Cell cycle-specific cleavage of Scc2 regulates its cohesin deposition activity.

Sister chromatid cohesion (SCC), efficient DNA repair, and the regulation of some metazoan genes require the association of cohesins with chromosomes. Cohesins are deposited by a conserved heterodimeric loading complex composed of the Scc2 and Scc4 proteins in Saccharomyces cerevisiae, but how the Scc2/Scc4 deposition complex regulates the spatiotemporal association of cohesin with chromosomes is not understood. We examined Scc2 chromatin association during the cell division cycle and found that the affinity of Scc2 for chromatin increases biphasically during the cell cycle, increasing first transiently in late G1 phase and then again later in G2/M. Inactivation of Scc2 following DNA replication reduces cellular viability, suggesting that this post S-phase increase in Scc2 chromatin binding affinity is biologically relevant. Interestingly, high and low Scc2 chromatin binding levels correlate strongly with the presence of full-length or amino-terminally cleaved forms of Scc2, respectively, and the appearance of the cleaved Scc2 species is promoted in vitro either by treatment with specific cell cycle-staged cellular extracts or by dephosphorylation. Importantly, Scc2 cleavage eliminates Scc2-Scc4 physical interactions, and an scc2 truncation mutant that mimics in vivo Scc2 cleavage is defective for cohesin deposition. These observations suggest a previously unidentified mechanism for the spatiotemporal regulation of cohesin association with chromosomes through cell cycle regulation of Scc2 cohesin deposition activity by Scc2 dephosphorylation and cleavage.

The Scc2/Scc4 cohesin loader determines the distribution of cohesin on budding yeast chromosomes.

Cohesins mediate sister chromatid cohesion and DNA repair and also function in gene regulation. Chromosomal cohesins are distributed nonrandomly, and their deposition requires the heterodimeric Scc2/Scc4 loader. Whether Scc2/Scc4 establishes nonrandom cohesin distributions on chromosomes is poorly characterized, however. To better understand the spatial regulation of cohesin association, we mapped budding yeast Scc2 and Scc4 chromosomal distributions. We find that Scc2/Scc4 resides at previously mapped cohesin-associated regions (CARs) in pericentromeric and arm regions, and that Scc2/Scc4-cohesin colocalization persists after the initial deposition of cohesins in G1/S phase. Pericentromeric Scc2/Scc4 enrichment is kinetochore-dependent, and both Scc2/Scc4 and cohesin associations are coordinately reduced in these regions following chromosome biorientation. Thus, these characteristics of Scc2/Scc4 binding closely recapitulate those of cohesin. Although present in G1, Scc2/Scc4 initially has a poor affinity for CARs, but its affinity increases as cells traverse the cell cycle. Scc2/Scc4 association with CARs is independent of cohesin, however. Taken together, these observations are inconsistent with a previous suggestion that cohesins are relocated by translocating RNA polymerases from separate loading sites to intergenic regions between convergently transcribed genes. Rather, our findings suggest that budding yeast cohesins are targeted to CARs largely by Scc2/Scc4 loader association at these locations.

Clinical, developmental and molecular update on Cornelia de Lange syndrome and the cohesin complex: abstracts from the 2014 Scientific and Educational Symposium.

Cornelia de Lange Syndrome (CdLS) is the most common example of disorders of the cohesin complex, or cohesinopathies. There are a myriad of clinical issues facing individuals with CdLS, particularly in the neurodevelopmental system, which also have implications for the parents and caretakers, involved professionals, therapists, and schools. Basic research in developmental and cell biology on cohesin is showing significant progress, with improved understanding of the mechanisms and the possibility of potential therapeutics. The following abstracts are presentations from the 6th Cornelia de Lange Syndrome Scientific and Educational Symposium, which took place on June 25-26, 2014, in conjunction with the Cornelia de Lange Syndrome Foundation National Meeting in Costa Mesa, CA. The Research Committee of the CdLS Foundation organizes the meeting, reviews and accepts abstracts, and subsequently disseminates the information to the families through members of the Clinical Advisory Board. In addition to the scientific and clinical discussions, there were educationally focused talks related to practical aspects of behavior and development. AMA CME credits were provided by Greater Baltimore Medical Center, Baltimore, MD.

Foundational and Clinical Science Integration in a Team-Based Learning Module Modeling Care of a Patient With Dyslipidemia.

