Maintain Sister Chromatid Cohesion Research Articles

CRISPR screens in sister chromatid cohesion defective cells reveal PAXIP1-PAGR1 as regulator of chromatin association of cohesin.

The cohesin complex regulates higher order chromosome architecture through maintaining sister chromatid cohesion and folding chromatin by DNA loop extrusion. Impaired cohesin function underlies a heterogeneous group of genetic syndromes and is associated with cancer. Here, we mapped the genetic dependencies of human cell lines defective of cohesion regulators DDX11 and ESCO2. The obtained synthetic lethality networks are strongly enriched for genes involved in DNA replication and mitosis and support the existence of parallel sister chromatid cohesion establishment pathways. Among the hits, we identify the chromatin binding, BRCT-domain containing protein PAXIP1 as a novel cohesin regulator. Depletion of PAXIP1 severely aggravates cohesion defects in ESCO2 mutant cells, leading to mitotic cell death. PAXIP1 promotes global chromatin association of cohesin, independent of DNA replication, a function that cannot be explained by indirect effects of PAXIP1 on transcription or DNA repair. Cohesin regulation by PAXIP1 requires its binding partner PAGR1 and a conserved FDF motif in PAGR1. PAXIP1 co-localizes with cohesin on multiple genomic loci, including active gene promoters and enhancers. Possibly, this newly identified role of PAXIP1-PAGR1 in regulating cohesin occupancy on chromatin is also relevant for previously described functions of PAXIP1 in transcription, immune cell maturation and DNA repair.

DNA Repair in Space and Time: Safeguarding the Genome with the Cohesin Complex.

DNA double-strand breaks (DSBs) are a deleterious form of DNA damage, which must be robustly addressed to ensure genome stability. Defective repair can result in chromosome loss, point mutations, loss of heterozygosity or chromosomal rearrangements, which could lead to oncogenesis or cell death. We explore the requirements for the successful repair of DNA DSBs by non-homologous end joining and homology-directed repair (HDR) mechanisms in relation to genome folding and dynamics. On the occurrence of a DSB, local and global chromatin composition and dynamics, as well as 3D genome organization and break localization within the nuclear space, influence how repair proceeds. The cohesin complex is increasingly implicated as a key regulator of the genome, influencing chromatin composition and dynamics, and crucially genome organization through folding chromosomes by an active loop extrusion mechanism, and maintaining sister chromatid cohesion. Here, we consider how this complex is now emerging as a key player in the DNA damage response, influencing repair pathway choice and efficiency.

Dot1l Reverses the Abnormal Self-Renewal Conferred on HSPCs through Cohesin Haploinsufficiency By Diminishing Expression of Hoxa7 and Hoxa9

