A novel mechanism for the establishment of sister chromatid cohesion by the ECO1 acetyltransferase

Cohesin complex mediates cohesion between sister chromatids, which promotes high-fidelity chromosome segregation. Eco1p acetylates the cohesin subunit Smc3p during S phase to establish cohesion. The current model posits that this Eco1p-mediated acetylation promotes establishment by abrogating the ability of Wpl1p to destabilize cohesin binding to chromosomes. Here we present data from budding yeast that is incompatible with this Wpl1p-centric model. Two independent in vivo assays show that a wpl1∆ fails to suppress cohesion defects of eco1∆ cells. Moreover, a wpl1∆ also fails to suppress cohesion defects engendered by blocking just the essential Eco1p acetylation sites on Smc3p (K112, K113). Thus removing WPL1 inhibition is insufficient for generating cohesion without ECO1 activity. To elucidate how ECO1 promotes cohesion, we conducted a genetic screen and identified a cohesion activator mutation in the SMC3 head domain (D1189H). Smc3-D1189H partially restores cohesion in eco1∆ wpl1∆ or eco1 mutant cells but robustly restores cohesion in cells blocked for Smc3p K112 K113 acetylation. These data support two important conclusions. First, acetylation of the K112 K113 region by Eco1p promotes cohesion establishment by altering Smc3p head function independent of its ability to antagonize Wpl1p. Second, Eco1p targets other than Smc3p K112 K113 are necessary for efficient establishment.     

A second Wpl1 anti‐cohesion pathway requires dephosphorylation of fission yeast kleisin Rad21 by PP4

Cohesin mediates sister chromatid cohesion which is essential for chromosome segregation and repair. Sister chromatid cohesion requires an acetyl‐transferase (Eso1 in fission yeast) counteracting Wpl1, promoting cohesin release from DNA. We report here that Wpl1 anti‐cohesion function includes an additional mechanism. A genetic screen uncovered that Protein Phosphatase 4 (PP4) mutants allowed cell survival in the complete absence of Eso1. PP4 co‐immunoprecipitated Wpl1 and cohesin and Wpl1 triggered Rad21 de‐phosphorylation in a PP4‐dependent manner. Relevant residues were identified and mapped within the central domain of Rad21. Phospho‐mimicking alleles dampened Wpl1 anti‐cohesion activity, while alanine mutants were neutral indicating that Rad21 phosphorylation would shelter cohesin from Wpl1 unless erased by PP4. Experiments in post‐replicative cells lacking Eso1 revealed two cohesin populations. Type 1 was released from DNA by Wpl1 in a PP4‐independent manner. Type 2 cohesin, however, remained DNA‐bound and lost its cohesiveness in a manner depending on Wpl1‐ and PP4‐mediated Rad21 de‐phosphorylation. These results reveal that Wpl1 antagonizes sister chromatid cohesion by a novel pathway regulated by the phosphorylation status of the cohesin kleisin subunit.     

Structural insights into the regulation of cohesion establishment by Wpl1

Correct segregation of duplicated chromosomes to daughter cells during mitosis requires the action of the cohesin complex. This tripartite ring‐shaped molecule is involved in holding replicated sister chromatids together from S phase until anaphase onset. Establishment of stable cohesion involves acetylation of the Smc3 component of cohesin during replication by the Eco1 acetyltransferase. This has been proposed to antagonise the activity of another member of the cohesin complex, Wpl1. Here, we describe the X‐ray structure of the conserved Wapl domain, and demonstrate that it binds the ATPase head of the Smc3 protein. We present data that suggest that Wpl1 may be involved in regulating the ATPase activity of cohesin, and that this may be subject to the acetylation state of Smc3. In addition, we present a structure of the Wapl domain bound to a functionally relevant segment of the Smc3 ATPase.     

Pds5 promotes cohesin acetylation and stable cohesin-chromosome interaction

Pds5 and Wpl1 act as anti-establishment factors preventing sister-chromatid cohesion until counteracted in S-phase by the cohesin acetyl-transferase Eso1. However, Pds5 is also required to maintain sister-chromatid cohesion in G2. Here, we show that Pds5 is essential for cohesin acetylation by Eso1 and ensures the maintenance of cohesion by promoting a stable cohesin interaction with replicated chromosomes. The latter requires Eso1 only in the presence of Wapl, indicating that cohesin stabilization relies on Eso1 only to neutralize the anti-establishment activity. We suggest that Eso1 requires Pds5 to counteract anti-establishment. This allows both cohesion establishment and Pds5-dependent stable cohesin binding to chromosomes.     

