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The Regulation of Centromere Function and Localization Alyssa Ferris
Summary Centromere function is essential for proper cell division, but the mechanism for determining the formation and location of the centromere is not well understood. Centromeres are a paradox because, while their basic function is very similar among eukaryotes, centromeric DNA sequences are significantly different, even between closely related species. (Lamb and James, 2010). This fact suggests that other forms of regulation are responsible for the location of the centromere, such as epigenetic regulation. This is one possible explanation for how centromere location can stay in the same location on a chromosme and how centromere function is independent of centromeric DNA; however, no such factor has been identified yet.
Background Information A centromere is a visible constriction of DNA located on a condensed chromosome, and where the kinetochore forms during cell division. The kinetochore forms on top of the centromere, and is composed of centromeric proteins (CENP) and other complexes (see figure 1). The most important protein is CENP-A, which serves as a base for the rest of the kinetochore and has been shown to influence where it forms (Buscaino et al., 2010). When a cell begins to divide, spindle fibers attach to either side of the kinetochore and separate the sister chromatids. Thus, the centromere ensures that the chromosomes are divided correctly among the resulting cells. Failure results in an uneven division of genetic material which usually results in cell death. The centromeric DNA is typically composed of repeating satellite sequences; each individual sequence is usually between 150 and 200bp (Henikoff et al., 2001).
Figure 1. http://www.edoc.hu-berlin.dae A diagram of a chromosome showing the centromere and the location of the kinetochore with microtubules attached.
In order for a cell to divide correctly each chromosome needs to have one centromere and cellular mechanisms ensure that its location is static. However, in some cases, the centromere forms in a different location on the chromosome for unknown reasons. These neocentromeres have the same general properties as a normal centromere, including a noticeable constriction of the chromosome and functioning kinetochore proteins, which serve as binding sites for microtubules during mitosis and meiosis (Buscaino et al., 2010). The Role of DNA Sequences Originally, DNA sequence were believed to determine the centromere’s location (Karpen and Allshire, 1997). Research in S. cerevisiae showed that the centromere consisted of three distinct satellite sequences which had unique motifs that boundd to centromeric proteins (Karpen and Allshire, 1997). However, research conducted in S. pombe indicated that the centromeric satellite repeats from S. cerevisiae were not conserved (Karpen and Allshire, 1997). More importantly, satellite sequences could be added or deleted without affecting the stability of the centromere (Karpen and Allshire, 1997). Later studies in Equus showed that evolutionarily new centromeres lacked satellite repeats and also identified locations with the past centromeric identity that still had satellite sequences (Piras et al., 2010). These findings imply that satellite sequences are a part of centromeric identity, but they are not the initial factors that determine centromere identity. Although, specific sequences in the centromere are not highly conserved, satellite sequences have been found in the DNA sequences of almost all centromeres. For example, human centromeres mainly consist of satellite sequences called alpha satellites, which consist of 178bp repeats (Lee et al., 1997). These repeats constitute approximately 62% of the genetic material surrounding the centromere, and an additional 24% consists of other satelVolume 1 | 2011-2012 | 21
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Figure 2. (Allshire and Karpen, 2008)a) In this model, CENP-A is maintained on the DNA by a chromatin-loading factor. There are two main theories about how this would work, either gaps would remain in the chromatin during replication and CENP-A would be inserted directly into the DNA or the old CENP-A locations would be temporarily replaced by H3 and then restored by a CENP-A-H3 exchange factor later in the cell cycle. b) In this process, centromeric chromatin is modified to include histone H3 which is tagged with specific epigenetic histone modifications, such as dimethylation of lysine 4, and these modifications would specify recruitment of CENP-A. c) In this method, preexisting CENP-A splits during DNA replications leading to the recruitment of additional CENP-A in order to restore chromatin integrity.
