact with large nuclear structures can be exported from nuclei; protein export was suggested not to require any export signals (Schmidt-Zachmann et al, 1993). However, recently a nuclear export sequence (NES) has been identified in proteins that are exported actively from the nucleus such as Rev and PKI (Fischer et al, 1995; Wen et al, 1995; reviewed by Gerace, 1995; Izaurralde and Mattaj, 1995). Several RNA-binding proteins have been suggested to be involved in RNA export. Export of 5SRNA requires interactions with ribosomal protein L5 or TFIIIA (Guddat et al, 1990). Export of influenza virus RNA-protein complex requires the viral protein M1 which also prevents the nuclear reimport of the viral RNA (Martin and Helenius, 1991).
Electron microscope tomography has examined the export of large RNPs in the salivary glands of the dipteran Chironomus. A 75S pre-mRNA is transcribed from the Balbiani ring granules in Chironomus tentans and is packed into RNP ribbon particles, 30-60 nm broad and 1015 nm thick, bent into a ring conformation; during export the particle is first oriented in a specific manner by specific recognition signals and subsequently the bent ribbon is gradually straightened and exported through the pore with the 5' end of RNA in the lead (Mehlin et al, 1992). Upon passage through pores, the proteins of the pre-mRNP dissociate; the protein composition of the particle in the nucleus and cytoplasm is different. The particle then unfolds and becomes associated with ribosomes (Mehlin et al, 1992).
Export of tRNA is a translocation process mediated by protein carrier(s) (Zasloff, 1983). tRNA molecules undergo a complex maturation processing involving trimming of the 5' and 3' ends, addition of three terminal CCA residues, and base modification; removal of introns (only 20% of tRNAs contain introns) is a highly regulated process occurring in association with the inner side of the nuclear envelope close to the pores (for references see Simos et al, 1996). Studies in yeast have shown that Los1 (required for pre-tRNA splicing) and Pus1 (involved in tRNA biogenesis) interact with the pore complex protein Nsp1; this involves pore proteins in the splicing of pre-tRNA (Simos et al, 1996).
Some early studies showed that no specific nuclear export signals are required. Schmidt-Zachmann et al (1993) arrived to the conclusion that a protein does not require positively acting export signals to be transported from nucleus to cytoplasm but instead, its shuttling ability is limited primarily by intranuclear interactions. Expression of some proteins can inhibit mRNA export; the mechanism could be exerted at the splicing step rather than on the actual translocation process. Expression of NS1 protein (which is encoded by the influenza virus RNA segment 8 along with NS2 produced from the same transcript as NS1 by differential splicing) in influenza virus-infected cells induced a generalized block of mRNA export from the nucleus; NS1 mRNA, NS2 mRNA and other mRNAs were retained in the nucleus of cells expressing NS1 protein, but no effect was observed when only NS2 protein was expressed (Fortes et al, 1994).
Ribosomal subunits are assembled in the nucleus from imported ribosomal proteins; export of ribosomal subunits to the site of their function (cytoplasm) is energydependent (Khanna-Gupta and Ware 1989; BataillĂŠ et al 1990). The nucleoside triphosphatase of nuclear envelope might be involved in the nucleocytoplasmic translocation of ribonucleoprotein (Agutter et al 1976). The mechanism of export of mRNA deduced by microinjection of Xenopus oocytes is also an energy-
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B. Identification of NLS-independent import pathways
bearing hnRNP proteins are mediators of mRNA export (Nakielny and Dreyfuss, 1996).
Proteins lacking basic type of NLS are thought to piggy-back into the nucleus in association with NLScontaining molecules. However, a new type of receptor molecule mediates nuclear import in a basic NLSindependent manner (Pollard et al, 1996; Aitchison et al, 1996; reviewed by Dingwall, 1996). This model applies to the import of hnRNPA1 molecules (Pollard et al, 1996) and to the import nuclear mRNA-binding proteins in Saccharomyces cerevisiae which have been isolated as a complex with Kap104 (Aitchison et al, 1996).
Microinjection into the nucleoplasm of Xenopus oocytes of PEG-coated gold particles showed that these were coated with protein containing nuclear export signals (NES) suggesting that the NES is not only required for translocation, but also for migration within the nucleoplasm (Feldherr and Akin, 1997). Nuclear trafficking of the catalytic (C) subunit of cAMP-dependent protein kinase (cAPK) is regulated by the heat-stable inhibitor (Pkl) of cAPK which contains a nuclear export signal (NES) (residues 35-49). Pkl has no obvious association with RNA. The core NES of Pkl comprises only residues 37-46, LALKLAGLDI and is able to trigger rapid, active net extrusion of the C-PKl complex from the nucleus.
The cytosolic yeast karyopherin, Kap104p, acts for returning mRNA binding proteins to the nucleus after mRNA export. Indeed, Kap104p binds directly to repeatcontaining nucleoporins and to the mRNA binding proteins, Nab2p and Nab4p, and functions for their nuclear import; depletion of Kap104p resulted in a rapid shift of Nab2p from the nucleus to the cytoplasm without affecting the localization of other nuclear proteins (Aitchison et al, 1996).
C. Nuclear export signals (NES) and export of mRNA: recent studies According to Guiochon-Mantel et al (1994) the NLS of the progesterone receptor or the NLS of T antigen were shown to impart to "-galactosidase the ability to shuttle between the nucleus and the cytoplasm; microinjected proteins devoid of a nuclear localization signal were unable to exit from the nucleus. The authors thought that the nuclear import requires energy whereas the nuclear export does not and this determines whether the NLS will function as an import or export signal. The discovery of nuclear export signals (NESs) in a number of proteins revealed the occurrence of signaldependent transport of proteins from the nucleus to the cytoplasm. The consensus motif of the NESs is a leucinerich, short amino-acid sequence. The NES is defined by its ability to translocate a protein from the nucleus to the cytoplasm when the two are tethered by a membranepermeable ligand (Klemm et al, 1997). The majority of proteins with NES are RNA-binding proteins which bind to and escort RNAs to the cytoplasm; nuclear export of RNA molecules is likely to be driven by protein-based nuclear export signals (reviewed by Nakielny and Dreyfuss, 1997; Lee and Silver, 1997). Nascent pre-mRNAs associate with the abundant hnRNPs and remain associated with them throughout the time they are in the nucleus. One group of HnRNPs is strictly nuclear in interphase cells (for example hnRNP C proteins), whereas the other group, although primarily nuclear at steady state, shuttles between the nucleus and the cytoplasm via NES; NES-
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The identification of NES have established a novel mechanism for regulation of gene expression: nuclear export of pre-mRNA can contribute to the regulation of gene expression. The processing of transcripts of TNF-" and "-globin was found to be regulated by the signal transduction pathway that includes the Src protein; Src seems to act on a general mechanism of splicing and/or mRNA transport. This regulation could involve RNAbinding proteins, which interact with Src (Neel et al, 1995).
