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REVIEW ARTICLE |
Howard Hughes Medical Institute, Department of Embryology, Carnegie Institution of Washington, Baltimore MD, 21210, USA
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Biochemical and electron microscopy (EM) studies indicate that cohesin forms a ring-like structure with Mcd1p considered to control ring opening and closing (Melby et al. 1998; Anderson et al. 2002; Haering et al. 2002; Gruber et al. 2003). A simple cohesin ring cleavage model has become popularized as explaining both the mechanism of cohesion establishment and its dissolution (Fig. 1; Nasmyth & Haering 2005). In this model, cohesin rings are loaded onto chromosomes prior to S phase. The rings are extremely static so that the DNA replication machinery must pass through them, which entraps sister chromatids and thereby establishes cohesion (Haering et al. 2002; Gruber et al. 2003). Consequently, cohesion would be maintained until Mcd1p is cleaved by separase at anaphase onset, causing cohesin ring dissociation from chromosomes and cohesion dissolution (Haering et al. 2002; Gruber et al. 2003). The strongest evidence for the ring cleavage model comes from early experiments in budding yeast. However, a further series of experiments in fission yeast, vertebrate cells and even budding yeast are incompatible with this simple ring cleavage model. These experiments reveal that cohesin is driven off chromosomes without separase activation or Mcd1p/Rad21p cleavage and that sister chromatid cohesion can be dissolved without cohesin dissociation from chromosomes. This review will address how these new findings reveal a multifaceted mechanism governing sister chromatid cohesion establishment, maintenance and dissolution during mitotic cell division.
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In this model, cohesion establishment is restricted to S phase because the replication fork moves through static cohesin rings already loaded onto DNA (Fig. 1). Cohesion is dissolved only when the Mcd1p/Rad21p subunit is cleaved by separase, which causes cohesin to dissociate from DNA. Cleavage ensures that cohesion dissolution is an irreversible step. This model makes several strong predictions. First, once sister chromatid cohesion has been established, the cohesin complex cannot dissociate from chromosomes unless Mcd1p/Rad21p is cleaved. Second, sister chromatid cohesion dissolution only occurs when cohesin has dissociated from chromosomes. Third, cleavage should be restricted to anaphase and most, if not all, Mcd1p should be cleaved. Initial experiments in budding yeast seemed to fit these expectations. Either mutation of separase (esp1) or mutation of the Mcd1p/Scc1p cohesin sub-unit to render it resistant to Esp1p proteolytic cleavage (non-cleavable Mcd1p) resulted in a failure of sister chromatid dissociation along the chromosome length (Uhlmann et al. 1999). Normally, budding yeast cohesin is bound to chromosomes in metaphase but lost from chromosomes during anaphase (Michaelis et al. 1997). In esp1 mutants or cells expressing non-cleavable Mcd1p, cohesin complex remains bound to chromosomes during anaphase (Ciosk et al. 1998; Uhlmann et al. 1999). Finally, a significant percentage of Mcd1p is cleaved during anaphase (Uhlmann et al. 1999). Because the ring model relies on an obligate Mcd1p cleavage at anaphase to dissolve cohesion, I refer to it as the static cohesin ring model.
The issue of how cohesin is initially loaded onto chromosomes prior to replication presents the first challenge to the static ring model. Biochemical data indicates that Smc1p and Smc3p hetero-dimerize via their hinges to form a V-shaped complex and Mcd1p binds to the SMC globular heads to form a ternary ring-like complex (Haering et al. 2002; Gruber et al. 2003). Scc3p associates with Mcd1p but not the SMC subunits to form the final complex (Haering et al. 2002). Soluble cohesin exists as an intact complex (Losada et al. 1998; Toth et al. 1999; Sumara et al. 2000) so how can the soluble complex be loaded onto DNA if the ring cannot be opened without Mcd1p/Rad21p cleavage? One proposed solution came from the identification of a complex containing Scc2p and Scc4p, whose function is required for cohesion establishment and cohesin localization to chromosomes (Furuya et al. 1998; Toth et al. 1999; Ciosk et al. 2000; Rollins et al. 2004; Bernard et al. 2006; Seitan et al. 2006). The Scc2/4p complex binds to cohesin and its function is required prior to or during S phase (Ciosk et al. 2000; Bernard et al. 2006). Therefore, it was proposed that the Mcd1p/Rad21p sub-unit is transiently displaced by the Scc2/4p complex, which opens the cohesin ring to enables a chromatid to enter and become trapped (Haering et al. 2002). This explanation means that the cohesin ring actually can be opened, but that Scc2/4p complex temporally restricts opening to S phase. Alternatively, Scc2/4p could simply facilitate cohesin complex association with chromosomes. Recent in vivo studies have implicated SMC1/3 hinge regions in efficient cohesin localization to DNA, illustrating the uncertainty in our understanding of how cohesin complex binds to chromosomes (Gruber et al. 2006; Milutinovich et al. 2007).
