Cohesin and Its Regulation: on the Logic of X-Shaped Chromosomes

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Cohesin and Its Regulation: On the Logic of X-Shaped Chromosomes

Judith H.I. Haarhuis,1,2 Ahmed M.O. Elbatsh,1,2 and Benjamin D. Rowland1,* 1Division of Cell Biology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, the Netherlands 2Co-ﬁrst author *Correspondence: [email protected] http://dx.doi.org/10.1016/j.devcel.2014.09.010

The X shape of chromosomes is one of the iconic images in biology. Cohesin actually connects the sister chromatids along their entire length, from S phase until mitosis. Then, cohesin’s antagonist Wapl allows the separation of chromosome arms by opening a DNA exit gate in cohesin rings. Centromeres are protected against this removal activity, resulting in the X shape of mitotic chromosomes. The destruction of the remaining centromeric cohesin by Separase triggers chromosome segregation. We review the two-phase regulation of cohesin removal and discuss how this affects chromosome alignment and decatenation in mitosis and cohesin reloading in the next cell cycle.

Introduction Chromatin Entrapment and Release by the Cohesin During each cell division, the entire genome must be duplicated Complex and separated in such a manner that each of the daughter cells The heart of the cohesin complex consists of three components inherits a complete copy of the genome without any alterations. (Smc1, Smc3, and Scc1) that together form a huge tripartite ring. Errors in these processes are believed to lie at the basis of can- The structural maintenance of chromosomes (Smc) subunits are cer and a range of genetic diseases. A key feature for controlling long rod-shaped proteins with ‘‘hinge’’ domains at one end, con- genomic integrity is sister chromatid cohesion, which is medi- nected by 50 nm long coiled coils to ‘‘head’’ (ATPase) domains at ated by the cohesin complex. the other end. Smc1 and Smc3 interact directly with one another Cohesin holds the sister chromatids together along their at their hinge interfaces, and their head domains are intercon- entire length, from S phase until the start of mitosis. In animal nected by the third subunit, sister chromatid cohesion 1 (Scc1, cells, cohesin is then removed from DNA in two distinct waves. also known as Mcd1 or Rad21). The Smc1 and Smc3 head do- The first step of cohesin removal is referred to as the ‘‘prophase mains together form a composite ATPase, which resembles pathway’’ and is dependent on cohesin’s antagonist Wapl. that of ABC transporters. Cohesin also has two subunits, Scc3 This pathway specifically removes cohesin from chromosome and Pds5, with less well-understood functions (Figure 2A) (Nas- arms. Importantly, centromeric cohesin is protected against myth and Haering, 2009; Peters et al., 2008). this removal activity, resulting in the well-known X shape of The finding that cohesin has a ring-shaped structure has led to mitotic chromosomes. Centromeric cohesin must resist the the model that cohesin may hold together the sister DNAs by pulling forces of microtubules up to the moment that the kinet- coentrapping them inside its ring-shaped structure (Haering ochores of all chromosomes are correctly attached to the et al., 2002). This ‘‘ring model’’ provides a tangible logic to the mitotic spindle in metaphase. Then, upon satisfaction of the principle of cohesion, and it explains how proteolytic cleavage spindle assembly checkpoint, the second wave of cohesin of cohesin’s Scc1 subunit can trigger the separation of sister removal is activated. This step is dependent on Separase, chromatids at anaphase onset. Although a number of alternative which proteolytically cleaves the remaining centromeric cohe- models have been proposed, the available evidence is in support sin and hereby triggers the separation of sister chromatids to of the ring model (Haering et al., 2008; Nasmyth and Haering, the opposite poles of the cell (Figure 1)(Nasmyth and Haering, 2009). The ring model has provided an important lead for uncov- 2009; Peters et al., 2008). ering the further regulation of cohesin through the cell cycle. Recent work has provided important insight into the regulation Cohesin complexes are assembled prior to their recruitment of cohesin’s association with chromatin. In this review, we to DNA (Losada et al., 1998; Waizenegger et al., 2000). Following outline our current understanding of the cycle of chromatin the logic of the ring model, cohesin rings must transiently entrapment and release by the cohesin complex. We discuss open up to allow the entry of DNA. Cohesin has three different the cellular mechanisms that drive cohesin from DNA in early interfaces that could in principle act as a DNA entry gate: two mitosis and how centromeres are protected against this removal interfaces at either end of Scc1 (which connect to Smc1 and activity. Finally, we address the purpose of having two distinct Smc3, respectively) and the hinge interface that connects cohesin removal pathways in mitosis. The two-phase cohesin Smc1 to Smc3. Intriguingly, the locking of the interfaces at removal process appears to be important for multiple key cellular either side of Scc1, by creating a fusion either between Scc1 processes, including the decatenation of intertwined sister and Smc1 or between Scc1 and Smc3, does not affect the chromatids, the correction of erroneous microtubule-kineto- viability of yeast cells. Locking of the hinge interface by contrast chore associations, and the recycling of cohesin rings for the does not support viability. Importantly, inducibly locking the following cell cycle. hinge interface using a Rapamycin-dependent FRB-FKBP12