D. CRM1 or exportin 1 binds NES A protein of 110 kDa (CRM1 or exportin 1 or XPO1) was identified in Xenopus oocyte extracts that binds to the intact NES but not to the mutated, non-functional NES. CRM1 is an essential mediator of the NES-dependent nuclear export of proteins in eukaryotic cells (Fukuda et al, 1997). CRM1 is an evolutionarily conserved protein, shown to , originally found as an essential nuclear protein in fission yeast; S. cerevisiae CRM1 shows homology to importin "-like transport factors and was shown to be an essential mediator of nuclear protein export in S. cerevisiae (Stade et al, 1997). The cytotoxin leptomycin B which inhibits the NES-mediated transport of Rev protein inhibited the binding of Xenopus CRM1 to NES (Fukuda et al, 1997). Overexpression of CRM1 in Xenopus oocytes stimulated Rev and U snRNA export from the nucleus and this process was inhibited by leptomycin B, a cytotoxin that was shown to bind to CRM1 protein; CRM1 was able to form a complex involving cooperative binding of both RanGTP and the nuclear export signal (NES) from either the Rev or PKI proteins implicating RanGTP in nuclear export (Stade et al, 1997; Fornerod et al, 1997). A mutation in the shuttling protein Crm1p affects not only protein export, but also mRNA export, indicating that
Boulikas: Nucleocytoplasmic trafficking these pathways are tightly coupled in S. cerevisiae (Stade et al, 1997). Retroviruses export unspliced, intron-containing RNA to the cytoplasm of infected cells despite the fact that intron-containing cellular RNAs cannot be exported; in HIV-1 this is accomplished by Rev which binds to elements in the viral RNA; in the absence of Rev, these intron-containing HIV-1 RNAs are retained in the nucleus (Zhang et al, 1996). The NES on Rev is the sequence LQLPPLERLTL (Wen et al, 1995). Visualization of viral transcripts using oligonucleotide probes specific for the unspliced or spliced forms of a particular HIV-1 viral RNA showed that in the absence of Rev, the unspliced HIV-1 viral RNAs were predominantly nuclear and were distributed (i) as approximately 10-20 intranuclear punctate signals of nascent transcripts and (ii) as a stable population of viral transcripts dispersed throughout the nucleoplasm excluding nucleoli (Zhang et al, 1996). Kim and coworkers (1996) have pinpointed the NES on HTLV-1 Rex that fully complements HIV-1 Rev as a stretch of 17 amino acids; four leucines within the minimal region were essential for NES function; this NES peptide could serve as nuclear export signal when conjugated with bovine serum albumin. The genome of the simpler retrovirus Mason-Pfizer monkey virus (MPMV) contains an element that serves as an autonomous nuclear export signal for intron-containing viral and cellular RNA through interaction with endogenous cellular factors; the same element is also essential for MPMV replication (Ernst et al, 1997).
polymerase II is inhibited, it seems that the M9 signal is a specific sensor for transcription-dependent nuclear transport. Consistent with in vitro data A1 dissociates from transportin 1 by RanGTP after nuclear import and becomes incorporated into hnRNP complexes, where A1 functions in pre-mRNA processing (Siomi et al, 1997). A novel human protein, termed MIP (101 kDa) which bears significant homology to human karyopherin/ importin-", binds M9 specifically; cytoplasmic microinjection of a truncated form of MIP that retains the M9 binding site blocked the in vivo nuclear import of a substrate containing the M9 without affecting the import of basic NLS-bearing substrates (Fridell et al, 1997). The shuttling hnRNP K protein contains also a novel shuttling domain (termed KNS) which has many of the characteristics of M9, in that it confers bi-directional transport across the nuclear envelope. KNS-mediated nuclear import is dependent on RNA polymerase II transcription, and a classical NLS can override this effect. Furthermore, KNS accesses a separate import pathway from either classical NLSs or M9 demonstrating the existence of a third protein import pathway into the nucleus (Michael et al, 1997).
IX. Regulated nuclear import and export A. Proteins with NLS and NES The transcription factor NF-ATc plays a key role in the activation of many early immune response genes and is regulated by subcellular localization. NF-ATc translocates from the cytoplasm to the nucleus in response to a rise in intracellular calcium. Calcineurin dephosphorylates conserved serine residues in the amino terminus of NF-AT, resulting in nuclear import (Beals et al, 1997). NF-ATc immediately returns to the cytoplasm when intracellular calcium levels fall a process mediated by a NES; glycogen synthase kinase-3 (GSK-3) phosphorylates conserved serines necessary for nuclear export and opposing Ca2+ /calcineurin signaling (Klemm et al, 1997; Beals et al, 1997).
E. Transportin is distinct from karyopherin (importin) A novel 38 amino acid transport signal was identified by Pollard and coworkers (1996) in the hnRNP A1 protein (which shuttles rapidly between the nucleus and the cytoplasm), termed M9, which confers bidirectional transport across the nuclear envelope. Furthermore, a specific M9-interacting protein, termed transportin, binds to wild-type M9. Transportin is a 90 kDa protein, distantly related to karyopherin " which also participates in mRNA export in a complex with hnRNP A1 and mRNA. Thus, it appears that there are at least two receptormediated nuclear protein import pathways.
The distribution of the v-Rel oncoprotein between the nucleus and the cytoplasm was experimentally manipulated using NLS and NES; the respective abilities of the v-Rel to localize to the nucleus in chicken embryo fibroblasts, to activate %B-dependent transcription in yeast, and to transform avian lymphoid cells were each markedly reduced by the fusion of a cis-acting NES onto v-Rel; the oncogenic properties of v-Rel were manifested only after a threshold of this protein in the nucleus was attained (Sachdev et al, 1997).
