Human tumor-derived p53 exhibit binding site selectivity and TS for transactivation by Charles J. DiComo, PhD

Human tumor-derived p53 proteins exhibit binding site selectivity and temperature sensitivity for transactivation in a yeast-based assay Charles J Di Como and Carol Prives Department of Biological Sciences, Columbia University, New York, New York 10027, USA

p53 is a sequence-speci®c transcriptional activator with a number of known target genes which contain p53responsive elements. Mutations in p53 have been identi®ed within its sequence-speci®c DNA binding domain in more than half of all human tumors, although a subset of tumor-derived p53 mutants have retained the ability to bind DNA and activate transcription under certain conditions. In order to broaden our understanding of this transactivating ability, we examined the e cacy by which p53 mutants bind to and activate reporters in an Saccharomyces cerevisiae-based assay. Analysis of 19 human tumor-derived p53 mutants, spanning the DNA binding domain of p53 and including the `hot-spot' class, revealed a broad array of transcriptional transactivation abilities at 248C, 308C and 378C, despite the fact that each mutant had originally been identi®ed as being inactive for transactivation in yeast against a single p53responsive RGC site-containing reporter. One class of mutants (P177L, R267W, C277Y and R283H) retained wild-type or near wild-type activity that is binding siteselective, even at physiological temperature (378C). Another class of mutants (V143A, M160I/A161T, H193R, Y220C and I254F), all positioned for maintaining the b-sca old of p53, also retained selective activity, but preferentially at sub-physiological temperatures (248 and 308C). Strikingly, however, in contrast to the other tumor derived mutants, all of the previously identi®ed `hot-spot' mutants were completely inactive with all sites tested. Moreover, a double mutant, L22E/W23S, located within the activation region and previously shown to be transcriptionally inactive in ®broblasts, retained wildtype or near wild-type binding site-selective activity in yeast. Finally, we found that transcriptional activity in vivo does not necessarily correlate with DNA binding in vitro. Keywords: p53; tumor-derived mutants; transactivation; Saccharomyces cerevisiae; FASAY

Introduction The p53 tumor suppressor gene encodes a protein which has been found to be mutated in over 50% of all human tumors (Friend, 1994; Hollstein et al., 1994). In response to genotoxic stress resulting in genomic instability, signals are transmitted to activate p53, e ectively leading to cell cycle arrest and/or apoptosis

Correspondence: C Prives Received 22 January 1998; revised 6 April 1998; accepted 7 April 1998

(reviewed in Gottlieb and Oren, 1996; Ko and Prives, 1996; Levine, 1997; and references therein). P53 is a nuclear phosphoprotein which functions as a transcription factor by binding DNA predominantly as a tetramer (a dimer of a dimer) to two pairs of a consensus sequence 5'-PuPuPuC(A/T)-3' arranged as inverted repeats (reviewed in Vogelstein and Kinzler, 1992; Prives, 1994). Transactivation by p53 may be mediated through direct contacts with components of the general transcription machinery, as interactions between the activation domain of p53 and p300/CBP (Avantaggiati et al., 1997; Gu and Roeder, 1997; Gu et al., 1997; Lill et al., 1997; Scolnick et al., 1997), TATAbinding protein (TBP) (Seto et al., 1992; Liu et al., 1993; Martin et al., 1993; Ragimov et al., 1993; Truant et al., 1993; Chang et al., 1995; Farmer et al., 1996), TFIIH components (p62, ERCC2 and ERCC3) (Xiao et al., 1994; Wang et al., 1995; Leveillard et al., 1996), and a number of TAFs (Drosophila TAFII40 and TAFII60, human TAFII31) (Lu and Levine, 1995; Thut et al., 1995; Farmer et al., 1996) have been demonstrated. Many of the target genes that contain p53-responsive cis-acting elements have been implicated in the regulation of the cell cycle, DNA synthesis and apoptosis, such as; p21 (El-Deiry et al., 1993; Harper et al., 1993; Xiong et al., 1993; Noda et al., 1994), mdm2 (Wu et al., 1993), GADD45 (Kastan et al., 1992), Cyclin G (Okamoto and Beach, 1994; Zauberman et al., 1995), Bax1 (Miyashita and Reed, 1995) and IGF-BP3 (Buckbinder et al., 1995). The p53 protein is modular and can be divided into distinct domains: (1) a transcriptional activation region at the amino terminus, comprising two subdomains (residues *1 ± 40 and *40 ± 70) (Fields and Jang, 1990; Raycroft et al., 1990; Unger et al., 1992; Chang et al., 1995; Candau et al., 1997); (2) a `PXXP' SH3-like domain (residues *61 ± 94) (Walker and Levine, 1996); (3) a sequence-speci®c DNA binding domain (residues *102 ± 292) (Bargonetti et al., 1993; Halazonetis and Kandil, 1993; Pavletich et al., 1993; Wang et al., 1993); and (4) a nonspeci®c- or damaged DNA-binding carboxyl terminal regulatory domain (residues *320 ± 393): containing a tetramerization domain (residues *320 ± 360); a basic domain (residues *363 ± 393); and two nuclear localization signals (Shaulsky et al., 1991; Wang et al., 1993; Brain and Jenkins, 1994; Bayle et al., 1995; Lee et al., 1995; Reed et al., 1995). A survey of the genomic mutations in p53 often reveals a deletion of one allele coincident with a missense mutation of the other allele. The vast majority of missense mutations identi®ed in over 6800 tumor-derived p53 alleles occur in the DNA binding domain (Hollstein et al., 1994; Levine et al., 1995; Hainaut et al., 1998). The solving of the crystal structure of the p53 DNA binding domain bound to its cognate site (Cho et al., 1994) has helped to

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explain how mutations in this domain interfere with DNA binding and has reinforced the importance of the DNA binding domain in the normal function of p53. Among the mutations in the p53 DNA binding domain are six amino acids residues (R175, G245, R248, R249, R273 and R282), often referred to as `hotspots', which are mutated with an unusually high frequency and together comprise about 40% of all p53 missense mutants (Nigro et al., 1989; Hollstein et al., 1991; 1994). These residues have been divided into two classes, `conformational' (e.g.: R175, G245, R249 and R282) and `contact' (e.g.: R248 and R273), depending on whether they contact DNA directly or aid in maintaining the structural integrity of the DNA binding domain (discussed in Cho et al., 1994). Many studies have addressed the DNA binding properties and the transcription transactivation function of tumor-derived p53 mutants with the assumption that mutations in the DNA binding domain would modify or destroy the ability of p53 to bind to DNA and activate transcription (reviewed in Gottlieb and Oren, 1996; Ko and Prives, 1996; Levine, 1997). The results of these experiments have upheld the assumption that tumor-derived p53 mutants exhibit aberrant DNA binding and transcriptional transactivation. These mutant proteins are also frequently expressed at high levels in most tumors due to increased stability. That increased levels of wild-type p53 can induce arrest or apoptosis in certain cell types has led to the strategy of trying to identify therapeutic agents that can restore DNA binding and transcriptional transactivation function to mutant p53 in vivo (Abarzua et al., 1995; 1996; Selivanova et al., 1997). A number of studies have characterized tumorderived p53 mutant function with respect to transactivation and sequence-speci®c DNA binding. Examining endogenous intact mutant p53 proteins in tumor cell lines (Park et al., 1994; Niewolik et al., 1995), transiently transfected tumor-derived p53 mutants in p53 null cell lines (Chen et al., 1993a; Chumakov et al., 1993; Friedlander et al., 1996b) and mutant p53/GAL4 DNA binding domain chimeras in CHO cells (Raycroft et al., 1991; Unger et al., 1993; Miller et al., 1993) has provided information that some mutants under restricted conditions display DNA binding or transactivation. For example, Friedlander et al. (1996b) found that at the physiological temperature (378C), p53 mutants are defective for sequence-speci®c DNA binding, whereas, at sub-physiological temperatures (25 ± 338C), mutants are capable of binding to DNA. Wild-type p53 is also temperature sensitive (Picksley et al., 1992; Hainaut et al., 1995; Friedlander et al., 1996b), but markedly less so than the mutants (Friedlander et al., 1996b). Although naturally occurring missense mutations have helped to elucidate and identify the central DNA binding domain, experimentally derived mutations in other regions of p53 have been informative as well. Particularly relevant to the work in our study is a double mutation L22E/W23S within the activation domain of p53 (Lin et al., 1994). Mutation of p53 at these two residues has been shown to dramatically reduce its ability to activate transcription in mammalian cells (Lin et al., 1994; Candau et al., 1997), as well as to bind to TAFs (Lu and Levine, 1995; Thut et al., 1995) and p300/CBP (Gu et al., 1997).