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Figure 1. Vertebrate Cohesin Is Removed from Chromosomes in Two Distinct Waves: First from the Arms and Then from Centromeres (A) Cohesin rings stably coentrap the sister chromatids throughout G2 until the start of mitosis. Then, cohesin is phosphorylated by the mitotic kinases CDK1, PLK1, and Aurora B. This allows Wapl to remove the bulk of cohesin from chromosome arms. Centromeres are protected by SGO1 (Shugoshin), which with the PP2A phosphatase counteracts the phosphorylation of centromeric cohesin rings and thus keeps them in a locked state. SGO1 also protects cohesin directly by preventing the binding of Wapl to cohesin. (B) Centromeric cohesion is maintained until it is destroyed by Separase at the metaphase-to-anaphase transition. This destruction is the trigger for chromosome segregation. Separase removes cohesin by proteolytically cleaving its Scc1 subunit. linkage system specifically prevents the establishment, but not difference lies in the timing of cohesin’s recruitment to DNA. In the maintenance, of cohesion. These findings have led to the yeast, cohesin is recruited to DNA in late G1 (To´ th et al., 1999), model that cohesin has a distinct DNA entry gate that is located whereas in animal cells, this recruitment already takes place in at the interface that connects Smc1 and Smc3 at their hinge do- telophase (Losada et al., 1998; Waizenegger et al., 2000). This mains (Figure 2B) (Gruber et al., 2006). This finding was recently difference is due to continued destruction of Scc1 by Separase also confirmed in human cells (Buheitel and Stemmann, 2013). through G1 in yeast (Uhlmann et al., 1999). The cohesin that as- Cohesin’s recruitment to DNA depends on its loader complex, sociates with DNA through G0 and G1 of animal cells has a rapid consisting of Scc2 and Scc4 (also known as the Kollerin complex, turnover, which is the consequence of a continuous cycle of or Nipbl and Mau2, respectively) (Ciosk et al., 2000). The molec- chromatin entrapment and release (Figure 2B) (Eichinger et al., ular role of the Scc2/Scc4 complex is only partially understood, 2013; Gerlich et al., 2006). and it is thought to regulate the opening or closing of the hinge If cohesin indeed entraps DNA inside its ring-shaped struc- interface in a manner that requires ATPase activity of the Smc1 ture, then the ring must subsequently open up to allow the and Smc3 head domains (Arumugam et al., 2003; Hu et al., release of DNA. Intriguingly, this release does not require the 2011; Murayama and Uhlmann, 2014). Even though the ATPase opening of the hinge interface but rather of the interface connect- domain of cohesin is its best conserved region, our understanding Smc3’s ATPase domain and the N terminus of Scc1. Cohesin ing of this crucial domain barely goes beyond the notion that rings with fusions between Smc3 and Scc1 do load onto DNA but ATP binding is required for the formation of cohesin rings and barely, if at all, turn over on the DNA (Buheitel and Stemmann, that ATP hydrolysis is essential for cohesin’s stable association 2013; Chan et al., 2012; Eichinger et al., 2013). This finding sup- with DNA (Arumugam et al., 2003, 2006; Hu et al., 2011; Ladurner ports the model that cohesin has a distinct DNA exit gate at its et al., 2014; Murayama and Uhlmann, 2014; Weitzer et al., 2003). Smc3-Scc1 interface (Figure 2B). Recent in vitro work shows that fission yeast cohesin can also Why cohesin might use distinct gates for DNA entry and entrap DNA in the absence of Scc2/Scc4, albeit inefficiently. The release is unknown. A likely explanation is that having two gates addition of Scc2 significantly stimulates the efficiency of DNA allows for a more specific level of regulation. It is worth pointing entrapment and of ATP hydrolysis, but Scc4 remarkably has out that ABC transporters also have dedicated entry gates and no effect (Murayama and Uhlmann, 2014). This is unexpected exit gates. The dedicated gates might in fact be a consequence because both Scc2 and Scc4 are essential for cohesin loading of the ABC-like ATPase machinery of cohesin. It is a possibility in vivo (Ciosk et al., 2000). A possible explanation for this conun- that this type of machinery confers a sense of directionality to drum is that Scc4 may specifically have a role in the loading of the transport. Cohesin could therefore be seen as a ‘‘chromatin cohesin onto chromosomal DNA (Murayama and Uhlmann, transporter.’’ In this particular case, the transport does not drive 2014). Intriguingly, the Scc2/Scc4 complex is recruited to nucle- a compound through a membrane but rather it allows cohesin osome-free DNA in vivo and appears to be important for the rings to entrap and release DNA (Figure 2C). maintenance of such naked DNA regions. This raises the possi- Cohesin’s turnover at chromatin is highly dependent on cohe- bility that Scc2/Scc4 also promotes DNA entrapment by cohesin sin’s antagonist Wapl. If Scc1 is artificially expressed in G1 of through the creation of an accessible DNA template (Lopez- yeast cells, this turnover requires Wapl, just like in vertebrate Serra et al., 2014). cells (Chan et al., 2012; Eichinger et al., 2013; Kueng et al., Most of cohesin’s subunits and regulators are conserved from 2006; Lopez-Serra et al., 2013; Tedeschi et al., 2013). The finding yeast to humans, but there are notable differences in the tempo- that Wapl inactivation in essence yields the same phenotype as ral and local control of cohesin’s association with DNA. The first the locking of cohesin’s exit gate has led to the model that Wapl