Transportin mediates the nuclear import of additional hnRNP proteins, including hnRNP F. A novel transportin homolog, transportin 2, which may differ from transportin 1 in its substrate specificity has also been identified and sequenced (Siomi et al, 1997). Because transportin 1 is localized both in the cytoplasm and the nucleoplasm and a pyruvate kinase-M9 fusion, which normally localizes in the nucleus, accumulates in the cytoplasm when RNA
Fluorescein iodoacetamide-labeled human p53, injected into the cytoplasm or nuclei of 3T3 cells, was imported
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Gene Therapy and Molecular Biology Vol 1, page 731 into or exported from nuclei within minutes. Import was inhibited by co-injection of the lectin wheat germ agglutinine (WGA). In contrast, the protein HSA conjugated with the T antigen NLS was only imported but not exported. These studies demonstrate the presence of NES on p53 in addition to import signals (NLS) and provide new views for its implication in carcinogenesis (Middeler et al, 1997). The subcellular localization and activity of c-Abl nonreceptor tyrosine kinase is regulated by cell adhesion. Upon adhesion of fibroblasts to fibronectin (an extracellular matrix protein) there is a coincident export of c-Abl from the nucleus to the cytoplasm. The cytoplasmic pool of c-Abl is reactivated within 5 min of adhesion and the activated cytoplasmic c-Abl becomes nuclear after 30 min. Thus, c-Abl can transmit integrin signals to the nucleus where it may integrate these to cell cycle signals (Lewis et al, 1996). Fragile X syndrome, a leading cause of inherited mental retardation, is attributable to the unstable expansion of a CGG-repeat within the FMR1 (Fragile X syndrome Mental Retardation) gene; the encoded protein (FMRP) is a ribosome-associated RNA-binding protein that contains both NLS and NES. Immuno-gold studies provided evidence of nucleocytoplasmic shuttling of FMRP, which was localized in neuronal nucleoplasm and within nuclear pores. FMRP was highly expressed in neurons but not glia throughout the rat brain; the dendritic localization of FMRP implicated this ribosomal protein in the translation of proteins involved in dendritic structure or function that could relate to the mental retardation occurring in fragile X syndrome (Feng et al, 1997).
The total number of hnRNPA1 and hnRNPA2 molecules in each HeLa cell nucleus is in the order of 7090 millions; during mitosis these proteins are released into the cytoplasm and are reimported after biogenesis of the new nuclear envelopes around the daughter cell nuclei. The import rate for these molecules could be 500 molecules per pore per minute assuming one hour for re-accumulation of the hnRNPA molecules in the nucleus (Dingwall, 1996). HnRNPA1 is one of a set of hnRNP proteins that do not posses an NLS. A stretch of 38 amino acids in A1, which interacts with a human protein called transportin, is both necessary and sufficient for nuclear import. Yeast Kap104 seems to be the analog of transportin and both display a region with homology to a domain in importin-" (Görlich and Mattaj, 1996) which might interact with similar domain in nucleoporins to mediate docking of the transportin-hnRNPA1 or Kap104-protein complex through the pore (Aitchison et al, 1996).
X. Import and export of U snRNPs In contrast to the concept of export of mRNPs there are cases where RNA-protein complexes are imported into nuclei. Small nuclear ribonucleoprotein particles (snRNPs) in particular U1 snRNPs, are assembled in the cytoplasm and are then imported into nuclei to facilitate splicing. Import of snRNPS and proteins may involve distinct pathways (Fischer et al 1991; Michaud and Goldfarb, 1992). The import of U1 snRNPs requires a trimethyl-G cap structure as well as protein binding to the Sm domain of U1 snRNA (Hamm et al, 1990; Fischer and Lührmann, 1990). Recently a role of the yeast importin-! (SRP1p) in nuclear export of capped U snRNAs has been unraveled in a remarkable series of events. Approximately 30% of SRP1p were found in a nuclear complex with the Saccharomyces cerevisiae nuclear cap-binding protein complex (CBC) which promotes nuclear export of capped U snRNAs and shuttles between nucleus and cytoplasm. Xenopus CBC is associated with importin-! in the nucleus and CBC might shuttle while bound to importin!. Binding of importin-" in the cytoplasm, a binding which displaces the RNA from the CBC-importin-! complex, and the commitment of CBC for nuclear reentry trigger and promote the release of capped U snRNAs into the cytoplasm (Görlich et al, 1996).
B. Import/export of HnRNPs Heterogeneous nuclear ribonucleoproteins (HnRNPs) is a group of 20 different hnRNP proteins designated RNPA to RNPU. Among these the C1, C2, and U molecules possess the basic NLS. However, the group A molecules do not. In spite of this, a major group of hnRNP proteins constantly shuttle between the nucleus and the cytoplasm (Michael et al, 1995; Pollard et al, 1996). HnRNPs can be divided into those that remain always nuclear and those that shuttle between the cytoplasm and the nucleus; the association of mRNA with those that posses NES and shuttle is believed to be largely responsible for mRNA export from the nucleus. hnRNP C proteins are restricted to the nucleus not because they lack an NES, but because they bear a nuclear retention sequence (NRS) that is capable of overriding NESs (Nakielny and Dreyfuss, 1996). The NRS in hnRNP C1 is a stretch of 78 amino acids; it was proposed that removal of NRS-containing hnRNP proteins from pre-mRNA is an essential step for mRNA export (Nakielny and Dreyfuss, 1996).
XI. Observations on nucleocytoplasmic trafficking pertaining to plasmid import A model was proposed (Boulikas, 1997a) for the import of plasmid DNA by taking into consideration the following observations or ideas:
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Boulikas: Nucleocytoplasmic trafficking (i ) DNA microinjected into Xenopus eggs or naked DNA incubated with frog oocyte extracts is spontaneously condensed into nucleosomes and chromatin and is then assembled into nuclei in vitro by the formation of a double nuclear envelope around the condensed DNA; the egg or egg extracts contain all the essential components for this process (Newport, 1987). We expect plasmid DNA to be complexed with a number of DNA-binding proteins in the cytoplasm and after nuclear import to be converted into chromatin structures; attachment to the nuclear matrix is a prerequisite for transcription and replication.
transferred DNA (T-DNA) portion of Ti (tumor-inducing) plasmid which enters infected plant cells and integrates into plant nuclear DNA (direct repeats define the T-DNA ends on Ti plasmid). Transfer begins when the VirD2 endonuclease produces a site-specific nick. Two Agrobacterium proteins, VirD2 and VirE2 containing NLS associate directly with T plasmid and mediate its nuclear import. VirE2 alone, which has been shown to actively transport ssDNA into the plant cell nucleus, packages ssDNA into semi-rigid, hollow cylindrical filaments with a telephone cord-like coiled structure as was shown by scanning transmission electron microscopy (STEM); these complexes were proposed to be actively imported through pore complexes (Citovsky et al, 1997). This is a clear example of plasmid import mediated by plasmid-associated proteins possessing NLS.