Introduction: Foundational and clinical science integration, a long-standing goal of undergraduate medical education, benefits learners by promoting retention of critical knowledge and skills as well as their transfer to the clinical setting. We implemented a team-based learning (TBL) module in which foundational knowledge and skills from the disciplines of biochemistry, nutrition, and genetics were leveraged in a simulated patient encounter for diagnosis and management of a patient with dyslipidemia. Methods: The TBL was deployed in a first-year medical student cardiovascular system course with 125 students over three academic years. Following individual and team readiness assurance tests (iRAT and tRAT, respectively), teams participated in an initial application exercise requiring consideration of clinical and laboratory data and other risk factors to engage the patient in a shared decision-making process. Using dietary and family history narratives in subsequent application exercises, teams completed recommendations for an individualized diet plan and an assessment of potential disease inheritance patterns to formulate appropriate patient care management strategies. Results: Student engagement with prelearning materials and session team activities was high as judged by RAT performance and application exercise outcomes: iRAT question performance ranged from 89% to 99% for individual items, and tRAT performance was routinely 100%. Learners reported that the exercises were impactful and believed the learned foundational knowledge and skills were transferable to future patient care. Discussion: The dyslipidemia TBL module provides an illustration for early clinical learners of how foundational knowledge and skills can be operationalized and transferred for optimal patient care.

The kinetochore is an enhancer of pericentric cohesin binding.

The recruitment of cohesins to pericentric chromatin in some organisms appears to require heterochromatin associated with repetitive DNA. However, neocentromeres and budding yeast centromeres lack flanking repetitive DNA, indicating that cohesin recruitment occurs through an alternative pathway. Here, we demonstrate that all budding yeast chromosomes assemble cohesin domains that extend over 20-50 kb of unique pericentric sequences flanking the conserved 120-bp centromeric DNA. The assembly of these cohesin domains requires the presence of a functional kinetochore in every cell cycle. A similar enhancement of cohesin binding was also observed in regions flanking an ectopic centromere. At both endogenous and ectopic locations, the centromeric enhancer amplified the inherent levels of cohesin binding that are unique to each region. Thus, kinetochores are enhancers of cohesin association that act over tens of kilobases to assemble pericentric cohesin domains. These domains are larger than the pericentric regions stretched by microtubule attachments, and thus are likely to counter microtubule-dependent forces. Kinetochores mediate two essential segregation functions: chromosome movement through microtubule attachment and biorientation of sister chromatids through the recruitment of high levels of cohesin to pericentric regions. We suggest that the coordination of chromosome movement and biorientation makes the kinetochore an autonomous segregation unit.

Genome-wide mapping of the cohesin complex in the yeast Saccharomyces cerevisiae.

In eukaryotic cells, cohesin holds sister chromatids together until they separate into daughter cells during mitosis. We have used chromatin immunoprecipitation coupled with microarray analysis (ChIP chip) to produce a genome-wide description of cohesin binding to meiotic and mitotic chromosomes of Saccharomyces cerevisiae. A computer program, PeakFinder, enables flexible, automated identification and annotation of cohesin binding peaks in ChIP chip data. Cohesin sites are highly conserved in meiosis and mitosis, suggesting that chromosomes share a common underlying structure during different developmental programs. These sites occur with a semiperiodic spacing of 11 kb that correlates with AT content. The number of sites correlates with chromosome size; however, binding to neighboring sites does not appear to be cooperative. We observed a very strong correlation between cohesin sites and regions between convergent transcription units. The apparent incompatibility between transcription and cohesin binding exists in both meiosis and mitosis. Further experiments reveal that transcript elongation into a cohesin-binding site removes cohesin. A negative correlation between cohesin sites and meiotic recombination sites suggests meiotic exchange is sensitive to the chromosome structure provided by cohesin. The genome-wide view of mitotic and meiotic cohesin binding provides an important framework for the exploration of cohesins and cohesion in other genomes.

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.

Gene-specific RNA polymerase II phosphorylation and the CTD code.

