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.     

Psm3 acetylation on conserved lysine residues is dispensable for viability in fission yeast but contributes to Eso1-mediated sister chromatid cohesion by antagonizing Wpl1

In budding yeast and humans, cohesion establishment during S phase requires the acetyltransferase Eco1/Esco1-2, which acetylates the cohesin subunit Smc3 on two conserved lysine residues. Whether Smc3 is the sole Eco1/Esco1-2 effector and how Smc3 acetylation promotes cohesion are unknown. In fission yeast (Schizosaccharomyces pombe), as in humans, cohesin binding to G(1) chromosomes is dynamic and the unloading reaction is stimulated by Wpl1 (human ortholog, Wapl). During S phase, a subpopulation of cohesin becomes stably bound to chromatin in an Eso1 (fission yeast Eco1/Esco1-2)-dependent manner. Cohesin stabilization occurs unevenly along chromosomes. Cohesin remains largely labile at the rDNA repeats but binds mostly in the stable mode to pericentromere regions. This pattern is largely unchanged in eso1Δ wpl1Δ cells, and cohesion is unaffected, indicating that the main Eso1 role is counteracting Wpl1. A mutant of Psm3 (fission yeast Smc3) that mimics its acetylated state renders cohesin less sensitive to Wpl1-dependent unloading and partially bypasses the Eso1 requirement but cannot generate the stable mode of cohesin binding in the absence of Eso1. Conversely, nonacetylatable Psm3 reduces the stable cohesin fraction and affects cohesion in a Wpl1-dependent manner, but cells are viable. We propose that Psm3 acetylation contributes to Eso1 counteracting of Wpl1 to secure stable cohesin interaction with postreplicative chromosomes but that it is not the sole molecular event by which this occurs.     

Phosphorylation of the Scc2 cohesin deposition complex subunit regulates chromosome condensation through cohesin integrity

The cohesion of replicated sister chromatids promotes chromosome biorientation, gene regulation, DNA repair, and chromosome condensation. Cohesion is mediated by cohesin, which is deposited on chromosomes by a separate conserved loading complex composed of Scc2 and Scc4 in Saccharomyces cerevisiae. Although it is known to be required, the role of Scc2/Scc4 in cohesin deposition remains enigmatic. Scc2 is a phosphoprotein, although the functions of phosphorylation in deposition are unknown. We identified 11 phosphorylated residues in Scc2 by mass spectrometry. Mutants of SCC2 with substitutions that mimic constitutive phosphorylation retain normal Scc2–Scc4 interactions and chromatin association but exhibit decreased viability, sensitivity to genotoxic agents, and decreased stability of the Mcd1 cohesin subunit in mitotic cells. Cohesin association on chromosome arms, but not pericentromeric regions, is reduced in the phosphomimetic mutants but remains above a key threshold, as cohesion is only modestly perturbed. However, these scc2 phosphomimetic mutants exhibit dramatic chromosome condensation defects that are likely responsible for their high inviability. From these data, we conclude that normal Scc2 function requires modulation of its phosphorylation state and suggest that scc2 phosphomimetic mutants cause an increased incidence of abortive cohesin deposition events that result in compromised cohesin complex integrity and Mcd1 turnover.     

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.     

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.     

Cohesin interaction with centromeric minichromosomes shows a multi-complex rod-shaped structure

Cohesin is the protein complex responsible for maintaining sister chromatid cohesion. Cohesin interacts with centromeres and specific loci along chromosome arms known as Chromosome Attachment Regions (CARs). The cohesin holocomplex contains four subunits. Two of them, Smc1p (Structural maintenance of chromosome 1 protein) and Smc3p, are long coiled-coil proteins, which heterodimerize with each other at one end. They are joined together at the other end by a third subunit, Scc1p, which also binds to the fourth subunit, Scc3p. How cohesin interacts with chromosomes is not known, although several models have been proposed, in part on the basis of in vitro assembly of purified cohesin proteins. To be able to observe in vivo cohesin-chromatin interactions, we have modified a Minichromosome Affinity Purification (MAP) method to isolate a CAR-containing centromeric minichromosome attached to in vivo assembled cohesin. Transmission Electron Microscopy (TEM) analysis of these minichromosomes suggests that cohesin assumes a rod shape and interacts with replicated minichromosome at one end of that rod. Additionally, our data implies that more than one cohesin molecule interacts with each pair of replicated minichromsomes. These molecules seem to be packed into a single thick rod, suggesting that the Smc1p and Smc3p subunits may interact extensively.     

