Chromosome Cohesion – Rings, Knots, Orcs and Fellowship

Commentary 2107 Chromosome cohesion – rings, knots, orcs and fellowship

Laura A. Díaz-Martínez1,*, Juan F. Giménez-Abián2 and Duncan J. Clarke3 1Department of Pharmacology, UT-Southwestern Medical Center, 6001 Forest Park Rd, Dallas, TX75390, USA 2Proliferación Celular, CSIC, Ramiro de Maeztu 9, 28040-Madrid, Spain 3Department of Genetics, Cell Biology and Development, University of Minnesota, 420 Washington Avenue SE, Minneapolis, MN55455, USA *Author for correspondence (e-mail: [email protected])

Accepted 6 May 2008 Journal of Cell Science 121, 2107-2114 Published by The Company of Biologists 2008 doi:10.1242/jcs.029132

Summary Sister-chromatid cohesion is essential for accurate chromosome the onset of anaphase allows sister-chromatid separation. segregation. A key discovery towards our understanding of However, recent studies have revealed activities that provide sister-chromatid cohesion was made 10 years ago with the cohesion in the absence of cohesin. Here we review these identification of cohesins. Since then, cohesins have been shown advances and propose an integrative model in which to be involved in cohesion in numerous organisms, from yeast to chromatid cohesion is a result of the combined activities of mammals. Studies of the composition, regulation and structure multiple cohesion mechanisms. of the cohesin complex led to a model in which cohesin loading during S-phase establishes cohesion, and cohesin cleavage at Key words: Cohesin, Chromosome cohesion, Catenations

Introduction et al., 1997), as measured using the LacO/LacI-GFP system Accurate chromosome segregation is the crux of mitosis – failure (Straight et al., 1996). A cohered locus is normally visualized as a causes genetic disorders, spontaneous abortions and cancer (Kops discrete fluorescent spot in early mitosis, but this was observed to et al., 2005; Rubio et al., 2005; Shah et al., 2003; Weaver and prematurely separate into two discrete spots in cohesin mutants. Cleveland, 2006; Weaver et al., 2007). To allow accurate Second, the association of cohesin with DNA mirrors the cohesion segregation, sister chromatids must remain cohered from their cycle: cohesin is bound to chromosomes from S-phase, when inception, which occurs in S-phase, until anaphase (Fig. 1). Sister- cohesion is established, until anaphase, when cohesion is abolished chromatid cohesion has been proposed to depend on a group of (Michaelis et al., 1997). Other experiments indicate that cleavage proteins called cohesins, which were first identified in yeast (Guacci of Mcd1 is necessary for loss of cohesion. Sister chromatids cannot

Journal of Cell Science et al., 1997; Michaelis et al., 1997), that form a tetra-subunit complex separate in yeast cells that express non-cleavable Mcd1 that is of Mcd1 (also known as Rad21 in Schizosaccharomyces pombe and resistant to Esp1 (separase) (Uhlmann et al., 1999). Conversely, Scc1 in Saccharomyces cerevisiae), Smc1, Smc3 and Scc3 (also artificial Mcd1 cleavage (not by Esp1) leads to premature sister- known as SA1 and SA2 in mammals). This ‘cohesin complex’ can chromatid separation (Uhlmann et al., 2000). Together, these studies form a 35-nm ring that tethers sister duplexes. Two main models form the foundation of a compelling model in which cohesion have been proposed to explain how this ring provides cohesion: the depends on the cohesin complex and cleavage of the ring results ring might entrap both duplexes (Haering et al., 2002; Nasmyth in loss of cohesion. and Haering, 2005) or two rings might interact, each one entrapping Here we review the role of cohesin in sister-chromatid cohesion a duplex (Anderson et al., 2002; Guacci, 2007; Huang et al., 2005). and discuss recent discoveries of cohesin-independent mechanisms Conserved orthologs of the cohesins are found in metazoans of cohesion. We will not, however, discuss recent evidence (Darwiche et al., 1999; Losada et al., 1998; Rollins et al., 1999; indicating that the regulation of the onset of anaphase by the spindle- Sumara et al., 2000), indicating that cohesin might be the universal assembly checkpoint is more complex than had been previously mechanism of sister-chromatid cohesion. Accessory proteins appreciated (Chestukhin et al., 2003; Gimenez-Abian et al., 2005a; regulate the loading of cohesin onto chromosomes (reviewed in Gimenez-Abian et al., 2005b; Papi et al., 2005; Zur and Brandeis, Huang and Laurent, 2004; Lee and Orr-Weaver, 2001; Nasmyth 2001) because this topic has been reviewed in detail elsewhere and Schleiffer, 2004; Riedel et al., 2004; Skibbens, 2005; Uhlmann, (Clarke et al., 2005). In this Commentary, we propose a model in 2004) and a protease, separase, cleaves Mcd1, presumably leading which cohesion depends on multiple mechanisms that collaborate to sister-chromatid separation (Ciosk et al., 1998; Uhlmann et al., to ensure cohesion along the length of the whole chromosome. 1999; Uhlmann et al., 2000). Thus, it is thought that cohesin-loading factors establish cohesion and separase triggers anaphase. Cohesion without cohesin in yeast The first evidence that protein complexes provide cohesion came Although it has been widely documented in budding yeast that from studies of circular minichromosomes in budding yeast; these cohesins can provide cohesion, the penetrance of the loss-of- minichromosomes remain cohered in the absence of DNA cohesion phenotype in cohesin mutants depends on the locus that intertwinings (Guacci et al., 1994; Koshland and Hartwell, 1987). is observed (Antoniacci and Skibbens, 2006; Baetz et al., 2004; Two lines of investigation have indicated that cohesin provides this Ciosk et al., 2000; D’Amours et al., 2004; Guacci et al., 1997; activity. First, mutant cohesins have been found to cause an Lam et al., 2006; Mayer et al., 2001; Michaelis et al., 1997; Strom increased distance between sister loci (Guacci et al., 1997; Michaelis et al., 2007; Sullivan et al., 2004; Suter et al., 2004; Toth et al., 2108 Journal of Cell Science 121 (13)