A screen for cohesion mutants uncovers Ssl3, the fission yeast counterpart of the cohesin loading factor Scc4

Sister-chromatid cohesion is mediated by cohesin, a ring-shape complex made of four core subunits called Scc1, Scc3, Smc1, and Smc3 in Saccharomyces cerevisiae (Rad21, Psc3, Psm1, and Psm3 in Schizosaccharomyces pombe). How cohesin ensures cohesion is unknown, although its ring shape suggests that it may tether sister DNA strands by encircling them . Cohesion establishment is a two-step process. Cohesin is loaded on chromosomes before replication and cohesion is subsequently established during S phase. In S. cerevisiae, cohesin loading requires a separate complex containing the Scc2 and Scc4 proteins. Cohesin rings fail to associate with chromatin and cohesion can not establish when Scc2 is impaired . The mechanism of loading is unknown, although some data suggest that hydrolysis of ATP bound to Smc1/3 is required . Scc2 homologs exist in fission yeast (Mis4), Drosophila, Xenopus, and human . By contrast, no homolog of Scc4 has been identified so far. We report here on the identification of fission yeast Ssl3 as a Scc4-like factor. Ssl3 is in complex with Mis4 and, as a bona fide loading factor, Ssl3 is required in G1 for cohesin binding to chromosomes but dispensable in G2 when cohesion is established. The discovery of a functional homolog of Scc4 indicates that the machinery of cohesin loading is conserved among eukaryotes.     

Cohesin rings devoid of scc3 and pds5 maintain their stable association with the DNA

Cohesin is a protein complex that forms a ring around sister chromatids thus holding them together. The ring is composed of three proteins: Smc1, Smc3 and Scc1. The roles of three additional proteins that associate with the ring, Scc3, Pds5 and Wpl1, are not well understood. It has been proposed that these three factors form a complex that stabilizes the ring and prevents it from opening. This activity promotes sister chromatid cohesion but at the same time poses an obstacle for the initial entrapment of sister DNAs. This hindrance to cohesion establishment is overcome during DNA replication via acetylation of the Smc3 subunit by the Eco1 acetyltransferase. However, the full mechanistic consequences of Smc3 acetylation remain unknown. In the current work, we test the requirement of Scc3 and Pds5 for the stable association of cohesin with DNA. We investigated the consequences of Scc3 and Pds5 depletion in vivo using degron tagging in budding yeast. The previously described DHFR-based N-terminal degron as well as a novel Eco1-derived C-terminal degron were employed in our study. Scc3 and Pds5 associate with cohesin complexes independently of each other and require the Scc1 "core" subunit for their association with chromosomes. Contrary to previous data for Scc1 downregulation, depletion of either Scc3 or Pds5 had a strong effect on sister chromatid cohesion but not on cohesin binding to DNA. Quantity, stability and genome-wide distribution of cohesin complexes remained mostly unchanged after the depletion of Scc3 and Pds5. Our findings are inconsistent with a previously proposed model that Scc3 and Pds5 are cohesin maintenance factors required for cohesin ring stability or for maintaining its association with DNA. We propose that Scc3 and Pds5 specifically function during cohesion establishment in S phase.     

Acetylation regulates monopolar attachment at multiple levels during meiosis I in fission yeast

In fission yeast, meiotic mono-orientation of sister kinetochores is established by cohesion at the core centromere, which is established by a meiotic cohesin complex and the kinetochore protein Moa1. The cohesin subunit Psm3 is acetylated by Eso1 and deacetylated by Clr6. We show that in meiosis, Eso1 is required for establishing core centromere cohesion during S phase, whereas Moa1 is required for maintaining this cohesion after S phase. The clr6-1 mutation suppresses the mono-orientation defect of moa1Δ cells, although the Clr6 target for this suppression is not Psm3. Thus, several acetylations are crucial for establishing and maintaining core centromere cohesion.     