(i i ) RNA is exported from nuclei in the form of a complex with proteins (Mehlin et al, 1992); ribosomal subunits are preformed in the nucleus from imported ribosomal proteins and are then exported as large RNAprotein complexes. Some U snRNPs are imported into nuclei (Hamm et al, 1990). We expect expulsion of plasmid DNA through pore complexes to the cytoplasm after its nuclear import to be negligible.
(v i i i ) Binding NLSs from SV40 T antigen to luciferase plasmid DNA promoted transgene expression following injection of DNA-NLS complexes into the cytoplasm of zebra fish eggs; NLS peptides, but not nuclear-import-deficient peptides, mediated import of DNA from the cytoplasm into embryo nuclei, under conditions in which naked DNA was not imported. Thus, use of NLS may reduce the need for elevated DNA copy numbers in some gene transfer applications (Collas et al, 1996; Collas and Alestrom, 1997).
(i i i ) Condensation of plasmid DNA with histones increases several-fold the efficiency of expression of foreign genes (Wagner et al, 1991; Fritz et al, 1996). (i v ) Complexation of the DNA with HMG proteins shortens significantly the time required for gene expression after transfection (Kaneda et al, 1989). According to this procedure Sendai virus was used to fuse DNA-loaded ganglioside liposomes with protein-containing membrane vesicles purified from red blood cells; cointroduction of HMG-1 protein showed rapid uptake of plasmids by nuclei; replacement of HMG-1 by BSA resulted in localization of the grains of the in situ hybridization in the cytoplasm after 6 h reaching the nucleus only after about 24h (Kaneda et al, 1989).
(i x ) Intact, protein-free SV40 DNA was localized to the nucleus after it was cytoplasmically injected into cells in a process which was inhibited (i) by wheat germ agglutinin (ii) by an anti-nucleoporin antibody which block the nuclear pore complex and (iii) by energy depletion. During this process the DNA accumulated at the nuclear periphery before its import and, as opposed to protein import, DNA import required transcription; furthermore, imported DNA colocalized with the SC-35 splicing complex antigen, suggesting localization to areas of transcription or message processing. The SV40 origin of replication and the early and late promoters supported import, whereas bacterial sequences alone and other SV40derived sequences did not (Dean, 1997).
(v) Plasmid DNA condensation with polylysine also enhances transfection of cell cultures; however, polylysine (18-24 kDa), microinjected into the cytoplasm of Tetrahymena, remained cytoplasmic; polylysine (5-9 kDa) was evenly distributed between the cytoplasm and the micro- and macronuclei of Tetrahymena by diffusion following microinjection; thus, large polylysine molecules cannot be imported into nuclei (White et al, 1989). Polylysine-plasmid complexes are proposed to be uncomplexed in the cytoplasm followed by binding of nascent nuclear proteins before plasmid import can take place. Plasmid complexation with polylysine may only help internalization through the cell membrane but not nuclear import.
(x) Fusion of liposomes with the cell membrane will release the encapsulated DNA into the cytoplasm; this mechanism is rather rare and liposomes seem to be internalized by receptor mediated endocytosis if appropriate ligands are exposed on its surface, by poration, especially when cationic lipids are present, or via phagocytosis ending into endosomes and lysosomes (Martin and Boulikas, 1997). Cationic lipids destabilize the biological membranes (both cytoplasmic and lysosomal) and mediate rapid delivery of plasmid to the cytoplasm (reviewed by Boulikas, 1998 page 1-172, this volume). Lysis of the liposome in the endosome or caveolae will release DNA
(v i ) Oligonucleotides tagged with NLS target the nucleus more efficiently than free oligos (Seibel et al, 1995); the same oligos tagged with mitochondrial signals enter mitochondria (Seibel et al, 1995). (v i i ) Agrobacterium tumefaciens elicits tumors on plant hosts by transporting a single-stranded (ss) copy of
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Gene Therapy and Molecular Biology Vol 1, page 733 inside vesicles; fusion of the liposome with the endosome or caveolae membranes will release the DNA into the cytoplasm; the presence of fusogenic peptides on the liposome will promote lysis of the endosomal membrane and release of the liposome-plasmid complex into the cytoplasm.
proteins in the cytoplasm as a prerequisite for its import. The binding efficiency and the type of proteins that are complexed in the cytoplasm with the plasmid DNA are matters of speculation. A number of studies support the model that nascent cytoplasmic proteins containing nuclear localization signals are complexed with the transfected DNA and mediate its nuclear import. What type of nascent nuclear proteins might be responsible for mediating plasmid translocation into nuclei in vivo? Certainly histones are abundantly synthesized in dividing cells and histone H1 has been shown to display an affinity for supercoiled over relaxed DNA plasmids (Singer and Singer, 1976). A number of transcription factors (TFs) and other nuclear proteins are synthesized de novo in actively proliferating cells (but at lower rates in terminally differentiated cells); these proteins could bind to the plasmid, especially to promoters and enhancers in a sequence-specific manner, and mediate the import of the plasmid-TF complex.
(x i ) Viruses have evolved different mechanisms for entry into cells (and into nuclei). For example, after entry into the cytoplasm the adenoviral particle is attached to the cytoplasmic side of pore complexes and the DNA is released to the interior of pore annuli entering the nucleoplasm. These highly ordered processes are accompanied by losses or protease degradation of specific proteins on the viral particles; a viral protease, L3/p23, located inside the capsid at 10 copies per virion, plays a key role in the stepwise dismantling and in the weakening of the capsid structure culminating with the release of the adenovirus DNA by degrading of the viral capsid protein VI (Greber et al, 1996). (x i i ) Spliced mRNAs are exported from nuclei via interaction with RNA-binding proteins (mainly from the HnRNP family) which contain nuclear export signals. Since these interactions are specific we expect the export of plasmid DNA, once it is imported to the nucleus, to be negligible.