2007; Losada et al., 2005; Rankin et al., 2005; Toyoda and Yanagida, 2006; Watrin et al., 2006) cells. Typically, increased distance between sister chromatids was observed in these experiments (Losada et al., 2005; Toyoda and Yanagida, 2006), but none of these approaches resulted in complete separation of the ion chromatids. Briefly, Losada and co-workers observed that ~30% of Cohes Chromosome Accurate biorientation segregation mitotic chromosomes that were assembled in cohesin-depleted S phase No cohes Xenopus egg extracts had unpaired regions and that the distance ion between sister chromatids was increased, but without complete separation (Losada et al., 2005). These findings were corroborated by Kenney and Heald with the additional important observation that centromere separation in cohesin-depleted Xenopus egg extracts is promoted only upon bipolar attachment to the mitotic spindle

No biorientation Aneuploidy (Kenney and Heald, 2006). It could be argued that these depleted extracts have small amounts of cohesin, which are sufficient for Fig. 1. Sister-chromatid cohesion is required for accurate chromosome centromere cohesion. However, Kenney and Heald simultaneously segregation. During a mitotic cell cycle, the genome is duplicated in S phase depleted two cohesin subunits almost to completion. and each identical copy is then segregated into the daughter cells. In These data partly agree with phenotypes that have been observed eukaryotes, this process is complex owing to the fragmentation of the genome in Scc1–/–/– chicken cells (the chicken Scc1 gene is located on in several chromosomes. Eukaryotic cells have evolved a mechanism, termed sister-chromatid cohesion, that keeps the two copies of a chromosome (sister chromosome 2, which is trisomic in DT40 cells), which exhibit an chromatids) together from the moment of duplication to the onset of anaphase. increased distance between, but retained association of, the sister This mechanism ensures the accurate segregation of one and only one copy of chromatids (Sonoda et al., 2001). Unlike in the Xenopus egg extracts, each chromosome to each daughter cell. When sister-chromatid cohesion is some cohesion was retained even after chromosomes attached to defective, mitotic processes such as chromosome biorientation and chromosome segregation are disrupted, resulting in aneuploidy, a hallmark of the spindle because the Scc1-depleted chicken cells aligned most most cancers. of their chromosomes into a metaphase plate. Consistent with the Xenopus data, chromosome segregation was abnormal (Sonoda et al., 2001; Vagnarelli et al., 2004). Chromosome congression to a metaphase plate followed by aberrant chromosome segregation 1999) (reviewed in Koshland and Guacci, 2000; Nasmyth and were also observed in cohesin-depleted Drosophila cells (Vass et al., Schleiffer, 2004; Skibbens, 2000). Only telomeres lose cohesion in 2003). Cells with separated sister chromatids and aberrant anaphases 100% of cohesin-mutant cells, whereas loci at the chromosome arms were observed after Rad21 knockdown but not after depletion of and pericentromere remain cohered in 40-75% of cells and the stromal antigen (SA), another cohesin subunit. SA-depleted cells ribosomal DNA (rDNA) locus remains cohered in the majority of had cohered sister chromatids (although the distance between the cohesin-mutant cells (Fig. 2). This indicates that cohesin is sufficient two chromatids was slightly increased) and were able to perform

