Here we study the roles of Smc3p acetylation, the Eco1p acetyltransferase, Wpl1p, and cohesin in cohesion establishment, as well as in the greater context of sister chromatid disjunction, condensation, and cell viability. We show that eco1Δ wpl1Δ
cells establish cohesion very poorly, if at all. This failure to establish cohesion contradicts the prevailing Wpl1p-centric model, which was based upon the incorrect assumption that the ability of a wpl1Δ
to restore viability to an eco1Δ
mutant serves as a surrogate marker for cohesion establishment (Rolef Ben-Shahar et al., 2008
; Rowland et al., 2009
; Sutani et al., 2009
). Our result demonstrates that Eco1p promotes S phase cohesion establishment by a mechanism other than, or in addition to, antagonizing Wpl1p. One additional function for acetylation may be modulating the Smc3p ATPase (Unal et al., 2008
). Consistent with this, Smc3p K113 residue is predicted to be in close proximity to the Walker A/B domain (Unal et al., 2008
). Genetic analyses support this view, as K113 acetyl-mimics limit the toxicity of Smc3p hydrolysis mutants (Heidinger-Pauli et al., 2010b
). Our data do not rule out the possibility that Eco1p-mediated acetylation of other cohesin subunits contributes to establishment.
A second insight into the mechanism responsible for cohesion establishment comes from our observation that smc3
acetyl-mimic alleles fail to establish cohesion when present as the sole functional Smc3p in cells. This failure could reflect a role for temporal regulation in establishment, as Smc3p acetylation normally occurs at the onset of S phase, and Eco1p has been genetically and biochemically linked with the replication fork and fork components (Onn et al., 2008
; Skibbens, 2011
). However, in both human and yeast cells, the cohesion defect of the acetyl-mimic can be partially overcome by the presence of a second copy of wild-type Smc3p (Unal et al., 2008
; Zhang et al., 2008
). It is important that, in these cells, Smc3p is unable to undergo temporal acetylation because of abrogation of Eco1p activity. These results suggest that limiting the amount of Smc3p acetylation is as important as, if not more important than, temporal regulation. Indeed, only a subset of Smc3p is acetylated in human and yeast cells, consistent with a regulation to limit acetylation levels (Zhang et al., 2008
). Furthermore, HOS1
has been identified as an Smc3p deacetylase in budding yeast, and hos1Δ
mutant cells have a slightly increased pool of acetylated Smc3p along with minor defects in cohesion (Beckouet et al., 2010
; Borges et al., 2010
; Xiong et al., 2010
). Thus the ratio of acetylated to nonacetylated Smc3p likely is an important contributor to cohesion establishment whose mechanism needs to be elucidated.
Studies of cohesin regulators such as Eco1p acetylation and Wpl1p have focused on their roles in sister chromatid cohesion. Yet it is known that cohesin and the cohesin regulators Eco1p and Pds5p are required for proper mitotic chromosome condensation in budding yeast (Guacci et al., 1997
; Hartman et al., 2000
; Lavoie et al., 2002
; Skibbens et al., 1999
). Here we show that although smc3-RQ
, eco1Δ wpl1Δ
, and smc3-K113Q
strains are equally defective for cohesion, only the smc3-RQ
and eco1Δ wpl1Δ
strains are competent for condensation of the rDNA
locus and are viable. This suggests that Eco1p antagonizes Wpl1p to allow condensation. Smc3p acetylation/removal of Wpl1p inhibition may represent a bifurcation step in which cohesin's fate subsequently diverges, followed by differential regulation of cohesin's cohesive and condensive functions (). The difference in the ability of the RQ
alleles to promote condensation suggests that, like cohesion establishment, the timing or amount of acetylation may be important for condensation.
FIGURE 8: (A) Model for regulation of cohesin's roles in cohesion and condensation. Cohesin in noncohesive form (circle). Eco1p acetylates Smc3p to remove Wpl1p inhibition to form activated cohesin (square). Activated cohesin can be used for either cohesion (oval) (more ...)
The correlation between viability and ability to condense rDNA
is consistent with condensation being the essential function executed in the smc3-RQ
and eco1Δ wpl1Δ
strains. However, Smc3p acetylation could also regulate other, noncohesive functions of cohesin. In fact the smc3-RQ
strains are sensitive to the DNA-damaging agent camptothecin, suggesting this acetylation may inhibit repair (this study). Budding yeast cohesin has also been shown to play a role in transcriptional regulation and to promote intramolecular loops at pericentric heterochromatin that are believed to help promote segregation (Yeh et al., 2008
; Skibbens et al., 2010
). We did not assess these functions and so cannot rule out that they contribute to the restoration of viability.