A Conserved Domain in the Scc3 Subunit of Cohesin Mediates the Interaction with Both Mcd1 and the Cohesin Loader Complex

The Structural Maintenance of Chromosome (SMC) complex, termed cohesin, is essential for sister chromatid cohesion. Cohesin is also important for chromosome condensation, DNA repair, and gene expression. Cohesin is comprised of Scc3, Mcd1, Smc1, and Smc3. Scc3 also binds Pds5 and Wpl1, cohesin-associated proteins that regulate cohesin function, and to the Scc2/4 cohesin loader. We mutagenized SCC3 to elucidate its role in cohesin function. A 5 amino acid insertion after Scc3 residue I358, or a missense mutation of residue D373 in the adjacent stromalin conservative domain (SCD) induce inviability and defects in both cohesion and cohesin binding to chromosomes. The I358 and D373 mutants abrogate Scc3 binding to Mcd1. These results define an Scc3 region extending from I358 through the SCD required for binding Mcd1, cohesin localization to chromosomes and cohesion. Scc3 binding to the cohesin loader, Pds5 and Wpl1 are unaffected in I358 mutant and the loader still binds the cohesin core trimer (Mcd1, Smc1 and Smc3). Thus, Scc3 plays a critical role in cohesin binding to chromosomes and cohesion at a step distinct from loader binding to the cohesin trimer. We show that residues Y371 and K372 within the SCD are critical for viability and chromosome condensation but dispensable for cohesion. However, scc3 Y371A and scc3 K372A bind normally to Mcd1. These alleles also provide evidence that Scc3 has distinct mechanisms of cohesin loading to different loci. The cohesion-competence, condensation-incompetence of Y371 and K372 mutants suggests that cohesin has at least one activity required specifically for condensation.     

An Eco1-independent sister chromatid cohesion establishment pathway in S. cerevisiae

Cohesion between sister chromatids, mediated by the chromosomal cohesin complex, is a prerequisite for their alignment on the spindle apparatus and segregation in mitosis. Budding yeast cohesin first associates with chromosomes in G1. Then, during DNA replication in S-phase, the replication fork-associated acetyltransferase Eco1 acetylates the cohesin subunit Smc3 to make cohesin’s DNA binding resistant to destabilization by the Wapl protein. Whether stabilization of cohesin molecules that happen to link sister chromatids is sufficient to build sister chromatid cohesion, or whether additional reactions are required to establish these links, is not known. In addition to Eco1, several other factors contribute to cohesion establishment, including Ctf4, Ctf18, Tof1, Csm3, Chl1 and Mrc1, but little is known about their roles. Here, we show that each of these factors facilitates cohesin acetylation. Moreover, the absence of Ctf4 and Chl1, but not of the other factors, causes a synthetic growth defect in cells lacking Eco1. Distinct from acetylation defects, sister chromatid cohesion in ctf4Δ and chl1Δ cells is not improved by removing Wapl. Unlike previously thought, we do not find evidence for a role of Ctf4 and Chl1 in Okazaki fragment processing, or of Okazaki fragment processing in sister chromatid cohesion. Thus, Ctf4 and Chl1 delineate an additional acetylation-independent pathway that might hold important clues as to the mechanism of sister chromatid cohesion establishment.     

A SUMO-Dependent Step during Establishment of Sister Chromatid Cohesion

Cohesin is a protein complex that ties sister DNA molecules from the time of DNA replication until the metaphase to anaphase transition. Current models propose that the association of the Smc1, Smc3, and Scc1/Mcd1 subunits creates a ring-shaped structure that entraps the two sister DNAs [1]. Cohesin is essential for correct chromosome segregation and recombinational repair. Its activity is therefore controlled by several posttranslational modifications, including acetylation, phosphorylation, sumoylation, and site-specific proteolysis. Here we show that cohesin sumoylation occurs at the time of cohesion establishment, after cohesin loading and ATP binding, and independently from Eco1-mediated cohesin acetylation. In order to test the functional relevance of cohesin sumoylation, we have developed a novel approach in budding yeast to deplete SUMO from all subunits in the cohesin complex, based on fusion of the Scc1 subunit to a SUMO peptidase Ulp domain (UD). Downregulation of cohesin sumoylation is lethal, and the Scc1-UD chimeras have a failure in sister chromatid cohesion. Strikingly, the unsumoylated cohesin rings are acetylated. Our findings indicate that SUMO is a novel molecular determinant for the establishment of sister chromatid cohesion, and we propose that SUMO is required for the entrapment of sister chromatids during the acetylation-mediated closure of the cohesin ring.     