Prior results have demonstrated that mammalian p53 can function as a transcription factor in yeast (Scharer and Iggo, 1992). To further study mutant p53, we have used an S. cerevisiae-based assay ®rst described by Ishioka et al. (1993) into which either wild-type or mutant forms of p53 are introduced into a His7 yeast strain carrying a reporter with a p53 binding site (RGC) driving the HIS3 gene. Yeast grown in the absence of histidine will only grow if the reporter is activated. While this assay was originally devised as a diagnostic tool, it has other advantages as well. It allows for rapid and quantitative assessment of the transcriptional activity of di erent forms of p53 in an essentially isogenic background, avoiding possible complications resulting from transient transfection of mammalian cells such as overexpression of p53 at nonphysiological levels. Furthermore, the process of DNA transfection itself can activate or stabilize endogenous p53 (Siegel et al., 1995) suggesting that transfected p53 might be similarly altered. Since Ishioka et al. (1993) found that all tumor-derived mutants tested were inactive with the RGC-containing HIS3 reporter, it was of interest to determine whether a number of alternative p53 binding sites within this reporter would be able to serve as response elements for wild-type and mutant forms of p53. Furthermore, the fact that yeast grows well at temperatures ranging from 24 ± 378C has allowed us to further study temperature sensitive transactivation by p53. Our results indicate that wildtype and some mutant forms of p53 are transcriptionally active with a number of di erent p53-responsive reporters. We discuss the possible consequences a cell may face by maintaining a p53 mutant which retains selective transcriptional activity.

Results Human wild-type p53 in yeast activates p53-responsive reporters Strains were constructed which contained the HIS3 gene (see Materials and methods) under the control of one of the following derived p53-responsive human or murine target gene cis-acting elements: p21 (El-Deiry et al., 1993); mdm2 (Wu et al., 1993); GADD45 (Kastan et al., 1992); Cyclin G (Okamoto and Beach, 1994); Bax (Miyashita and Reed, 1995); IGF-BP3 Box A and Box B (Buckbinder et al., 1995); ribosomal gene cluster (termed RGC) (Kern et al., 1991b); and an arti®cial high a nity binding-p53 consensus element (termed SCS) (Halazonetis et al., 1993) (see Figure 1 and Materials and methods). Each of the above reporter strains were transformed with plasmids expressing either human wild-type or mutant p53 under control of the constitutive alcohol dehydrogenase (ADH1 ) minimal promoter (Ishioka et al., 1993). We chose the constitutive ADH1 promoter to express wild-type and mutant p53 proteins which does not express extremely high levels of p53, since previous work has demonstrated that high level expression of wild type p53 and certain tumor-derived mutants imparts a slow growth phenotype in both S. cerevisiae (Nigro et al., 1992) and Schizosaccharomyces pombe (Wagner et al., 1991, 1993; Bischo et al., 1992). The growth assay utilized for our phenotypic analysis relies on the fact

Target gene selectivity by mutant p53 proteins CJ DiComo and C Prives

that the HIS3 gene is under the control of an inactive GAL1 promoter (lacking an upstream activating sequence or UAS). This promoter is activated and HIS3 expressed only when bound by a transcriptional activator, such as p53, at sites placed upstream of the minimal GAL1 promoter. We score transactivation as growth or lack thereof on histidine-de®cient media. Con®rming the results of Scharer and Iggo (1992), we observed p53-dependent HIS3 transcription of the RGC-containing reporter as assayed by growth on histidine-de®cient media (Figure 2b). Additionally, wild-type p53 transactivated to varying degrees all reporters, with the exception of those containing IGFBP3 Box A and Box B (Figure 2b and not shown). As a control, isogenic strains expressing either: (1) wildtype p53 and containing a HIS3 reporter with no p53responsive cis-acting element; or (2) vector control and any one of the p53-responsive cis-acting element reporters did not grow on histidine-de®cient media (Figure 2b, Tables 1, 2 and 3 (scored as `7'), and not shown), demonstrating the speci®city of the assay. Interestingly, the extent to which wild-type p53 was able to transactivate each reporter was dependent on the cis-acting element present upstream of the HIS3 coding sequence. Whereas the RGC- and Bax-containing reporter strains grew slowly on histidine-de®cient media (Figure 2b and Tables 1, 2 and 3, scored as `+'), the p21-, SCS-, mdm2-, GADD45- and Cyclin Gcontaining reporter strains grew with a growth rate scored as `+++' (hereinafter referred to as `wildtype') (Figure 2b and Tables 1, 2 and 3). While IGFBP3 Box A and Box B have been shown to be p53responsive cis-acting elements in mammalian cells

(Buckbinder et al., 1995; Friedlander et al., 1996b; Ludwig et al., 1996), we detected no such activation in yeast (Tables 1, 2 and 3 (scored as `7'), and not shown). Moreover, the relative activation of these reporters by wild-type p53 was equal at all temperatures (248C, 308C and 378C), with two exceptions: (1) wild-type p53 activated the GADD45-containing reporter to a lesser extent than the p21-, SCS-, mdm2- and Cyclin G-containing reporters at 248C, but yet greater than the Bax- and RGC-containing reporters at the same temperature; and (2) wild-type p53 did not activate the Bax-containing reporter at 248C (not shown). To determine if the extent to which wild-type p53 transactivated the p53-responsive reporters was due to the expression level of p53 in the cell, each of the strains shown in Figure 2a were grown to log phase and total cell extracts were prepared. As detected by Western blot analysis with a mixture of anti-p53 antibodies, the protein levels of wild-type p53 in each strain were readily detected and were quite similar (Figure 3). Therefore, the growth rate di erences are not due to variations in the levels of p53 protein, but are due to the ability of wild-type p53 to transactivate the cis-acting element present in each reporter. The DNA binding properties of yeast-expressed human wild-type p53 are similar to immunopuri®ed human wildtype p53 Using the electrophoretic mobility shift assay (EMSA), we compared DNA binding by either immunopuri®edor yeast-expressed human wild-type p53 proteins at