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Figure 2. A Cycle of DNA Entrapment and Release by the Cohesin Complex (A) The cohesin complex forms a gigantic ring-shaped structure that is thought to hold together sister chromatids by coentrapping sister DNAs inside cohesin rings. (B) Cohesin rings transiently entrap DNA strands in G1 of animal cells. Cohesin has an entrance gate for DNA at its interface connecting the ‘‘hinge’’ domains of Smc1 and Smc3. Cohesin is then removed from DNA by the opening of its DNA exit gate at the interface connecting Smc3 and Scc1. Cohesin’s turnover on DNA is dependent on Wapl and on the opening of the exit gate. (C) Cohesin could be seen as a ‘‘chromatin transporter.’’ Cohesin’s distinct DNA entrance and exit gates (left) are reminiscent of ABC-like transporters (right). Both cohesin and these transporters harbor ABC-like ATPase domains. This machinery may ensure the directionality of the transport of DNA through cohesin and of a compound through the transporter. (D) The cycle of DNA entrapment and release is blocked in S phase, when Eco1 acetylates two lysines on Smc3. In animal cells, this allows the recruitment of Sororin, which locks cohesin rings in a closed state and renders them insensitive to Wapl. Yeast have no known Sororin ortholog. (E) In prophase and prometaphase of animal cells, cohesin rings are unlocked along chromosome arms, but not at centromeres. This unlocking occurs through the phosphorylation of Sororin and SA2 (one of two somatic vertebrate Scc3 subunits), which allows Wapl-dependent cohesin removal from DNA and causes the separation of chromosome arms. In yeast, all cohesion is maintained until anaphase and is destroyed by Separase at the metaphase-to-anaphase transition. releases cohesin from DNA by opening up cohesin’s exit gate locking of cohesin’s exit gate through the fusion of Smc3 to (Figure 2B). How Wapl might achieve this feat is discussed in Scc1 allows stable cohesion in the absence of Eco1 (Chan more detail later on in this review. et al., 2012). Antiestablishment activity turned out to be the same as Wapl-mediated cohesin release from DNA, and Eco1- Locking Cohesin Rings around the Sister Chromatids in mediated Smc3 acetylation apparently allows the establishment S Phase of cohesion by locking cohesin’s exit gate (Figure 2D). The cohe- The seemingly futile cycle of DNA entrapment and release is sin rings that provide cohesion are thought to remain locked disrupted in S phase, when sister chromatid cohesion is estab- through G2. This model is supported by the observation that lished. Cohesion establishment is dependent on the acetyltrans- from S phase onward, there is a subpool of chromatin-associ- ferase Eco1 (also known as Ctf7) (Ivanov et al., 2002; Skibbens ated cohesin complexes that has little to no turnover (Chan et al., 1999; To´ th et al., 1999), which acetylates Smc3 at its et al., 2012; Gerlich et al., 2006; Lopez-Serra et al., 2013). ATPase domain and hereby renders cohesin rings resistant to Eco1-dependent Smc3 acetylation is conserved from yeast to Wapl (Rolef Ben-Shahar et al., 2008; Rowland et al., 2009; Sutani humans (which have two homologs, ESCO1 and ESCO2) (Zhang et al., 2009; Unal et al., 2008; Zhang et al., 2008). Initially, it was et al., 2008). The mechanism by which Smc3 acetylation renders unclear how Wapl counteracts the establishment of cohesion. cohesin rings insensitive to Wapl is only partially understood. At Wapl apparently had an ‘‘antiestablishment’’ activity, which this point, there appears to be another difference between yeast was proposed to either prevent the entry of DNA into cohesin and animal cells because in the latter, Smc3 acetylation causes rings or rather cause the release of DNA from cohesin (Rolef the recruitment of Sororin, which counteracts Wapl by prevent- Ben-Shahar et al., 2008; Rowland et al., 2009; Sutani et al., ing its binding to Pds5 (Lafont et al., 2010; Nishiyama et al., 2009). This notion was resolved by the crucial ﬁnding that the 2010). Yeast, however, have no known Sororin ortholog, and

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its function has been proposed to be performed by specified anaphase transition. In animal cells, however, the vast bulk of co- domains of another cohesin subunit (Chan et al., 2013). hesin rings along chromosome arms is removed from DNA in How cohesin manages to specifically coentrap the sister prophase and prometaphase by an activity that is referred to chromatids of each individual chromosome is one of the main as the prophase pathway (Losada et al., 1998; Waizenegger outstanding questions in the field. This process is likely depen- et al., 2000). Prophase cohesin removal is highly dependent on dent on the local regulation of Eco1 activity because this may Wapl and requires the dissociation of cohesin’s DNA exit gate be what determines which cohesin rings are locked around at the Smc3-Scc1 interface (Buheitel and Stemmann, 2013; Ei- DNA. The cohesion establishment process is intimately con- chinger et al., 2013; Gandhi et al., 2006; Kueng et al., 2006). nected to DNA replication. Indeed, Eco1 is specifically required Importantly, centromeric cohesin is protected against the pro- in S phase (Skibbens et al., 1999; To´ th et al., 1999). Eco1 can phase pathway, which results in the separation of chromosome bind to multiple factors involved in DNA replication, including arms, but not centromeres. When chromosomes condense in PCNA, and it has been found enriched at sites of DNA replication mitosis, the persistence of cohesin specifically at the centro- (Kenna and Skibbens, 2003; Lengronne et al., 2006; Moldovan meres leads to the classical X shape of mitotic chromosomes et al., 2006; Song et al., 2012). An intuitive model is that cohesin (Figure 1A) (Losada et al., 1998; Waizenegger et al., 2000). The entraps single DNAs before DNA replication and that the replica- condensation process in turn is to a large part dependent on tion fork passes through cohesin rings (Haering et al., 2002). This the condensin complex (Hirano, 2012). model has the pleasing simplicity that this automatically ensures The X shape of chromosomes is particularly clear in cells that the coentrapment of the sister DNAs. A variant of this model is have been artificially arrested in mitosis. During an unperturbed that cohesin rings stay put but transiently open up to allow pas- mitosis, the prophase pathway removes most, but not all, cohe- sage of the fork and subsequently reclose to coentrap the sisters sin rings from chromosome arms. In prometaphase, chromo- (Lengronne et al., 2006). A fundamentally different model, how- some arms become visually discernable, but the arms do remain ever, is that cohesin can de novo coentrap two DNAs at once partially connected. These remaining connections may have after the passage of replication forks. In each of these scenarios, multiple molecular causes, including a small amount of persist- the proximity of Eco1 to the replication fork could help ensure ing cohesin rings, sister DNA intertwinings, or both (Gime´ nez- that cohesin rings coentrap both sister DNAs. Abia´ n et al., 2004; Paliulis and Nicklas, 2004). When cells are There are multiple additional connections between cohesion artificially arrested in mitosis with spindle poisons, the prophase establishment and DNA replication, but not all of these are pathway removes all detectable arm cohesin, and the arms conserved through eukaryotes. An important example is the separate completely (Gime´ nez-Abia´ n et al., 2004). The cohesin finding that cohesin recruitment to DNA requires prereplication rings that are removed from DNA by Wapl in prophase do not complexes in vertebrates (Gillespie and Hirano, 2004; Guillou reload until telophase (Losada et al., 1998; Waizenegger et al., et al., 2010; Higashi et al., 2012; Takahashi et al., 2008, 2004). 2000). This postponed reloading is thought to be the conse- Yeast prereplication complexes, by contrast, are dispensable quence of CDK1-mediated inactivation of the Scc2/Scc4 loader for cohesin loading (Uhlmann and Nasmyth, 1998). The logic complex (Gillespie and Hirano, 2004; Watrin et al., 2006). behind this difference is not understood, but it may be related This brings us to the question of what causes the loss of to the fact that yeast cohesin does not load onto DNA until late protection against Wapl in early mitosis. Cohesin’s removal G1. Another example is the report that Smc3 acetylation speeds from chromosome arms turns out to be the consequence of the up DNA replication in cultured human cells (Terret et al., 2009). phosphorylation of Sororin and SA2 (one of two somatic verte- This activity is not conserved through eukaryotes because brate Scc3 homologs) by the mitotic kinases Aurora B, PLK1, Smc3 acetylation does not affect replication speed in yeast (Lo- and CDK1 (Figures 1A and 2E) (Dreier et al., 2011; Gime´ nez-Abia´ n pez-Serra et al., 2013). Intriguingly, Wapl inactivation does speed et al., 2004; Hauf et al., 2005; Liu et al., 2013b; Losada et al., 2002; up DNA replication in both yeast and human cells (Lopez-Serra Nishiyama et al., 2013; Sumara et al., 2002; Zhang et al., 2011). et al., 2013; Manning et al., 2014), but it is unclear whether this How these phosphorylations cause the loss of protection against phenotype affects the cohesion establishment process. Wapl is not fully understood. Sororin phosphorylation causes Eco1 is degraded after S phase, and, as a consequence, its dissociation from Pds5, which in turn allows Wapl’s binding cohesion can normally only be established in S phase. However, to Pds5 (Nishiyama et al., 2010). How SA2 phosphorylation when Eco1 degradation is inhibited by, for example, DNA dam- promotes Wapl-dependent ring opening is currently unknown. age, cohesion can also be established in G2 (Lyons and Morgan, Notably, the prophase pathway does not involve deacetylation 2011; Stro¨ m et al., 2007; Unal et al., 2007). This indicates that the of Smc3, which takes place after cohesin has been removed coentrapment of sister DNAs does not strictly require DNA repli- from chromatin. The deacetylation of Smc3 is nevertheless cation. Interestingly, DNA damage-induced cohesion does not very important because efficient cohesion establishment in the involve Smc3 acetylation but rather appears to require Eco1- subsequent S phase requires de novo acetylation of Smc3. dependent acetylation of Scc1 (Heidinger-Pauli et al., 2009). Hereby, the deacetylation of Smc3 (by Hos1 in yeast and by Why these different settings of cohesion establishment might HDAC8 in humans) prepares cohesin complexes for their next call for differential acetylation is currently unknown. acetylation cycle (Beckoue¨ t et al., 2010; Borges et al., 2010; Deardorff et al., 2012; Xiong et al., 2010). Cohesin Removal from Chromosome Arms in Prophase The cohesin rings that have been locked in S phase stably hold How Does Wapl Open Up Cohesin Rings? the sister chromatids together until the start of mitosis. In yeast, How Wapl opens up cohesin rings is largely a mystery. Wapl all cohesive cohesin is maintained until the metaphase-to- may directly cause the disengagement of Scc1 from Smc3,