The data assessing whether cohesin forms a ring entrapping DNA is not definitive
The notion that cohesin complex forms a static ring around sisters stems, in part, from biochemical studies in budding yeast. Cohesin binding to bulk chromatin was reportedly so stable that it cannot be dissociated at even 1.6 M KCl, which was proposed to provide support for a static ring entrapping DNA (Ciosk et al. 2000). However, histone binding to DNA is also salt resistant (Kornberg 1977) so this parameter alone doesn't provide any real support for ring entrapment. Two examples indicate that cohesin binding to DNA may be less robust that initially reported. First, cohesin binding to bulk chromatin begins to be disrupted at 250 mM KCl and is mostly lost at only 500 mM KCl (V. Guacci, unpublished data). The second example comes from studies of circular mini-chromosomes isolated from metaphase-arrested cells, and subsequent assays of the ability of chromosomal proteins to precipitate mini-chromosome DNA (Ivanov & Nasmyth 2005). Antibodies to histones, either histone H2B or to the specialized centromeric histone H3 Cse4p (CENPA), precipitated almost all the mini-chromosomal DNA, whereas cohesin antibodies could only precipitate 10% of this DNA (Ivanov & Nasmyth 2005). This difference suggests that cohesin binds DNA significantly less tightly than histones. The minor population of mini-chromosomal DNA precipitated by cohesin was greatly reduced by linearization, which led to the proposal that cohesin rings slid off the DNA ends (Ivanov & Nasmyth 2005). However, these linearization experiments also showed that Cse4p binding of mini-chromosome DNA was significantly reduced (Ivanov & Nasmyth 2005), casting doubt on a ring sliding interpretation. Moreover, most mini-chromosomes cannot be precipitated by cohesin, raising questions as to whether the residual 10% that does precipitate is representative of the behavior of all cohesin. Finally, nicking the circular mini-chromosomes also reduces cohesin binding although not as dramatically as linearization, indicating that DNA topology influences cohesin binding (Ivanov & Nasmyth 2005). This raises the possibility that other topological changes could have more dramatic effects on cohesin binding.
The observations that cohesin dissociates from DNA at more moderate salt concentrations and that cohesin binding to DNA is weaker than histones do not rule out a cohesin ring entrapping DNA. However, they suggest that a putative ring could be opened without Mcd1p cleavage or cohesin association with chromosomes can be disrupted. Thus, the question of whether cohesin forms a ring around DNA or merely associates with DNA remains unresolved. In any event, these experiments do not assess how sister chromatids are held together.
Cohesion establishment requires one or more steps after cohesin deposition
The cell cycle window where cohesin is chromosomally bound varies depending on the organism. Fission yeast cohesin is chromosomally bound at all cell cycle stages (Tomonaga et al. 2000). Vertebrate cohesin is bound to chromosomes at all times except for a small window from anaphase onset though early telophase (Losada et al. 1998, 2000; Sumara et al. 2000). Budding yeast cohesin is bound from the G1/S phase transition through metaphase (Michaelis et al. 1997). One common feature is that cohesin is bound to chromosomes prior to DNA replication. However, cohesin deposition prior to replication is not sufficient to establish cohesion. DNA replication/repair factors such as PCNA and alternative RFC subunits play a role because when mutated, cause a modest (10%–20%) defect in establishment (Skibbens et al. 1999; Hanna et al. 2001; Mayer et al. 2001; Kenna & Skibbens 2003; Moldovan et al. 2006). Ctf7p/Eso1p is the most important factor as loss of its function during S phase causes precocious sister dissociation at levels comparable to cohesin complex mutants (Skibbens et al. 1999; Toth et al. 1999). Ctf7p has physical and genetic interaction with many efficiency factors, including PCNA and alternative RFC proteins (Skibbens et al. 1999; Mayer et al. 2001; Kenna & Skibbens 2003; Moldovan et al. 2006). This connection supported the idea that cohesion establishment is coupled with DNA replication (Skibbens et al. 1999; Moldovan et al. 2006).
In yeast, the precise chromosomal sites where cohesin complex binds, termed cohesin-associated regions (CARs), have been determined using chromatin immunoprecipitation (ChIP) (Blat & Kleckner 1999; Megee et al. 1999; Laloraya et al. 2000; Glynn et al. 2004; Lengronne et al. 2004; Weber et al. 2004). Despite the large cohesion defect of budding and fission yeast ctf7/eco1 mutants, the levels of cohesin and distribution at CARs are similar to that of wild-type cells (Tanaka et al. 2001; Noble et al. 2006; Milutinovich et al. 2007). These data support a model where cohesin complex is loaded at CARs and the nascent sister chromatids become paired by a Ctf7p-dependent step during or soon after replication fork passage. A similar conclusion was drawn from experiments using non-cleavable Mcd1p. Wild-type cells were arrested at metaphase and then cohesin complex containing non-cleavable Mcd1p was loaded onto chromosomes (Haering et al. 2004). Upon release from metaphase, anaphase chromosome segregation is normal even though non-degradable Mcd1p remained on chromosomes, indicating that the metaphase loaded cohesin did not generate cohesion (Haering et al. 2004). If sister chromatid cohesion were formed by a single cohesin ring entrapping both sister chromatids, one would expect that loading cohesin at metaphase would result in the complex wrapping around the already paired sisters at some frequency. Moreover, once the static cohesin ring had been loaded before replication, it is hard to see why DNA replication factors would subsequently be needed for efficient establishment.