Journal of Cell Science to provide cohesion in yeast, because it seems to be the only anaphase normally (Vass et al., 2003). mechanism that operates at telomeres, but also that cohesin- In human cells, the specific conditions in which cohesin independent mechanisms might contribute to cohesion at other loci. depletions have been performed have led to different interpretations In a scenario in which cohesins were the sole mechanism of the data. Here we describe our attempt to unify these observations. providing cohesion, non-functional cohesin or the absence of a Several reports describe premature loss of cohesion in 40-80% of cohesin subunit should lead to loss of cohesion upon replication of Rad21-depleted mitotic cells (Losada et al., 2005; Rankin et al., any locus. In studies that timed the loss of cohesion in mcd1 mutants, 2005; Toyoda and Yanagida, 2006; Watrin et al., 2006). In all of some cells did lose cohesion as soon as DNA replication was these studies, following Rad21 depletion using RNAi in observed (Noble et al., 2006). However, despite synchronous DNA asynchronous cultures, mitotic cells were isolated and sister- replication within populations of yeast cells in such experiments, chromatid separation scored as a percentage of mitotic cells (rather loss of cohesion typically occurred asynchronously in mcd1 mutants than as a percentage of total cells). In our analyses of cohesin- after the release from G1 arrest. The maximum percentage of mcd1 depleted cells, using the same small interfering (si)RNAs for RNAi, cells that displayed a loss-of-cohesion phenotype was only achieved we observed that ~4% of the total cells both arrested in mitosis and after all the cells reached G2 (Michaelis et al., 1997; Noble et al., had separated sister chromatids (Diaz-Martinez et al., 2007). This 2006). The fact that loss of cohesion in cohesin-defective cells can small percentage of the total cells corresponds to the 40-80% of be delayed for some time after replication suggests that other the mitotic cells described above. mechanisms of cohesion exist. The key issues thus become identifying the source of these arrested mitotic cells that have separated sister chromatids and Cohesin in other eukaryotes determining at which point cohesion was lost. By examining Cohesins are conserved from yeast to humans, which suggests that complete cell cycles in synchronized cells, we observed that ~100% their role in cohesion is conserved. Efforts to directly assess the of cohesin-depleted cells reach mitosis with normally cohered sister role of cohesin in sister-chromatid cohesion in higher eukaryotes chromatids and that all of these are able to form a metaphase plate include the depletion of cohesins from Xenopus laevis egg extracts (Diaz-Martinez et al., 2007). Some cells appeared to lose cohesion (Kenney and Heald, 2006; Losada et al., 1998), an inducible knock- asynchronously, usually after a prolonged metaphase delay, and out of Rad21 in chicken cells (Sonoda et al., 2001), and the use of some of these cells then arrested in mitosis (Diaz-Martinez et al., RNA interference (RNAi) to deplete cohesins from Drosophila 2007). These arrested cells presumably account for the previous melanogaster (Vass et al., 2003) and human (Diaz-Martinez et al., observations of mitotic cells with separated sister chromatids that Multiple cohesion mechanisms 2109

remains unclear, but one possibility is that cleavage of Rad21 has Telomere separation: a signaling role that serves to inactivate the spindle-assembly 1 86% mcd1-1 (NOC) checkpoint, triggering coordinated and efficient loss of cohesion. 0 k b

3 BMH1-locus separation: Upon checkpoint silencing, multiple pathways that drive 24% mcd1-1 (NOC)2 synchronous anaphase might become active. Consistent with the observation that most cohesin-depleted VTC3-locus separation: human cells retain centromere cohesion until metaphase or 56% mcd1-1 (NOC)2 anaphase, the frequency of cells with separated sister chromatids Centromere separation: following RNAi of cohesin dramatically diminishes to less than

~50% mcd1-1 (NOC)3 20% of mitotic cells after 3 hours in colcemid (Inoue et al., 2007) 2

53% mcd1-1 (NOC) and 4% after 16 hours (Diaz-Martinez et al., 2007). By contrast, 40 k b

k b the percentage of cells with separated sister chromatids that is 3 2 URA3-locus separation: observed after a long mitotic arrest in the presence of nocodazole <5% wild type (NOC)4 20% wild type (cdc16)4 remains high (>65% after 16 hours) in cells that have been depleted 25% wild type (NOC)5 of the centromere-guardian Shugoshin (Sgo1). Therefore, Sgo1, a 15% scc1-73 (NOC)6 protein that is thought to protect centromeric cohesin from removal, 2 5 k b 40% mcd1-1 (NOC) might regulate cohesin and additional cohesion factors at the 3 40% smc1-259 (cdc20)7 centromere. Together, these results indicate that sister-chromatid b 40% scc1-73 (NOC)8 cohesion can be maintained in a manner that is independent of 50% scc1-73 (NOC)4 129 k cohesin, but that cohesin is needed for faithful anaphase. Thus, 60% smc1-259 (cdc16)4 80% scc1-73 (cdc16)4 studies in yeast cells and higher eukaryotes have left open the