Acute myelogenous leukemia (AML) is a high-risk hematopoietic malignancy with a poor prognosis. Developing novel treatments for AML has been difficult due to the heterogeneity of genetic mutations observed across patients. Recent genomic sequencing of AML patients have identified recurrent somatic mutations in cohesin complex genes in a subset of patients (10-20%). The canonical function of the cohesin complex is to maintain sister chromatid cohesion permitting proper segregation of genomic information to each daughter cell in mitosis. Complete loss of cohesin function causes an early embryonic lethal phenotype in knock-out mice, implying that some level of cohesin function is absolutely required for normal development. Interestingly, somatic cohesin mutations in AML are uniformly heterozygous and not associated with dramatic changes in chromosomal number, implying that cohesin mutations promote AML through a function independent of their role during mitosis.Recent investigations have demonstrated that cohesin haploinsufficiency leads to enhanced self-renewal in Hematopoietic Stem and Progenitor Cells (HSPCs). Previous studies from our lab identified significant increases in the HSPC self-renewal factors Hoxa7 and Hoxa9 in Rad21 -depleted HSPCs concomitant with decreased levels of the repressive histone mark trimethylated lysine 27 on histone H3 (H3K27me3) at the Hoxa7 and Hoxa9 promoters, indicating that Rad21 plays a role in proper PRC2-mediated silencing of these genes. Immunoprecipitation and ChIP experiments revealed that cohesin and PRC2 directly interact and are bound in close proximity to Hoxa7 and Hoxa9 . Interestingly genome-wide levels of PRC2's repressive histone mark H3K27me3 were reduced. Importantly, H3K27me3 levels were significantly decreased at the Hoxa7 / Hoxa9 locus. Knockdown of either Hoxa7 or Hoxa9 in Rad21 -depleted HSPCs suppressed their self-renewal, implying both are critical downstream effectors of reduced cohesin levels.In the present study we sought to delineate additional molecular mechanisms by which cohesin mutations promote enhanced HSPC self-renewal and evaluate possible therapeutic interventions to reverse the phenotype conferred on HSPCs by cohesin haploinsufficiency. We find that reduction of the core cohesin subunit Rad21 using RNAi increases levels of dimethylated lysine 79 on histone H3 (H3K79me2) genome-wide in contrast to the genome-wide reduction of H3K27me3 levels. H3K79me2 is deposited by the Dot1l chromatin modifying complex and is correlated with active gene expression. Given the established role of Dot1l in regulating Hoxa9 expression, we sought to evaluate whether inhibiting Dot1l methyltransferase activity would reverse the phenotype caused by cohesin haploinsufficiency. As anticipated, we observed that inhibition of Dot1l function, using either of two commercially available inhibitors or RNAi mediated knockdown, reverses the enhanced self-renewal capacity of cohesin haploinsufficient HSPCs. Consistent with our proposed mechanism, this reduced self-renewal occurs with a concomitant reduction in Hoxa7 and Hoxa9 expression. We observe a similar trend using HSPCs from Smc3 conditional knockout mice, a more elegant genetic model. RNA-sequencing and ChIP-sequencing studies are ongoing to evaluate the transcriptome and epigenomic changes imparted by cohesin haploinsufficiency and how these changes are affected by Dot1l inhibition. Our preliminary studies are intriguing because cohesin haploinsufficiency appears to have contrasting effects on genome-wide levels H3K27me3 and H3K79me2, two opposing epigenetic marks. In conclusion, our data demonstrate that Dot1l inhibition negatively regulates self-renewal of cohesin haploinsufficient HSPCs, but not control HSPCs, by abbrogating the abberant expression of Hoxa7 and Hoxa9 . This study establishes feasibility for the use of Dot1l inhibition as a targeted therapeutic treatment for AML patients harboring cohesin mutations. DisclosuresLevine:Qiagen: Equity Ownership; Roche: Research Funding; Celgene: Research Funding; Roche: Research Funding; Qiagen: Equity Ownership; Celgene: Research Funding.

Cohesin Is Out for Stalled Replication Fork Restart.

Replication stress underlies many genomic alterations in cancer cells. In this issue of Developmental Cell, Benedict etal. show that WAPL-dependent cohesin removal is needed to restart DNA synthesis at stalled forks and promote survival following replication stress, uncovering an unexpected link between stress and sister chromatid cohesion loss.

STAG2 Loss Gives Rise to Therapeutically Targetable DNA Damage Repair Defects and Altered Replication Fork Dynamics in Acute Myeloid Leukaemia