黏着素与基因表达调控

黏着素(cohesin)是一种多亚基蛋白复合体,在进化上相当保守。在真核生物细胞中,黏着素主要功能是将复制产生的姐妹染色单体连接在一起,直到细胞分裂的后期,黏着素亚基Scc1水解最终导致染色单体的分离。但是最近研究表明,黏着素在基因表达、染色质结构变化和发育调节等方面也起着非常重要的作用,并且发现黏着素对基因的调节作用与其对染色体的黏着功能无关。在酵母中,黏着素最初定位于其装载蛋白Scc2的DNA结合位点上,但是在细胞周期的G2期,黏着素聚集于转录汇集区之间进而调控转录终止。在果蝇染色体上,黏着素与装载蛋白Scc2的同源物Nipped-B共定位,其作用是阻抑增强子和启动子的远距离接触。而在哺乳动物中,黏着素与CTCF隔离子蛋白共定位,并以依赖于CTCF的方式调控转录。本文概述了黏着素在不同真核生物染色体上的定位与分布,并对其在基因表达调控中的功能机制及其研究现状进行了重点阐述。     

Cohesin's ATPase activity is stimulated by the C-terminal Winged-Helix domain of its kleisin subunit

BACKGROUND: Cohesin, a multisubunit protein complex conserved from yeast to humans, holds sister chromatids together from the onset of replication to their separation during anaphase. Cohesin consists of four core subunits, namely Smc1, Smc3, Scc1, and Scc3. Smc1 and Smc3 proteins are characterized by 50-nm-long anti-parallel coiled coils flanked by a globular hinge domain and an ABC-like ATPase head domain. Whereas Smc1 and Smc3 heterodimerize via their hinge domains, the kleisin subunit Scc1 connects their ATPase heads, and this results in the formation of a large ring. Biochemical studies suggest that cohesin might trap sister chromatids within its ring, and genetic evidence suggests that ATP hydrolysis is required for the stable association of cohesin with chromosomes. However, the precise role of the ATPase domains remains enigmatic. RESULTS: Characterization of cohesin's ATPase activity suggests that hydrolysis depends on the binding of ATP to both Smc1 and Smc3 heads. However, ATP hydrolysis at the two active sites is not per se cooperative. We show that the C-terminal winged-helix domain of Scc1 stimulates the ATPase activity of the Smc1/Smc3 heterodimer by promoting ATP binding to Smc1's head. In contrast, we do not detect any effect of Scc1's N-terminal domain on Smc1/Smc3 ATPase activity. CONCLUSIONS: Our studies reveal that Scc1 not only connects the Smc1 and Smc3 ATPase heads but also regulates their ATPase activity.     

Evidence that loading of cohesin onto chromosomes involves opening of its SMC hinge

Cohesin is a multisubunit complex that mediates sister-chromatid cohesion. Its Smc1 and Smc3 subunits possess ABC-like ATPases at one end of 50 nm long coiled coils. At the other ends are pseudosymmetrical hinge domains that interact to create V-shaped Smc1/Smc3 heterodimers. N- and C-terminal domains within cohesin's kleisin subunit Scc1 bind to Smc3 and Smc1 ATPase heads respectively, thereby creating a huge tripartite ring. It has been suggested that cohesin associates with chromosomes by trapping DNA within its ring. Opening of the ring due to cleavage of Scc1 by separase destroys sister-chromatid cohesion and triggers anaphase. We show that cohesin's hinges are not merely dimerization domains. They are essential for cohesin's association with chromosomes, which is blocked by artificially holding hinge domains together but not by preventing Scc1's dissociation from SMC ATPase heads. Our results suggest that entry of DNA into cohesin's ring requires transient dissociation of Smc1 and Smc3 hinge domains.     