Might acetylation of cohesin regulate condensation in other organisms? Cohesin has been shown to be important in meiotic chromosome condensation in budding and fission yeasts, as well as in mice (Ding et al., 2006
; Revenkova et al., 2004
; Jin et al., 2009
). A recent study using Xenopus
egg extracts indicated that cohesin plays a role in condensation when chromosomes were converted into a more mitotic structure by altering the ratio of condensins I and II (Shintomi and Hirano, 2011
). Together these results suggest that cohesin plays a conserved role in templating chromosome structure to enable proper mitotic and meiotic condensation in eukaryotes. Therefore it will be interesting to examine the role of cohesin acetylation and Wpl1p in condensation in other organisms. However, metazoans also contain additional proteins that modulate chromosome structure (condensin II complex and sororin) that are not found in budding yeast (Ono et al., 2003
; Rankin et al., 2005
). These additional proteins may contribute to or alter how acetylation or Wpl1p regulates cohesin's role in condensation.
The ability to condense chromosomes in the smc3-RQ and eco1Δ wpl1Δ strains cannot explain their viability with gross defects in cohesion establishment, since cohesin is assumed to be required for bipolar attachments and segregation. Contrary to this dogma, we show that these strains, as well as those abrogated for Mcd1p and Smc3p function, disjoin sister chromatids reasonably well. This cohesin-independent disjunction likely stems from the unusual biology of budding yeast, in which sister kinetochores and the spindle are assembled early in S phase. The unreplicated DNA around the sister kinetochores acts a surrogate for cohesion, enabling bipolar attachments (). As replication is completed, the sister chromatids precociously separate, yet segregate properly. As predicted by this model, delaying spindle assembly until mitosis abrogates cohesin-independent segregation (this study).
Is this cohesin-independent pathway sufficient on its own to promote viability, or is it supplemented by a small amount of residual cohesin-mediated cohesion that persists in our mutants? We cannot rule out the latter possibility. However, it is clear from the inviability and gross chromosome missegregation that result from inhibiting the S phase–coupled pathway that the cohesin-independent mechanism contributes significantly to viability. In fact our analysis likely underestimates the efficacy of disjunction via the cohesin-independent mechanism. The RQ
shows disjunction of 85% of chromosome IV by our assay. If this were the true rate of disjunction on all chromosomes, one would not expect any viable cells, so our assay must underestimate disjunction. One possibility is that smaller chromosomes may disjoin better than chromosome IV, which is the second longest yeast chromosome. Because smc3-RQ
and eco1Δ wpl1Δ
strains only partially restore condensation, larger chromosomes may be more prone to tangling and delayed segregation. Even though we underestimated the fidelity of the cohesin-independent mechanism for disjunction under unperturbed growth, this pathway is very sensitive to microtubule inhibitors (this study; Sutani et al., 2009
). Therefore cohesin-independent segregation is unlikely to be robust enough to enable viability when confronted with environmental stresses found commonly in nature, such as spindle damage or low temperatures.
Is cohesin-independent segregation unique to budding yeast? This replication-coupled mechanism is reminiscent of bacteria, where early-replicating regions segregate before replication is complete (Draper and Gober, 2002
). Although spindle assembly in S phase is an unusual feature of budding yeast, the principle of cohesin-independent segregation is worth considering in other eukaryotic organisms as well. For example, DNA catenation can link sisters without cohesin (Vagnarelli et al., 2004
), and so, in organisms in which catenation persists into mitosis, such as fission yeast (Uemura et al., 1987
), cohesin-independent segregation may exist and influence chromosome segregation and viability.
Mutations in cohesin and its regulators have been implicated in cancer and in developmental diseases, making the elucidation of cohesin regulation of medical relevance (Wang et al., 2004
; Dorsett, 2007
). For developmental diseases, the severity of the disease state does not correlate with the level of defective cohesion, implicating cohesin's noncohesive functions and illustrating the importance of elucidating their regulation (Dorsett and Krantz, 2009
). However, the essential role of cohesin in sister chromatid cohesion rendered it difficult to assess the importance of cohesin's noncohesive functions to cell viability. Our discovery that budding yeast cells are viable without efficient cohesion establishment provides a platform with which to genetically dissect noncohesive functions of cohesin.