I have depicted three models to explain a post-deposition step for establishment. One model posits that cohesin forms a dynamic ring around both sisters, which is initially opened to load cohesin on chromosomes and then re-opened to establish cohesion (Fig. 2A). The second version is a single cohesin ring that is opened to entrap only one sister chromatid, then after replication there are two complexes at sister CARs which associate to form cohesion (Fig. 2B). The third model is similar to the second except that cohesin only associates with DNA rather than entrapping it (Fig. 2C). In these models, the coupling of establishment with replication is a consequence of factors associated with the replication machinery serving to either re-open the cohesin ring or to promote cohesin dimerization. Alternatively, the temporal restraint of establishment could also be mediated by restricting the expression or activity of such factors to S phase, rather than a coupling to replication per se.
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Other observations in yeast and vertebrates are best explained using the alternative models described in Fig. 2 rather than the static cohesin ring depicted in Fig. 1. Pds5p is another evolutionarily conserved protein required for sister chromatid cohesion (Denison et al. 1993; van Heemst et al. 1999; Hartman et al. 2000; Panizza et al. 2000; Sumara et al. 2000; Tanaka et al. 2001; Wang et al. 2002; Stead et al. 2003; Dorsett et al. 2005). Pds5p is considered to function primarily as a regulator that promotes sister chromatid cohesion maintenance, rather than serving a structural role in cohesion (Tanaka et al. 2001; Stead et al. 2003). It co-localizes temporally and spatially with cohesin at CARs in yeast (Hartman et al. 2000; Panizza et al. 2000; Tanaka et al. 2001). Cohesin and Pds5p co-immunoprecipitate but the interaction is salt sensitive so Pds5p is not considered a cohesin sub-unit (Sumara et al. 2000). In budding and fission yeast pds5 mutants, most sister chromatids do establish cohesion, but exhibit precocious sister dissociation at centromere proximal and distal loci during mitosis at levels similar to cohesin complex mutants (Fig. 3A; Hartman et al. 2000; Tanaka et al. 2001; Stead et al. 2003). There is a minor defect in establishment in budding yeast pds5 mutants (Fig. 3A; see 45 min time point), which will be discussed below (Noble et al. 2006). Similarly, both human PDS5 orthologs are required for the maintenance of sister chromatid cohesion at metaphase, but there seems to be some specialization (Losada et al. 2005). Depletion of human Pds5A alone or Pds5B alone caused more pronounced loss of either arm cohesion or centromeric cohesion, respectively (Losada et al. 2005). Neither single PDS5 depletion gave cohesion defects as pronounced as Rad21 depletion, but it was reported that the Pds5A and Pds5B double depletion was not possible for technical reasons, so there may be some functional overlap (Losada et al. 2005). Cohesin still remains broadly bound to chromosomes in yeast, Drosophila and human cells when PDS5 function is compromised (Fig. 3B; Hartman et al. 2000; Tanaka et al. 2001; Dorsett et al. 2005; Losada et al. 2005). The fact that cohesion is established but then dissolved without cohesin complex dissociation is incompatible with the static cohesin ring model.
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Thus, sister chromatid cohesion can be dissolved even when cohesin remains abound to chromosomes. In addition, there are region specific differences in the regulation of cohesin binding and sister chromatid cohesion that occur at times when separase is not active. It is difficult to reconcile these results with the notion that separase mediated Mcd1p/Rad21p cleavage induces cohesin dissociation to drive cohesion dissolution. In the case of a dynamic cohesin complex, the ring would transiently reopen and allow one sister to diffuse away followed by ring reclosure or the open ring dissociates and then rebinds a single chromatid (Fig. 4A). Alternatively, the association between adjacent complexes is weakened so that sisters dissociate but cohesin binding to DNA is unaffected (Fig. 4B,C).