Figure 1 The p53-responsive cis-acting elements. Sequences are derived from the naturally occurring cis-acting elements found in p53-responsive target genes: p21 (El-Deiry et al., 1993); mdm2 (Wu et al., 1993); GADD45 (Kastan et al., 1992); Cyclin G (Okamoto and Beach, 1994); Bax1 (Miyashita and Reed, 1995); and IGF-BP3 (Buckbinder et al., 1995). The `H' refers to human origin, while the `M' refers to murine origin. The SCS high a nity binding site was identi®ed by Halazonetis et al. (1993) and represents the perfect copy of the consensus deduced by El-Deiry et al. (1992)

2529

Target gene selectivity by mutant p53 proteins CJ DiComo and C Prives

2530

248C to radiolabelled oligoduplex DNA containing the p53-responsive cis-acting element from p21 (CUO3/4, see Materials and methods). Human p53 expressed in

Figure 2 Expression of human wild-type p53 in yeast activates p53-responsive reporters. (a) Strains expressing wild-type p53 (pADH1 : p53 on a LEU2/CEN plasmid) and containing one of the following p53-responsive reporters (SCS : HIS3, RGC : HIS3, p21 : HIS3, mdm2 : HIS3, GADD45 : HIS3, Cyclin G : HIS3 and Bax : HIS3 on a TRP1/CEN plasmid) or control reporter (DUASpGAL1 : HIS3 on a TRP1/CEN plasmid) were streaked out for single colonies onto SC minus leucine minus tryptophan plates and grown for 2 days at 308C. (b) Colonies in (a) were streaked out for singles onto SC minus leucine minus tryptophan minus histidine plates and grown for 2 days at 308C

Table 1 Cis-acting element none RGC SCS p21 mdm2 Cyclin G GADD45 Bax IGF (A) IGF (B)

308 308 308 378 308 378 308 378 308 378 308 378 308 378 308 308

whole yeast cell extracts bound to the p21 probe (Figure 4, lane 6). The monoclonal antibody PAb421 (Harlow et al., 1981), which interacts with an epitope (amino acids 373 ± 381) within the C-terminus of p53 (Wade-Evans and Jenkins, 1985) and enhances the DNA binding function of wild-type and certain mutant forms of p53 (Hupp et al., 1992, 1993; Halazonetis and Kandil, 1993; Jayaraman and Prives, 1995) also stimulated binding by yeast-expressed p53 (see supershifted complex, Figure 4, lane 7). Additionally, the monoclonal antibody PAb1801 (Banks et al., 1986), which interacts with an epitope (amino acids 46 ± 55) (Legros et al., 1994) within the N-terminus of p53, enhanced DNA binding by yeast-expressed p53 proteins, although to a lesser extent than PAb421 (Figure 4, lane 9). Moreover, a peptide corresponding to the PAb421 epitope of p53, shown previously to activate DNA binding by p53 (Hupp et al., 1995; Shaw et al., 1996), stimulated DNA binding by yeast-expressed wild-type p53 (Figure 4, lane 8). An isogenic whole cell extract containing a control vector (no human p53 expression) exhibited no detectable DNA binding to the p21 probe, demonstrating the speci®city of the assay for human p53 expressed in yeast (Figure 4, lane 5). As expected, human wild-type p53, immunopuri®ed from baculovirus-infected insect cells, was also stimulated by PAb421 (Figure 4, lane 2), 421 peptide (Figure 4, lane 3), and PAb1801 (Figure 4, lane 4). A longer exposure of this autoradiogram revealed the DNA binding complex in lane 1 of immunopuri®ed p53 in the absence of peptide or antibodies (not shown). Note that approximately 10 ± 30 times more yeastexpressed p53 protein than immunopuri®ed p53 protein was required in order to detect the p53/DNA complex. Whether this is because yeast-expressed p53 binding occurs in the context of a crude whole cell extract is not yet established. We conclude from the above results that the DNA binding properties, as measured by EMSA, of yeast-expressed human wildtype p53 to the p21 probe are similar to immunopuri®ed human wild-type p53. p53 `hot-spot' mutants are transcriptionally inactive We examined the transcription-activating potential of a panel of human tumor-derived p53 mutants. All of

Activation of reporters by `hot-spot' mutants

Cont

R175H .

G245D .

R248W *

R249S .

R273H *

R282A .

7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7

7 + +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ + + 7 7

7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7

`7'=no growth; `+'=growth; `++'=moderate growth; `+++'=wild-type growth; `*'=contact mutant; `.'=structural mutant

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2531

Table 2 Activation of reporters at 378C Cis-acting element none RGC SCS p21 mdm2 Cyclin G GADD45 Bax IGF (A) IGF (B)

308 308 308 378 308 378 308 378 308 378 308 378 308 378 308 308

Cont

L22E W23S

P177L

R267W

C277Y*

R283H*

7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7

7 + +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ + + 7 7

7 7 +++ +++ +++ +++ 7 7 +++ +++ ++ ++ 7 7 7 7

7 7 +++ +++ +++ ++ 7 7 7 7 7 7 7 7 7 7

7 7 +++ +++ +++ +++ 7 7 7 7 7 7 7 7 7 7

7 7 7 7 +++ +++ 7 7 7 7 7 7 7 7 7 7

7 7 +++ +++ +++ +++ + + +++ ++ + + 7 7 7 7

`7'=no growth; `+'=growth; `++'=moderate growth; `+++'=wild-type growth; `*'=contact mutant

Table 3 Activation at sub-physiological temperatures Cis-acting element none RGC SCS p21 mdm2 Cyclin G GADD45 Bax IGF (A) IGF (B)

248 248 248 308 248 308 248 308 248 308 248 308 248 308 248 248

Cont

V143A

M1601 A161T b

H193R

Y220C b

1254F b

7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7

7 + +++ +++ +++ +++ +++ +++ +++ +++ ++ +++ 7 + 7 7

7 7 +++ +++ +++ ++ 7 7 7 7 7 7 7 7 7 7

7 7 +++ +++ +++ +++ + 7 +++ +++ + 7 7 7 7 7

7 7 + 7 + 7 7 7 7 7 7 7 7 7 7 7

7 7 +++ +++ +++ +++ + 7 + 7 + 7 7 7 7 7

`7'=no growth; `+'=growth; `++'=moderate growth; `+++'=wild-type growth; `b '=b-sca old maintenance

these mutants, with the exception of V143A, R175H, R248W, R249S and R282A were isolated by FASAY (functional analysis of separated alleles in yeast), which allows the detection of p53 mutations from tumors or germline p53 mutations from patients' lymphocytes (Ishioka et al., 1993). Those p53 mutants isolated by FASAY by Ishioka et al. (1993) were identi®ed as such by being defective for transactivation of the identical RGC-containing reporter utilized in this survey (Tables 1, 2 and 3, scored as `7'). We constructed the remaining ®ve alleles from human tumor-derived p53 cDNA (see Materials and methods). We determined whether the p53 mutants tested above were capable of activating transcription from reporters containing p53-responsive cis-acting elements other than RGC. This analysis was carried out at various temperatures: 248C (sub-physiological); 308C (optimal growth condition for S. cerevisiae); and 378C (physiological temperature of mammalian cells); and by di erent growth-measuring techniques (replica plating and streak-out from histidine-containing media to histidine-de®cient media). The results of this survey