XIII. Perspectives
(x i i i ) Fluorescently labeled oligonucleotides, after delivery using DOTAP liposomes, entered the cell using an endocytic pathway and redistributed from punctate cytoplasmic regions into the nucleus; nuclear uptake took place only with positively charged complexes; DOTAP increased over 100 fold the antisense activity of a specific anti-luciferase oligonucleotide (Zelphati and Szoka, 1997). The nuclear membrane was found to pose a barrier against nuclear import of oligonucleotides which accumulated in the perinuclear area; although DOSPA/DOPE liposomes could deliver ODNs into the cytosol, these liposomes were unable to mediate nuclear import of ODNs; on the contrary oligonucleotide-DDAB/DOPE complexes with a net positive charge were released from vesicles into the cytoplasm and mediated nuclear import of the oligos (Lappalainen et al, 1997). Labeled oligonucleotides delivered to animals by tail vein injection in complexes with DC-Chol:DOPE liposomes were localized primarily to phagocytic vacuoles of Kupffer cells at 24 h postinjection; nuclear delivery of oligonucleotide in vivo was not observed (Litzinger et al, 1996).
XII. A model for the nuclear import of plasmid DNA Taking into account these observations we have proposed a plausible model for the nuclear import of plasmid DNA after its cytoplasmic localization (Boulikas, 1997a; F i g u r e 5 ). Plasmid is complexed with nuclear
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Antisense and triplex-forming oligonucleotides, in their single- or double-stranded form, as well as RNA or DNA oligonucleotides have been extensively used in targeting nuclear DNA. The chemistry for covalent coupling of oligonucleotides to peptides has been established. Linkage of oligonucleotides to NLS peptides or to mitochondrial import peptides resulted in nuclear or mitochondrial targeting, respectively (Seibel et al, 1995). Oligo-nucleotides may enter nuclei after their crosscomplexation with nuclear proteins in the cytoplasm. Studies with fluorescent-labeled single-stranded oligonucleotides show binding to RPA in vitro (Costas Koumenis, Stanford, Personal communication). RPA is the main single-stranded DNA-binding activity present in mammalian cells. Understanding the rules that govern trafficking through the pore complex is instructive to our comprehension of plasmid uptake by nuclei during somatic gene transfer and for developing strategies to overcome obstacles for foreign gene expression by enhancing the nuclear import. Because of their increase rates of proliferation and protein import, cancer cells are expected to be more susceptible to nuclear import of plasmid and to uptake transfected plasmid at higher rates compared with terminally differentiated cells. However, cancer cells especially solid tumors of epithelial origin (lung, colon, head & neck, brain tumors) do not readily internalize particles such as liposomes (Martin and Boulikas, 1997); the step of translocation across the cell membrane, and not the step of nuclear import, is expected to be the rate limiting step in the overall gene transfer procedure in these cancer cells.
Boulikas: Nucleocytoplasmic trafficking
Figure 5. A model for the import of proteins into nuclei. The pore complex is shown with its octagonal symmetry. S t e p 1 . A complex of the plasmid DNA with one or more proteins possessing NLS (NLS-protein) is formed in the cytoplasm; NLS proteins might include histones, HMGs, transcription factors, or other DNA-binding proteins after their de novo synthesis on polyribosomes; the NLS-protein then binds to karyopherins !/". 2 . The complex is docked by binding to multiple sites on nucleoporins (structural proteins of the pore complex). 3 . The p10 and Ran-GTP dissociate karyopherin !/NLS-protein-plasmid complex which is expelled to the nucleoplasm resulting in plasmid DNA nuclear import. Adapted from Boulikas T (1 9 9 7 a ) Nuclear localization signal peptides for the import of plasmid DNA in gene therapy. I n t J O n c o l 10, 301-309. Reproduced with kind permission from the International Journal of Oncology.
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Boulikas: Nucleocytoplasmic trafficking Cancer is a disease of the control of the cell cycle and cell signaling involving mutations in a number of oncogenes and tumor suppressor genes (Spandidos, 1985). Our prediction that tumor cells will import plasmidprotein complexes across the nuclear envelope more efficiently than nondividing cells provides a basis for the preferential targeting of cancer cells and might have important implications in human gene therapy.
Acknowledgments Special thanks to Emile Zuckerkandl for stimulating discussions.
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The ATP-driven protein translocation-motor of mitochondria Chitkala Satyanarayana and Martin Horst* Zentrum für Biochemie und Molekulare Zellbiologie der Universität Göttingen, Abtl. Biochemie 2, Gosslerstr. 12d, D37073 Göttingen, Germany __________________________________________________________________________________________________ *
Corresponding author, tel: (49) 551 39 5949, fax: (49) 551 39 5979; e-mail: horst@uni-bc2.gwdg.de
Summary The majority o f mitochondrial proteins are encoded i n the nucleus. In the c y t o s o l they are synthesized as precursor proteins which are transported into mitochondria. Protein import into mitochondria requires a concerted action of a variety of different proteins. For the transport of precursor proteins across the mitochondrial inner membrane and their folding in the matrix the mitochondrial hsp70 (mhsp70) chaperone plays an essential role. Mhsp70 is found in at least two protein complexes within mitochondria: together with Tim44 and mGrpE, mhsp70 forms the import-complex and together with Mdj1 and mGrpE i t forms a folding-complex. This review focuses on the function of the import-complex. It is believed that mhsp70 can act as a mechanochemical enzyme that actively pulls precursor proteins across the inner membrane.
peptidase. Finally, the precursor protein folds with or without the help of the matrix localized chaperones. Mitochondrial hsp70 (mhsp70) is essential for the translocation across the inner membrane for all matrix targeted precusor proteins as well as for the folding of some precursor proteins in the matrix space.
I. Introduction Mitochondria do contain their own DNA. However, this DNA encodes only about a dozen proteins. The majority of the mitochondrial proteins are encoded in the nucleus and are imported from the cytosol. These proteins are synthesized in the cytosol as precursor proteins containing usually an N-terminal mitochondrial targeting signal. They are co- or post-translationally transported into mitochondria. Cytosolic members of the hsp70 family as well as a mitochondrial import stimulating factor facilitate the transfer of the precursor to the receptor complex (Hachiya et al., 1995). Different precursor proteins bind to different subcomplexes of the heterotetrameric receptor. These precursors are then delivered to the insertion pore (Hachiya et al., 1995; Komiya et al., 1997). The translocation of precursor proteins across both mitochondrial membranes is facilitated by the components of the translocase found in the outer membrane (Tom) and the translocase found in the inner membrane (Tim). Translocation requires a loosely folded conformation of the precursor protein during import, an electrochemical potential across the mitochondrial inner membrane and ATP in the matrix space. Following translocation, the presequence is removed by the matrix localized processing
One of the most fascinating questions about protein import into mitochondria is how a matrix localized mhsp70 chaperone can facilitate import of precursor proteins from the cytosol into mitochondria. In this review we will focus on this aspect of mhsp70 function.