Acute Myeloid Leukaemia (AML) is a genetically heterogeneous disease of the blood and bone marrow primarily affecting older populations. Chemotherapy has been the standard of care for four decades, just 10% of elderly patients show a response and it will be curative in only a third of patients aged 18-60. Therefore, it's necessary to identify new therapeutic targets for improved treatment options. The search for novel mutations within the genomes of karyotypically normal AML patients identified Cohesin's STAG2 as a recurrently mutated gene (6%). Cohesin is a ring-shaped multimeric protein complex which has a multitude of roles including maintaining sister chromatid cohesion, DNA replication and repair, gene expression and chromatin architecture. STAG2 remains the most understudied Cohesin gene, how a mutation affects the ability of Cohesin to fulfil certain roles needs elucidation as well as how STAG2 functions as part of Cohesin to repair DNA breaks, promote faithful replication and control gene expression. Therefore, we sought to explore the consequences of loss-of-function STAG2 mutations on DNA repair and replication using a STAG2 knockout (ΔSTAG2) isogenic cell line model created using CRISPR/Cas9. Immunofluorescent staining of DNA damage repair proteins 53BP1 and RAD51 following 2Gy irradiation (IR) suggests ΔSTAG2 cells have a significant DNA repair defect. The average 53BP1 and RAD51 foci counts were significantly higher (p=0.001) in mutant cells which had three times as many foci at 24 hours after IR. In addition, 40% of ΔSTAG2 cells stain positive for 53BP1 and RAD51 respectively at 24 hours post-IR. This damage persists at 48 and 72 hours, whereas wild type (WT) damage returns to basal levels. Given that our data suggests a HR defect, we assessed the response of WT and ΔSTAG2 cells to PARP inhibitors (PARPi). Interestingly, ΔSTAG2 cells showed significantly greater sensitivity than WT cells to highly potent PARPi Talazoparib with an IC50 of 62nM. We next assayed single DNA fibres to evaluate the implications of a STAG2 mutation on the dynamics of individual replication forks. Interestingly, we observed a significantly reduced number of ongoing replication forks compared to WT cells (p=0.01) with a concomitant increase in the number of stalled forks (p=0.01) in the absence of exogeneous DNA damage. Following treatment with hydroxyurea (HU) this phenotype was exacerbated and ΔSTAG2 cells exhibit significantly more stalled replication forks than WTs (p=0.001). This indicates a role for STAG2 in maintaining replication fork stability and promoting fork restart following replication stress. Importantly, our data identifies a potential DNA damage repair deficiency that sensitises ΔSTAG2 cells to PARPi monotherapy as well as unearthing an essential role for STAG2 in replication fork progression and fork stability. This mutation could therefore be exploited therapeutically and represents a novel therapeutic target/approach for AML patients with mutated STAG2. Disclosures No relevant conflicts of interest to declare.

Maize Dek15 Encodes the Cohesin-Loading Complex Subunit SCC4 and Is Essential for Chromosome Segregation and Kernel Development.

Cohesin complexes maintain sister chromatid cohesion to ensure proper chromosome segregation during mitosis and meiosis. In plants, the exact components and functions of the cohesin complex remain poorly understood. Here, we positionally cloned the classic maize (Zea mays) mutant defective kernel 15 (dek15), revealing that it encodes a homolog of SISTER CHROMATID COHESION PROTEIN 4 (SCC4), a loader subunit of the cohesin ring. Developing dek15 kernels contained fewer cells than the wild type, but had a highly variable cell size. The dek15 mutation was found to disrupt the mitotic cell cycle and endoreduplication, resulting in a reduced endosperm and embryo lethality. The cells in the dek15 endosperm and embryo exhibited precocious sister chromatid separation and other chromosome segregation errors, including misaligned chromosomes, lagging chromosomes, and micronuclei, resulting in a high percentage of aneuploid cells. The loss of Dek15/Scc4 function upregulated the expression of genes involved in cell cycle progression and stress responses, and downregulated key genes involved in organic synthesis during maize endosperm development. Our yeast two-hybrid screen identified the chromatin remodeling proteins chromatin remodeling factor 4, chromatin remodeling complex subunit B (CHB)102, CHB105, and CHB106 as SCC4-interacting proteins, suggesting a possible mechanism by which the cohesin ring is loaded onto chromatin in plant cells. This study revealed biological functions for DEK15/SCC4 in mitotic chromosome segregation and kernel development in maize.

Research progress of cohesin complex mutation in acute myeloid leukemia

Acute myeloid leukemia (AML) is a heterogenous hematopoietic malignancy that arises from clonal expansion and abnormal differentiation of myeloid precursor cells. Recently, recurrent mutation of cohesin complex has been discovered in AML based on next generation sequencing. Cohesin complex mainly functions as maintaining sister chromatid cohesion during mitosis, which also plays a prominent role in gene expression. Cohesin mutations confer both enhanced self-renewal and altered differtiation to hematopoietic stem and progenitor cells. Of note, cohesin mutations are associated with Down syndrome-associated acute megakaryocytic leukemia (DS-AMKL). Cohesin mutation provides potential target for clinical AML treatment. In this paper, the pathogenesis and progress of cohesin mutation in AML are reviewed. Key words: Leukemia, myeloid, acute; Mutation; Cohesin; Down syndrome-associated acute megakaryocytic leukemia (DS-AMKL)

Connecting GCN5's centromeric SAGA to the mitotic tension-sensing checkpoint.