Figure 3 The expression levels of human wild-type p53 in yeast. Human wild-type p53-expressing yeast strains denoted in Figure 2a were grown to log phase, extracts prepared, and subjected to Western blot analysis, where 50 mg of total cell extract was loaded. The Western blot was probed with a mixture of anti-p53 antibodies at a 1/3000 dilution (upper panel) and an anti-btubulin antibody at a 1/3000 dilution (lower panel). As a control for anti-p53 antibody speci®city, a strain containing a control vector (pADH1 on a LEU2/CEN plasmid) and the p21 : HIS3 reporter was used (lane 9)

Target gene selectivity by mutant p53 proteins CJ DiComo and C Prives

2532

Figure 4 Electrophoretic mobility shift assays using immunopuri®ed and yeast-expressed human wild-type p53. DNA binding by immunopuri®ed human wild-type p53 (30 ng) to a 32P-labeled p21 probe (6 ng) at 248C (lanes 1 ± 4) and by yeast-expressed wild-type p53 (160 mg whole cell extract) to a 32P-labeled p21 probe (6 ng) at 248C (lanes 6 ± 9) in the absence of antibody (lanes 1 and 6) or in the presence of PAb421 (200 ng, lanes 2 and 7) or of PAb1801 (200 ng, lanes 4 and 9) or of PAb421 epitope peptide (200 ng, lanes 3 and 8). Lane 5 (C) contains 160 mg of whole yeast cell extract containing a control vector (pADH1 on a LEU2/CEN plasmid) expressing no p53. `Flu-p53' denotes immunopurifed human wild-type p53; `yeast-p53' denotes yeast-expressed human wild-type p53; `C' denotes the control whole yeast cell extract. It should be noted that *160 mg of yeast extract contains approximately 800 ng of p53

are described in detail below and summarized in Tables 1, 2 and 3. With respect to the `hot-spot' alleles, we found that the two contact mutants (R248W and R273H) and four conformational mutants (R175H, G245D, R249S, and R282A) were incapable of transactivating each of the reporters at all temperatures, including 248C (Table 1). It is worth reiterating that these six residues are critical for p53/DNA interactions (Cho et al., 1994) and are among the most frequently mutated residues found in human tumors (Nigro et al., 1989; Hollstein et al., 1991; 1994). It is noteworthy that these `hot-spot' p53 mutants were the only ones that were completely defective in transactivation. A class of p53 mutants which retain wild-type activity and binding-site selectivity at 378C A second class identi®ed in our survey are those mutants which retained transcriptional activity at all temperatures, including 378C. These alleles are: P177L (located on the L2 loop, a region where a majority of the p53 mutations occur); R267W (located in the S10 strand of the b-sandwich); C277Y (which contacts DNA on the pyrimidine-rich strand); and R283H (which contacts the phosphate backbone on the purine-rich strand) (Cho et al., 1994). All of these mutants transactivated the SCS- and p21-containing reporter plasmids and no other reporter constructs, with the exception of C277Y, which did not transactivate the SCS-containing reporter, and R283H, which transactivated all reporters, but to varying degrees (Table 2, scored as `+' to `+++'), excluding Bax and IGF-BP3 Box A and Box B, which

were either weak or inactive p53 cis-acting elements, respectively, with wild-type p53 (Tables 1, 2 and 3). In addition, we detected weak binding by the p53P177L and p53R267W proteins to the p21 probe by EMSA. In contrast, two independently isolated mutants (H179L and H179R), located on the H1 helix of Loop 2 and which aid in coordinating a zinc atom, were completely inactive (not shown). The C277Y mutant, ®rst identi®ed in a Ewing tumor, as well as in breast and colon cancer (Kovar et al., 1993; Hainaut et al., 1997), has recently been described as being a true speci®city mutant, in that it is capable of binding to an altered p53 consensus binding site (5'-TTTCATGAAA-3') better than to a wild-type site (5'-AGGCATGTCT-3') (Gagnebin et al., 1998). We found, by replica plating, that the C277Y mutant also transactivated the p21-containing reporter as well as wild-type p53, but no other (Table 2, scored as `+++'). However, under more stringent conditions, that is, by streak-out for singles on histidine-de®cient media, the ability of the C277Y mutant to transactivate was greatly reduced (scored as `+', not shown). Interestingly, C277Y was the only mutant surveyed capable of transactivating the p21-containing reporter, but not the SCS-containing reporter. The SCS sequence was identi®ed by Halazonetis et al. (1993) as a sequence which binds better to wild-type p53 than other consensus sites. All other mutants which were capable of activating the p21-containing reporter demonstrated at least comparable activity with the SCS-containing reporter. The R283H allele was the most wild-type-like mutant tested, in that it retained transcriptional activity towards many of the 53-responsive reporters at the physiological temperature (Table 2). This is the only tumor-derived mutant surveyed to retain this level of activity. The crystal structure of p53 bound to its cognate site (Cho et al., 1994) reveals that R283 contacts the phosphate backbone of DNA at a distance from the primary DNA contacts made by R175 and R273. This might explain why we did not detect a dramatic diminution in transactivating function. Interestingly, the p53R283H protein bound more e ciently (®vefold) than wild-type p53 to both p21 and SCS probes by EMSA. A class of p53 mutants which are binding-site selective at sub-physiological temperature A third class identi®ed in our survey did not transactivate or displayed very weak activation (V143A) at 378C. However, these mutants retained wild-type or near-wild-type activity towards the SCSand p21-containing reporters at 308C. Additionally, they displayed extreme temperature sensitivity towards the remaining reporter constructs, such that activation was only demonstrated when the temperature was reduced even further to 248C. These alleles are: V143A, (located in the S3 strand of the b-sandwich); the double mutant M160I/A161T, (located on the S4 strand); H193R (located on the L2 loop near the S5 strand); Y220C (located on the short loop between the S7 and S8 strand); and I254F (located on the S9 strand) (Cho et al., 1994). A feature shared by these residues is that they are positioned to maintain the structural integrity of the b-sca old, although they are at a distance from