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Another result contrary to the static ring model can be seen during the normal progression from prophase to metaphase. In vertebrates, cohesin complex is found between sister chromatids along the length of prophase chromosomes (Losada et al. 1998, 2000; Sumara et al. 2000). By metaphase, the amount cohesin complex is dramatically decreased along chromosome arms but remains high over the centromeric heterochromatin, a process termed the prophase removal pathway (Losada et al. 1998, 2000; Sumara et al. 2000). This process is not dependent on separase proteolysis since it occurs prior to securin destruction and no Rad21p cleavage products are detected (Losada et al. 1998, 2000; Sumara et al. 2000; Hauf et al. 2001). Even when cohesin contains non-cleavable Rad21p, prophase removal occurs normally (Hauf et al. 2005). These data clearly demonstrate that cohesin is removed from chromosomes without Rad21p cleavage during the normal course of mitotic chromosome morphogenesis.
To avoid violating the static ring model, one could postulate that the cohesin population removed during prophase does not function in cohesion so its properties do not reflect how sister chromatid cohesion is dissolved. However, several experiments demonstrate that this is not the case. Prophase removal requires the CDC5/POLO kinase and both RAD21 and SA2 proteins are POLO (Plk1) kinase substrates (Waizenegger et al. 2000; Losada et al. 2002; Sumara et al. 2002; Gimenez-Abian et al. 2004). SA2 is the key target because its phosphorylation increases during mitosis and the pool of soluble cohesin complex contains the phosphorylated SA2 (Sumara et al. 2002; Kueng et al. 2006). Cells expressing the SA2-12xA allele, which lacks Plk1 phosphorylation sites, are inhibited for prophase removal so similar levels of cohesin are found between sisters at centromeres and chromosome arms on metaphase chromosomes (Hauf et al. 2005). More definitively, in contrast to wild-type cells, chromosome arms now remain associated after prolonged metaphase arrest (Hauf et al. 2005). Wapl and sororin are two other proteins that also regulate the prophase removal pathway in vertebrate cells (Rankin et al. 2005; Gandhi et al. 2006; Kueng et al. 2006). Wapl depletion by siRNA results in significantly higher levels of cohesin complex on metaphase chromosome arms and more robust arm cohesion (Gandhi et al. 2006; Kueng et al. 2006). Unlike in normal cells, phosphorylated SA2 remains bound to metaphase chromosomes in Wapl depleted cells (Kueng et al. 2006). These data indicate that Waplp promotes prophase removal and that cohesin dissociation is not simply a consequence of SA2 phosphorylation weakening its chromosomal binding. Sororin is less well characterized but when sororin levels are increased in Xenopus extracts, mitotic chromosomes assemble with somewhat higher cohesin levels and more tightly associated sister chromatids (Rankin et al. 2005). Thus, the cohesin population modulated by prophase removal is indeed functional for mediating cohesion, but can be removed without Rad21p cleavage.
Waplp and sororin have each been shown to physically interact with cohesin (Gandhi et al. 2006; Kueng et al. 2006). Either Wapl over-expression or sororin depletion causes precocious sister chromatid dissociation (Rankin et al. 2005; Gandhi et al. 2006). These data indicate that Waplp and sororin intimately interact with cohesin but that Wapl and Sororin serve opposite functions. However, the cohesin complex has not been assayed so it is not known whether Waplp over-expression or sororin depletion drives cohesin from chromosomes or recapitulates the pds5 mutant phenotype, cohesion loss with cohesin remaining bound.
Sister chromatid cohesion can be dissolved during anaphase when separase is impaired
The static ring model predicts that inhibiting the Mcd1p/Rad21p cleavage pathway will generate defects in sister chromatid separation, with regions containing the highest cohesin complex levels being most impaired. This simple expectation is not fulfilled in fission yeast. When the fission yeast separase mutant (cut1) is grown at non-permissive temperature, chromosome segregation is indeed defective (Hirano et al. 1986; Funabiki et al. 1993). However, most chromosomal DNA segregates in many cells and only a small region appears unable to separate so is ultimately cut by cytokinesis (Hirano et al. 1986; Funabiki et al. 1993). Precise characterization of centromeric loci using fluorescent in situ hybridization (FISH) reveals their complete sister dissociation and segregation to opposite poles (Funabiki et al. 1993). Even telomeric loci had patterns consistent with some level of sister dissociation but appeared delayed or trapped in the mid-zone. The highest levels of cohesin complex are found over large peri-centric domains, a pattern conserved from yeast to vertebrates (Losada et al. 1998, 2000; Blat & Kleckner 1999; Megee et al. 1999; Laloraya et al. 2000; Sumara et al. 2000; Tomonaga et al. 2000; Waizenegger et al. 2000; Glynn et al. 2004; Lengronne et al. 2004; Weber et al. 2004). Consistent with this distribution, cohesion is more robust at centromeric regions than at chromosomal arm regions in plant and animal cells (Rieder & Palazzo 1992) as well as in budding and fission yeast (Bernard et al. 2001; Eckert et al. 2007). The cut1 defect, where segregation is perturbed at centromere distal regions but not at centromeric regions, is the opposite of that expected for an obligate separase mediated Mcd1p/Rad21p cleavage model. The fact that only a very minor amount of fission yeast Rad21p is cleaved (< 5% of total Rad21p) at anaphase is consistent with a limited role for separase (Tomonaga et al. 2000).