Multiple interdependent mechanisms ensure faithful segregation of chromosomes during cell division. Among these, the spindle assembly checkpoint monitors attachment of spindle microtubules to the centromere of each chromosome, whereas the tension-sensing checkpoint monitors the opposing forces between sister chromatid centromeres for proper biorientation. We report here a new function for the deeply conserved Gcn5 acetyltransferase in the centromeric localization of Rts1, a key player in the tension-sensing checkpoint. Rts1 is a regulatory component of protein phopshatase 2A, a near universal phosphatase complex, which is recruited to centromeres by the Shugoshin (Sgo) checkpoint component under low-tension conditions to maintain sister chromatid cohesion. We report that loss of Gcn5 disrupts centromeric localization of Rts1. Increased RTS1 dosage robustly suppresses gcn5∆ cell cycle and chromosome segregation defects, including restoration of Rts1 to centromeres. Sgo1’s Rts1-binding function also plays a key role in RTS1 dosage suppression of gcn5∆ phenotypes. Notably, we have identified residues of the centromere histone H3 variant Cse4 that function in these chromosome segregation-related roles of RTS1. Together, these findings expand the understanding of the mechanistic roles of Gcn5 and Cse4 in chromosome segregation.

Hsp90 Is Essential for Chl1-Mediated Chromosome Segregation and Sister Chromatid Cohesion.

Recent studies have demonstrated that aberrant sister chromatid cohesion causes genomic instability and hence is responsible for the development of a tumor. The Chl1 (chromosome loss 1) protein (homolog of human ChlRl/DDX11 helicase) plays an essential role in the proper segregation of chromosomes during mitosis. The helicase activity of Chl1 is critical for sister chromatid cohesion. Our study demonstrates that Hsp90 interacts with Chl1 and is necessary for its stability. We observe that the Hsp90 nonfunctional condition (temperature-sensitive iG170Dhsp82 strain at restrictive temperature) induces proteasomal degradation of Chl1. We have mapped the domains of Chl1 and identified that the presence of domains II, III, and IV is essential for efficient interaction with Hsp90. We have demonstrated that Hsp90 inhibitor 17-AAG (17-allylamino-geldenamycin) causes destabilization of Chl1 protein and enhances significant disruption of sister chromatid cohesion, which is comparable to that observed under the Δchl1 condition. Our study also revealed that 17-AAG treatment causes an increased frequency of chromosome loss to a similar extent as that of the Δchl1 cells. Hsp90 functional loss has been earlier linked to aneuploidy with very poor mechanistic insight. Our result identifies Chl1 as a novel client of Hsp90, which could be further explored to gain mechanistic insight into aneuploidy.IMPORTANCE Recently, Hsp90 functional loss has been linked to aneuploidy; however, until now none of the components of sister chromatid cohesion (SCC) have been demonstrated as the putative clients of Hsp90. In this study, we have established that Chl1, the protein which is involved in maintaining sister chromatid cohesion as well as in preventing chromosome loss, is a direct client of Hsp90. Thus, with understanding of the molecular mechanism, how Hsp90 controls the cohesion machinery might reveal new insights which can be exploited further for attenuation of tumorigenesis.

Phosphorylation of the synaptonemal complex protein SYP-1 promotes meiotic chromosome segregation

Chromosomes that have undergone crossing over in meiotic prophase must maintain sister chromatid cohesion somewhere along their length between the first and second meiotic divisions. Although many eukaryotes use the centromere as a site to maintain cohesion, the holocentric organism Caenorhabditis elegans instead creates two chromosome domains of unequal length termed the short arm and long arm, which become the first and second site of cohesion loss at meiosis I and II. The mechanisms that confer distinct functions to the short and long arm domains remain poorly understood. Here, we show that phosphorylation of the synaptonemal complex protein SYP-1 is required to create these domains. Once crossover sites are designated, phosphorylated SYP-1 and PLK-2 become cooperatively confined to short arms and guide phosphorylated histone H3 and the chromosomal passenger complex to the site of meiosis I cohesion loss. Our results show that PLK-2 and phosphorylated SYP-1 ensure creation of the short arm subdomain, promoting disjunction of chromosomes in meiosis I.