Target gene selectivity by mutant p53 proteins CJ DiComo and C Prives

the DNA binding surface. While the mutations may cause local deformations in structure, they appear not to have that dramatic an e ect on p53's DNA binding surface, as all still retain some activity, albeit at nonphysiological temperatures (Table 3, scored as `+' and `+++' at 248C and 308C). We were able to detect binding by the p53Y220C protein to both p21 and SCS, whereas the p53H193R and p53I254F proteins only bound weakly to p21 by EMSA. Prior results have demonstrated that the V143A mutant activates transcription of p21-, mdm2-, GADD45-, and Cyclin G-promoter-containing reporters in a temperature-sensitive manner (at 328C, but not 378C) in transiently transfected H1299 human tumor cells (Friedlander et al., 1996a). In our examination of this same mutation, we observed transactivation of the SCS-containing reporter at 248C and 308C, but not at 378C (Table 3, scored as `+++', and not shown). Moreover, the V143A mutant transactivated the p21-containing reporter to near wild-type levels at 248C, and to a lesser extent at 308C and 378C (Table 3, scored as `+++' to `+', and not shown). However, we observed no such activation of the mdm2-, GADD45-, or Cyclin G-containing reporters by this mutant. To test whether this lack of transactivation ability was due to low protein levels, we overexpressed the V143A allele from the GAL1 promoter and found no di erences in transactivation of p21- and SCS-containing reporters from the ADH1 expressed constructs (not shown), and neither mdm2, GADD45-, nor Cyclin G-containing reporters were transactivated. Note, however, that the mdm2- and Cyclin G-containing reporters utilized by Friedlander et al. (1996a) contained large genomic segments of either intronic or promoter DNA, respectively. We can not explain the lack of transactivation of the GADD45-containing reporter, as our intronic GADD45 element is nearly `equivalent' to that used by Friedlander et al. (1996a).

(1994) as being critical for transcriptional transactivation, as well as for the binding of Hdm2 (the human homolog of mdm2) to the p53 amino terminus. Moreover, as described above, this transactivation by p53 appears to be mediated through direct contacts of the amino terminus of p53 with components of the general transcription machinery. While the double mutant L22E/W23S fails to activate transcription in mammalian cells, it still retains the ability to bind to p53-responsive cis-acting elements as well as wild-type p53 proteins (Lin et al., 1994; CJD and CP, unpublished). Unexpectedly, when we tested this same mutation, we observed transactivation of the SCS-, p21-, Cyclin G-, and GADD45-containing reporters (Table 2, scored as `++' to `+++'). The L22E/ W23S mutant was as active as wild-type p53 towards the SCS- and p21-containing reporters at 378C, but displayed lesser activity with Cyclin G and GADD45 by streak-out (Table 2, scored as `+' to `++' and not shown). Interestingly, L22E/W23S displayed extremely weak transactivation of the mdm2-containing reporter, and was virtually incapable of activating the Baxcontaining reporter at all temperatures (Table 2, scored as `7'). Additionally, we were able to detect DNA binding by the p53L22E/W23S protein to a p21 probe in the absence and presence of p53-speci®c monoclonal antibodies by EMSA. Transcriptional activation by mutant p53 proteins in yeast does not always correlate with DNA binding by EMSA Most tumor-derived mutations are found within the DNA binding domain of p53. Our analysis has demonstrated that many tumor-derived mutants retain transcriptional activity that is binding site selective and temperature sensitive (Tables 1, 2 and 3). We were therefore interested in comparing DNA binding by either wild-type or those mutant forms of p53, which had retained transcriptional activity towards the p21

Mutations which disrupt the p53 DNA binding domain by deletion or intron retention are inactive Two additional tumor-derived mutations surveyed are the S261R allele (which also retains intron 7 due to a mutation at the donor splicing site) and the Intron 7 allele (which retains intron 7 due to a six base deletion around the intron 7 splice donor site). These mutations, by including intron 7, result in truncated proteins terminating in intron 8 (as determined by sequence analysis and Western blot analysis, not shown) and excluding exons 9 and 10. While exons 9 and 10 do not encode residues crucial to DNA binding, they do encode the tetramerization domain (residues *320 ± 360), the basic domain (residues *363 ± 393); and one of the two nuclear localization signals (the signal located downstream of the tetramerization domain). Both mutants failed to transactivate every p53-responsive reporter at all temperatures (not shown). The double mutant L22E/W23S retains transcriptional activity in yeast towards p53-responsive reporters The hydrophobic residues at positions L22 and W23 of human p53 were originally identi®ed by Lin et al.

Table 4 Activation versus DNA binding p53 Control wild-type L22E/W23S V143A M1601/A161T R175H P177L H179L H179R H193R Y220C G245D R248W R249S 1254F S261R R267W R273H C277Y R282A R283H Intron 7

Txn

Bind

7 + + + + 7 + 7 7 + + 7 7 7 + 7 + 7 + 7 + 7

7 + + 7 7 7 7 7 ND 7 7 7 7 7 7 7 7 7 7 7 ++ ND

EMSA with p21 probe Bind+421 Bind+1801 7 + + 7 7 7 + 7 ND 7 + 7 7 7 7 7 7 7 + 7 ++ ND

7 + + 7 7 7 + 7 ND + + 7 7 7 + 7 + 7 + 7 ++ ND

`7': lack of transactivation and binding to p21; `+': ability to transactivate and bind to p21; `N.D.': not determined

2533

Target gene selectivity by mutant p53 proteins CJ DiComo and C Prives

2534

cis-acting element-containing reporter, in yeast whole cell extracts. Unexpectedly, we found that transcriptional activity in vivo did not necessarily correlate with DNA binding in vitro (Table 4). As seen in Figure 4, using EMSA, human wild-type p53 expressed in whole yeast cell extracts binds to a p21 probe, in the presence and absence of p53-speci®c antibodies or PAb421 peptide. Similarly, whole cell extracts were prepared from p53 mutant-expressing strains (V143A, M160I/A161T, I254F and R267W) which were found to transactivate the p21 : HIS3 reporter at wild-type levels. However, we were not able to detect binding by any of these mutants to a p21 probe with the same sequence as in the HIS3 reporter construct (Tables 2, 3 and 4). Moreover, no DNA binding was detectable even in the presence of the activating monoclonal antibody PAb421. Since it is possible that the epitope for this antibody, located in the C-terminus of p53, is masked in these mutants, we repeated the EMSA in the presence of PAb1801, whose epitope is located in the N-terminus of p53 (residues 46 ± 55). In this case, while no detectable binding to a p21 probe by V143A and M160I/A161T was observed, weak binding was detected by H193R, I254F, and R267W mutants with PAb1801 (Table 4). The results with C277Y were also unexpected: this mutant bound (with antibody) comparably to RGC, SCS, and p21 probes in vitro and yet was only capable of transactivating the p21containing reporter. With the notable exception of C277Y, our studies suggest that transcriptional activity in vivo requires a minimal level of wild-type or mutant p53 to be bound to it's cognate binding sites, which drive transcription of the DUASpGAL1 : HIS3 reporters.

tions, perhaps in a cell type speci®c manner. Mutants which have lost all transcriptional activity would then be more likely to be selected during tumor progression, and thus would appear more frequently in the database of p53 mutations. Arguing against this simple hypothesis, however, are the observations from a number of groups showing that some `hot-spot' mutations display transcriptional activity in transiently transfected mammalian cells, albeit with only a subset of p53-responsive reporters (Chen et al., 1993a; Chumakov et al., 1993; Friedlander et al., 1996b; Ludwig et al., 1996). Whether the yeast-based assay in fact re¯ects more accurately the true potential (or lack thereof) of various p53 mutants thus remains to be determined. It should be noted that Pientenpol et al. (1994) observed that R273H displayed partial transactivation of reporters containing two (but not single) copies of high a nity arti®cial binding sites (El-Deiry et al., 1992; Funk et al., 1992). Additionally, those authors used a galactose-inducible promoter which yields approximately 25 ± 30-fold more p53 protein than the ADH1 promoter used in our studies (not shown, CJD and CP, unpublished). Thus, at extremely high levels of protein, the R273H mutant shows very restricted transactivation function. In our survey of the literature related to mutant p53 function in mammalian cells (Chen et al., 1993a; Chumakov et al., 1993; Miller et al., 1993; Park et al., 1994; Niewolik et al., 1995; Friedlander et al., 1996b), R273H appears to be the mutant most commonly associated with some transcriptional activity.