Analysis of human cells perturbed for cohesion dissolution also fails to fit the separase cleavage model for cohesion dissolution. In human cells, the role of separase has been characterized by use of siRNA (Waizenegger et al. 2002; Gimenez-Abian et al. 2005; Papi et al. 2005). The analysis is complicated by the fact that separase plays multiple roles such as being important for progression the G2 to M phase, prophase to metaphase and spindle elongation (Gimenez-Abian et al. 2005; Papi et al. 2005). Despite the complications, anaphase cells can be found and most chromosomal DNA is segregated, including centromeric regions, but small regions on some chromosome arms remained connected (Gimenez-Abian et al. 2005). The phenotype of separation at centromeres but connections at chromosome arms is similar to that seen in fission yeast. Even if residual separase activity remained in the fission yeast cut1 mutant or siRNA depleted human cells, one would expect that centromeres would be the most resistant to separation due to their high levels of cohesin and more robust cohesion.
Recently, mouse embryonic fibroblast (MEF) cells were created where separase could be deleted using Cre/lox mediated recombination, which allowed the consequences of multiple rounds of cell division without separase to be examined. After separase deletion, ploidy increased with time so that by 48 h and 72 h cells with twice the normal DNA content (8C) and this further increased to > 16C by 96 h (Kumada et al. 2006; Wirth et al. 2006). To characterize chromosomes, separase deleted MEFs were arrested in metaphase using colcemide or nocodazole. In cells examined after 48 h without separase, nearly all cells contained a number of diplo-chromosomes, defined as one chromosome with paired sisters being closely associated with another chromosome with paired sisters (Kumada et al. 2006; Wirth et al. 2006). The diplo-chromosomes had variable sites mediating their association, but were often associated at centromeric regions (Kumada et al. 2006; Wirth et al. 2006). At 72 h post-separase deletion, cells frequently contained quadruple chromosomes with centromeric associations (Kumada et al. 2006; Wirth et al. 2006). FISH analysis demonstrated diplo-chromosomes contained the same chromosomes not random chromosomes (Kumada et al. 2006). Budding yeast separase mutants have defective spindles that collapse during mitosis, and in the subsequent division, reduplication of the spindle poles results in multi-polar spindles (Baum et al. 1988; McGrew et al. 1992; Funabiki et al. 1996a; Kumada et al. 1998; Jensen et al. 2001). The post-separase deleted MEFs also had multi-polar spindle (Kumada et al. 2006; Wirth et al. 2006). The simplest interpretation of the mouse data was that in separase deleted MEFs, sister chromatid cohesion was not completely dissolved during anaphase so that in the subsequent S phase, DNA replication and spindle poles duplication to generated chromosomes with four attached sister chromatids and multi-polar spindles (Kumada et al. 2006; Wirth et al. 2006).
At first glance, the phenotype of separase deleted mouse MEFs seems to fit that predicted by the separase cleavage model. However, the connections between the diplo-chromosomes are more tenuous than those between sister chromatids (Kumada et al. 2006; Wirth et al. 2006). Most importantly, by 96 h, even though ploidy increases to > 16C, most chromosomes are now individual chromosomes with paired sisters and very infrequently are diplo-chromosomes or quadro-chromosomes detected (Wirth et al. 2006). This result demonstrates that the aberrant connections between most chromosomes have been resolved with increasing time in the absence of separase. It is not known whether this resolution occurs gradually throughout the cell cycle, or at specific stages in mitosis, such as prophase or anaphase. Surprisingly, in separase-depleted cells, whereas robust levels of cohesin are detected between sister chromatids at centromeres and fainter levels detected between sisters along the arms, cohesin complex could not be detected at the regions connecting diplo-chromosomes (Wirth et al. 2006). We cannot rule out that residual cohesin complex remains in the diplo-chromosome connecting regions, but it is below the limits of detection. It may be most cohesion between sisters was actually dissolved in the absence of separase during anaphase, but that residual cohesion is sufficient to prevent complete sister separation. In the succeeding chromosome cycles, separase independent mechanisms for releasing cohesin or dissolving cohesion promote release of the residual cohesion. Alternatively, it may be that the connections between diplo-chromosomes are not the same as those between sister chromatids, so are eventually resolved by another mechanism unrelated to cohesion dissolution. There is evidence that separase depletion can indeed generate abnormal chromosomes in human cells. When G2 phase HeLa cells are fused with metaphase cells, the G2 chromosomes prematurely condense to form slender single rods reminiscent of early prophase chromosomes (Gimenez-Abian et al. 2005). In contrast, G2 chromosomes from HeLa cells depleted for separase by siRNA form discontinuous and diffuse structures following cell fusion (Gimenez-Abian et al. 2005). This result suggests that the failure of sister segregation in separase deleted cells may be due, at least in part, to chromosome structural abnormalities present in interphase cells, rather than due solely to a failure to dissolve cohesion at anaphase.