The Cohesin Subunit Rad21 Is a Negative Regulator of Hematopoietic Self-Renewal through Epigenetic Repression of HoxA7 and HoxA9

Abstract Acute myelogenous leukemia (AML) is a high-risk hematopoietic malignancy with a poor prognosis. Developing novel treatments for AML has been difficult due to the heterogeneity of genetic mutations observed across patients. Recent genomic sequencing of AML patients have identified recurrent somatic mutations in cohesin complex genes in a subset of patients (10-20%). The canonical function of the cohesin complex is to maintain sister chromatid cohesion permitting proper segregation of genomic information to each daughter cell in mitosis. Interestingly, somatic cohesin mutations in AML are uniformly heterozygous and not associated with dramatic changes in chromosomal number, implying that cohesin mutations promote AML through a function independent of their role during mitosis. Recent studies have demonstrated that haploinsufficiency of the cohesin complex leads to enhanced self-renewal in Hematopoeitic Stem and Progenitor Cells (HSPCs). We sought to delineate the molecular mechanisms by which cohesin mutations promote enhanced HSPC self-renewal since this represents an early step of oncogenic transformation. In the present study we confirmed previous studies showing that reduced cohesin levels elicit enhanced self-renewal of murine HPSCs in vitro in serial-replating assays. Gene arrays and RT-qPCR identified significant increases in the HSPC self-renewal factors Hoxa7 and Hoxa9. We confirmed the gene array data by RT-PCR and observed that Hoxa9 levels increase within 24 hours following Rad21 depletion. Consistent with these data, multiple approaches demonstrated a high correlation between Rad21 depletion and Hoxa9 target gene expression programs. We next sought to identify the mechanism that leads to this abberant expression of Hoxa9. GSEA identified derepression of Polycomb Repressive Complex 2 (PRC2) target genes in Rad21-depleted HSPCs. Given the well-described silencing of Hox clusters by the PRC2 complex, we examined if Rad21-depletion-induced HoxA9 gene activation could be mediated by epigenetic changes. Rad21 depletion resulted in decreased levels of H3K27me3 at the Hoxa7 and Hoxa9 promoters, indicating that Rad21 plays a role in proper PRC2 mediated silencing of these genes. Using immunoprecipitation experiments we further demonstrate that cohesin and PRC2 interact and are bound in close proximity to Hoxa7 and Hoxa9, arguing that Hoxa7 and Hoxa9 derepression is directly mediated by Rad21-targeting of PRC2. Consistent with this finding we observed reduced global levels of PRC2's repressive histone mark H3K27me3 arguing that PRC2 requires the cohesin complex for proper targeting. Importantly, knockdown of either Hoxa7 or Hoxa9 suppressed self-renewal, implying both are critical downstream effectors of reduced cohesin levels. Interestingly, inhibition of Dot1L function also reduced the enhanced self-renewal capacity elicited by cohesin depletion, consistent with the Hoxa locus being regulated by H3K79 methylation. Our data demonstrate that the cohesin complex regulates PRC2 targeting to silence Hoxa7 and Hoxa9 and negatively regulate self-renewal. Our studies identify a novel epigenetic mechanism underlying leukemogenesis and provide a possible therapeutic treatment for AML patients harboring cohesin mutations. Disclosures No relevant conflicts of interest to declare.

Mitotic cohesin subunit Mcd1 regulates the progression of meiotic recombination in budding yeast.

The cohesin complex holds sister chromatids together and prevents premature chromosome segregation until the onset of anaphase. Mcd1 (also known as Scc1), the α-kleisin subunit of cohesin, is a key regulatory subunit of the mitotic cohesin complex and is required for maintaining sister chromatid cohesion, chromosome organization, and DNA repair. We investigated the function of Mcd1 in meiosis by ectopically expressing Mcd1 during early meiotic prophase I in Saccharomyces cerevisiae. Mcd1 partially regulated the progression of meiotic recombination, sister chromatid separation, and nuclear division. DNA physical analysis during meiotic recombination showed that Mcd1 induced double-strand breaks (DSBs) but negatively regulated homologous recombination during DSB repair; Mcd1 expression delayed post-DSB stages, leading to inefficiencies in the DSB-to-joint molecule (JM) transition and subsequent crossover formation. These findings indicate that meiotic cells undergo Mcd1-mediated DSB formation during prophase I, and that residual Mcd1 could regulate the progression of JM formation during meiotic recombination.