lite repeats (Lamb and Birchler, 2010). Studies have also shown that the presence of alpha DNA repeats is positively correlated with centromere stability. Also, if alpha satellites are inserted into ectopic DNA, noncentromeric chromosome sites, an artificial centromere will form. However, a centromere will not form if neocentromeric DNA is inserted into a chromosome (Lamb and Birchler, 2003). Sequencing data from other species confirms this trend. For example, Arabidopsis centromeres contain satellite sequences and other repeats similar to those found in humans and which are in the same general locations on the chromosome. These satellite sequences are 178bp long and resemble alpha satellites; however, they consist of entirely different DNA sequences. The only commonalities between satellite sequences are their approximate length, between 150bp and 200bp, and the fact that the sequences tend to be rich in adenine and thiamine (Karpen and Allshire, 2003). Additionally, although satellite sequences are often found at the centromere, they can also be present in ectopic DNA (Allshire and Karpen, 2008). Therefore, satellite sequences are neither necessary nor sufficient for centromere formation, and there must be an additional mechanism influencing centromere location. The Role of Proteins Centromeric proteins are important because, unlike satellite sequences, they are found exclusively at the centromere, and many are only found there during mitosis when the kinetochore forms (Henikoff et al., 2001). CENPA, C and E are especially important because they have been found at active centromeres and neocentromeres but 22 | 2011-2012 | Volume 1
not at old or nonfunctioning centromeres (Henikoff et al., 2001). CENP-A has been shown in previous studies to be the most likely protein to maintain the centromere. Its absence causes most kinetochore proteins to be misplaced, but the absence of other centromere proteins does not affect the deposition of CENP-A. In addition, the overexpression of CENP-A results in its random integration into DNA which causes the formation of ectopic centromeres (Allshire and Karpen, 2008). CENP-A has been found to mark the centromere location by replacing the H3 protein in the histones of a centromeric nucleosome (Henikoff et al., 2001). It has also been found to act as a heritable molecule located on the chromosome during DNA replication, which could allow it to regulate centromere location over multiple generations of cells (Allshire and Karpen, 2008). Although CENP-A plays an essential part in the formation of the kinetochore, another mechanism must be responsible for its original recruitment and incorporation into centromeric DNA. Allshire and Karpen (2008) proposed three different models for the recruitment of CENP-A. One model is that during DNA replication, a chromatin-loading factor that recognizes CENP-A could deposit it into an identical location in the new DNA strand. This would explain why centromeres are at a single site and why neocentromere locations are maintained once they have been established. Another possible system proposes that modifications in H3 recruit specific CENP-A assembly proteins, which are responsible for recruiting the proteins that form the kinetochore. This system would also require the presence
Review of an enzyme or binding proteins in the cell which are able to modify the H3. A third model hypothesizes that the intrinsic properties of the CENP-A nucleosomes provide the signals for CENP-A recruitment. For example, the octamer structure could split into two tetramers, and recruitment of CENP-A would be a reconstitution of the tetramers. This model would require proteins to facilitate the division of CENP-A and other proteins to recruit CENP-A when it is reconstituted. Although one of these three models may be responsible for CENP-A deposition in the centromere, none has significant experimental support. Abnormal Centromeres Some studies have shown that neocentromeres are more likely to form in close proximity to the current centromere, suggesting that either the centromere influences the surrounding DNA or that latent chromosomes maintain some remnants of centromeric identity (Karpen and Allshire, 1997). In Drosophilia, studies have shown that acentric chromosome fragments can gain centromeric function. These neocentromeres are usually formed at the tip of the X chromosome where the chromosome splits, which is approximately 40Mb away from the normal centromere (Karpen and Allshire, 1997). Similar studies in human cells have also identified three locations in the karyotype where neocentromeres are most likely to form, indicating that the location for neocentromere formation is nonrandom (O’Neill and Carone, 2009)(Ketel et al., 2009). Studies have shown that in some organisms, such as nematodes, centromeres are holocentric, the microtubules attach along whole chromosomes (Karpen and Allshire, 1997). It is possible that in the past most organisms had holocentric centromeres, but they evolved into a single, larger centromere. If the DNA from the holocentric microtubule binding sites had maintained some of its previous identity, neocentromeres would result when factors in the cell, such as CENP-A, had been incorrectly recruited to these sequences. Carone et al. (2008) found that in Macropus eugenii, a retrovirus called KERV was located near transcription sites in the centromere and that it promoted transcription of centromeric DNA. The study also showed that the ncRNA was used to recruit proteins to the centromere to form the kinetochore. Other studies have found that previous centromere locations are located next to KERV sequences (O’Neill and Carone, 2009), and evidence was found that higher order DNA-protein structures suppress alternate centromere locations when a functioning centromere is present (Karpen and Allshire, 1997). It is possible that these retroviral promoters are located by holocentric chromosome locations when they are activated. As a result, the production of a ncRNA could cause recruitment of the structures necessary to form a functioning kinetochore and hence a neocentromere.