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but this could also be an important step further downstream in What Is the Role of Scc3 and Pds5? the cohesin ring opening reaction. The structure of cohesin’s Scc3 and Pds5 are arguably cohesin’s least well-understood exit gate is unknown. However, the structure of the equivalent subunits. This may be due to the fact that each of these factors interface in the related bacterial Smc complex was solved has roles in multiple stages through the cycle of DNA entrapment recently (Bu¨ rmann et al., 2013) and suggests that cohesin’s and release by cohesin. As a consequence, these proteins exit gate may consist of two alpha helices of the N terminus cannot be labeled as being merely cohesion establishment or of Scc1 that run along the coiled coil just above Smc3’s maintenance factors, and different mutants of these proteins ATPase domain (Figure 2A). It is an exciting thought that frequently yield very different results. Both Scc3 and Pds5 bind Wapl may open up the exit gate by regulating ATPase activity. to cohesin’s Scc1 subunit (Chan et al., 2013; Haering et al., Cohesin’s ATPase cycle involves ATP binding and hydrolysis of 2002; Shintomi and Hirano, 2009). Scc3 is generally viewed as two ATPs. Because each of these ATPs needs to be hydro- a core cohesin subunit because it is copurified with Smc1, lyzed to achieve stable binding of cohesin to chromatin (Hu Smc3, and Scc1 in a stoichiometric manner from cell extracts et al., 2011), it will be difficult to dissect the role of the (To´ th et al., 1999). Pds5 is also a stoichiometric cohesin subunit ATPase cycle in DNA release by cohesin. If the ATPase cycle at chromatin in G2 (Chan et al., 2012), but its association to the does turn out to have such a role, this would introduce a other subunits is less tight in solution (Sumara et al., 2000). new puzzle. How could Wapl-induced ATPase activity specif- This may indicate that Pds5’s association to cohesin is somehow ically open the exit gate while Scc2/Scc4-induced ATPase regulated. Pds5 also forms a subcomplex with Wapl in extracts, activity controls the entrance gate? One explanation could be but whether or not this is of any physiological importance is that these gates are differentially regulated as a consequence unknown (Kueng et al., 2006). of the DNA entrapment status. Another possibility indeed Scc3 is required for cohesin’s recruitment to DNA and binds to is that cohesin’s ABC-like ATPase machinery confers a one- the Scc2/Scc4 loader complex (Hu et al., 2011; Murayama and way directionality to cohesin’s gates. It is, however, also Uhlmann, 2014), it is required for the maintenance of cohesion possible that Wapl opens up cohesin rings in a manner that (Roig et al., 2014), and it also has domains that are important does not involve ATPase activity. for cohesin’s removal from DNA by Wapl (Chan et al., 2012; Row- Recent structural work shows that Wapl has a conserved land et al., 2009). Pds5 is not required for cohesin’s recruitment C terminus consisting of HEAT repeats that bind to multiple co- to DNA (Hu et al., 2011), but it is important for Smc3 acetylation hesin subunits, but, remarkably, these interactions appear to by Eco1 (Carretero et al., 2013; Chan et al., 2013; Vaur et al., differ between fungi and vertebrates (Chatterjee et al., 2013; 2012). Pds5 maintains cohesion in some, but not all, species. Ouyang et al., 2013). In both systems, Wapl’s flexible N terminus In fission yeast, for example, Pds5 is a nonessential gene, and binds directly to Pds5. Wapl’s association to Pds5 is essential for it primarily has a role in DNA release (Tanaka et al., 2001). In its cohesin removal activity (Chan et al., 2012; Rowland et al., budding yeast, by contrast, Pds5 is essential for maintenance 2009), and Sororin is thought to protect vertebrate cohesin of cohesion, and its release function is restrained to specific sub- against Wapl by occupying its binding site on Pds5 (Nishiyama domains of this protein (Chan et al., 2012, 2013; D’Ambrosio and et al., 2010). Wapl also binds to Scc3/SA2 in both systems, but Lavoie, 2014; Hartman et al., 2000; Panizza et al., 2000; Rowland in animal cells, this interaction requires the presence of Scc1 et al., 2009). Likewise, in Xenopus extracts, Pds5 is primarily (Gandhi et al., 2006; Kueng et al., 2006; Ouyang et al., 2013; important for DNA release, whereas in mouse cells, Pds5 is Rowland et al., 2009; Shintomi and Hirano, 2009). Remarkably, important for both cohesion and release (Carretero et al., 2013; only yeast Wapl appears to bind to the ATPase domain of Shintomi and Hirano, 2009). Both Scc3 and Pds5 have two verte- Smc3, and the tightness of this interaction seems to be partially brate homologs in somatic cells (SA1 and SA2 and Pds5A and dependent on the acetylation status of Smc3 (Chatterjee et al., Pds5B, respectively) that appear to have distinct roles in regu- 2013). The reason for the different binding modes between these lating cohesion at centromeres, arms, and telomeres (Losada, very different species is unknown. 2014). We should note that it is not clear whether cohesin rings in yeast are ever removed from DNA through their ‘‘unlocking.’’ Cohesin Protection at Centromeres until Anaphase The available evidence rather suggests that in yeast, all the cohe- Centromeres are protected against Wapl-dependent cohesin sive cohesin rings are cleaved by Separase at anaphase onset. removal by SGO1 (or Shugoshin, in full, which is Japanese for Wapl can indeed remove cohesin rings from chromatin if Scc1 ‘‘guardian spirit’’) (McGuinness et al., 2005; Salic et al., 2004). is artificially expressed in yeast G1 (Chan et al., 2012; Lopez- SGO1 protects cohesin in two distinct ways. The first involves Serra et al., 2013), but it appears that the acetylated cohesin SGO1’s binding to the PP2A phosphatase (Kitajima et al., rings remain stably associated with DNA until their cleavage by 2006; Riedel et al., 2006; Tang et al., 2006). The SGO1/PP2A Separase (Beckoue¨ t et al., 2010; Borges et al., 2010). This is in complex protects centromeres by counteracting the phosphory- stark contrast to animal cells, in which most acetylated cohesin lation of Sororin and SA2 (Figures 1A and 1B) (Kitajima et al., rings are unlocked by mitotic phosphorylation and then removed 2006; Liu et al., 2013b; Nishiyama et al., 2013). SGO1 also pro- by Wapl (Figure 1A) (Deardorff et al., 2012; Nishiyama et al., tects against Wapl by its direct association to cohesin. Through 2013; Whelan et al., 2012). It is therefore possible that the ability competitive binding, this prevents the recruitment of Wapl to to unlock cohesin rings lies at the basis of the reported differ- SA2/Scc1 (Hara et al., 2014). ences between vertebrates and fungi. Likewise, Sororin may The protection of centromeric cohesin is crucial because this exist in animal cells solely to allow the reversible locking of confers the cohesion that must resist the pulling forces of cohesin rings. microtubules until anaphase onset. The remaining centromeric