400 k b 400 90% scc1-73 (cdc20)9 possibility that cohesin and additional factors collaborate to ensure the association between sister chromatids until anaphase. HOR2-locus separation: 27% mcd1-1 (NOC)2 Cohesin-independent cohesion DNA catenations rDNA-locus separation: 57% mcd1-1 (cdc20) 2 DNA catenation (the physical intertwining of sister chromatids) was <10% mcd1-1 (NOC)10 the first mechanism of cohesion to be formally proposed (Murray <10% scc1-73 (cdc20)11 and Szostak, 1985). Catenations are a byproduct of semi- conservative replication (Sundin and Varshavsky, 1980) and Fig. 2. Contribution of cohesin to cohesion in budding yeast depends on the physically couple replication to cohesion – one being the inevitable locus involved. A summary of studies in which a loss of cohesion was consequence of the other (Fig. 3). By contrast, cohesin-mediated observed at different loci in cohesin mutants is shown. These results indicate cohesion is biochemically coupled to replication, because genomic that the specific contribution of the cohesin complex to the cohesion of any DNA can be replicated to completion in the absence of cohesin. two sister-chromatid loci is locus dependent. All the data refer to the loss of Journal of Cell Science cohesion that has been measured in G2-M cells and the methods that were Logically, cells probably co-evolved their mechanisms of genome used to arrest the cells in mitosis are shown in brackets. Note that cdc16 and replication and sister-chromatid cohesion. As a mechanism of cdc20 cells arrest in mitosis in the presence of a functional spindle, whereas cohesion, catenation fulfils the expected physical dependency on nocodazole (NOC) disrupts the spindle. The chromosome depicted in the replication, whereas cohesin-mediated cohesion does not. figure is not drawn to scale and does not represent a particular yeast The removal of catenations is performed by topoisomerase chromosome: the loci that are listed belong to different chromosomes. The α colors reflect the level of penetrance of the loss-of-cohesion phenotype when type II enzymes (Top2 in budding yeast and topoisomerase II cohesin mutations are present. Loci listed in red boxes show more loss of and IIβ in higher eukaryotes), the activity of which is required for cohesion, meaning that cohesion at these loci depends mostly on the cohesin chromosome individualization (Gimenez-Abian et al., 2000), sister- complex. Loci in the green boxes show an ~50% loss of cohesion, suggesting chromatid resolution (Gimenez-Abian et al., 1995) and chromosome that cohesin partially contributes to cohesion at these loci. Loci in the blue boxes show a very low loss of cohesion after cohesin mutation, meaning that segregation (Clarke et al., 1993; Holm et al., 1985; Holm et al., chromosome cohesion at these loci depends mainly on mechanisms other than 1989; Sakaguchi and Kikuchi, 2004; Uemura et al., 1987; Uemura cohesin. Studies referenced in this schematic: 1(Antoniacci and Skibbens, and Yanagida, 1984). That catenations can maintain an association 2006), 2(Lam et al., 2006), 3(Guacci et al., 1997), 4(Michaelis et al., 1997), between chromatids is evident from the failed sister-chromatid 5 6 7 8 (Mayer et al., 2001), (Suter et al., 2004), (Strom et al., 2007), (Toth et al., separation, which leads to a cut (cells untimely torn) phenotype in 1999), 9(Ciosk et al., 2000), 10(D’Amours et al., 2004), 11(Sullivan et al., 2004). the absence of Top2 (Holm et al., 1985; Uemura et al., 1987). What appear to be catenations that link sister-chromatid centromeres are present in human cells that are arrested in mitosis by microtubule are seen after depletion of cohesin (Losada et al., 2005; Rankin poisons (Bickmore and Oghene, 1996). Furthermore, some evidence et al., 2005; Toyoda and Yanagida, 2006; Watrin et al., 2006). indicates that catenation-mediated cohesion is sufficient for cohesion However, because such cells first form metaphase plates before independent of cohesin (Toyoda and Yanagida, 2006; Vagnarelli sister-chromatid separation (Diaz-Martinez et al., 2007), we suggest et al., 2004; Diaz-Martinez et al., 2006), and it has been suggested that this phenotype is not the result of a complete failure in the that the removal of both cohesin and catenations contributes to establishment of cohesion, but is due to uncoordinated loss of regulate sister-chromatid separation (Diaz-Martinez et al., 2006; cohesion during anaphase. We propose that cohesin accounts for Kenney and Heald, 2006; Toyoda and Yanagida, 2006). synchronous sister-chromatid separation at anaphase but is not The requirement of topoisomerase II at different stages of the required for centromere cohesion in early mitosis. The mechanism chromosome cycle is presumably why it has been an effective target by which cohesin promotes synchronous sister-chromatid separation of anti-cancer drugs (Nelson et al., 1984; Tewey et al., 1984). 2110 Journal of Cell Science 121 (13)