Discussion

Many of the tumor-derived p53 mutants we surveyed were originally identi®ed as being inactive for transactivation of an RGC-containing HIS3 reporter at 308C (Ishioka et al., 1993). Surprisingly, when we tested these tumor-derived mutants against other physiologically relevant target gene binding sitecontaining reporters, we found that many retained activity. We divided the mutants into two classes: (1) those that retain activity which is binding site selective at sub-physiological temperatures (V143A, M160I/ A161T, H193R, Y220C and I254F); and (2) those that retain activity which is binding site selective, even at physiological temperatures (L22E/W23S, P177L, R267W, C277Y and R283H). The class of mutants which retain activity at sub-physiological temperatures are all poised to maintain the integrity of the b-sca old (Cho et al., 1994). While these mutations a ect residues located at a distance from the DNA binding surface, their detrimental e ect on transactivation reinforces the importance of the b-sca old structure in supporting the DNA binding surface of p53. The ®ve mutants which retain activity at physiological temperature merit further discussion. Presumably, the tumor-derived mutants (P177L, R267W, C277Y and R283H) were all selected for at 378C. Why would these residues be selected for in tumors, given that they retain partial wild-type activity? Do these alleles possess a novel function and might these proteins regulate gene(s) involved in the fate of a cell? Friedlander et al. (1996a) and Ludwig et al. (1996) have shown that V143A and R175P, respectively, are

In this report we examined the ability of human wildtype and mutant p53 proteins to activate a variety of p53-responsive binding site-containing reporters in S. cerevisiae. This assay allowed us: (1) to survey of a broad panel of tumor-derived and non-tumor-derived p53 mutants; (2) to measure the transactivation of naturally occurring (RGC, p21, mdm2, GADD45, Cyclin G, Bax, IGF-BP3 Box A and IGF-BP3 Box B) and synthetic (SCS) p53-responsive elements by wild-type and mutant p53; and (3) to determine the e cacy of this ability at a variety of temperatures (248C, 308C and 378C). Yeast-expressed human p53 `hot-spot' mutants are transcriptionally inactive, even at reduced temperatures It is remarkable that of all mutants that were tested, only the six `hot-spot' mutants were completely defective with all reporters and at all temperatures. From these data, we might speculate that the high frequency of mutation at these codons in p53 re¯ects a more profoundly defective phenotype. P53 has a number of di erent transcriptional targets in cells whose functions regulate cell cycle and cell death. Di erent types of cells may require unique combinations of such targets being activated as part of the tumor suppression regimen of p53. A mutant which retains partial activity would be more likely to conserve some tumor suppressor activity under certain condi-

Tumor-derived p53 mutants, once thought to be transcriptionally inactive, retain activity that is binding site selective and temperature dependent

Target gene selectivity by mutant p53 proteins CJ DiComo and C Prives

defective for inducing apoptosis, yet each can activate transcription from a subset of p53-responsive reporters. It would be interesting to examine the apoptotic and transactivation spectrum of P177L, R267W, C277Y and R283H. The L22E/W23S mutant reveals a fundamental di erence between yeast and mammalian cells The L22E/W23S double mutant, when expressed in yeast, retained almost full wild-type activity towards many of the p53-responsive reporters. This mutant, however, di ers signi®cantly from wild-type p53 in that it only very weakly transactivates the mdm2-containing reporter at all temperatures tested, and can not activate the Bax-containing reporter at all. Recently, Candau et al. (1997), in examining chimeras containing the p53 amino terminus and the GAL4 DNA binding domain in yeast, identi®ed a new transactivation subdomain in p53 located between amino acids 40 ± 83. Moreover, residues W53 and F54 were found to be critical for function in both yeast and mammalian cells. That the L22E/W23S double mutant retains almost full wildtype activity in a binding site-selective manner, lends support to the ®ndings of Candau et al. (1997) and further suggests that the subdomain between 40 ± 83 may be the primary region in yeast that supports transactivation, even in the context of the full length p53 protein. It has been demonstrated that residues L22 and W23 are crucial for p53's ability to communicate with the general transcriptional machinery in mammalian cells. Clearly, however, the requirement for these residues is relaxed in yeast. DNA binding properties of p53 in yeast We were able to show that yeast-expressed human wildtype p53 is capable of binding to sequences which are cloned into those reporters. Not only did human p53 in yeast whole cell extracts bind to a p21 probe by EMSA, these complexes were supershifted upon addition of p53-speci®c monoclonal antibodies and DNA binding was enhanced by a p53-speci®c peptide, similar to immunopuri®ed p53. Although these results suggest that the transactivation measured in our growth assays is mostly due to p53 binding to and activating the binding site-containing reporters, there were discrepancies noted upon examination of mutant p53 binding. The ®rst discrepancy was that, in contrast to wild-type p53, a number of mutants were capable of transactivation of a subset of reporters but either did not bind detectably to p21 DNA by EMSA (V143A and M160I/ A161T) or bound weakly only in the presence of antibody (P177L, H193R, I254F and R267W). Addressing the lack of detectable DNA binding by certain p53 mutants with EMSA, we put forth as possible explanations the following: (1) despite their approximate equivalence with wild-type p53 protein levels, the quantities of certain p53 mutants in the extracts might have been limiting; (2) the epitopes for the antibodies we utilized to detect supershifts might be masked; (3) the DNA binding activity of some p53 mutants might be susceptible to the conditions employed in preparing the whole cell extracts; or (4) certain p53 mutants might require a de®ned chromatin structure when binding to the reporter plasmids in vivo,