Pds5B is required for cohesion establishment and Aurora B accumulation at centromeres

Cohesin mediates sister chromatid cohesion and contributes to the organization of interphase chromatin through DNA looping. In vertebrate somatic cells, cohesin consists of Smc1, Smc3, Rad21, and either SA1 or SA2. Three additional factors Pds5, Wapl, and Sororin bind to cohesin and modulate its dynamic association with chromatin. There are two Pds5 proteins in vertebrates, Pds5A and Pds5B, but their functional specificity remains unclear. Here, we demonstrate that Pds5 proteins are essential for cohesion establishment by allowing Smc3 acetylation by the cohesin acetyl transferases (CoATs) Esco1/2 and binding of Sororin. While both proteins contribute to telomere and arm cohesion, Pds5B is specifically required for centromeric cohesion. Furthermore, reduced accumulation of Aurora B at the inner centromere region in cells lacking Pds5B impairs its error correction function, promoting chromosome mis‐segregation and aneuploidy. Our work supports a model in which the composition and function of cohesin complexes differs between different chromosomal regions.     

Cohesin Acetylation Promotes Sister Chromatid Cohesion Only in Association with the Replication Machinery

Acetylation of the Smc3 subunit of cohesin is essential to establish functional cohesion between sister chromatids. Smc3 acetylation is catalyzed by members of the Eco family of acetyltransferases, although the mechanism by which acetylation is regulated and how it promotes cohesion are largely unknown. In vertebrates, the cohesin complex binds to chromatin during mitotic exit and is converted to a functional form during or shortly after DNA replication. The conserved proliferating cell nuclear antigen-interacting protein box motif in yeast Eco1 is required for function, and cohesin is acetylated during the S phase. This has led to the notion that acetylation of cohesin is stimulated by interaction of Eco1 with the replication machinery. Here we show that in vertebrates Smc3 acetylation occurs independently of DNA replication. Smc3 is readily acetylated before replication is initiated and after DNA replication is complete. However, we also show that functional acetylation occurs only in association with the replication machinery: disruption of the interaction between XEco2 and proliferating cell nuclear antigen prevents cohesion establishment while having little impact on the overall levels of Smc3 acetylation. These results demonstrate that Smc3 acetylation can occur throughout interphase but that only acetylation in association with the replication fork promotes sister chromatid cohesion. These data reveal how the generation of cohesion is limited to the appropriate time and place during the cell cycle and provide insight into the mechanism by which acetylation ensures cohesion.     

Cell-cycle regulation of cohesin stability along fission yeast chromosomes

Sister chromatid cohesion is mediated by cohesin, but the process of cohesion establishment during S-phase is still enigmatic. In mammalian cells, cohesin binding to chromatin is dynamic in G1, but becomes stabilized during S-phase. Whether the regulation of cohesin stability is integral to the process of cohesion establishment is unknown. Here, we provide evidence that fission yeast cohesin also displays dynamic behavior. Cohesin association with G1 chromosomes requires continued activity of the cohesin loader Mis4/Ssl3, suggesting that repeated loading cycles maintain cohesin binding. Cohesin instability in G1 depends on wpl1, the fission yeast ortholog of mammalian Wapl, suggestive of a conserved mechanism that controls cohesin stability on chromosomes. wpl1 is nonessential, indicating that a change in wpl1-dependent cohesin dynamics is dispensable for cohesion establishment. Instead, we find that cohesin stability increases at the time of S-phase in a reaction that can be uncoupled from DNA replication. Hence, cohesin stabilization might be a pre-requisite for cohesion establishment rather than its consequence.     

Cohesin's DNA Exit Gate Is Distinct from Its Entrance Gate and Is Regulated by Acetylation

Sister chromatid cohesion is mediated by entrapment of sister DNAs by a tripartite ring composed of cohesin's Smc1, Smc3, and α-kleisin subunits. Cohesion requires acetylation of Smc3 by Eco1, whose role is to counteract an inhibitory (antiestablishment) activity associated with cohesin's Wapl subunit. We show that mutations abrogating antiestablishment activity also reduce turnover of cohesin on pericentric chromatin. Our results reveal?a "releasing" activity inherent to cohesin complexes transiently associated with Wapl that catalyzes their dissociation from chromosomes. Fusion of Smc3's nucleotide binding domain to α-kleisin's N-terminal domain also reduces cohesin turnover within pericentric chromatin and permits establishment of Wapl-resistant cohesion in the absence of Eco1. We suggest that releasing activity opens the Smc3/α-kleisin interface, creating a DNA exit gate distinct from its proposed entry gate at the Smc1/3 interface. According to this notion, the function of Smc3 acetylation is to block its dissociation from α-kleisin. The functional implications of regulated ring opening are discussed.     