II. The HSP70 chaperone family Heat shock proteins of the Hsp70 family are found in nearly all organisms. These proteins play an essential role in protein folding, transport into different cellular compartments, and regulation of the heat-shock response (reviewed in: Hightower et al., 1994; McKay et al., 1994). The members of this protein family are highly conserved from bacteria to man (Lindquist and Craig 1988; Boorstein et al., 1994). Some members of the hsp70 family are constitutively expressed whereas others are expressed only under stress conditions (Lindquist and Craig, 1988; Boorstein et al., 1994). Hsp70 proteins bind to unfolded, 741
Satyanarayana and Horst: The ATP-driven protein translocation-motor of mitochondria hydrophobic surface-exposed segments of polypeptide chains (Pelham, 1986; Blond Elguindi et al., 1993; Flynn et al., 1991; Landry et al., 1992; Zhu et al., 1996). However, the actual mechanism of hsp70 function is still not completely understood. Binding and release of substrates (polypeptides or peptides) by hsp70 is regulated by adenine nucleotides. The conformation of hsp70 changes during the cyle of ATP-binding, ATP-hydrolysis, and release of ADP and Pi (Flaherty et al., 1990; Hightower et al., 1994; von Ahsen et al., 1996). Studies on DnaK, the hsp70 protein of E. coli, demonstrated that optimal functioning of this chaperone requires the cochaperones DnaJ and GrpE (reviewed in Georgopoulos and Welch, 1993). DnaJ accelerates ATP hydrolysis by DnaK, whereas GrpE acts as an adenine nucleotide-exchange factor for DnaK (Liberek et al., 1991; McCarty et al., 1995). Homologs of DnaJ and/or GrpE cooperate with hsp70 proteins in the eukaryotic cytosol and in the lumenal spaces of mitochondria and the endoplasmic reticulum (Bolliger et al., 1994; Caplan et al., 1993; Ikeda et al., 1994; Laloraya et al., 1994; Schlenstedt et al., 1995; Horst et al., 1997a).
A. HSP70 chaperones involved in protein translocation a. BiP The lumen of the endoplasmic reticulum (ER) contains an abundant hsp70 chaperone: BiP (heavy chain binding protein) in mammals and Kar2p in yeast. The protein was initially identified because of its key role in the folding of newly-imported ER proteins (Brodsky and Schekman, 1993). Later it was found that this protein also mediates the ATP-dependent translocation of proteins into the yeast ER, and that this function involves the ATP-regulated interaction of BiP/Kar2p with the membrane protein Sec63p (reviewed in: Brodsky and Schekman, 1994; Brodsky, 1996). Kar2p is needed for co- as well as posttranslational translocation into the yeast ER (Brodsky et al., 1995). When ER membranes are solubilized and fractionated in the absence of ATP, Kar2p copurifies with Sec63p (Brodsky and Schekman, 1993). Genetic studies have confirmed the functional importance of the Kar2pSec63p interaction (Scidmore et al., 1993). Kar2p appears to recognize a region in Sec63p that is homologous to the conserved "J domain" in members of the DnaJ protein family (Sadler et al., 1989; Ang et al., 1991). b. Mitochondrial HSP70 In Saccharomyces cerevisiae the product of the SSC1 gene (mitochondrial hsp70) is located within mitochondria. Genetic and biochemical evidence implicate mhsp70 as a component of the mitochondrial protein import machinery. 742
Mhsp70 is essential for growth (Craig et al., 1987). In vitro import studies of mitochondria isolated from a temperature sensitive mhsp70 mutant showed defects in protein import and an accumulation of precursor proteins at contact sites (Kang et al., 1990). Furthermore, mhsp70 can be crosslinked to or co-immunoprecipitated with precursor proteins on their way to the matrix (Kang et al., 1990; Scherer et al., 1990). Subsequent experiments have shown that mhsp70 forms a transient, ATP-dependent interaction with newly imported precursor proteins (Manning-Krieg et al., 1991). Interestingly, it has been shown that a temperature sensitive mhsp70 allele fails to bind and to complete import of a partially translocated precursor protein (Gambill et al., 1993). These data and others suggest that mhsp70 is playing a major role in mitochondrial protein import and protein folding (reviewed in: Langer and Neupert, 1994; Rassow et al., 1996). Mhsp70 performs these different functions together with several different partner proteins. The first identified mhsp70 complex was the sitespecific endonuclease Endo.SceI, which is a dimer consisting of mhsp70 and a 50-kDa nuclease subunit (Morishima et al., 1990). In the heterotetrameric protein folding complex, mhsp70 works with two partner proteins (Horst et al., 1997a): Mdj1p, a homolog of bacterial DnaJ (Rowley et al., 1994); and a mitochondrial GrpE (mGrpE) dimer, a homolog of bacterial GrpE (bGrpE) (Bolliger et al., 1994; Laloraya et al., 1994; Nakai et al., 1994). Mdj1 is itself a chaperone (Prip-Buus et al., 1996). However, it also stimulates the ATPase-activity of mhsp70 suggesting that it supports mhsp70 function during protein folding (Horst et al., 1997a). MGrpE functions as a mitochondrial ADP-ATP exchange factor for mhsp70 (Azem et al., 1997). In E. coli the hsp70 chaperone system comprises of three proteins: DnaK, DnaJ, and bGrpE (Georgopoulos and Welch, 1993; Szabo et al., 1994). The mitochondrial hsp70 folding system is the only one in eukaryotes that contains a DnaJ as well as a bGrpE homolog, suggesting that the mitochondrial system is the closest eukaryotic homolog of the bacterial system (Bolliger et al., 1994; Laloraya et al., 1994; Nakai et al., 1994; Rowley et al.,1994; Westermann et al., 1995; Prip-Buus et al., 1996; Horst et al. 1997a). In the import complex, mhsp70 forms a heterotetrameric complex with Tim44 and a mGrpE dimer (Kronidou et al., 1994; Rassow et al., 1994; Schneider et al., 1994). Tim44 functions in this complex as a membrane anchor for mhsp70. Tim44 spans the mitochondrial inner membrane, appears to be intimately associated with the translocation channel (Maarse et al., 1992; Scherer et al., 1992) and contains a domain that is weakly homologous to the J domain of Sec63p. It was suggested that the J-like domain of Tim44 is the binding site for mhsp70 (Rassow et al., 1994). If so, the
Gene Therapy and Molecular Biology Vol 1, page 743 translocation systems of mitochondria and the ER would be rather similar (see however, Brodsky, 1996).
the one for the opposite movement of the polypeptide chain.
