Cohesin dysfunction results in cell wall defects in budding yeast

Cohesin is a conserved chromatin-binding multisubunit protein complex involved in diverse chromosomal transactions such as sister-chromatid cohesion, chromosome condensation, regulation of gene expression, DNA replication, and repair. While working with a budding yeast temperature-sensitive mutant, mcd1-1, defective in a cohesin subunit, we observed that it was resistant to zymolyase, indicating an altered cell wall organization. The budding yeast cell wall is a strong but elastic structure essential for maintenance of cell shape and protection from extreme environmental challenges. Here, we show that the cohesin complex plays an important role in cell wall maintenance. Cohesin mutants showed high chitin content in the cell wall and sensitivity to multiple cell wall stress-inducing agents. Interestingly, temperature-dependent lethality of cohesin mutants was osmoremedial, in a HOG1-MAPK pathway-dependent manner, suggesting that the temperature sensitivity of these mutants may arise partially from cell wall defects. Moreover, Mpk1 hyper-phosphorylation indicated activation of the cell wall integrity (CWI) signaling pathway in cohesin mutants. Genetic interaction analysis revealed that the CWI pathway is essential for survival of mcd1-1 upon additional cell wall stress. The cell wall defect was independent of the cohesion function and accompanied by misregulation of expression of several genes having cell wall-related functions. Our findings reveal a requirement of cohesin in maintenance of CWI that is independent of the CWI pathway, and that may arise from cohesin’s role in regulating the expression of multiple genes encoding proteins involved in cell wall organization and biosynthesis.     

Cohesin acetylation and Wapl‐Pds5 oppositely regulate translocation of cohesin along DNA

Cohesin is a ring‐shaped protein complex that plays a crucial role in sister chromatid cohesion and gene expression. The dynamic association of cohesin with chromatin is essential for these functions. However, the exact nature of cohesin dynamics, particularly cohesin translocation, remains unclear. We evaluated the dynamics of individual cohesin molecules on DNA and found that the cohesin core complex possesses an intrinsic ability to traverse DNA in an adenosine triphosphatase (ATPase)‐dependent manner. Translocation ability is suppressed in the presence of Wapl‐Pds5 and Sororin; this suppression is alleviated by the acetylation of cohesin and the action of mitotic kinases. In Xenopus laevis egg extracts, cohesin is translocated on unreplicated DNA in an ATPase‐ and Smc3 acetylation‐dependent manner. Cohesin movement changes from bidirectional to unidirectional when cohesin faces DNA replication; otherwise, it is incorporated into replicating DNA without being translocated or is dissociated from replicating DNA. This study provides insight into the nature of individual cohesin dynamics and the mechanisms by which cohesin achieves cohesion in different chromatin contexts.     

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.     

Cut1/separase-dependent roles of multiple phosphorylation of fission yeast cohesin subunit Rad21 in post-replicative damage repair and mitosis

Cohesin is a multiprotein complex essential for sister-chromatid cohesion. It plays a pivotal role in proper chromosome segregation and DNA damage repair. The mitotic behavior of cohesin is controlled through its phosphorylation, which possibly induces the dissociation of cohesin from chromosomes and enhances its susceptibility to separase. Here, we report using mass spectrometry and anti-phospho antibodies that the central domain of Rad21, the separase-target subunit of Schizosaccharomyces pombe cohesin, is regulated by various kinase-induced phosphorylation at nine residues, indicating the multiple roles for S. pombe cohesin. In vegetative and non-dividing G0 cells, Rad21 is phosphorylated by unknown S/TP-consensus kinases, in mitotic and non-mitotic cells by polo/Plo1 and CDK, and in DNA-damaged cells by Rad3/ATR. While mitotic phosphorylation is implicated in the dissociation of Rad21 and its cleavage by separase in anaphase, the Rad3/ATR-dependent damage-induced phosphorylation occurs intensively at the time of repair completion, and only in post-replicative cells. This damage-induced Rad21 phosphorylation is involved in the recovery process of cells from checkpoint arrest, and needed for the removal of cohesin by separase after the completion of damage repair. These complex phospho-regulations of Rad21 indicate the functional significance of cohesin in cell adaptation to a variety of cellular conditions.     

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。     

Identification of cohesin association sites at centromeres and along chromosome arms.

A multisubunit cohesin complex holds sister chromatids together after DNA replication. Using chromatin immunoprecipitation, we detected cohesin association with centromeres and with discrete sites along chromosome arms from S phase until metaphase in S. cerevisiae. Short DNA sequences (130-280 bp) are sufficient to confer cohesin association. Cohesin association with a centromere depends on Mif2p, the centromere binding factor CBF3, and a centromere-specific histone variant, Cse4p. Because only active centromeres confer cohesin association with centromeric DNA, we suggest that cohesin is recruited by the same chromatin structure that confers the attachment of microtubules. Propagation of this structure might be partly epigenetic. Finally, cohesion associated with "minimal" centromeres is insufficient to resist the splitting force exerted by microtubules and appears to be reinforced by cohesion provided by their flanking DNA sequences.     

On the choreography of genome folding: A grand pas de deux of cohesin and CTCF

Cohesin and CTCF are key to the 3D folding of interphase chromosomes. Cohesin forms chromatin loops via loop extrusion, a process that involves the formation and enlargement of DNA loops. The architectural protein CTCF controls this process by acting as an anchor for chromatin looping. How CTCF controls cohesin has long been a mystery. Recent work shows that CTCF dictates chromatin looping via a direct interaction of its N-terminus with cohesin. CTCF's ability to regulate chromatin looping turns out to also be partially dependent on several RNA-binding domains. In this review, we discuss recent insights and consider how cohesin and CTCF together may orchestrate the folding of the genome into chromosomal loops.     

Pericentric chromatin is organized into an intramolecular loop in mitosis

BACKGROUND: Cohesin proteins link sister chromatids and provide the basis for tension between bioriented sister chomatids in mitosis. Cohesin is concentrated at the centromere region of the chromosome despite the fact that sister centromeres can be separated by 800 nm in vivo. The function of cohesin at sites of separated DNA is unknown. RESULTS: We provide evidence that the kinetochore promotes the organization of pericentric chromatin into a cruciform in mitosis such that centromere-flanking DNA adopts an intramolecular loop, whereas sister-chromatid arms are paired intermolecularly. Visualization of cohesin subunits by fluorescence microscopy revealed a cylindrical structure that encircles the central spindle and spans the distance between sister kinetochores. Kinetochore assembly at the apex of the loop initiates intrastrand loop formation that extends approximately 25 kb (12.5 kb on either side of the centromere). Two centromere loops (one from each sister chromatid) are stretched between the ends of sister-kinetochore microtubules along the spindle axis. At the base of the loop there is a transition to intermolecular sister-chromatid pairing. CONCLUSIONS: The C loop conformation reveals the structural basis for sister-kinetochore clustering in budding yeast and for kinetochore biorientation and thus resolves the paradox of maximal interstrand separation in regions of highest cohesin concentration.