Acetylation of Smc3 by Eco1 is required for S phase sister chromatid cohesion in both human and yeast

Sister chromatid cohesion is normally established in S phase in a process that depends on the cohesion establishment factor Eco1, a conserved acetyltransferase. However, due to the lack of known in vivo substrates, how Eco1 regulates cohesion is not understood. Here we report that yeast Eco1 and its human ortholog, ESCO1, both acetylate Smc3, a component of the cohesin complex that physically holds the sister chromatid together, at two conserved lysine residues. Mutating these lysine residues to a nonacetylatable form leads to increased loss of sister chromatid cohesion and genome instability in both yeast and human. In addition, we clarified that the acetyltransferase activity of Eco1 is essential for its function. Our study thus identified a molecular target for the acetyltransferase Eco1 and revealed that Smc3 acetylation is a conserved mechanism in regulating sister chromatid cohesion.     

Epitope tag-induced synthetic lethality between cohesin subunits and Ctf7/Eco1 acetyltransferase

Ctf7/Eco1-dependent acetylation of Smc3 is essential for sister chromatid cohesion. Here, we use epitope tag-induced lethality in cells diminished for Ctf7/Eco1 activity to map cohesin architecture in vivo. Tagging either Smc1 or Mcd1/Scc1, but not Scc3/Irr1, appears to abolish access to Smc3 in ctf7/eco1 mutant cells, suggesting that Smc1 and Smc3 head domains are in direct contact with each other and also with Mcd1/Scc1. Thus, cohesin complexes may be much more compact than commonly portrayed. We further demonstrate that mutation in ELG1 or RFC5 anti-establishment genes suppress tag-induced lethality, consistent with the notion that the replication fork regulates Ctf7/Eco1.     

Rtt101‐Mms1‐Mms22 coordinates replication‐coupled sister chromatid cohesion and nucleosome assembly

Two sister chromatids must be held together by a cohesion process from their synthesis during S phase to segregation in anaphase. Despite its pivotal role in accurate chromosome segregation, how cohesion is established remains elusive. Here, we demonstrate that yeast Rtt101‐Mms1, Cul4 family E3 ubiquitin ligases are stronger dosage suppressors of loss‐of‐function eco1 mutants than PCNA. The essential cohesion reaction, Eco1‐catalyzed Smc3 acetylation is reduced in the absence of Rtt101‐Mms1. One of the adaptor subunits, Mms22, associates directly with Eco1. Point mutations (L61D/G63D) in Eco1 that abolish the interaction with Mms22 impair Smc3 acetylation. Importantly, an eco1LGpol30A251V double mutant displays additive Smc3ac reduction. Moreover, Smc3 acetylation and cohesion defects also occur in the mutants of other replication‐coupled nucleosome assembly (RCNA) factors upstream or downstream of Rtt101‐Mms1, indicating unanticipated cross talk between histone modifications and cohesin acetylation. These data suggest that fork‐associated Cul4‐Ddb1 E3s, together with PCNA, coordinate chromatin reassembly and cohesion establishment on the newly replicated sister chromatids, which are crucial for maintaining genome and chromosome stability.     

Cohesin acetyltransferase Esco2 is a cell viability factor and is required for cohesion in pericentric heterochromatin

Sister chromatid cohesion, mediated by cohesin and regulated by Sororin, is essential for chromosome segregation. In mammalian cells, cohesion establishment and Sororin recruitment to chromatin-bound cohesin depends on the acetyltransferases Esco1 and Esco2. Mutations in Esco2 cause Roberts syndrome, a developmental disease in which mitotic chromosomes have a 'railroad' track morphology. Here, we show that Esco2 deficiency leads to termination of mouse development at pre- and post-implantation stages, indicating that Esco2 functions non-redundantly with Esco1. Esco2 is transiently expressed during S-phase when it localizes to pericentric heterochromatin (PCH). In interphase, Esco2 depletion leads to a reduction in cohesin acetylation and Sororin recruitment to chromatin. In early mitosis, Esco2 deficiency causes changes in the chromosomal localization of cohesin and its protector Sgo1. Our results suggest that Esco2 is needed for cohesin acetylation in PCH and that this modification is required for the proper distribution of cohesin on mitotic chromosomes and for centromeric cohesion.