Distinct developmental function of two Caenorhabditis elegans homologs of the cohesin subunit Scc1/Rad21

Cohesin, which mediates sister chromatid cohesion, is composed of four subunits, named Scc1/Rad21, Scc3, Smc1, and Smc3 in yeast. Caenorhabditis elegans has a single homolog for each of Scc3, Smc1, and Smc3, but as many as four for Scc1/Rad21 (COH-1, SCC-1/COH-2, COH-3, and REC-8). Except for REC-8 required for meiosis, function of these C. elegans proteins remains largely unknown. Herein, we examined their possible involvement in mitosis and development. Embryos depleted of the homolog of either Scc3, or Smc1, or Smc3 by RNA interference revealed a defect in mitotic chromosome segregation but not in chromosome condensation and cytokinesis. Depletion of SCC-1/COH-2 caused similar phenotypes. SCC-1/COH-2 was present in cells destined to divide. It localized to chromosomes in a cell cycle-dependent manner. Worms depleted of COH-1 arrested at either the late embryonic or the larval stage, with no indication of mitotic dysfunction. COH-1 associated chromosomes throughout the cell cycle in all somatic cells undergoing late embryogenesis or larval development. Thus, SCC-1/COH-2 and the homologs of Scc3, Smc1, and Smc3 facilitate mitotic chromosome segregation during the development, presumably by forming a cohesin complex, whereas COH-1 seems to play a role important for development but unrelated to mitosis.     

ATP hydrolysis is required for cohesin's association with chromosomes

BACKGROUND: A multi-subunit protein complex called cohesin is involved in holding sister chromatids together after DNA replication. Cohesin contains four core subunits: Smc1, Smc3, Scc1, and Scc3. Biochemical studies suggest that Smc1 and Smc3 each form 50 nm-long antiparallel coiled coils (arms) and bind to each other to form V-shaped heterodimers with globular ABC-like ATPases (created by the juxtaposition of N- and C-terminal domains) at their apices. These Smc "heads" are connected by Scc1, creating a tripartite proteinaceous ring. RESULTS: To investigate the role of Smc1 and Smc3's ATPase domains, we engineered smc1 and smc3 mutations predicted to abolish either ATP binding or hydrolysis. All mutations abolished Smc protein function. The binding of ATP to Smc1, but not Smc3, was essential for Scc1's association with Smc1/3 heterodimers. In contrast, mutations predicted to prevent hydrolysis of ATP bound to either head abolished cohesin's association with chromatin but not Scc1's ability to connect Smc1's head with that of Smc3. Inactivation of the Scc2/4 complex had a similar if not identical effect; namely, the production of tripartite cohesin rings that cannot associate with chromosomes. CONCLUSIONS: Cohesin complexes whose heads have been connected by Scc1 must hydrolyze ATP in order to associate stably with chromosomes. If chromosomal association is mediated by the topological entrapment of DNA inside cohesin's ring, then ATP hydrolysis may be responsible for creating a gate through which DNA can enter. We suggest that ATP hydrolysis drives the temporary disconnection of Scc1 from Smc heads that are needed for DNA entrapment and that this process is promoted by Scc2/4.     

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.     

In vivo analysis of cohesin architecture using FRET in the budding yeast Saccharomyces cerevisiae

Cohesion between sister chromatids in eukaryotes is mediated by the evolutionarily conserved cohesin complex. Cohesin forms a proteinaceous ring, large enough to trap pairs of replicated sister chromatids. The circumference consists of the Smc1 and Smc3 subunits, while Scc1 is thought to close the ring by bridging the Smc (structural maintenance of chromosomes) ATPase head domains. Little is known about two additional subunits, Scc3 and Pds5, and about possible conformational changes of the complex during the cell cycle. We have employed fluorescence resonance energy transfer (FRET) to analyse interactions within the cohesin complex in live budding yeast. These experiments reveal an unexpected geometry of Scc1 at the Smc heads, and suggest that Pds5 plays a role at the Smc hinge on the opposite side of the ring. Key subunit interactions, including close proximity of the two ATPase heads, are constitutive throughout the cell cycle. This depicts cohesin as a stable molecular machine undergoing only transient conformational changes during binding and dissociation from chromosomes. Using FRET, we did not observe interactions between more than one cohesin complex in vivo.     

The cohesin complex: sequence homologies, interaction networks and shared motifs

Background  

Cohesin is a macromolecular complex that links sister chromatids together at the metaphase plate during mitosis. The links are formed during DNA replication and destroyed during the metaphase-to-anaphase transition. In budding yeast, the 14S cohesin complex comprises at least two classes of SMC (structural maintenance of chromosomes) proteins - Smc1 and Smc3 - and two SCC (sister-chromatid cohesion) proteins - Scc1 and Scc3. The exact function of these proteins is unknown.     

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.     

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.