III. Protein translocation models What is the mechanism for the translocation of precursor proteins across the mitochondrial membranes? Two translocation mechanisms were suggested: a "Brownian-Ratchet" and "Translocation-Motor" (Neupert et al., 1990; Simon et al., 1992; Glick, 1995; Pfanner and Meijer, 1995; Horst et al., 1997b).
A. The "Brownian-Ratchet" model In the "Brownian-Ratchet" model, precursor polypeptides randomly oscillate within the translocation channel. In the first version of the "Brownian-Ratchet" model this random oscillation was suggested to be only due to Brownian-molecular motion. When the precursor protein moves inward, a mhsp70 molecule binds to the emerging segment of the polypeptide chain in the matrix, thereby preventing its reverse movement. Repeating such a binding event will finally lead to the translocation of the entire precursor protein across the inner membrane into the matrix space (Figure 1). Tim44 can have two different functions: a more passive function just as a membrane anchor for mhsp70 or a more active one as a promotor for binding of mhsp70 molecules to the precursor chain. Tim44 could perform the latter function by acting in a DnaJ-like fashion to catalyze ATP hydrolysis by mhsp70. The observation that precursor chains can slide bidirectionally in the mitochondrial translocation channel strongly supports the "Brownian-Ratchet" model (Ungermann et al., 1994). This "sliding" was suggested to be due to random thermal motion of the polypeptide chain in the translocation channel (Neupert et al., 1990; Simon et al., 1992; Glick, 1995; Pfanner and Meijer, 1995; Horst et al., 1997b).
An extended version of the "Brownian-Ratchet" model has been proposed to explain the import of precursor proteins containing a tightly folded domain. In this model the folded domain spontaneously unfolds at the mitochondrial surface due to random thermal movements ("breathing" of the molecule). This would allow an inward movement of the precursor chain (Stuart et al., 1994). Thus, the rate of import of such a folded precursor protein is limited by its spontaneous unfolding rate. For example, fusion proteins containing mouse dihydrofolate-reductase (DHFR) are imported into mitochondria within 10-20 minutes at room temperature (Endo and Schatz, 1988); and DHFR spontaneously unfolds, on average, once every few minutes at this temperature (Viitanen et al., 1991). Such import kinetics can easily be explained by the "BrownianRatchet" model. However, the DHFR unfolds spontaneously very fast, much faster than the majority of other proteins. A single unfolding transition usually requires hours or even days (Creighton, 1993). For example, this is true for the heme-binding domain of cytochrome b2, which is already tightly folded after release of the nascent chain from the ribosome. Nevertheless, the precursor is imported in vitro within only a few minutes (Glick et al., 1993). Interestingly, in contrast to DHFR fusion proteins, cytochrome b2 absolutely requires both matrix ATP and functional mhsp70 for import (Glick et al., 1993; Voos et al., 1993; Stuart et al., 1994), suggesting that mhsp70 uses the energy of ATP hydrolysis to accelerate unfolding of the heme-binding domain at the mitochondrial surface.
B. The "ATP-dependent translocationmotor" model The above mentioned results and others have lead to another model for the translocation across the mitochondrial inner membrane, the so called "ATPdependent Translocation-Motor" model (Glick, 1995; Pfanner and Meijer, 1995, Horst et al., 1997). In this model the precursor bound mhsp70 undergoes a conformational change upon ATP hydrolysis, thereby generating an inward force on the precursor chain. As a result the polypeptide is pulled into the matrix space (F i g u r e 1 ). The role of Tim44 in both translocation models would be different: in the "Brownian-Ratchet" model Tim44 positions mhsp70 just close to the import site, whereas in the "Translocation-Motor" model Tim44 would not only position mhsp70 close to the import pore, but would furthermore allow for force generation by serving as a membrane anchor for mhsp70 during its "powerstroke". In both models, Tim44 could also promote
If Brownian motion was the sole factor involved in protein translocation a precursor protein which can not be fully imported due to a tightly folded domain should slide back out of the translocation channel in a few milliseconds. In reality this process takes some minutes (Ungermann et al., 1996). This suggests that some sort of interaction occurs between the translocating polypeptide and the components of the translocation channel, hindering free diffusion of the polypeptide chain in the translocation channel. The created "friction" between the polypeptide chain in the translocation channel could partly be responsible for the unidirectionality of the translocation process. This would assume that the "friction" for the inward movement of the polypeptide chain is smaller than
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Gene Therapy and Molecular Biology Vol 1, page 744 F i g . 1 Comparison of the "Brownian-Ratchet" and the "Translocation-Motor" model for the translocation of polypeptides across the mitochondrial inner membrane (adapted from: Glick et al, 1995). Dark grey: translocation channel; light grey: mhsp70; IM: Mitochondrial inner membrane.
membrane close to the import site and the heme-binding domain remains stably folded (Glick et al., 1993).
the binding of mhsp70 to the precursor chain. In the "ATP-dependent Translocation-Motor", the action of mhsp70 during protein import is analogous to the ATPdependent translocation of actin filaments by myosin (Spudich, 1994).