There is a difference in the frequency of centromeric connections detected in separase knockout MEFs as compared to separase deficient fission yeast and HeLa cells. The MEF diplo-chromosomes were examined in the absence of spindles (nocodazole or colcemide arrest) whereas the HeLa and fission yeast cells contained spindles. This raises the possibility that the diplo-chromosome centromeric connections are generally too weak to resist poleward microtubule forces. In summary, separase appears to serve a less important role in cohesion dissolution in fission yeast and vertebrate cells than in budding yeast.
Sister chromatid cohesion can be dissolved during anaphase when cohesin contains non-cleavable Rad21p or when Mcd1p cleavage is reduced
Experiments where non-cleavable Rad21p is expressed in human cells also indicate that sister chromatids can still separate. Only about 10% of these cells appeared to have thick bridge of unseparated DNA in the cytokinetic furrow, whereas 90% of anaphase cells had segregated chromosomal DNA connected by a thin DNA thread (Hauf et al. 2001; Yalon et al. 2004). More than half of these threads contained telomeric proximal loci but only rarely did they contain centromeric regions (Yalon et al. 2004). Monitoring of condensed chromosomes as they enter anaphase reveals that centromeric regions indeed often separate in HeLa cells expressing non-cleavable Rad21p but chromosome arms have more difficulty separating (Diaz-Martinez et al. 2007). If cohesion could not be dissolved, one expected outcome is that following a new round of replication, four sister chromatids would be tightly associated. Only a subset of metaphase chromosomes exhibited connections to other chromosomes, even after several days of Rad21p expression (Hauf et al. 2001; Diaz-Martinez et al. 2007). These connections were often at discrete arm or telomeric regions rather than being predominantly at centromeric regions (Hauf et al. 2001; Diaz-Martinez et al. 2007). Thus, the dissolution of centromeric cohesion appears less perturbed by non-cleavable Rad21p than arm cohesion, yet even arm cohesion must often get dissolved at some cell cycle stage. These results are similar to that obtained with separase deficient cells.
Even in budding yeast, an obligatory role for cohesin cleavage in cohesion dissolution is contradicted. The CDC5 kinase is required for Mcd1p phosphorylation and in vitro studies reveal that Mcd1p is a better substrate for separase when phosphorylated (Uhlmann et al. 2000; Alexandru et al. 2001). One would expect that inhibiting the CDC5 kinase should impair or block cohesion dissolution, with peri-centric regions being the most dramatically affected. In budding yeast cdc5 mutants, Mcd1p is unphosphorylated and cleavage is significantly reduced (Alexandru et al. 2001). However, the dissolution of cohesion occurs with essentially wild-type kinetics at peri-centric regions (35 kb from CEN5) (Alexandru et al. 2001). Telomeric loci also separate in most cdc5 mutant cells but do so only after an approximately 1-h delay (Alexandru et al. 2001). Chromosome spreads of cdc5 anaphase cells show that cohesin complex is still present at high levels at peri-centric regions, but has a more punctate pattern over the bulk DNA mass (Alexandru et al. 2001). Sister centromere separation during anaphase with cohesin still bound to centromeric regions provides another example where cohesion dissolution does not require cohesin dissociation. Moreover, it appears centromere proximal cohesion in budding yeast, like that in human cells, appears less dependent on Mcd1p/Rad21p cleavage than arm cohesion.
Cohesin association with chromosomes is rapidly modulated by chromatin changes
Rather than being a static ring, cohesin is likely to be dynamic in its ability to bind chromosome or mediate cohesion. One study from human cells used FRAP to show that there is in fact some level of dynamic association of cohesin complex with DNA (Gerlich et al. 2006). Data from budding yeast is also consistent with some dynamism. CARs are usually, but not always, found at sites of convergent transcription on chromosome arms in yeast (Laloraya et al. 2000; Glynn et al. 2004; Lengronne et al. 2004). Induction of a promoter causes a decrease in cohesin complex at the most proximal CAR, but has no effect on the adjacent CAR (Glynn et al. 2004; Lengronne et al. 2004). These results led to the proposal that cohesin rings entrapping sister chromatids are pushed by transcription and end up at convergent regions (Lengronne et al. 2004). However, cohesin binding over the large peri-centric domain is not restricted to such convergent regions, but rather localizes equally well over transcribed regions as at intergenic regions (Glynn et al. 2004). Moreover, budding yeast kinetochores function as a bidirectional enhancer that can rapidly modulate cohesin binding over the broad peri-centric domain within a single cell-cycle or even during metaphase arrest (Megee et al. 1999; Weber et al. 2004; Eckert et al. 2007). Since cohesin binding can be rapidly increased or decreased over broad domains, some aspect of chromatin structure is likely modulating binding rather than transcription per se.