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cohesin persists until the kinetochores of all chromosomes are removal pathway that drives cohesin from chromosome arms attached by microtubules and the spindle assembly checkpoint in early mitosis? This prophase pathway appears to be unique (SAC) is satisfied. This causes the destruction of Securin, which to metazoan cells because yeast indeed do not have this unleashes Separase that in turn proteolytically cleaves Scc1 and pathway and separate their sisters by Separase-mediated cleav- hereby triggers the synchronous separation of sister chromatids age of cohesin along the entire length of their chromosomes. in anaphase (Figure 1B) (Nasmyth and Haering, 2009; Peters Remarkably, inactivation of the prophase pathway in animal cells et al., 2008). by small interfering RNA (siRNA)-mediated depletion of Wapl SGO1 localization to chromatin is dependent on two different does not prevent chromosome segregation per se. In this phosphorylation marks. The first entails CDK1-dependent phos- setting, Separase efficiently cleaves the cohesin rings along phorylation of SGO1, which allows it to bind to cohesin along the the entire length of chromosomes at anaphase onset (Gandhi entire length of chromosomes (Liu et al., 2013b). The second is et al., 2006; Kueng et al., 2006). What then is the purpose of hav- Bub1-mediated phosphorylation of histone H2A. As Bub1 (a ki- ing two distinct cohesin removal steps in animal cells? Recent nase important for chromosome alignment and the SAC) locates work shows that the two-step cohesin removal process is in to centromeres, this allows the concentration of SGO1 at centro- fact crucial for three key cellular events: sister DNA decatena- meres (Kawashima et al., 2010). tion, correction of erroneous microtubule-kinetochore attach- When the kinetochores of one chromosome are properly ments, and preservation of cohesin rings for reloading in the sub- attached to microtubules emanating from opposite poles of the sequent cell cycle. cell, this creates tension that causes SGO1 to relocalize from co- Sister DNA Decatenation hesin at the inner centromeres to phosphorylated H2A at the The sister DNAs of each chromosome are held together not only outer centromeres (Lee et al., 2008; Liu et al., 2013a). This shift by cohesin but also by DNA intertwinings, or catenanes. These is dependent on the dephosphorylation of SGO1 and is required structures are a natural consequence of DNA replication, and for faithful chromosome segregation, but the underlying mecha- many catenanes persist until mitosis. Catenanes can be resolved nisms are unknown (Liu et al., 2013a). The SGO1 shift may be by topoisomerase II via its DNA strand-passing activity (Nitiss, required to pull away PP2A from the vicinity of centromeric cohe- 2009). It could be argued that catenanes confer a type of sin and thereby allow Scc1 phosphorylation by PLK1, which cohesion. However, in contrast to cohesin, catenanes cannot makes it a better substrate for Separase (Alexandru et al., resist the pulling forces of microtubules because topoisomerase 2001; Hauf et al., 2005). SGO1’s displacement could indeed allows DNA strand passing upon tension (Farcas et al., 2011). also cause loss of protection against Wapl. It is clear that Topoisomerase-dependent decatenation is nevertheless essen- SGO1 and PP2A control an intricate network of phosphorylation tial for proper chromosome segregation (Nitiss, 2009). The marks that is currently only partially understood (Liu et al., dependency on topoisomerase for proper chromosome segre- 2013b). Despite the fact that yeast have no prophase pathway, gation decreases from prophase to metaphase, which indicates SGO1 also localizes to centromeres in mitosis of this species. that the loss of catenanes roughly follows the rate of cohesin Here, SGO1 is not required for protection of cohesin, but it is removal (Oliveira et al., 2010). important for chromosome biorientation (Peplowska et al., Interestingly, recent work shows that catenanes are main- 2014; Verzijlbergen et al., 2014). tained by cohesin (Farcas et al., 2011; Wang et al., 2010b) and When cells are artificially arrested in metaphase for multiple that cohesin arm removal by the prophase pathway is important hours, chromosomes eventually suffer from loss of cohesion. for the timely decatenation of chromosomes (Haarhuis et al., The cause of this cohesion loss is not fully understood and has 2013; Tedeschi et al., 2013). Together, these results show that been referred to as ‘‘cohesion fatigue’’ (Daum et al., 2011). It cohesin removal may be a prerequisite for decatenation in has been proposed that the centromeric cohesin rings are prophase. Catenanes are normally primarily detected between ruptured due to the pulling forces of microtubules. However, sister centromeres in early anaphase (Chan and Hickson, 2009) this particular form of cohesion loss can be prevented by deple- and are thought to be remnants of the catenanes that were main- tion of Wapl or by the expression of SA2 mutants that cannot tained by centromeric cohesin until anaphase onset (Wang et al., bind Wapl (Daum et al., 2011; Hara et al., 2014). In addition, as 2010b). Importantly, inhibition of the prophase pathway by Wapl mentioned above, the pulling forces create tension that causes depletion causes an increase in the number of catenanes that the shift of SGO1 from cohesin to outer kinetochores (Lee persist in late anaphase (Figures 3A and 3B) (Haarhuis et al., et al., 2008; Liu et al., 2013a). Together, this indicates that cohe- 2013; Tedeschi et al., 2013). This supports the model that the sion fatigue could also rather be a consequence of the loss of prophase pathway acts to limit the amount of catenanes that protection against Wapl-dependent cohesin removal. Cohesion must be resolved at anaphase onset. Too many catenanes can fatigue, however, only takes place when cells are arrested in pose a risk at this point because topoisomerase may not manage metaphase for prolonged periods. It is therefore unlikely that to resolve all catenanes prior to cytokinesis. Wapl will remove cohesin from centromeres upon tension in an Correction of Erroneous Microtubule-Kinetochore unperturbed mitosis. Attachments Cohesin also has a number of functions that are cohesion inde- What Is the Purpose of Having Two Cohesin Removal pendent. An important example is cohesin’s role in the recruit- Steps? ment of the chromosome passenger complex (CPC). This The purpose of Separase-mediated cohesin destruction is complex, which consists of the Aurora B kinase, INCENP, Survi- evident because this is the trigger for chromosome segregation. vin, and Borealin, is essential for the correction of erroneous But what is actually the biological purpose of the first cohesin microtubule-kinetochore attachments (van der Waal et al.,