Another approach to test the two different models is to characterize the nucleotide-dependent interactions (ATPasecycle) of mhsp70 with Tim44 and a precursor protein. According to the "Translocation-Motor" model the generation of a force on the translocating precursor requires that at some point hsp70 must be bound simultaneously to its membrane anchor and to the translocating polypeptide chain. In contrast the "Brownian-Ratchet" model requires that mhsp70 dissociates from the membrane anchor after ATP-hydrolysis so that the translocating polypeptide chain can continue to oscillate in the channel. ATP promotes the dissociation of hsp70 proteins from unfolded polypeptides due to nucleotide dependant conformational changes (Hightower et al., 1994). ATP also disrupts the Tim44-mhsp70 and Sec63p-BiP/Kar2p complexes (Brodsky and Schekmann, 1993; Kronidou et al., 1994; Schneider et al., 1994; Rassow et al., 1994). Recent kinetic studies have suggested that ATP binding rather than ATP hydrolysis leads to the release of hsp70 proteins from peptide substrates (Palleros et al., 1993; Prasad et al., 1994; Schmid et al., 1994; Azem et al., 1997). Therfore the effect of poorly hydrolysable ATP analogs on the interactions between mhsp70 and Tim44 was investigated (Horst et al., 1996). These analogs promote dissociation of mhsp70 from Tim44, whereas mhsp70 remains bound to Tim44 in the presence of ADP. Thus, the current evidence implies that the ATP-bound form of mhsp70 reversibly associates with Tim44 and incoming precursor proteins, whereas ATP hydrolysis
To test the prediction that mhsp70 can accelerate unfolding of a precursor protein the following experiments using a precursor protein containing the DHFR-domain fused to the cytochrome b2 presequence of variable length were performed. Precursor whose presequence is not long enough to span both mitochondrial membranes, and having a folded DHFR-domain, are imported slowly. Precursor proteins with a longer presequence are imported orders of magnitude faster (Matouschek et al., 1997). It seems therefore that if the presequence of these precursor proteins is long enough to span both mitochondrial membranes, mhsp70 can actively unfold the tightly folded heme-binding domain on the mitochondrial surface. Precursor proteins with a short presequence can not be unfolded as the presequence can not interact with mhsp70. Mhsp70 may have an unfolding activity linked to a conformational change which generats a power stroke on the precursor. If the "Translocation-Motor" model should hold true it has to be ruled out that the environment at the entrance of the mitochondrial import channel can unfold precursor proteins. There is some evidence against that: import of DHFR fusion proteins with short presequences is not faster than the spontaneous unfolding of DHFR in solution. Furthermore, in ATP-depleted mitochondria the cytochrome b2 presequence inserts proper into the outer 744
Gene Therapy and Molecular Biology Vol 1, page 745 generates an ADP-bound form of mhsp70 that associates tightly with Tim44 and the precursor. A conformational change of the ADP-bound mhsp70 molecule would then exert an inward pulling force on the precursor chain
Taken together these results have lead us to propose the following schematic representation of the mhsp70 action (Glick, 1995; Pfanner and Meijer, 1995, Horst et al., 1997b; Figure 2).
The presequence of a mitochondrial precursor is first translocated across both membranes into the matrix where it interacts with mhsp70. Experiments with different length precursors have shown that at least 50 residues are needed to span the two mitochondrial membranes (Rassow et al., 1990). Therefore some process other than mhsp70dependent pulling must initiate the translocation process. It was suggested that the electrochemical potential across the inner membrane is essential for insertion of the mitochondrial presequence across the inner membrane (Schleyer and Neupert, 1985; Cyr et al., 1993). Many mitochondrial precursor proteins like DHFR fusion proteins with presequences as short as 12 amino acids can be imported, even if the DHFR moieties are initially folded (Hurt et al., 1985). In these cases the DHFR-domain is most likely spontaneously unfolded on the mitochondrial surface ("breathing" of the DHFR domain) followed by the translocation of the presequence into the matrix where it comes into contact with mhsp70. The
IV. The function of the "translocationmotor" Based on the observation that ATP is initially needed for the interaction of mhsp70 with the incoming precursor chain (Schneider et al., 1994) in stage 1, mhsp70-ATP associates transiently with Tim44 and the precursor. In the next stage mhsp70 hydrolyzes ATP and mhsp70-ADP associates stably with the precursor and Tim44. This is consistent with the observation that in the absence of ATP, mhsp70 copurifies with Tim44 (Kronidou et al., 1994; Schneider et al., 1994; Rassow et al., 1994) and that mhsp70, the precursor and Tim44 form a stable complex under these conditions (Horst et al., 1996). In stage 3, mhsp70 undergoes a conformational change thereby pulling a stretch of the precursor inwards. Indeed, it was recently shown that upon ATP-binding mhsp70 undergoes a conformational change (von Ahsen et al., 1996). In stage 4, an ADP-ATP exchange reaction, which is facilitated by mGrpE (Azem et al., 1997) takes place. Following the conformational change and the ATP-ADP exchange, mhsp70-ATP dissociates from Tim44 and the precursor (stage 5). This cycle (Figure 2) is repeated until the precursor protein is completely imported into the matrix. According to this model mhsp70 together with its associated proteins function remarkably similar to the proposed ATP-dependent mechanism of other hsp70 chaperones (Greene et al., 1995; McCarty et al., 1995) and to the conventional force generating systems such as the interaction found between myosin and actin cables (Spudich et al., 1994).
V. Outlook Future experiments on the structure of this translocation complex and the interaction of its subunits at the molecular level should allow us to distinguish between the two translocation models. Most interestingly, in the intermembrane space of chloroplasts there is an hsp70 like protein anchored to the inner face of the outer membrane (Marshall et al., 1990; Schnell et al., 1994). This protein may also function by pulling precursor proteins across the chloroplast outer membrane. The "Translocation-Motor" may therefore be an universal mechanism for translocating proteins across organellar membranes.
F i g . 2 Model of the reaction cycle of the ATP-dependent "Translocation-Motor" (modified from: Horst et al., 1996). For details see paragraph IV. Dark grey: translocation channel; light grey: mhsp70 and mGrpE; IM: Mitochondrial inner membrane.
It could also be possible to use techniques originally invented for studies in the motor protein field (Walker and Sheetz, 1993): mhsp70 could be coupled to a plane solid support so that all molecules are aligned. If mhsp70
"Brownian Ratchet" model may therefore account for this initial step in the import process.
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functions as a molecular motor a precursor protein may move along the mhsp70 molecules.
Acknowledgements
Craig, E.A., Kramer, J., Kosic-Smithers, J. ( 1 9 8 7 ) . SSC1, a member of the 70-kDa heat shock protein multigene family of Saccharomyces cerevisiae, is essential for growth. P r o c . N a t l . A c a d . S c i . U S A 84, 4156-4160.
We thank W. Oppliger and S. Fedkenhauer for excellent technical assistence. We are indebted to members of our laboratories for helpful discussions. We would especially like to thank Drs. N.G. Kronidou and P.V. Schu for comments on the manuscript. M. Horst was supported by a Heisenberg fellowship and by grants from the German Research Society (DFG).
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