It has long been known that cohesin's Scc1 and Scc3/SA1/SA2 subunits are specifically phosphorylated in mitosis [2
]. In budding yeast it has been shown that phosphorylation of Scc1 by Cdc5 enhances the cleavability of cohesin by separase [19
]. In vertebrates, however, the functional significance of these modifications has remained unclear. The circumstantial evidence in vertebrate systems that has existed so far points to a role of cohesin phosphorylation in controlling the ability of cohesin to bind chromatin [2
], not in modulating Scc1 cleavage by separase. It was also unclear whether Plk1, a kinase that is essential for cohesin dissociation from chromosome arms during prophase and prometaphase [16
], regulates cohesin in early mitosis by directly phosphorylating Scc1 or SA2, or by modifying other proteins that might be required for cohesin unloading.
Our analysis of mitotic cohesin regulation in human cells revealed distinct roles for the phosphorylation of Scc1 and SA2. Phosphorylation of human Scc1 enhances the cleavability of this protein by separase, at least at the second of Scc1's two cleavage sites (see ), and thereby shows that this mode of cohesin regulation is conserved from yeast to humans. Furthermore, our data imply that Scc1 phosphorylation is not required for the dissociation of cohesin from chromosome arms (see Figure S2
), again consistent with the situation in yeast where Scc1 is phosphorylated in mitosis [19
], yet cohesin complexes do not dissociate from chromosomes until separase is activated [34
In contrast to the data for Scc1, our results show that SA2 phosphorylation is essential for the dissociation of at least some cohesin complexes from chromosome arms (see ), but this modification does not seem to be necessary for the cleavage of cohesin complexes by separase (C and unpublished data). Cells that express nonphosphorylatable versions of SA2 are unable to remove all cohesin complexes from their chromosome arms during prometaphase (see A), even if mitosis is prolonged for many hours by treatment with spindle poisons (see B); presumably as a consequence, cohesion between sister chromatid arms is not lost in these cells during prolonged prometaphase arrest (see ). These phenotypes are virtually identical to the cohesin and cohesion phenotypes of cells in which Plk1 has been depleted [18
]. It has also been observed that Plk1 can phosphorylate Scc1 and SA2 in vitro, can thereby decrease the ability of cohesin to bind chromatin, and is required for the mitosis-specific phosphorylation of Scc1 and SA1/SA2 in Xenopus
egg extracts [16
]. Obviously none of these results can exclude the possibility that kinases other than Plk1 contribute to SA2 phosphorylation, nor the possibility that Plk1 may also have to phosphorylate proteins other than SA2 to allow cohesin dissociation, but the simplest interpretation of all the data is that Plk1 is essential for cohesin unloading because it is required for SA2 phosphorylation, which in turn is a prerequisite for cohesin dissociation.
It will be interesting to learn whether this type of regulation is restricted to human SA2, or whether it also applies to paralogs and orthologs of SA2. In addition to SA1 and SA2, a meiosis-specific paralog of the Scc3 family exists in mammals, called SA3 (or STAG3) [36
]. Most of the phosphorylation sites that we identified in SA2 are conserved in SA1, whereas SA3 diverges from SA1 and SA2 mostly in its C-terminal sequence (unpublished data). This difference could have important implications for the regulation of meiosis I, where arm cohesion needs to be protected to allow the separation of homologous chromosomes in anaphase I (reviewed in [37
]), and where chromosome arms do not separate even if cells are arrested by treatment with spindle poisons [38
]. It is possible that the replacement of SA1/2 by SA3 renders meiotic cohesin complexes resistant to Plk1-dependent removal from chromosome arms, and thereby allows the maintenance of arm cohesion until separase is activated. Likewise it will be interesting to analyze whether Scc3 is phosphorylated in budding yeast, in which cohesin dissociation from chromosome arms in early mitosis has not been detected.
How Important Are Scc1 and SA2 Phosphorylation In Vivo?