Phosphorylation of the RNA polymerase (Pol) II C-terminal domain (CTD) repeats (1-YSPTSPS-7) is coupled to transcription and may act as a 'code' that controls mRNA synthesis and processing. To examine the code in budding yeast, we mapped genome-wide CTD Ser2, Ser5 and Ser7 phosphorylations and the CTD-associated termination factors Nrd1 and Pcf11. Phospho-CTD dynamics are not scaled to gene length and are gene-specific, with highest Ser5 and Ser7 phosphorylation at the 5' ends of well-expressed genes with nucleosome-occupied promoters. The CTD kinases Kin28 and Ctk1 markedly affect Pol II distribution in a gene-specific way. The code is therefore written differently on different genes, probably under the control of promoters. Ser7 phosphorylation is enriched on introns and at sites of Nrd1 accumulation, suggesting links to splicing and Nrd1 recruitment. Nrd1 and Pcf11 frequently colocalize, suggesting functional overlap. Unexpectedly, Pcf11 is enriched at centromeres and Pol III-transcribed genes.

The enhancement of pericentromeric cohesin association by conserved kinetochore components promotes high-fidelity chromosome segregation and is sensitive to microtubule-based tension.

Sister chromatid cohesion, conferred by the evolutionarily conserved cohesin complex, is essential for proper chromosome segregation. Cohesin binds to discrete sites along chromosome arms, and is especially enriched surrounding centromeres, but past studies have not clearly defined the roles of arm and pericentromeric cohesion in chromosome segregation. To address this issue, we developed a technique that specifically reduced pericentromeric cohesin association on a single chromosome without affecting arm cohesin binding. Under these conditions, we observed more extensive stretching of centromeric chromatin and elevated frequencies of chromosome loss, suggesting that pericentromeric cohesin enrichment is essential for high-fidelity chromosome transmission. The magnitude of pericentromeric cohesin association was negatively correlated with tension between sister kinetochores, with the highest levels of association in cells lacking kinetochore-microtubule attachments. Pericentromeric cohesin recruitment required evolutionarily conserved components of the inner and central kinetochore. Together, these observations suggest that pericentromeric cohesin levels reflect the balance of opposing forces: the kinetochore-mediated enhancement of cohesin binding and the disruption of binding by mechanical tension at kinetochores. The involvement of conserved kinetochore components suggests that this pathway for pericentromeric cohesin enrichment may have been retained in higher eukaryotes to promote chromosome biorientation and accurate sister chromatid segregation.

The core centromere and Sgo1 establish a 50-kb cohesin-protected domain around centromeres during meiosis I.

The stepwise loss of cohesins, the complexes that hold sister chromatids together, is required for faithful meiotic chromosome segregation. Cohesins are removed from chromosome arms during meiosis I but are maintained around centromeres until meiosis II. Here we show that Sgo1, a protein required for protecting centromeric cohesins from removal during meiosis I, localizes to cohesin-associated regions (CARs) at the centromere and the 50-kb region surrounding it. Establishment of this Sgo1-binding domain requires the 120-base-pair (bp) core centromere, the kinetochore component Bub1, and the meiosis-specific factor Spo13. Interestingly, cohesins and the kinetochore proteins Iml3 and Chl4 are necessary for Sgo1 to associate with pericentric regions but less so for Sgo1 to associate with the core centromeric regions. Finally, we show that the 50-kb Sgo1-binding domain is the chromosomal region where cohesins are protected from removal during meiosis I. Our results identify the portions of chromosomes where cohesins are protected from removal during meiosis I and show that kinetochore components and cohesins themselves are required to establish this cohesin protective domain.

Acetylation of histone H4 by Esa1 is required for DNA double-strand break repair.

Although the acetylation of histones has a well-documented regulatory role in transcription, its role in other chromosomal functions remains largely unexplored. Here we show that distinct patterns of histone H4 acetylation are essential in two separate pathways of double-strand break repair. A budding yeast strain with mutations in wild-type H4 acetylation sites shows defects in nonhomologous end joining repair and in a newly described pathway of replication-coupled repair. Both pathways require the ESA1 histone acetyl transferase (HAT), which is responsible for acetylating all H4 tail lysines, including ectopic lysines that restore repair capacity to a mutant H4 tail. Arp4, a protein that binds histone H4 tails and is part of the Esa1-containing NuA4 HAT complex, is recruited specifically to DNA double-strand breaks that are generated in vivo. The purified Esa1-Arp4 HAT complex acetylates linear nucleosomal arrays with far greater efficiency than circular arrays in vitro, indicating that it preferentially acetylates nucleosomes near a break site. Together, our data show that histone tail acetylation is required directly for DNA repair and suggest that a related human HAT complex may function similarly.