12 Developmental Cell 31, October 13, 2014 ª2014 Elsevier Inc. Developmental Cell Review

Figure 3. Prophase Cohesin Removal from Chromosome Arms Is Required for Multiple Key Mitotic Processes (A) Cohesin’s two-step removal in vertebrate mitosis serves at least three purposes. (1) Decatenation of sister chromatids: cohesin rings maintain the intertwinings, or catenanes, of sister DNAs. Removal of cohesin rings in prophase ensures that topoisomerase II merely needs to decatenate centromeres after the destruction of centromeric cohesin by Separase at anaphase onset. (2) Chromosome passenger complex (CPC) focusing at centromeres: the CPC is important for the correction of erroneous microtubule-kinetochore attachments. Because the CPC to a large degree follows cohesin’s localization on chromatin, cohesin’s removal from chromosome arms allows the CPC to concentrate at centromeres. (3) Recycling of cohesin rings: the removal of cohesin rings from DNA by the prophase pathway involves the transient opening up of cohesin’s exit gate. These rings are intact and reload onto DNA in telophase and in G1 of the subsequent cell cycle. (B) When cohesin is not removed from chromosome arms in early mitosis, this can result in multiple defects: (1) catenanes are maintained by cohesin along the entire length of chromosomes until metaphase. After Separase-mediated cleavage of cohesin, topoisomerase needs to decatenate the sisters along their entire length. This increases the chance of a catenane persisting until cytokinesis, which is a threat to genomic stability. (2) The CPC does not sufﬁciently focus at centromeres, and misattachments are corrected less efﬁciently, resulting in segregation errors with lagging chromosomes. (3) All cohesin rings persist until they are removed by Separase, which proteolytically cleaves Scc1. These cohesin complexes require de novo Scc1 expression and cannot readily reload onto DNA in the subsequent G1.

2012). Cohesin controls the localization of the CPC in two ways. The CPC ensures faithful chromosome segregation through The first pathway involves SGO1, which upon phosphorylation Aurora B-mediated phosphorylation of those kinetochores that by CDK1 binds to cohesin (Liu et al., 2013b). In addition to its are incorrectly attached by microtubules. Aurora B phosphory- crucial role in the protection of cohesin against Wapl-mediated lates outer-kinetochore substrates involved in microtubule bind- removal, SGO1 is also important for the recruitment of the ing when kinetochores are incorrectly attached by microtubules CPC to chromatin through the binding to Borealin (Tsukahara (Liu et al., 2009; Welburn et al., 2010). The current model is that et al., 2010). The second pathway involves the kinase Haspin, Aurora B specifically phosphorylates substrates at incorrectly which phosphorylates Histone H3 at Threonine 3. This mark cre- attached kinetochores because the kinetochores that are ates a docking site for the CPC by binding to Survivin (Kelly et al., correctly attached are pulled out of Aurora B’s reach due to 2010; Wang et al., 2010a; Yamagishi et al., 2010). Interestingly, the tension exerted by the microtubules. Phosphorylation of Haspin-dependent phosphorylation is regulated by cohesin’s kinetochores causes the destabilization of attachments, which Pds5 subunit. In yeast, Pds5 recruits Haspin directly (Yamagishi allows for a new round of search-and-capture by the microtu- et al., 2010). Whether this is also the case in vertebrate cells re- bules (van der Waal et al., 2012). For this important function, mains to be shown, but Histone H3 phosphorylation and Aurora the mammalian CPC needs to act at centromeres. When the pro- B localization are strongly affected in Pds5B-deficient mouse phase pathway is inactivated through Wapl depletion, the CPC is cells (Carretero et al., 2013). As such, the CPC’s localization at recruited all along chromosomes and is insufficiently enriched at DNA is to a large degree dependent on cohesin. centromeres (Haarhuis et al., 2013; Shintomi and Hirano, 2009;