Deregulation of the Replisome Factor MCMBP Prompts Oncogenesis in Colorectal Carcinomas through Chromosomal Instability

Genetic instability has emerged as an important hallmark of human neoplasia. Although most types of cancers exhibit genetic instability to some extent, in colorectal cancers genetic instability is a distinctive characteristic. Recent studies have shown that deregulation of genes involved in sister chromatid cohesion can result in chromosomal instability in colorectal cancers. Here, we show that the replisome factor minichromosome maintenance complex–binding protein (MCMBP), which is directly involved in the dynamics of the minichromosome maintenance complex and contributes to maintaining sister chromatid cohesion, is transcriptionally misregulated in different types of carcinomas. Cellular studies revealed that both MCMBP knockdown and overexpression in different breast and colorectal cell lines is associated with the emergence of a subpopulation of cells with abnormal nuclear morphology that likely arise as a consequence of aberrant cohesion events. Association analysis integrating gene expression data with clinical information revealed that enhanced MCMBP transcript levels correlate with an increased probability of relapse risk in colorectal cancers and different types of carcinomas. Moreover, a detailed study of a cohort of colorectal tumors showed that the MCMBP protein accumulates to high levels in cancer cells, whereas in normal proliferating tissue its abundance is low, indicating that MCMBP could be exploited as a novel diagnostic marker for this type of carcinoma.

English

Sister chromatid cohesion denotes the phenomenon that sister chromatids are initially attached to each other in mitosis to guarantee the error-free distribution into the daughter cells. Cohesion is mediated by binding proteins and only resolved after mitotic chromosome condensation is completed. However, the amount of attachement points required to maintain sister chromatid cohesion while still allowing proper chromosome condensation is not known yet. Additionally the impact of cohesion on the mechanical properties of chromosomes also poses an interesting problem. In this work we study the conformational and mechanical properties of sister chromatids by means of computer simulations. We model both protein-mediated cohesion between sister chromatids and chromosome condensation with a dynamic binding mechanisms. We show in a phase diagram that only specific link concentrations lead to connected and fully condensed chromatids that do not intermingle with each other nor separate due to entropic forces. Furthermore we show that dynamic bonding between chromatids decrease the Young's modulus compared to non-bonded chromatids.

Scc1 sumoylation by Mms21 promotes sister chromatid recombination through counteracting Wapl

DNA double-strand breaks (DSBs) fuel cancer-driving chromosome translocations. Two related structural maintenance of chromosomes (Smc) complexes, cohesin and Smc5/6, promote DSB repair through sister chromatid homologous recombination (SCR). Here we show that the Smc5/6 subunit Mms21 sumoylates multiple lysines of the cohesin subunit Scc1. Mms21 promotes cohesin-dependent small ubiquitin-like modifier (SUMO) accumulation at laser-induced DNA damage sites in S/G2 human cells. Cells expressing the nonsumoylatable Scc1 mutant (15KR) maintain sister chromatid cohesion during mitosis but are defective in SCR and sensitive to ionizing radiation (IR). Scc1 15KR is recruited to DNA damage sites. Depletion of Wapl, a negative cohesin regulator, rescues SCR defects of Mms21-deficient or Scc1 15KR-expressing cells. Expression of the acetylation-mimicking Smc3 mutant does not bypass the requirement for Mms21 in SCR. We propose that Scc1 sumoylation by Mms21 promotes SCR by antagonizing Wapl at a step after cohesin loading at DSBs and in a way not solely dependent on Smc3 acetylation.

