In the current study, we provide significant insights into how Eco1p stimulates cohesion generation in G2/M in response to DSBs, how the DNA damage signal transduction pathway coordinate this response, and finally how DSB-induced cohesion compares to the cohesion generation that occurs during S phase. We provide multiple lines of evidence that Eco1p regulates DSB-induced cohesion by acetylating Mcd1-K84, K210 upon DNA damage-induced phosphorylation of Mcd1-S83 ((Heidinger-Pauli et al., 2008
), ). Nonacetylatable mutation of mcd1-K84, K210 phenocopies eco1
mutations. Both block DSB-induced cohesion at a step after cohesin binding to chromatin and both block constitutive cohesion of Mcd1-S83D, placing them downstream of Chk1p in the DNA damage kinase cascade. The acetyl-mimics of Mcd1 (Mcd1-K84Q, K210Q) also phenocopy Eco1p over-expression: both allow cohesion generation in G2/M in the absence of a DSB and Chk1p. Furthermore, the acetyl-mimics bypass the requirement for Eco1p in DSB-induced cohesion. These data provide strong support for the model that Mcd1-K84, K210 are Eco1p acetylation targets, although future biochemical studies will be required to confirm this conclusion in vivo
Model Illustrating the Regulation of Cohesion Generation in S Phase and G2/M Phase
Here we provide evidence that Eco1p’s ability to acetylate Mcd1p is enhanced by Mcd1p phosphorylation. Preventing Mcd1-S83 phosporylation, or the deleting its likely kinase, Chk1p, results in an identical phenotype to preventing acetylation of Mcd1p at residues of K84 and K210. Namely, DSB-induced cohesion, but not S phase cohesion is blocked at a step after cohesin binding ((Heidinger-Pauli et al., 2008
), this study). Acetyl-mimic and phospho-mimic mutants phenocopy elevated expression of Eco1p; they all generate cohesion in G2/M in the absence of a DSB. Finally epistasis analysis with the modification-null and modification–mimic alleles indicate that DSB-induced phosphorylation of Mcd1-S83 enhances Eco1p’s ability to acetylate Mcd1p in G2/M, thereby increasing the relative activity of Eco1p.
Based upon these observations, we speculate that Eco1p has a poor affinity for its substrates not only in G2/M, but also in S phase. Indeed, Eco1p interacts poorly with soluble cohesin and requires Scc2p/Scc4p (loading factors for cohesin) to acetylate Smc3p during S phase, leading to the suggestion that Eco1p preferentially acetylates cohesin bound to chromatin (Noble et al., 2006
; Unal et al., 2008
). During S phase, Eco1p is likely recruited to chromatin through PCNA associated with DNA replication, thereby ensuring the proper timing of cohesion generation(reviewed in(Skibbens et al., 2007
)). In G2/M, phosphorylation of Mcd1p may serve as a replication independent surrogate to facilitate Eco1p function. These results set the precedent that other unique facilitators of Eco1p may exist to modulate cohesin function in other contexts such as transcription.
We propose that Eco1p promotes DSB-induced cohesion by antagonizing Wpl1p. The putative targets of Eco1p for DNA damage, Mcd1-K84, K210, are no longer required for DSB-induced cohesion when Wpl1p is absent. Furthermore, wpl1Δ allows cohesion generation in G2/M in the absence of a DSB. These results strongly suggest that Wpl1p antagonizes cohesion generation in G2/M, explaining why cohesion generation is normally limited to S phase, and that the function of Mcd1-K84, K210 acetylation is to counteract Wpl1p ().
The role of Wpl1p as an antagonist in mitosis in budding yeast agrees with previous studies of its mammalian homolog, Wapl. During prophase, Wapl is required for the dissolution of S phase established cohesion from chromosome arms (Gandhi et al., 2006
; Kueng et al., 2006
). Wapl could also act immediately on any cohesion formed during G2/M thereby blocking cohesion generation. Indeed, depletion of Wapl in results in a larger amount of cohesin on chromosomes in prophase than would be expected by just inhibiting the normal removal of cohesin loaded on chromosome arms during S phase. Thus Wapl inactivation may allow new cohesion generation in G2/M in mammalian cells similar to what we have shown for Wpl1p in budding yeast (this study). Interestingly, Wapl-stimulated cohesion dissolution in mammalian cells leads to the removal of cohesin from chromosome arms as well as loss of cohesion. One intriguing possibility is that Wapl counteracts cohesion at a step independent from cohesin binding as Wpl1p does in budding yeast, but these non-cohesive cohesins are then more susceptible to being removed from chromosomes in mammalian cells.
Here we provide a model where Eco1p and Wpl1p act as a simple positive and negative regulatory switch controlling cohesion generation (). However, both the regulatory switch and the function of the Wpl1p and Eco1p are likely to be more complicated. The regulatory switch may contain additional components like Pds5p, a cohesion accessory factor that associates with Eco1p and possibly Wpl1p as well (Gandhi et al., 2006
; Noble et al., 2006
; Sutani et al., 2009
). Furthermore, Wpl1p may have additional functions beyond controlling cohesion generation. For example, wpl1
Δ cells are mildly sensitive to irradiation, a phenotype that cannot be explained by constitutive cohesion generation in G2/M (Game et al., 2003
). Similarly Eco1p may regulate other cohesin functions. In fact, Eco1p acetylates other residues on Smc3p and other cohesin subunits (Ivanov et al., 2002
; Unal et al., 2008
). The physiological relevance of these acetylation events will be determined when they are analyzed in their proper context.
Eco1p antagonizes Wpl1p likely by acetylating Mcd1-K84, K210 for DSB-induced cohesion and Smc3-K112, K113 for S phase cohesion in budding yeast (This study, (Ben-Shahar et al., 2008
)). These pathways are distinct and each modification is functional only in its specific context. These observations reveal a surprising complexity to the regulation of cohesion generation. To explain this complexity we envision at least two possibilities. Wpl1p may always inhibit the same step in cohesion generation, for example by binding to the Smc3p-Mcd1p subdomain and modulating Smc3p ATPase activity. However, the interaction between Wpl1p and this subdomain might change during the cell cycle such that Eco1p must acetylate different targets to antagonize the interaction. Alternatively, Wpl1p blocks steps in cohesion generation that are unique to the stages of the cell cycle, for example steps unique to the presence or absence of DNA replication. Our data provide a framework for future studies to build upon and distinguish these two models.