regulate cohesion solely via Top2. Smt4 is required for cohesion in the context of a circular minichromosome that lacks catenations, Semi- indicating that there is another Smt4 target that contributes to conservative cohesion. replication of DNA Interestingly, Smt4-dependent cohesion shares three important features with cohesin-mediated cohesion. First, as observed in cohesin mutants, cohesion is lost only in a fraction of smt4 cells. Catenated Second, similar to cohesin-mediated cohesion, Smt4-dependent sister cohesion varies from locus to locus (Bachant et al., 2002). Consistent chromatids with a sufficiency for cohesin-dependent cohesion at telomeres (Antoniacci and Skibbens, 2006), smt4 mutants do not show loss Decatenation of cohesion at telomeres (Bachant et al., 2002), whereas cohesion by topoisomerase II at the URA3 locus is partially lost both in smt4 mutants (Bachant et al., 2002) and in cohesin mutants (Weitzer et al., 2003). These results suggest that these two mechanisms of cohesion have additive Decatenated roles in sister-chromatid cohesion. A systematic analysis to sister understand the specific contributions of each cohesion mechanism chromatids at different loci is needed. Third, the separation that is observed after smt4 or cohesin inactivation occurs after the S-phase is Fig. 3. The formation of catenations and their resolution by topoisomerase II. completed, at least in a subset of cells. These results suggest that Catenations are a byproduct of DNA replication and they form at the points at sister-chromatid cohesion at a specific locus is the result of a which two replication forks collide. Removal of catenations is performed by complex balance between different cohesion mechanisms – absence type-II topoisomerases, which produce a double-strand break in one of of any one of these mechanisms might lead to weakened cohesion the chromatids and pass the other through the break. Two resolved sister that, although able to initially provide association and perhaps chromatids are the result of the strand-passing process and re-ligation reaction. provide cohesion in most of the cells during an unperturbed mitosis, is not sufficient to maintain pairing after a prolonged mitotic arrest. However, the idea that there is a progressive removal of catenations Studies to determine whether this is true, and to test the specific during the cell cycle led to the idea that, although catenations remain contribution of each cohesion mechanism at different loci, are at centromeres until anaphase, they are maintained as a consequence needed. of cohesion and their removal is not regulated. This hypothesis is A cohesion-related role for sumoylation of topoisomerase II has supported by the dispensability of catenations for minichromosome also been revealed in higher eukaryotes. Depletion of the SUMO E3- segregation in yeast (Guacci et al., 1994; Koshland and Hartwell, ligase PIASγ from human cells leads to prolonged metaphase arrest 1987). However, as pointed out by Guacci et al. (Guacci et al., 1994), and lack of enrichment of topoisomerase IIα at centromeres (Diaz- these data do not rule out a function of catenation in the Martinez et al., 2006). Xenopus PIASγ is required for the formation

Journal of Cell Science establishment of cohesion. Moreover, minichromosomes are of a unique SUMO focus at the inner centromere (Azuma et al., 2005) segregated 100-fold less efficiently than endogenous yeast and is responsible for topoisomerase-IIα sumoylation. Disrupting chromosomes (10–3 versus 10–5 errors per cell division) (Koshland PIASγ activity in egg extracts leads to failed chromosome segregation and Hartwell, 1987). Whether this decrease in the faithfulness of (Azuma et al., 2005). Topoisomerase IIα is sumoylated in human chromosome segregation is due to reduced cohesion in the absence cells (Mao et al., 2003) and evidence suggests that PIASγ is the of catenations remains unknown. relevant E3 ligase (Diaz-Martinez et al., 2006; Agostinho et al., 2008). The view that decatenation is unregulated has begun to change Possible redundancy or cooperation between SUMO ligases is in light of studies indicating that topoisomerase IIα is regulated suggested by the dispensability of PIASγ in mice (Wong et al., 2004), by sumoylation during mitosis in yeast (Bachant et al., 2002; which in turn rely on the SUMO-ligase activity of RanBP2 for Takahashi et al., 2006) (reviewed in Porter and Farr, 2004), Xenopus topoisomerase-II enrichment at the centromere (Dawlaty et al., (Azuma et al., 2005; Azuma et al., 2003), mice (Dawlaty et al., 2008). Interestingly, regardless of the particular SUMO ligase that is 2008) and human (Diaz-Martinez et al., 2006) cells. In budding involved, sumoylation of topoisomerase II is required for its yeast, mutation of the SUMO-isopeptidase Smt4 leads to premature enrichment at the centromere and defects in this process can result sister-chromatid separation. This cohesion defect is probably caused in failure of sister-chromatid segregation (Azuma et al., 2005; Diaz- by defects in the regulation of decatenation, because Top2 Martinez et al., 2006; Dawlaty et al., 2008). Furthermore, PIASγ sumoylation was increased in smt4 mutants, a non-sumoylatable most probably regulates a cohesin-independent form of cohesion, top2-SNM mutant rescued the cohesion defect of smt4 mutants because the centromeres of human cells simultaneously depleted of (Bachant et al., 2002) and constitutive Top2 sumoylation induced PIASγ and the cohesin-protector Sgo1 remain cohered but lack enrichment of Top2 at pericentromeric regions (Takahashi et al., cohesin at the centromere (Diaz-Martinez et al., 2006). 2006). These results suggest that sumoylation increases Top2 These data have revealed a mechanism that specifically regulates activity at the centromeres by promoting its recruitment to these topoisomerase IIα at the centromere during mitosis in metazoans. loci, which leads to premature decatenation. Thus, centromeric catenations might be actively maintained until That Smt4-dependent regulation of cohesion is independent of anaphase, in a checkpoint-dependent manner, by modulation of cohesin is indicated by the unchanged distribution of cohesin in the activities of SUMO ligases. However, in addition to smt4 mutants and the sister-chromatid separation observed in these topoisomerase II, other proteins that have cohesion-related mutants in the absence of separase (Bachant et al., 2002). Despite functions, such as Pds5 (Stead et al., 2003) and Ycs4 (D’Amours these intriguing data, other facts indicate that Smt4 is unlikely to et al., 2004), are known to be sumoylated. Therefore, the SUMO Multiple cohesion mechanisms 2111