Street Broad Scientific There is no one explanation for the stability of centromere location and kinetochore formation. Centromeric DNA sequences are not highly conserved, but satellite sequences that are approximately the same length occur in most eukaryotes. CENP-A has been found to recruit other proteins and complexes to the centromere in order to form the kinetochore; however, no distinct regulation of the production or deposition of CENP-A has been identified. Additionally, neocentromeres form in ectopic DNA, proving that satellite sequences and centromeric DNA is not necessary for centromeric function. However, neocentromeres tend to form at the same locations on chromosomes, indicating that some factor is causing centromere localization. References Allshire, R. and G. Karpen. 2008. Epigenetic regulation of centromeric chromatin: old dogs, new tricks? Nature Reviews 9:923-937.
Bergmann, H. J., M. G. Rodríguez, N. M. C. Martins, H. Kimura, D. A. Kelly, H. Masumoto, V. Larionov, L. E. T. Jansen, and W. C. Earnshaw. 2011. Epigenetic engineering shows H3K4me2 is required for HJURP targeting and CENP-A assembly on a synthetic human kinetochore. The EMBO Journal 30: 328 – 340. Buscaino, A., R. Allshire, and A. Pidoux. 2010. Building centromeres: home sweet home or a nomadic existence? Current Opinion in Genetics and Development 20:118-126.
Carone, D., M. Longo, G. Ferreri, H. Hall, M. Harris, N. Shook, K. Bulazel, B. Carone, C. Obergfell, M. O’Neill, and R. O’Neill. 2008. A new class of retroviral and satellite encoded small RNAs emanates from mammalian centromeres. Cromosoma 118:113-125. Piras F., S. Nergadze, E. Magnani, L. Bertoni, C. Attolini, L. Khoriauli, E. Raimondi, and E. Giulotto. 2010. Uncoupling of satellite DNA and centromeric function in the genus Equus. PLoS Genetics 6:1-10.
Karberg M., R. J. Leavitt, D. A. A. Cabaya, M. E. Van Eden, and X. Y. Jia. 2009. A Method for Quantifying DNA Methylation Percentage Without Chemical Modification. Zymo Research Corporation. Karpen, G. and R. Allshire. 1997. The case for epigenetic effects on centromere identity and function. Trends in Genetics 13: 489-96. Henikoff, S., K. Ahmad, and H. Malik. 2001. Centromere paradox: stable inheritance with rapidly evolving DNA. Science 293: 1098-102.
Ketel, C. H. Wang, M. McClellan, K. Bouchonville, A. Selmecki, T. Lahav, M. Gerami-Nejad, and J. Berman. 2009. Neocentromeres form efficiently at multiple possible loci in Candida albicans. PLoS Genetics 5: 1–18. Lamb, J. and J. Birchler. 2003. The role of DNA sequence in centromere formation. Genome Biology 4:1-4.
Lee, C., R. Weverick, R. Fisher, M. Ferguson-Smith, and C. Lin. 1997. Human centromeric DNAs. Human Genetics 100: 291-304. O’Neill. R. and D. Carone. 2009. The role of ncRNA in centromeres: a lesson from marsupials. Progress in Molecular and Subcellular Biology 48: 77-101.
Stimpson, K. M, Sullivan B. A. 2010. Epigenomics of Q1centromere assembly and function. Current Opinion in Cell Biology 22: 1-9.
Sullivan, B. A. and G. H. Karpen. 2004. Centromeric chromatin exhibits a histone modification pattern that is distinct from both euchromatin and heterochromatin. Nature Structural & Molecular Biology 11: 1076 – 1083.
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