and this structure would be absent in the oligoduplex probes utilized in the electrophoretic mobility shift assays in vitro. Indeed, recent studies examining the in¯uence of DNA conformation on sequence-speci®c DNA binding by p53 showed that wild-type p53 binds to certain DNA structures, such as Holliday junctions, in a non-speci®c manner (Lee et al., 1997), as well as non-B-DNA, in a sequence-speci®c manner (Kim et al., 1997). It is noteworthy that Chen et al. (1993b) found that TFIID can stabilize the ability of p53 to bind to its cognate site in DNA. Furthermore, Friedlander et al. (1996b) showed that antibodies to the amino terminus of p53 can stabilize mutant p53 binding to DNA. It is attractive to speculate that certain factors, such as components of the general transcription machinery or as yet unidenti®ed cellular proteins, stabilize or enhance DNA binding by p53 in vivo. Such interactions with mutant p53 might be disrupted by lysis of yeast cells. A ®fth possibility is that sequence-speci®c DNA binding by certain p53 mutants may be abrogated by the excess double-stranded poly(d(I-C)) present in the EMSA reaction bu er, in addition to the cellular DNA/RNA present in the yeast cell extracts. A recent study presented evidence that binding of large, but not small, DNAs by the C-terminus of p53 interfered with sequence-speci®c DNA binding by p53 (Anderson et al., 1997). It should be noted that we tested a variety of EMSA conditions (including varying the ratio of poly(d(I-C)) to probe, the amount of spermidine and/ or BSA, and the concentration of yeast cell extract) in order to optimize binding by wild-type p53. Note that it is the binding (or lack thereof) by the tumor-derived mutants relative to wild-type p53 that we are assaying. Finally, it may be that full transactivation (scored as `+++' in our assay) requires a threshold of binding by p53. Wild-type p53 may be capable of DNA binding that is markedly higher than the required threshold, and the rate limiting factor(s) might then be one or more interactions with components of the general transcription machinery. Another incongruity was the surprising fact that C277Y cannot activate SCS (or RGC) and yet binds detectably to both SCS and RGC by EMSA (albeit only in the presence of p53 antibodies). The C277Y mutant, recently identi®ed as an altered speci®city mutant (Gagnebin et al., 1998), is clearly worthy of further attention in order to understand how it binds to DNA and activates transcription. Finally, that more tumor-derived p53 mutants than once thought have the intrinsic ability to bind DNA and activate transcription and that antibodies and small chemically modi®ed peptides can restore this DNA binding activity to certain mutants, in vitro as well as in vivo (Halazonetis and Kandil, 1993; Hupp et al., 1995; Abarzua et al., 1995, 1996; Friedlander et al., 1996b; Selivanova et al., 1997), lends support to the strategies of identifying molecules that can mimic and extend the stabilizing e ects of antibodies and peptides. Materials and methods Yeast strains and media All yeast strains were isogenic with S288C, except that they were wild-type at GAL2. Prior to the introduction of the wild-type or mutant p53 constructs, CUY5 (trp1D1 ura3-52

2535

Target gene selectivity by mutant p53 proteins CJ DiComo and C Prives

2536

his3D200 leu2-3, -112 lys2-801) was transformed with one of the HIS3 reporter plasmids or control plasmid. Rich (YP) and synthetic complete (SC) media were constituted as described elsewhere (Rose et al., 1990), except that the YP media also contained 0.1 g of tryptophan per liter and 0.1 g of adenine per liter and the SC media contained 0.2 g of leucine, 0.1 g of all other amino acids, 0.1 g of uracil, and 0.1 g of adenine per liter. All strains were grown in glucose to a ®nal concentration of 2%. Transformation of yeast Yeast were transformed by a modi®ed version of the lithium acetate (LiOAc) method (Guthrie and Fink, 1991). An overnight culture was diluted in 100 ml of media and grown for two doublings. The cells were collected by centrifugation at 5000 r.p.m. for 5 min and washed once with sterile water and once with 0.1 M LiOAc. The cell pellet was resuspended in 1/100 of the initial culture volume in 0.1 M LiOAc. For each transformation, 100 ml of cells were mixed with plasmid DNA, carrier DNA (2 ml of 10 mg/ml boiled, sonicated salmon sperm (Sigma)), and incubated in 800 ml of a 40% polyethylene glycol/0.1 M LiOAc solution for 30 min. The samples were heat shocked at 428C for 5 min and 10 ml of sterile water was added. The cells were centrifuged at 5000 r.p.m. for 5 min, the pellets resuspended, and the cells spread on selective plates. Plates were incubated at 308C until colonies appeared. Plasmids Expression of p53 in yeast pCUB7 (pLS76) expresses full length p53 cDNA from the ADH1 promoter with the CYC1 terminator downstream of the p53 cDNA, CEN6 and ARSH4 for stable, low copy number replication, and the LEU2 gene for plasmid maintenance (Ishioka et al., 1993). The plasmids expressing full length p53 mutants were isolated by FASAY (Ishioka et al., 1993), except for L22E/W23S, V143A, R175H, R248W, R249S and R282A. In order to obtain pADH1p53L22E/W23S, a 1.6 kb HindIII/ XbaI cDNA fragment in pUC119 was subcloned into the HindIII/SacI site of pCB1153 (pADH1 on a LEU2/2m plasmid, (Di Como et al., 1995). In order to obtain pADH1p53V143A, a PshAI/NcoI fragment spanning codons 60 ± 159 was swapped into pCUB7 from a cDNA clone (pCUB2: pRS314-CX3, Pientenpol et al., 1994) harboring the missense mutation. In order to obtain pADH1p53R175H and pADH1p53R248W, a NcoI/StuI fragment spanning codons 160 ± 346 was swapped into pCUB7 from cDNA clones (pCUB12: pC53-CX22 (Kern et al., 1991a) and pCUB14: pC53-248 (Kern et al., 1991a), respectively) harboring the missense mutation. In order to obtain pADH1p53R249S and pADH1p53R282A, an Eco47III fragment spanning codons 181 ± 336 was swapped into pCUB7 from cDNA clones (pCUB11: pC53-249S (Kern et al., 1992) and pCUB15: pGEMhp53-282A (Waterman et al., 1995), respectively) harboring the missense mutation. All constructs were sequenced to con®rm that the p53 cDNA contained the missense mutation(s). Reporter plasmids p53-responsive reporter plasmids were constructed as follows: a duplex oligonucleotide encoding one of the p53-responsive cis-acting elements (see below) was cloned upstream of the inactive GAL1 promoter (DUASpGAL1) at a unique BamHI site in pCUB79 (a PstI/SacI fragment in pUC119) and the resulting plasmids were sequenced for the orientation and insert number of the oligoduplex. From these constructs, an AatII/SacI fragment was swapped into pCUB53 (pBM2389, Ishioka et al., 1993) containing the HIS3 coding sequence on a TRP1/ CEN plasmid. The synthesized oligonucleotides (Operon Technologies, Inc.) encoding the p53-responsive cis-acting elements (shown in bold) were as follows: CUO3