Somewhat unexpectedly, we found that expression of neither nonphosphorylatable Scc1 nor nonphosphorylatable SA2 blocked progression through mitosis. Although it remains formally possible that our experiments did not identify all mitosis-specific phosphorylation sites and that we therefore did not mutate all critical sites, we consider it more plausible to think that phosphorylation of these proteins is not absolutely essential for progression through mitosis, at least in transformed cultured cells. This notion is supported by the finding that cohesin can be removed from chromosomes (presumably by separase), even in cells in which Plk1 has been depleted and Aurora-B has been inhibited—i.e., under conditions where the early mitotic dissociation of cohesin from chromosome arms is inhibited [18
]. The implication is that the early mitotic dissociation of cohesin from chromosomes is not absolutely essential for mitosis, because separase is able to cleave all cohesin complexes that reside on chromosomes at the metaphase-anaphase transition. In this respect, human cells therefore appear to be more similar to budding yeast than previously suspected, in that HeLa cells can also initiate anaphase without first having to remove cohesin from chromosome arms.
Likewise, there are similarities between yeast and HeLa cells in the regulation of Scc1. In both systems, Scc1 phosphorylation enhances its cleavability by separase, but in neither case is this modification essential for viability (this study) [19
]. An interesting hint to the possible function of Scc1 phosphorylation comes from the observation that budding yeast cells lacking the securin Pds1 are viable and are able to undergo anaphase, but this ability is dramatically decreased if phosphorylation sites in Scc1 are mutated [19
]. Since securin not only inhibits separase but is also required for its activation, yeast cells lacking securin may not have enough separase activity to cleave cohesin if Scc1 is not phosphorylated. Phosphorylation of Scc1 might increase its affinity for separase, and this effect may simply enhance the fidelity of anaphase initiation. Securin is also not essential for viability in human cells, but in its absence the specific activity of separase is decreased [39
]. It would be interesting to test whether human cells lacking securin require Scc1 phosphorylation for viability.
Similarly, it is possible that SA2 phosphorylation and the resulting dissociation of cohesin from chromosomes in early mitosis, albeit not being essential, increase the fidelity of chromosome segregation. It is also conceivable that removal of cohesin prior to cleavage is not important for mitosis but for the next interphase. Separase-dependent cohesin removal destroys the Scc1 subunit and thereby renders cohesin nonfunctional. In contrast, phosphorylation-dependent dissociation appears to leave cohesin intact and might thereby enable the rapid reloading of cohesin onto chromatin in telophase, i.e., without the necessity for new Scc1 transcription and translation, which is inhibited during mitosis.
How Does SA2 Phosphorylation Lead to Cohesin Dissociation?
Cohesin is bound to chromatin in an extremely stable manner ([8
]; E.R. and J.M.P, unpublished data), and this may be related to the fact that Smc1, Smc3, and Scc1 form a ring-like complex, at least in budding yeast [40
]. It has been proposed that this protein ring establishes cohesion by encircling the sister chromatid strands [40
]. In this model, it is easy to imagine how cleavage of Scc1 releases cohesin from chromatin. However, Scc3 binds to Scc1 and is not required for formation of the ring-like complex, and it is therefore not immediately obvious how phosphorylation of SA2 could lead to dissociation of cohesin from chromosomes. One possibility is that SA2 phosphorylation induces a conformational change in cohesin that opens the ring. Bulk phosphorylation of SA2's C terminus, for example, might considerably change its surface charge, thereby affecting interactions between Scc1 and the Smc1/3 subunits. In its simplest form, this model would predict that SA2 phosphorylation is sufficient for opening of the cohesin ring and thus is sufficient for cohesin dissociation. However, in preliminary experiments, we have been unable to observe cohesin dissociation when we added purified active Plk1 to chromatin (I. Sumara and J.M.P, unpublished data), whereas the simultaneous addition of Plk1 and Xenopus
egg extracts to chromatin did enable cohesin dissociation [16
]. It is therefore also possible that phosphorylation of SA2 recruits cohesin unloading factors to chromatin (which in the above experiment might have been contributed by the Xenopus
extract), which then somehow enable the dissociation of cohesin from chromosomes. In budding yeast and C. elegans,
cohesin needs additional factors for its loading onto chromatin [42
]. Cohesin might similarly need aid for unloading, at least in the absence of Scc1 cleavage. If such additional factors exist and interact with SA2, the cell lines we created might provide a means to isolate the relevant molecules by differential purification of cohesin complexes containing wild-type SA2 and SA2–12xA.
