Communication between distinct subunit interfaces of the cohesin complex promotes its topological entrapment of DNA.

Cohesin mediates higher order chromosome structure. Its biological activities require topological entrapment of DNA within a lumen(s) formed by cohesin subunits. The reversible dissociation of cohesins Smc3p and Mcd1p subunits is postulated to form a regulated gate that allows DNA entry and exit into the lumen. We assessed gate-independent functions of this interface in yeast using a fusion protein that joins Smc3p to Mcd1p. We show that in vivo all the regulators of cohesin promote DNA binding of cohesin by mechanisms independent of opening this gate. Furthermore, we show that this interface has a gate-independent activity essential for cohesin to bind chromosomes. We propose that this interface regulates DNA entrapment by controlling the opening and closing of one or more distal interfaces formed by cohesin subunits, likely by inducing a conformation change in cohesin. Furthermore, cohesin regulators modulate the interface to control both DNA entrapment and cohesin functions after DNA binding

Chromosomal Addresses of the Cohesin Component Mcd1p

We identified the chromosomal addresses of a cohesin subunit, Mcd1p, in vivo by chromatin immunoprecipitation coupled with high resolution PCR-based chromosomal walking. The mapping of new Mcd1p-binding sites (cohesin-associated regions [CARs]) in single-copy sequences of several chromosomes establish their spacing (∼9 kb), their sequestration to intergenic regions, and their association with AT-rich sequences as general genomic properties of CARs. We show that cohesins are not excluded from telomere proximal regions, and the enrichment of cohesins at the centromere at mitosis reflects de novo loading. The average size of a CAR is 0.8–1.0 kb. They lie at the boundaries of transcriptionally silenced regions, suggesting they play a direct role in defining the silent chromatin domain. Finally, we identify CARs in tandem (rDNA) and interspersed repetitive DNA (Ty2 and subtelomeric repeats). Each 9-kb rDNA repeat has a single CAR proximal to the 5S gene. Thus, the periodicity of CARs in single-copy regions and the rDNA repeats is conserved. The presence and spacing of CARs in repetitive DNA has important implications for genomic stability and chromosome packaging/condensation

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.National Institutes of Health (U.S.) (Grant R01GM092813

Recombination protein Tid1p controls resolution of cohesin-dependent linkages in meiosis in Saccharomyces cerevisiae

Sister chromatid cohesion and interhomologue recombination are coordinated to promote the segregation of homologous chromosomes instead of sister chromatids at the first meiotic division. During meiotic prophase in Saccharomyces cerevisiae, the meiosis-specific cohesin Rec8p localizes along chromosome axes and mediates most of the cohesion. The mitotic cohesin Mcd1p/Scc1p localizes to discrete spots along chromosome arms, and its function is not clear. In cells lacking Tid1p, which is a member of the SWI2/SNF2 family of helicase-like proteins that are involved in chromatin remodeling, Mcd1p and Rec8p persist abnormally through both meiotic divisions, and chromosome segregation fails in the majority of cells. Genetic results indicate that the primary defect in these cells is a failure to resolve Mcd1p-mediated connections. Tid1p interacts with recombination enzymes Dmc1p and Rad51p and has an established role in recombination repair. We propose that Tid1p remodels Mcd1p-mediated cohesion early in meiotic prophase to facilitate interhomologue recombination and the subsequent segregation of homologous chromosomes

Pds5p regulates the maintenance of sister chromatid cohesion and is sumoylated to promote the dissolution of cohesion

Pds5p and the cohesin complex are required for sister chromatid cohesion and localize to the same chromosomal loci over the same cell cycle window. However, Pds5p and the cohesin complex likely have distinct roles in cohesion. We report that pds5 mutants establish cohesion, but during mitosis exhibit precocious sister dissociation. Thus, unlike the cohesin complex, which is required for cohesion establishment and maintenance, Pds5p is required only for maintenance. We identified SMT4, which encodes a SUMO isopeptidase, as a high copy suppressor of both the temperature sensitivity and precocious sister dissociation of pds5 mutants. In contrast, SMT4 does not suppress temperature sensitivity of cohesin complex mutants. Pds5p is SUMO conjugated, with sumoylation peaking during mitosis. SMT4 overexpression reduces Pds5p sumoylation, whereas smt4 mutants have increased Pds5p sumoylation. smt4 mutants were previously shown to be defective in cohesion maintenance during mitosis. These data provide the first link between a protein required for cohesion, Pds5p, and sumoylation, and suggest that Pds5p sumoylation promotes the dissolution of cohesion

PIASγ Is Required for Faithful Chromosome Segregation in Human Cells

BACKGROUND: The precision of the metaphase-anaphase transition ensures stable genetic inheritance. The spindle checkpoint blocks anaphase onset until the last chromosome biorients at metaphase plate, then the bonds between sister chromatids are removed and disjoined chromatids segregate to the spindle poles. But, how sister separation is triggered is not fully understood. PRINCIPAL FINDINGS: We identify PIASγ as a human E3 sumo ligase required for timely and efficient sister chromatid separation. In cells lacking PIASγ, normal metaphase plates form, but the spindle checkpoint is activated, leading to a prolonged metaphase block. Sister chromatids remain cohered even if cohesin is removed by depletion of hSgo1, because DNA catenations persist at centromeres. PIASγ-depleted cells cannot properly localize Topoisomerase II at centromeres or in the cores of mitotic chromosomes, providing a functional link between PIASγ and Topoisomerase II. CONCLUSIONS: PIASγ directs Topoisomerase II to specific chromosome regions that require efficient removal of DNA catenations prior to anaphase. The lack of this activity activates the spindle checkpoint, protecting cells from non-disjunction. Because DNA catenations persist without PIASγ in the absence of cohesin, removal of catenations and cohesin rings must be regulated in parallel