The cycle of sister chromatid cohesion is tightly woven into the fabric of the cell division cycle. In this study we demonstrate the direct regulation of cohesion establishment by the master cell cycle regulator Cdk1. Our results suggest that Clb2-Cdk1-dependent phosphorylation of Eco1 from late S phase to mitosis targets nuclear Eco1 for ubiquitination by SCFCdc4
and subsequent degradation by the proteasome. This reduces the amount of Eco1 acetyltransferase activity below some threshold required to establish cohesion, which, together with reduced ECO1
transcription in G1 (Spellman et al., 1998
), prevents the establishment of new cohesion until DNA replication occurs in S phase of the next cell cycle. In cells containing a nonphosphorylatable Eco1, or upon activation of the DNA damage response, Eco1 is stabilized and cohesion can be established in metaphase.
The regulation of cohesion establishment, like that of all critical processes, is a finely tuned balance of opposing positive and negative influences. In this case, the acetyltransferase activity of Eco1 during DNA replication facilitates the establishment of sister chromatid cohesion. In metaphase, however, Cdk1 kinase activity inhibits Eco1, allowing the anti-establishment activities of Wpl1 and Pds5 to predominate, thus preventing establishment after replication. By modulating the positive input (establishment), Cdk1 helps generate a level of chromosomal cohesion that is optimal for efficient sister chromatid separation in anaphase.
We uncovered the regulation of Eco1 by Cdk1 using a new method of creating constitutively phosphorylated mutants. Linking Cdk1 to Eco1 (via Cln3) inhibited Eco1 function, greatly reducing cell viability. Significantly, ECO1-4D
did not mimic the phosphorylated state, revealing the usefulness of the fusion technique for detecting phenotypes that are not seen with traditional phosphomimics. The effect of Cdk1 phosphorylation on the majority of its substrates is unknown; the technique presented here, combined with previously created yeast library strains (Ghaemmaghami et al., 2003
) and the recent identification of hundreds of Cdk1 substrates (Ubersax et al., 2003
; Holt et al., 2009
), could facilitate a better understanding of the role of Cdk1 in regulating cell cycle events.
Eco1 undergoes an interesting pattern of phosphorylation by Cdk1 over the cell cycle (). Given that Eco1 is not a Clb5-specific substrate () (Loog and Morgan, 2005
), the phosphorylation seen in early S phase is likely to be incomplete. This partial phosphorylation might not be sufficient for degradation, given that Cdc4 only has appreciable affinity for multiply phosphorylated substrates. Instead, early phosphorylation by Clb5-Cdk1 may prime Eco1 to be rapidly phosphorylated when Clb2 activity rises in late S phase. It is also possible that phosphorylated Eco1 exists in S phase cells because some other mechanism prevents its degradation at that stage of the cell cycle.
In a cell experiencing double-strand DNA breaks (DSBs), new cohesion aids DNA repair and is established by the reactivation of Eco1 (Sjogren and Nasmyth, 2001
; Strom et al., 2004
; Unal et al., 2004
; Unal et al., 2007
). This appears to occur by two separate mechanisms: the stabilization of Eco1 () and the phosphorylation of Scc1 by Chk1 (Heidinger-Pauli et al., 2008
). Either mechanism is sufficient to establish cohesion in metaphase independent of DNA damage signaling () (Heidinger-Pauli et al., 2009
). Once the mechanism of Eco1 stabilization in damage is elucidated, it will be possible to test whether stabilization, like Scc1 phosphorylation (Heidinger-Pauli et al., 2008
), is necessary for establishment of damage-induced cohesion. Interestingly, we found that Eco1 is stabilized not only after induction of DSBs, but also following inhibition of DNA replication with hydroxyurea (). Perhaps Eco1 stabilization facilitates cohesion establishment upon removal of replication stress.
The substrate of Eco1 during S phase is the Smc3 subunit of cohesin, whereas damage-induced Eco1 activity is thought to target the Scc1 subunit (Heidinger-Pauli et al., 2009
). It is likely that Eco1-4A also targets Scc1, since the metaphase cohesion that results from ECO1
overexpression depends on lysines in Scc1, not Smc3 (Heidinger-Pauli et al., 2009
), and does not promote excess Smc3 acetylation (Borges et al., 2010
). Mechanisms therefore exist to focus Eco1 activity on Smc3 in S phase and on Scc1 thereafter. The molecular basis of this selectivity is not known, although it seems likely that other factors, such as the replication fork or mitotic chromatin structure, influence the choice of targets. Structural changes in cohesin or cohesin-associated factors throughout the cell cycle are also possible. Interestingly, acetylation of Smc3 in S phase can occur even when Eco1 levels are severely reduced (Breslow et al., 2008
) or when replication is delayed until late S phase (Beckouet et al., 2010
), possibly due to a high local concentration of Eco1 at the replication fork. Scc1 acetylation, on the other hand, is more sensitive to enzyme concentration, as the degradation promoted by Cdk1 in mitosis is sufficient to prevent cohesion establishment at this stage.
Given that excess cohesion establishment activity is not grossly detrimental to yeast cell viability, why has an intricate system evolved to inhibit it? Since cells establish new genome-wide cohesion upon DNA damage, the cohesion removal system has likely developed a tolerance to some excess cohesion. This could be achieved by an excess of separase activity, for example. Indeed, we found that a reduction in separase activity (the esp1-2 mutation) had little effect on the viability of ECO1-4A cells (). However, we did observe defects in ECO1-4A esp1-2 cells delayed in mitosis by the spindle checkpoint, suggesting that separase activity becomes limiting when ECO1-4A cells accumulate too much cohesion during a metaphase delay. Moreover, we observed a loss of sister separation synchrony in ECO1-4A cells that was greatly enhanced when ECO1-4A was combined with a separase mutation. Yeast cells have therefore evolved both an excess of separase activity and a mechanism to temporally inhibit cohesion establishment to ensure robust and efficient cohesion removal at the onset of anaphase.
The regulatory mechanism we uncovered might be conserved in other eukaryotes. Vertebrate homologs of Eco1 are phosphorylated during mitosis (Hou and Zou, 2005
; Lafont et al., 2010
; Olsen et al., 2010
). The positions of Cdk1 consensus sites are not precisely conserved beyond the most closely related yeasts, but multiple Cdk1 motifs are present at some location in the N-terminus of most homologs. Given that the phosphorylation of Eco1 seems to serve only as an interaction motif for Cdc4, the exact position of the Cdk1 sites would be under less selective pressure to remain fixed through evolution (Holt et al., 2009
). If the regulation of cohesion establishment in the cell cycle is conserved, it would be interesting to study the effect of an Eco1 phosphomutant in organisms that use a prophase pathway to remove cohesion from chromosome arms prior to anaphase (Sumara et al., 2000
), as these cells may be more sensitive to persistent establishment activity. Additionally, because mutations in human Eco1 homologs have been implicated in certain cancers (Ryu et al., 2007
; Luedeke et al., 2009
), it will also be important to pursue the intriguing possibility that defects in Eco1 regulation generate chromosomal instability and thereby facilitate tumor evolution.