(5'-GATCCTCGAGGAACATGTCCCAACATGTTGCTCGAG -3') and CUO4 (5'-GATCCTCGAGCAACATGTTGGGACATGTTCCTCGAG-3') for p21; CUO11 (5'-GATCCTCGAGGGTCAAGTTGGGACACGTCCCTCGAG-3') and CUO12 (5'-GATCCTCGAGGGACGTGTCCCAACTTGACCCTCGAG-3') for mdm2; CUO13 (5'-GATCCTCGAGGAACATGTCTAAGCATGCTGCTCGAG - 3') and CUO14 (5'-GATCCTCGAGCAGCATGCTTAGACATGTTCCTCGAG-3') for GADD45; CUO15 (5'-GATCCTCGAGAGACCTGCCCGGGCAAGCCTCTCGAG - 3') and CUO16 (5'-GATCCTCGAGAGGCTTGCCCGGGCAGGTCTCTCGAG-3') for Cyclin G; CUO17 (5'-GATCCTCGAGTCACAAGT TAGAGACAAGCCTGGG CGTG GGCTATATTCTCGAG-3') and CUO18 (5'-GATCCTCGAGAATATAGCCCACGCCCAGGCT TGTCTCTAACT TGTGACTCGAG-3') for Bax; CUO24 (5'-GATCCTCGAGGGGCAAGACCTGCCAAGCCTCTCGAG-3') and CUO25 (5' - GATCCTCGAGAGGCT TGGCAGGTCT TGCCCCTCGAG-3') for IGF-BP3 (Box B); CUO26 (5'-GATCCTCGAGAAACAAGCCACCAACATGCTTCTCGAG - 3') and CUO27 (5' - GATCCTCGAGAAGCATGTTGGTGGCTTGTTTCTCGAG-3') for IGF-BP3 (Box A); and CUO28 (5'GATCCTCGAGGGGCATGTCCGGGCATGTCCCT-CGAG-3') and CUO29 (5'-GATCCTCGAGGGACATGCCCGGACATGCCCCTCGAG-3') for SCS. The RGC-containing p53-responsive reporter plasmid (pSS1) is as described (Ishioka et al., 1993), where a duplex oligonucleotide 5'-TCGACCTTGCCTGGACTTGCCTGGCCTTGCCTTTTTCGA-3' with XhoI ends was cloned into the BamHI site of pCUB53 (pBM2389) after half-®lling the ends with Klenow. Preparation of protein extracts and detection of p53 protein by Western immunoblot analysis Cellular extracts and Western immunoblots were prepared as described, except for minor changes (Sutton et al., 1991; Di Como and Arndt, 1996). Brie¯y, exponentially growing cells were harvested by centrifugation and washed with ice cold lysis bu er (100 mM Tris-HCl (pH 7.5), 200 mM NaCl, 1 mM EDTA, 5% glycerol, 0.5 mM dithiothreitol). Cells were resuspended in 100 ml of lysis bu er containing PI (1 mM phenylmethylsulfonyl ¯uoride and 1.2 mg each of leupeptin, antipain, chymostatin, and pepstatin per ml (Sigma)). The cells were lysed by vortexing four times for 15 s in the presence of glass beads. An additional 50 ml of lysis bu er containing PI was added, the cells vortexed once for 10 s and the free liquid removed. This step was repeated and the free liquids (extracts) were combined and centrifuged at 16 000 g for 20 min to remove cell debris. Protein concentrations were determined using the Bio-Rad assay using bovine globulin as a standard (Bio-Rad Lab, CA). An equal volume of 26gel sample bu er (Silhavy et al., 1984) was added to the extracts. Samples were heated to 958C for 5 min, centrifuged for 3 min at 16 000 g, and electrophoresed through a 10% SDS-polyacrylamide gel. Protein gels were transferred to polyvinylidene ¯uoride (PVDF) membranes (Millipore). For p53 detection, a mixture of puri®ed p53 monoclonal antibodies was used containing PAb421, PAb1801, PAb240, and DO-1, each at a 1/3000 dilution of a 50 ng/ml stock; for b-tubulin detection, the primary monoclonal antibody was B1BE2 at a 1/3000 dilution. Proteins were visualized with an enhanced chemiluminescence detection system (Amersham). Electrophoretic mobility shift assays with immunopuri®ed p53 and whole yeast cell extracts expressing p53 Electrophoretic mobility shift assays were performed as described, except for minor changes (Freeman et al., 1994; Friedlander et al., 1996b). Brie¯y, for whole yeast cell extracts, exponentially growing cells were harvested by

Target gene selectivity by mutant p53 proteins CJ DiComo and C Prives

centrifugation and washed with ice cold lysis Bu er A (20 mM Tris-HCl (pH 7.2). 100 mM NaCl, 5 mM EDTA, 10% glycerol, 0.1% Nonidet P-40, 2 m M dithiothreitol). Cells were resuspended in 100 ml of lysis Bu er A containing PI (1 mM phenylmethylsulfonyl ¯uoride and 1.2 mg each of leupeptin, antipain, chymostatin and pepstatin per ml (Sigma)). The cells were lysed by vortexing four times for 15 s in the presence of glass beads. An additional 50 ml of lysis Bu er A containing PI was added, the cells vortexed once for 10 s and the free liquid removed. This step was repeated and the free liquids (extracts) were combined and centrifuged at 16 000 g for 20 min to remove cell debris. Protein concentrations were determined using the Bio-Rad assay using bovine globulin as a standard (Bio-Rad Lab, CA). When necessary, total cell extracts were frozen in a dry ice/alcohol bath and stored at 7808C. Recombinant baculovirus expressing wild-type p53 has been described (Friedman et al., 1990; Bargonetti et al., 1992). Extracts of infected sf9 insect cells were prepared and p53 was puri®ed from lysates by immunoa nity procedures (Wang et al., 1989). Puri®ed p53 protein was prepared with protein A-Sepharose columns cross-linked with the p53speci®c monoclonal antibody PAb421 (Harlow et al., 1981). The proteins were eluted with a molar excess of PAb421 epitope-containing peptide (KKGQSTSRHKK-OH) (WadeEvans and Jenkins, 1985) or with 50% ethylene glycol. The protein was dialyzed into Dialysis Bu er (10 mM HEPES (pH 7.5), 5 mM NaCl, 0.1 mM EDTA, 1 mM dithiothreitol and 50% glycerol). Probes were made as follows: synthesized oligonucleotides encoding the p53-responsive elements (40-mers) were selfannealed and extended with Klenow in the presence of [a-32P]dATP.

Binding reaction mixtures contained either 160 mg of whole yeast cell extract or 30 ng of immunopuri®ed wildtype p53 in Gel Shift Bu er (20 mM HEPES (pH 7.9), 25 mM KCl, 0.1 mM EDTA, 2 mM MgCl2, 0.5 mM dithiothreitol, 0.025% Nonidet P-40, 2 mM spermidine (Sigma), 10% glycerol, 0.1 mg acetylated bovine serum albumin (NEB) and double-stranded poly(d(I-C)); 120 ng (Boehringher Mannheim) to a ®nal volume of 20 ml. In all cases, volumes were equalized with Dialysis Bu er for immunopuri®ed p53 or Bu er A for whole yeast cell extract-containing p53. Reactions were incubated for 10 min at 248C, after which 6 ng of 32P-labeled oligonucleotide was added and the incubation was continued at 248C for 15 min. Samples were run on 4.5% native polyacrylamide gels at 180 ± 200 V (not exceeding 40 mA current) in 0.56TBE bu er at 248C for 2 h. Gels were dried under vacuum at 808C and exposed to Kodak XAR ®lm overnight at 7808C. Acknowledgements We gratefully thank R Iggo for the human tumor-derived p53 expression plasmids, for the pSS1 and pBM2389 reporter constructs, and for his insightful discussions and comments. We also thank G Petsko for the S288C yeast strain (CUY5) and helpful comments. P Friedlander, L Jayaraman, L Ko, K Okamoto, and S-Y Shieh are thanked for additional plasmids. Finally, we thank E Freulich for the preparation of the anti-p53 antibodies and B Futcher for the anti-b-tubulin antibody. Critical comments and discussion on the manuscript were made by J Ahn, C Gaiddon and K Okamato. CJD is supported by the Cancer Research Fund of the Damon Runyon-Walter Winchell Foundation Fellowship, DRG-1427. CP is supported by a grant from the US Army, (DAMD17-94)4275.

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