If SA2 phosphorylation results in cohesin unloading by somehow enabling the opening of the cohesin ring without its cleavage, it would be conceivable that this reaction is simply the reverse of the loading process, during which the cohesin ring presumably also has to be opened transiently [44
]. However, a prediction of this model would be that SA2 phosphorylation would also be required for the loading of the cohesin complex, whereas we find that complexes containing nonphosphorylatable cohesin can efficiently associate with chromatin and even establish functional cohesion. It is therefore more plausible to hypothesize that SA2 phosphorylation is a modification that is specifically used to remove cohesin from chromosomes in early mitosis by enabling a reaction that is not simply the reverse of the loading reaction.
Which Cohesin Complexes Are Regulated by SA2 Phosphorylation?
SA2 phosphorylation is required for the dissociation of cohesin from chromosome arms, but it does not seem to affect the behavior of cohesin at centromeres. As a consequence, sister chromatid arms are resolved much farther from each other than centromeres during a normal prometaphase, and they can lose cohesion completely if prometaphase is prolonged, whereas cohesion at centromeres is protected. How is this regulation achieved? Recent work in fission yeast has shown that members of the Mei-S332 family of proteins [45
] are required for the persistence of cohesin at centromeres in meiosis I [46
]. These proteins, called shugoshins or Sgo1, are thought to protect centromeric cohesin in anaphase I from premature cleavage by separase, but they are also found at centromeres in mitotic Drosophila
and budding yeast cells [49
]. In an associated paper by McGuinness et al. [51
], we show that an ortholog of Sgo1 is also required for the persistence of cohesin at centromeres and for the maintenance of sister chromatid cohesion during prometaphase in human cells. Remarkably, Sgo1-depleted cells do not show cohesion defects if a nonphosphorylatable form of SA2 is expressed. This observation implies that cohesin normally persists at centromeres because Sgo1 protects cohesin in this chromosomal domain from phosphorylation. To test this hypothesis it will be important to determine whether SA2 phosphorylation does occur at centromeres. Our identification of in vivo phosphorylation sites on SA2 may be an important prerequisite for achieving this goal, because it should enable the generation of phosphospecific antibodies. Likewise, it will be interesting to learn how Sgo1 prevents or antagonizes SA2 phosphorylation, and whether the same mechanism is able to protect centromeric cohesin from separase in meiosis I.
Previous work has highlighted the difference in the regulation of cohesin complexes between chromosome arms and centromeres [10
], but several observations suggest that there may also be important differences among different populations of cohesin complexes on chromosome arms. When we compared Scc1 cleavage in cells expressing either wild-type or nonphosphorylatable SA2, we noticed that the levels of Scc1 cleavage products in the latter cells were only slightly increased, if at all (unpublished data). If all complexes containing nonphosphorylatable SA2 remained on chromosome arms until prometaphase, we would instead expect to see more Scc1 cleavage in cells containing these complexes than in cells containing wild-type SA2. Furthermore, we noticed that the immunofluorescence intensity of SA2–12xA-myc in interphase cells from which soluble cohesin complexes had been removed by preextraction was clearly higher than the intensity of SA2–12xA-myc on prometaphase and metaphase chromosomes, and in subcellular fractionation and immunoblotting experiments we found that a fraction of SA2–12xA still became soluble in nocodazole-arrested cells (unpublished data). We made similar observations in immunofluorescence experiments in which we analyzed the chromosome association of Scc1-myc in Plk1-depleted cells. Also in these cells, some cohesin still seemed to dissociate from chromosome arms, despite the depletion of Plk1 (T. Hirota and J.M.P, unpublished data). The observation that some cohesin complexes do dissociate from chromosome arms even if Plk1 is depleted or if these complexes contain nonphosphorylatable SA2, whereas others do not, cannot simply be explained by slow dissociation kinetics of cohesin under these conditions, because those complexes that persist on chromosome arms can still be found there after 10 h of prometaphase arrest (see B). We therefore favor the hypothesis that there are, in fact, two distinct populations of cohesin on chromosome arms: one whose dissociation depends on Plk1 activity and SA2 phosphorylation, and one whose dissociation does not. Since the population of cohesin complexes whose dissociation depends on Plk1 and SA2 is able to establish cohesion (see ), it is possible that those complexes that seem to be able to dissociate without Plk1 and SA2 phosphorylation are bound to chromatin in a manner that does not establish cohesion. Such binding modes must exist, because cohesin rebinds to chromatin in telophase [3
], i.e., long before sister chromatids have been generated by DNA replication. This speculative model makes important predictions, for example that cohesin dissociation from unreplicated DNA should not depend on SA2 phosphorylation; we will attempt to test this prediction in the future.