Developmental Cell 31, October 13, 2014 ª2014 Elsevier Inc. 13 Developmental Cell Review

Tedeschi et al., 2013). Correspondingly, error correction is morphology in G0 that is marked by an axis of cohesin along hampered and chromosomes missegregate in anaphase (Fig- the entire length of chromosomes, which is referred to as ‘‘vermi- ures 3A and 3B) (Haarhuis et al., 2013). celli’’ (Tedeschi et al., 2013). One explanation for this finding is The finding that the prophase pathway is important for deca- that in the absence of Wapl, cohesin cannot let go of in cis tenation and error correction pinpoints an important role for DNA loops once they have been formed. this pathway in chromosome segregation. This raises the possi- Considering the broad range of cellular functions that are bility that it acts to protect against aneuploidy. Wapl depletion in controlled by cohesin, it is perhaps not surprising that cohesin’s untransformed cells, however, causes a p53-dependent cell- subunits and regulators are found mutated in many human cycle arrest, which presumably is a consequence of missegrega- diseases, ranging from developmental disorders (referred to as tion-induced DNA damage (Crasta et al., 2012; Janssen et al., ‘‘cohesinopathies’’) to various types of cancer (Losada, 2014). 2011). p53-deficient cells, by contrast, proliferate well in the In some cohesinopathies, such as Roberts syndrome and War- absence of Wapl and concomitantly become highly aneuploid. saw breakage syndrome, the mutations cause cohesion defects The two-step cohesin removal process apparently does protect (van der Lelij et al., 2010; Vega et al., 2005). Cornelia de Lange against aneuploidy (Haarhuis et al., 2013). syndrome mutations, however, do not cause obvious cohesion Preserving Cohesin Rings for Reloading in the defects but rather appear to affect transcription (Remeseiro Subsequent Cell Cycle and Losada, 2013). We should note that in yeast, cohesin’s Prophase cohesin removal drives cohesin from DNA by tran- different functions require different levels of cohesin complexes. siently opening its exit gate. These cohesin rings are intact and DNA repair, for example, requires a significantly higher threshold can in principle reload onto DNA. This is in contrast to Sepa- than is needed for sister chromatid cohesion (Heidinger-Pauli rase-mediated cohesin cleavage, which destroys the Scc1 sub- et al., 2010). It is therefore well possible that some mutations unit of specifically DNA-bound cohesin complexes (Sun et al., affect one function of cohesin without affecting the other. 2009). Cohesin complexes that have been removed from DNA by Separase can only reload upon the de novo binding to an Why Do Yeast Have No Prophase Pathway? uncleaved Scc1 subunit. Therefore, the prophase pathway is If the two-phase cohesin removal process is so important for important to save cohesin rings from Separase-mediated cleav- faithful chromosome segregation, it is curious that yeast have age. Recent work shows that cohesin reloading in telophase and not evolved a prophase pathway. We can only speculate why, early G1 is severely affected if the prophase pathway is inacti- but one possibility is that the prophase pathway is primarily vated by Wapl depletion (Tedeschi et al., 2013). The prophase important in species that require transcriptional regulation by co- pathway apparently is crucial to allow timely cohesin reloading hesin in G1. This clearly is not the case in budding yeast because in the subsequent cell cycle (Figures 3A and 3B). these cells have no cohesin complexes through G1. Cohesin’s turnover at DNA also appears to be less important for transcrip- Why Is Cohesin Recruited to Chromosome Arms At All? tional regulation in budding yeast because transcription is barely This brings us to the question of why cohesin is recruited to chro- affected in Wapl-deficient yeast cells (Lopez-Serra et al., 2013). mosome arms in the first place. If cohesin is essential at centro- Another possibility is that the relatively short chromosomes of meres and must be absent from arms, then why is it not merely yeast harbor fewer catenanes than vertebrate chromosomes recruited to centromeres? This presumably is due to one or mul- and that topoisomerase can efficiently resolve these arm cate- tiple important roles of cohesin at chromosome arms. Cohesion nanes after Separase-mediated cleavage of cohesin. is, for example, also required for DNA repair (Sjo¨ gren and Nas- We should note that the centromeric structure of budding myth, 2001). Although the mechanisms are not fully understood, yeast is very different from vertebrate centromeres. This species cohesin is thought to be necessary to keep the sisters in each has small dot centromeres that can stretch apart upon bipolar other’s proximity to allow high fidelity repair through homologous attachment to distances that are considerably larger than the recombination. A second important role for cohesin at chromo- diameter of cohesin rings. The cohesion between the sister chro- some arms is transcriptional regulation. Cohesin affects tran- matids is therefore thought to be conferred by cohesin rings that scription in multiple ways. For example, cohesin appears to be hold together the ‘‘pericentromeres’’ (Yeh et al., 2008). Intrigu- required for CTCF-mediated in cis long-range DNA interactions. ingly, budding yeast Aurora B does not need to focus at centro- This has led to the interesting concept that cohesin may regulate meres to enforce error correction (Campbell and Desai, 2013). transcription through the in cis coentrapment of two DNA fibers This finding may be related to the fact that the dot centromeres (Merkenschlager and Odom, 2013). This hypothesis, however, of budding yeast can only be attached by one microtubule at remains to be tested. once. As a consequence, merotelic attachments (misattach- If cohesin indeed controls transcription through the entrap- ments in which two microtubules from different poles attach to ment of in cis DNA loops, it will be equally important that these one kinetochore) are impossible in this species. This latter expla- loops can be removed in a controlled manner. Interestingly, nation, however, cannot be the sole reason of the absence of the Wapl inactivation has major effects on transcription. It is an prophase pathway in yeast because merotelic attachments are exciting prospect that transcription may be regulated through possible in fission yeast, which also have no prophase pathway the dynamic formation and dissolution of such cohesin-medi- (Tomonaga et al., 2000). ated in cis DNA loops. This possibility is supported by the remarkable finding that Wapl deficiency has severe conse- Conclusions and Perspective quences for chromosomal architecture. Mouse cells devoid of The X shape of chromosomes was first described in the late Wapl display a striking partially condensed chromosomal 19th century (Boveri, 1890). It has taken well over a century to