PP2A

CK2 P SUMO PIAS␥

Catenated sister chromatids Inter-chromatid catenations Topoisomerase II␣

PP2A

Plk1 ? SUMO Pds5 Aurora-B P SA1/2 Haspin Rad21 Separase mc1 S

mc3 S Inter-chromatid Sister cohesin chromatids

Cohesin complex

Fig. 4. Multiple mechanisms of cohesion. Cohesion between any sister-chromatid loci is the result of cooperation between at least two mechanisms of cohesion: DNA catenations (top) and cohesin (bottom). On one hand, the cohesin complex – formed by Smc1, Smc3, Mcd1/Rad21/Scc1 and Scc3/SA1/SA2 – is thought to tether the two sister chromatids together by physical entrapment. DNA catenations, on the other hand, provide sister-chromatid cohesion by the intertwinement of the two chromatids. The contribution of each mechanism at a specific locus might be influenced by factors such as the spacing of catenations, the location of cohesin-binding regions, chromatin structure and changes in chromatid cohesion that are induced after DNA replication (e.g. DNA damage or de novo cohesin loading) (Kim et al., 2002; Nagao et al., 2004; Potts et al., 2006; Sjogren and Nasmyth, 2001; Strom et al., 2004; Strom et al., 2007; Unal et al., 2007). The existence of different cohesion mechanisms is advantageous because it allows differential regulation of cohesion at specific chromosome regions. Both cohesin and catenations are subject to complex regulatory mechanisms and have to be concertedly removed during anaphase, possibly by post-translational modifications such as phosphorylation and sumoylation. Some of these regulatory mechanisms are depicted here.

Journal of Cell Science ligases have the potential to drive different aspects of sister- occurred in ~30% of cells. Interestingly, orc2 mcd1 and orc5 mcd1 chromatid separation and perhaps direct the concerted dissolution of mutants showed an additive defect (Shimada and Gasser, 2007), different cohesion mechanisms (Fig. 4). Further understanding suggesting that ORC and cohesin act in parallel to provide cohesion. of these enzymes will clarify their roles in sister-chromatid cohesion. ORC-mediated cohesion can be dynamically re-established in G2-M after loss of cohesion has occurred (Shimada and Gasser, ORC and condensin 2007), but this cohesion depends on cohesin, suggesting that some During DNA replication, the cohesin loading factors interact with degree of sister-chromatid association is required. This is consistent components of the replication machinery such as PCNA and RFC with a model in which tight sister-chromatid association depends (reviewed in Guacci, 2007; Skibbens et al., 2007); thus, components on cooperative activities. of the replication fork might activate these loading mechanisms. In A role of ORC in mitosis seems to be conserved in metazoans, addition, replication proteins might have a direct role in cohesion. because Orc2 and Orc5 mutants in Drosophila cells (Pflumm and The origin recognition complex (ORC) comprises six subunits, Botchan, 2001), as well as Orc2- or Orc6-depleted human cells marks replication origins in eukaryotic cells and facilitates the (Prasanth et al., 2004; Prasanth et al., 2002), have mitotic defects binding of other replication proteins (Quintana and Dutta, 1999). that increase ploidy, induce mitotic arrest and spindle abnormalities, ORC mutants in budding yeast arrest in mitosis with a normal 2C and affect chromosome congression and condensation (Pflumm and DNA content and show an increased loss of centromeric plasmids Botchan, 2001; Prasanth et al., 2004; Prasanth et al., 2002), which (Dillin and Rine, 1998; Loo et al., 1995). Genetic interactions is consistent with the localization of ORC to centromeres and between ORC genes and MCD1 have been described in budding centrosomes during mitosis (Prasanth et al., 2004; Prasanth et al., yeast, and mutations in orc5 enhance the cohesion defect of mcd1 2002). However, none of these studies has yet described defects in (scc1-73) cells (Suter et al., 2004). Cells that are depleted of Orc2 cohesion (Pflumm and Botchan, 2001; Prasanth et al., 2004; lose cohesion even though the binding of cohesin to chromosomes Prasanth et al., 2002). does not change. Increased targeting of ORC to the URA3 locus Recent studies have also linked the condensin complex decreased the loss of cohesion that was induced by MCD1 mutation with cohesion. Drosophila mutants of a condensin subunit (Shimada and Gasser, 2007). Thus, ORC-mediated cohesion is (Cap-G) have cohesion defects at centromeres, whereas the sufficient for cohesion when ORC is present in high quantities, and chromosome arms remain cohered even in anaphase, which it does not require cohesin. However, the loss of cohesion after causes chromosome missegregation (Dej et al., 2004). In yeast, Orc2 depletion, similar to that seen after cohesin depletion, only mutations in condensin subunits have been associated with loss 2112 Journal of Cell Science 121 (13)