Synaptonemal Complex-Dependent Centromeric Clustering and the Initiation of Synapsis in Drosophila Oocytes

The pairing of homologous chromosomes and the intimate synapsis of the paired homologs by the synaptonemal complex (SC) are essential for subsequent meiotic processes including recombination and chromosome segregation. Here we show that the centromere clustering plays an important role in initiating homolog synapsis during meiosis in Drosophila females. Although centromeres are not clustered prior to the onset of meiosis, all four pairs of centromeres are actively clustered into one or two masses during early meiotic prophase. Within the 16-cell cyst, centromeric clustering appears to define the first step in the initiation of synapsis. Clustering is restricted to the nuclei that form the SC and is dependent on all known SC proteins. Surprisingly, both centromeric clusters and the SC components associated with them persist long after the disassembly of the euchromatic SC at the end of pachytene. The initiation of homologous recombination through the formation of programmed double-strand breaks (DSBs) is not required for either the formation or the maintenance of the centromeric clusters. Our data support a view in which the SC-mediated clustering at the centromeres is the initiating event for meiotic synapsis.

Shugoshin–PP2A counteracts casein-kinase-1-dependent cleavage of Rec8 by separase

During meiosis, the cohesin complexes that maintain sister chromatid cohesion are lost in a stepwise manner. At meiosis I the cohesin subunit Rec8 is cleaved only along the chromosome arms; until meiosis II it is protected at centromeres by the action of shugoshin (Sgo1)-protein phosphatase 2A (PP2A). Although this regulation hypothetically involves phosphorylation that is antagonized by Sgo1-PP2A, the kinase and substrate that are responsible are as yet unknown. Using a genetic screen for 'anti-shugoshin', we identify Hhp2, an orthologue of casein kinase 1delta/epsilon (CK1), as a factor required for Rec8 cleavage in fission yeast. We show that CK1, rather than a Polo-like kinase that is widely believed to do so, acts as the cohesin kinase to promote this cleavage during meiosis. Crucially, forced localization of excess Hhp2 at the pericentromeric region abrogates the ability of Sgo1-PP2A to protect centromeric Rec8. Thus, our studies prove the key notion that the balance between Rec8 phosphorylation and its dephosphorylation by Sgo1-PP2A regulates the step-wise loss of chromosomal cohesion in meiosis.

SOLO: a meiotic protein required for centromere cohesion, coorientation, and SMC1 localization in Drosophila melanogaster

Sister chromatid cohesion is essential to maintain stable connections between homologues and sister chromatids during meiosis and to establish correct centromere orientation patterns on the meiosis I and II spindles. However, the meiotic cohesion apparatus in Drosophila melanogaster remains largely uncharacterized. We describe a novel protein, sisters on the loose (SOLO), which is essential for meiotic cohesion in Drosophila. In solo mutants, sister centromeres separate before prometaphase I, disrupting meiosis I centromere orientation and causing nondisjunction of both homologous and sister chromatids. Centromeric foci of the cohesin protein SMC1 are absent in solo mutants at all meiotic stages. SOLO and SMC1 colocalize to meiotic centromeres from early prophase I until anaphase II in wild-type males, but both proteins disappear prematurely at anaphase I in mutants for mei-S332, which encodes the Drosophila homologue of the cohesin protector protein shugoshin. The solo mutant phenotypes and the localization patterns of SOLO and SMC1 indicate that they function together to maintain sister chromatid cohesion in Drosophila meiosis.

Cohesin's dual role in the DNA damage response: repair and checkpoint activation

Cohesin's dual role in the DNA damage response: repair and checkpoint activation

Cohesin regulatesVSGmonoallelic expression in trypanosomes

Antigenic variation allows Trypanosoma brucei to evade the host immune response by switching the expression of 1 out of ∼15 telomeric variant surface glycoprotein (VSG) expression sites (ESs). VSG ES transcription is mediated by RNA polymerase I in a discrete nuclear site named the ES body (ESB). However, nothing is known about how the monoallelic VSG ES transcriptional state is maintained over generations. In this study, we show that during S and G2 phases and early mitosis, the active VSG ES locus remains associated with the single ESB and exhibits a delay in the separation of sister chromatids relative to control loci. This delay is dependent on the cohesin complex, as partial knockdown of cohesin subunits resulted in premature separation of sister chromatids of the active VSG ES. Cohesin depletion also prompted transcriptional switching from the active to previously inactive VSG ESs. Thus, in addition to maintaining sister chromatid cohesion during mitosis, the cohesin complex plays an essential role in the correct epigenetic inheritance of the active transcriptional VSG ES state.