14 Developmental Cell 31, October 13, 2014 ª2014 Elsevier Inc. Developmental Cell Review

uncover the purpose of this morphology. The finding that the Boveri, T. (1890). Zellen-Studien. (Jena: G. Fischer). two-phase cohesin removal process is important for decatena- Buheitel, J., and Stemmann, O. (2013). Prophase pathway-dependent removal tion, error correction, and cohesin reloading sheds light on the of cohesin from human chromosomes requires opening of the Smc3-Scc1 interconnected nature of these very diverse processes. Cohesin gate. EMBO J. 32, 666–676. must persist at centromeres to resist the pulling forces of micro- Bu¨ rmann, F., Shin, H.-C., Basquin, J., Soh, Y.-M., Gime´ nez-Oya, V., Kim, Y.- tubules until the satisfaction of the SAC. By resisting these G., Oh, B.-H., and Gruber, S. (2013). An asymmetric SMC-kleisin bridge in 20 forces, cohesin also ensures the correction of erroneous micro- prokaryotic condensin. Nat. Struct. Mol. Biol. , 371–379. tubule-kinetochore attachments. Remarkably, cohesin must Campbell, C.S., and Desai, A. (2013). Tension sensing by Aurora B kinase is also be removed from chromosome arms to allow the efficient independent of survivin-based centromere localization. Nature 497, 118–121. correction of these errors. This removal is also crucial for the Carretero, M., Ruiz-Torres, M., Rodrı´guez-Corsino, M., Barthelemy, I., and timely decatenation of sister chromatids. The local protection Losada, A. (2013). Pds5B is required for cohesion establishment and Aurora 32 versus removal of cohesin apparently is a highly delicate balance B accumulation at centromeres. EMBO J. , 2938–2949. that must be tightly controlled. Chan, K.L., and Hickson, I.D. (2009). On the origins of ultra-fine anaphase We have learned a lot over the last 15 years about cohesin and bridges. Cell Cycle 8, 3065–3066. its regulation. The finding that cohesin has dedicated DNA entry Chan, K.L., Roig, M.B., Hu, B., Beckoue¨ t, F., Metson, J., and Nasmyth, K. and exit gates may have significant consequences beyond the (2012). Cohesin’s DNA exit gate is distinct from its entrance gate and is 150 field of cohesion. Chromosome condensation and DNA repair regulated by acetylation. Cell , 961–974. are regulated by Smc protein complexes that are closely related Chan, K.L., Gligoris, T., Upcher, W., Kato, Y., Shirahige, K., Nasmyth, K., and to cohesin (condensin and the Smc5/Smc6 complex, respec- Beckoue¨ t, F. (2013). Pds5 promotes and protects cohesin acetylation. Proc. Natl. Acad. Sci. USA 110, 13020–13025. tively) (Nasmyth and Haering, 2005). Whether the analogous interfaces in these complexes act as such gates remains to be Chatterjee, A., Zakian, S., Hu, X.-W., and Singleton, M.R. (2013). Structural 32 seen. It is an exciting thought that this family of complexes insights into the regulation of cohesion establishment by Wpl1. EMBO J. , 677–687. may in fact constitute a class of ‘‘chromatin transporters.’’ If so, these complexes could be likewise regulated at the level of Ciosk, R., Shirayama, M., Shevchenko, A., Tanaka, T.U., To´ th, A., Shev- chenko, A., and Nasmyth, K. (2000). Cohesin’s binding to chromosomes de- the controlled local and temporal locking of their DNA entry pends on a separate complex consisting of Scc2 and Scc4 proteins. Mol. and exit gates. Cell 5, 243–254. Many important unanswered questions remain regarding the Crasta, K., Ganem, N.J., Dagher, R., Lantermann, A.B., Ivanova, E.V., Pan, Y., regulation of cohesin’s association with DNA. For example, we Nezi, L., Protopopov, A., Chowdhury, D., and Pellman, D. (2012). DNA breaks only partially understand how Scc2/Scc4 loads cohesin onto and chromosome pulverization from errors in mitosis. Nature 482, 53–58. DNA. Cohesin’s ATPase activity clearly has a vital role in the D’Ambrosio, L.M., and Lavoie, B.D. (2014). Pds5 prevents the PolySUMO- entrapment and release cycle, but our current understanding dependent separation of sister chromatids. Curr. Biol. 24, 361–371. of this role is rudimental. We also do not know how Wapl opens Daum, J.R., Potapova, T.A., Sivakumar, S., Daniel, J.J., Flynn, J.N., Rankin, S., up cohesin rings or what determines which cohesin rings are and Gorbsky, G.J. (2011). Cohesion fatigue induces chromatid separation in locked around the sister DNAs by Eco1. These are only some cells delayed at metaphase. Curr. Biol. 21, 1018–1024. of the many fascinating questions regarding the cohesin com- Deardorff, M.A., Bando, M., Nakato, R., Watrin, E., Itoh, T., Minamino, M., plex that need to be answered in the future. Saitoh, K., Komata, M., Katou, Y., Clark, D., et al. (2012). HDAC8 mutations in Cornelia de Lange syndrome affect the cohesin acetylation cycle. Nature 489, 313–317. ACKNOWLEDGMENTS Dreier, M.R., Bekier, M.E., 2nd, and Taylor, W.R. (2011). 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