of cohesion (Lam et al., 2006; Vas et al., 2007) as well as with As is evident from the yeast data, the contribution of different lack of separation at the rDNA locus (D’Amours et al., 2004; cohesion mechanisms varies from locus to locus, perhaps being Freeman et al., 2000; Strunnikov et al., 1995). Whether this is influenced by the locations in which catenations arise during due to independent roles of condensin, namely cohesion and replication, and the sites of cohesin, condensin and ORC binding, condensation, or two manifestations of the same activity that lead as well as the surrounding chromatin environment. to opposite effects, perhaps owing to the special structure of the rDNA locus, remains to be tested. The contribution of condensin Different needs, different solutions to cohesion, similar to the contribution of cohesin, varies from The existence of collaborative mechanisms of cohesion makes the locus to locus and the effects are additive in a condensin-cohesin need for multiple pathways that inactivate cohesion in anaphase double mutant (Lam et al., 2006). The possibility that condensin obligatory. Two events that are necessary for sister-chromatid maintains catenation-mediated cohesion remains to be tested, but separation, namely cohesin removal (promoted by separase and the condensin has been shown to be involved in recruiting kinases of the prophase pathway) and decatenation (promoted by topoisomerase II to the chromosome scaffold (Coelho et al., 2003). topoisomerase II), have been widely documented, but further This suggests a link between condensin and catenations. research is needed to understand the mechanism that regulates their Alternatively, the role of condensin in cohesion might be a concerted activity in anaphase. Two types of mechanism for secondary consequence of its role in condensation. This might be the concerted removal of multiple cohesion devices can be particularly important in regions that have complex chromatin envisioned: a parallel and independent activation of all cohesion- structures such as centromeres and rDNA. removal mechanisms upon the generation of a single ‘go’ signal, or a network-type regulation in which activation of one separation Multiple mechanisms of cohesion mechanism influences the activity of the others. It is also possible An integrative model of cohesion that a mixture of these two mechanisms exists so that a single ‘go’ Based on the data discussed above, we propose a model in which signal serves as the initial activator of some or all of these pathways, cohesion is the result of a complex balance and coordination which, in turn, regulate the other pathways. Interestingly, recent between several mechanisms of cohesion. Two main mechanisms, research has shown that some cohesion mechanisms might share a provided by DNA catenations and cohesin, result in cohesion that common regulatory theme: sumoylation of both topoisomerase II is distributed along the whole chromosome in all eukaryotes and the cohesin-associated protein Pds5 have been shown to that have been studied (Fig. 4). However, these mechanisms are regulate cohesion (Azuma et al., 2005; Azuma et al., 2003; Bachant subject to differential regulation at centromeres, arms, telomeres et al., 2002; Diaz-Martinez et al., 2006; Stead et al., 2003). These and perhaps other loci. Both centromeric cohesin and catenations results suggest that SUMO-related processes might be responsible are maintained in a spindle-checkpoint-dependent manner in higher for tipping the balance in favor of loss of cohesion during anaphase. eukaryotes. Similarly, cohesin and catenations are removed from The existence of multiple mechanisms of cohesion creates the chromosomes coordinately in anaphase – complete and efficient difficulty of ensuring their concerted deactivation to allow complete removal of both of these cohesion systems is required for the sister-chromatid segregation during mitosis. However, it can also accurate segregation of chromosomes. explain the ability of cells to provide differential cohesion dynamics