Different chromatin remodeling complexes have connections with cohesin complex and cohesion. The budding yeast RSC chromatin remodeling complex is important for cohesin complex binding and establishment of sister chromatid cohesion at chromosome arms, but not at centromeric regions (Huang et al. 2004). The ISWI chromatin remodeling complex in fission yeast (Swi6) is required for generating high cohesin complex levels and sister chromatid cohesion at centromeric regions, but not at arm loci (Bernard et al. 2001). Similarly, human ISWI is required for cohesin localization to a sub-set of CARs (Hakimi et al. 2002). Both the RSC and ISWI complexes physically associate with cohesin complex in vivo (Hakimi et al. 2002; Nonaka et al. 2002; Huang et al. 2004). These attributes are similar to the Scc2/4 complex except they are region specific. Another region specific cohesion factor is the SIR2–4 complex, which generates region specific silent chromatin, including at the HMR mating cassette locus (Rusche et al. 2003). The SIR2–4 complex binds to the HMR locus and is required to recruit cohesin to this locus (Chang et al. 2005). Site specific recombination was used to generate intact circular mini-chromosomes containing HMR and demonstrate that cohesin bound at HMR does indeed promote cohesion (Chang et al. 2005).
Histone modifications also influence cohesin complex binding. Histone H2AX phosphorylation is required for cohesin complex recruitment to double strand breaks and to facilitate DNA repair (Unal et al. 2004). Phosphorylated histone H3 and cohesin complex co-localize between sister chromatids of human metaphase chromosomes (Dai et al. 2006). Haspin is the human histone H3 kinase responsible for this phosphorylation (Dai et al. 2006). Haspin activity also regulates prophase removal, stabilizing cohesin complex and arm cohesion when over-expressed or inducing precocious sister dissociation when depleted (Dai et al. 2006). Thus, chromatin likely plays a major role in multiple aspects of cohesin function. These include specifying where cohesin binds, its level of binding, whether cohesin remains bound or dissociates, and finally, its ability to mediate cohesion.
How is cohesion establishment regulated?
The three models are presented to explain how Ctf7p could promote establishment (Fig. 5). I placed cohesin complex closer to the DNA than Pds5p to reflect the fact that Pds5p localization is dependent on cohesin complex, but cohesin complex binding to chromosomes is largely independent of Pds5p function (Fig. 3B,C; Hartman et al. 2000; Tanaka et al. 2001; Dorsett et al. 2005; Losada et al. 2005). A generic open cohesin ring is depicted since it is unclear where the ring would be opened. Dimerization between two cohesin complexes has not yet been shown, but an interaction between the SMC hinges to form cohesin dimers has been modeled as being analogous to that of the SMC like Rad50p in the MRE complex (Milutinovich & Koshland 2003). How can Ctf7p promote establishment when it does not localize at CARs or co-immunoprecipitate (co-IP) with cohesin complex? Pds5p provides a connection. Genetic and biochemical data from budding and fission yeast indicate that Ctf7p and Pds5p interact to regulate establishment (Tanaka et al. 2001; Noble et al. 2006). The evidence indicates both co-operative and antagonistic roles so the models presented here incorporate both aspects of this regulation. Cohesin complex and Pds5p are at CARs prior to replication, but Pds5p inhibits establishment by either preventing cohesin ring opening or to block dimerization (Fig. 5). To promote establishment, Pds5p helps recruits Ctf7p to CARs, where it transiently displaces Pds5p. Ctf7p now acts on cohesin complex to enable entrapment of both sisters or facilitate cohesin complex interactions. Finally, Pds5p returns to form a protected sister of cohesion.
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How is cohesin removal during prophase regulated?
Pds5p is required for cohesion maintenance and Waplp is a key promoter of prophase removal so both figure prominently in the three models shown (Fig. 6). Waplp and Pds5p interact independently of cohesin complex, but seem to have opposite roles with regard to regulating sister chromatid cohesion even though cohesin remains bound when either is depleted (Losada et al. 2005; Gandhi et al. 2006; Kueng et al. 2006). Using the Ctf7p–Pds5p model as a paradigm, the Waplp–Pds5p interaction would have both positive and negative roles (Fig. 6). Pds5p protects cohesion by locking the cohesin ring (Fig. 6A) or stabilizing cohesin dimers (Fig. 6B,C). To promote prophase removal, Waplp displaces Pds5p to gain access to cohesin. Waplp interacts with a dimer of Rad21p and SA1p and it was proposed that Waplp opens the cohesin ring by altering Rad21p binding to Smc1p and Smc3p (Gandhi et al. 2006), so I have shown the ring opening (Fig. 6A,B). Alternatively, the action of Wapl on RAD21 and SA2 could weaken cohesin affinity for chromosomes (Fig. 6C).