Journal of Cell Science We propose that catenations are the default or primary within a chromosome. Examples of the locus-specific timing of mechanism of cohesion because they are physically coupled to separation exist in nature, such as the centromere-breathing replication. Cohesin is then loaded onto replicated (already phenomenon (the brief and reversible separation of centromeres catenated) sister chromatids. This process occurs concurrently with upon attachment to the spindle) and the late separation of the rDNA replication, because cohesin-loading mechanisms are active during locus in budding yeast. Separation of the rDNA locus seems to be S phase. But, we argue that the establishment of cohesin-mediated a clear example of specialized cohesion because cohesion at this cohesion in S phase might be a dynamic process. The observation locus is independent of cohesin and its separation depends on a that activation of cohesin-loading mechanisms during G2-M after functional condensin complex (D’Amours et al., 2004). Although a single double-strand break leads to genome-wide de novo parallel separation (at centromeres and chromosome arms) of the establishment of cohesion (Strom et al., 2007; Unal et al., 2007) sister chromatids during anaphase is the norm in human cells during raises the possibility that a similar mechanism operates during S undisturbed mitoses (Gimenez-Abian et al., 2005b), ~4% of HeLa phase. In this scenario, activation of cohesin loading would lead cells separate the centromeres before the chromosome arms to loading of cohesin at all loci. Some of these cohesin complexes (Gimenez-Abian et al., 2005b). This suggests that the mechanisms will land in unreplicated regions and will later be removed to allow of chromosome-arm and centromere separation, although normally passage of the replication machinery. Once that region is replicated acting in concert, can be uncoupled. This differential regulation is and held close to its sister chromatid (perhaps aided by perhaps more dramatically revealed by the separation of catenations), cohesin can be loaded again, this time providing chromosome arms but not centromeres during prolonged arrest in cohesion. It can be envisioned that this genome-wide loading of the presence of microtubule poisons (Gimenez-Abian et al., 2004). cohesin is a dynamic event that occurs throughout S phase. The observations that expression of non-cleavable cohesin or The alternative model is that cohesin loading is restricted to the depletion of separase results in failure to separate the chromosome replication forks. arms but that centromeres can separate (Diaz-Martinez et al., 2007; In addition to catenations and cohesin, other mechanisms of Gimenez-Abian et al., 2005a; Gimenez-Abian et al., 2005b; Papi cohesion that are contributed to by ORC and condensin might exist. et al., 2005; Yalon et al., 2004) suggest that cohesin-mediated However, further research is needed to determine whether these are cohesion, although present at the centromere, might be primarily bona fide cohesion factors or whether they are regulators of cohesin responsible for the cohesion of chromosome arms, whereas and catenations. ORC and condensin could also act by affecting centromeric cohesion could be mediated by a cohesin-independent cohesion indirectly – by providing specific chromatin structures. mechanism, both factors being synchronously removed in anaphase. Multiple cohesion mechanisms 2113

It is noteworthy that a step-wise loss of cohesion is observed during centromeres requires RanBP2-mediated SUMOylation of topoisomerase IIalpha. Cell 133, 103-115. meiosis, with cohesion at the chromosome arms being lost Dej, K. J., Ahn, C. and Orr-Weaver, T. L. (2004). Mutations in the Drosophila condensin during anaphase I, allowing the separation of homologous subunit dCAP-G: defining the role of condensin for chromosome condensation in mitosis chromosomes, followed by the dissolution of centromeric cohesion and gene expression in interphase. Genetics 168, 895-906. Diaz-Martinez, L. A., Gimenez-Abian, J. F., Azuma, Y., Guacci, V., Gimenez-Martin, during anaphase II to separate sister chromatids. The existence of G., Lanier, L. M. and Clarke, D. J. (2006). PIASgamma is required for faithful two mechanisms of cohesion that are differentially regulated at the chromosome segregation in human cells. PLoS ONE 1, e53. arms and centromeres could account for this step-wise regulation Diaz-Martinez, L. A., Gimenez-Abian, J. F. and Clarke, D. J. (2007). Cohesin is dispensable for centromere cohesion in human cells. PLoS ONE 2, e318. of cohesion during meiosis. Dillin, A. and Rine, J. (1998). Roles for ORC in M phase and S phase. Science 279, 1733- 1737. Freeman, L., Aragon, A. L. and Strunnikov, A. (2000). The condensin complex governs Conclusions chromosome condensation and mitotic transmission of rDNA. J. Cell Biol. 149, 811- A complex picture has emerged in which diverse mechanisms 824. collaborate to provide cohesion along the chromosome. These Gimenez-Abian, J. F., Clarke, D. J., Mullinger, A. M., Downes, C. S. and Johnson, R. T. (1995). A postprophase topoisomerase II-dependent chromatid core separation step mechanisms of cohesion include, but might not be limited to, in the formation of metaphase chromosomes. J. Cell Biol. 131, 7-17. cohesin and DNA catenations. Renewed effort to understand the Gimenez-Abian, J. F., Clarke, D. J., Devlin, J., Gimenez-Abian, M. I., De la Torre, nature, regulation and complex interactions of the multiple C., Johnson, R. T., Mullinger, A. M. and Downes, C. S. (2000). Premitotic chromosome individualization in mammalian cells depends on topoisomerase II activity. Chromosoma mechanisms of cohesion is needed to attain a comprehensive 109, 235-244. knowledge of these systems. Gimenez-Abian, J. F., Sumara, I., Hirota, T., Hauf, S., Gerlich, D., de la Torre, C., Ellenberg, J. and Peters, J. M. (2004). Regulation of sister chromatid cohesion between chromosome arms. Curr. Biol. 14, 1187-1193. We thank V. Guacci, H. Yu, J. Bachant, P. R. Potts and M. Potts for Gimenez-Abian, J. F., Diaz-Martinez, L. A., Waizenegger, I. C., Gimenez-Martin, G. critical reading of this manuscript, and members of the Clarke and Yu and Clarke, D. J. (2005a). Separase is required at multiple pre-anaphase cell cycle labs for discussions. 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