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What is the role of separase in anaphase?
The emerging picture is that cohesin is regulated at multiple cell cycle stages, initially to load onto chromosomes, then to establish cohesion and again for prophase removal. At all these stages, either the cohesin ring is opened or its association with chromosomes is altered. An obligate separase cleavage model requires that at anaphase onset, none of these other mechanisms operate to remove cohesin. This specialization does not seem plausible unless one assumed that the cohesin remaining on metaphase chromosomes is fundamentally distinct from cohesin removed at prophase. However, the metaphase cohesins are not distinct because SGO1 depletion in vertebrate cells induces complete loss of cohesin binding and sister chromatid cohesion by early mitosis in a separase independent manner (Salic et al. 2004; Tang et al. 2004; Kitajima et al. 2005). The fact that most vertebrate cohesin is not chromosomally bound at metaphase has been invoked to explain why only a minor population of total Rad21p is detected as cleavage products (Sumara et al. 2000; Waizenegger et al. 2000). The obvious prediction of the obligate separase cleavage model is that when prophase removal is inhibited, there should be a significant increase in RAD21 cleavage at anaphase onset in order to remove the additional chromosomally bound cohesins. However, SA2-12xA expressing cells rapidly remove the extra cohesions at anaphase onset without any obvious increase in Rad21p cleavage, and undergo normal chromosome segregation (Hauf et al. 2005). Because Rad21p cleavage did not increase in SA2-12xA expressing cells, it appears that the population of cohesin destined to be cleaved is predetermined rather than representing an obligate removal of functional cohesin from metaphase chromosomes. Therefore, it is likely that multiple mechanisms operate concurrently during the metaphase to anaphase transition.
Why would cells require multiple mechanisms to dissolve cohesion at anaphase onset?
Earlier in the cell cycle, the processes which modulate cohesin loading onto chromosomes, cohesion establishment and prophase removal do not have to function with 100% efficiency. CAR sites are distributed along the entire length of yeast chromosomes (Blat & Kleckner 1999; Megee et al. 1999; Laloraya et al. 2000; Glynn et al. 2004; Lengronne et al. 2004; Weber et al. 2004). It has been estimated that in budding yeast, there are roughly 5–20 cohesin complexes at each CAR site (Weitzer et al. 2003). This broad distribution of CARs and the multiple cohesins at each CAR provide a level of redundancy such that even if only the efficiency of cohesin loading or establishment was only 80%, it should not be detrimental to cells. In contrast, even if only 5% or 10% of cohesion sites were not dissolved at anaphase, sister chromatid non-disjunction would likely result and generate detrimental or lethal events. The use of multiple mechanisms may be necessary to ensure that 100% of cohesion is dissolved in a very short time interval at anaphase onset. Separase mediated cleavage would be only one of several ways to promote cohesion dissolution. Other mechanisms include altering cohesin binding to chromosomes by either by opening the cohesin ring or altering its affinity for chromosomes and cohesion dissolution without cohesin complex dissociation. The differences observed from organism to organism would simply reflect which mechanism is predominant.
Separase is clearly required for chromosome segregation. Separase localizes to the mitotic spindle and spindle poles and is required for spindle integrity (Baum et al. 1988; McGrew et al. 1992; Funabiki et al. 1996a; Kumada et al. 1998; Jensen et al. 2001). The catastrophic anaphases seen in separase-depleted cells could reflect dual defects in cohesion dissolution and spindle function. One possibility is that separase could serve a regulatory role to co-ordinate spindle elongation with cohesion dissolution at anaphase onset. It has been suggested that separase mediated cleavage makes the dissolution of cohesion irreversible (Uhlmann 2003). However, establishment occurs only during S phase. The fact that cohesin complex loaded onto chromosomes in metaphase does not generate cohesion clearly illustrates this restriction (Haering et al. 2004). Thus, once cohesion is dissolved during mitosis, by whatever mechanism, it cannot be re-established even if cohesin rebound chromosomes. This makes Mcd1p cleavage, or lack thereof, irrelevant with respect to irreversibility. It may be that Mcd1p/Rad21p cleavage occurs at CAR sites where lower levels of ring opening or affinity modifying factors are found. Alternatively, separase could serve as a fail-safe function to remove residual cohesion not dissolved by other mechanisms. A more intriguing notion is that cleaved cohesins are performing a specialized biological role, either as part of their role in cohesion role, or independent of cohesion. Cohesin complex has been implicated in multiple roles, including gene silencing, chromosome condensation and recombination (Jessberger et al. 1996; Guacci et al. 1997; Donze et al. 1999; Hartman et al. 2000; Unal et al. 2004). It is possible that the various mechanisms for removing cohesin or modulating its function evolved to more efficiently regulate these different processes.
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* Correspondence: E-mail: guacci{at}ciwemb.edu
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Accepted: 7 April 2007
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