Histone chaperone NAP1 mediates sister chromatid resolution by counteracting protein phosphatase 2A.
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ABSTRACT:
SUBMITTER: Moshkin YM
PROVIDER: S-EPMC3784504 | biostudies-literature | 2013
REPOSITORIES: biostudies-literature
Publications
Histone chaperone NAP1 mediates sister chromatid resolution by counteracting protein phosphatase 2A.
Moshkin Yuri M YM Doyen Cecile M CM Kan Tsung-Wai TW Chalkley Gillian E GE Sap Karen K Bezstarosti Karel K Demmers Jeroen A JA Ozgur Zeliha Z van Ijcken Wilfred F J WF Verrijzer C Peter CP
PLoS genetics 20130926 9
Chromosome duplication and transmission into daughter cells requires the precisely orchestrated binding and release of cohesin. We found that the Drosophila histone chaperone NAP1 is required for cohesin release and sister chromatid resolution during mitosis. Genome-wide surveys revealed that NAP1 and cohesin co-localize at multiple genomic loci. Proteomic and biochemical analysis established that NAP1 associates with the full cohesin complex, but it also forms a separate complex with the cohesi ...[more]
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Project description:The fidelity of chromosome duplication through cell divisions requires timely binding and release of the cohesin. Cohesin is a ring-shaped protein complex linking newly replicated sister chromatids to ensure their appropriate transmission through mitosis. Upon commencement of mitosis cohesin is removed from DNA in two steps: first, from chromosome arms resulting in sister chromatid resolution, and, second, from centromers leading to sister chromatid segregation. As DNA of eukaryotic chromosomes is assembled into chromatin, regulation of sister chromatid cohesion-segregation may involve chromatin modifying machinery, but this link is not well understood. Here we report that H2A-H2B histone chaperone NAP1, a factor, which is primarily implicated in chromatin assembly, is required for cohesin release from mitotic chromosome arms. NAP1 and cohesin protein complex interact directly and share multiple binding sites on chromatin. Depletion of the NAP1 hinders cohesin removal during mitosis resulting in accumulation of unresolved sister chromatids. Thus, in addition to its well established functions in chromatin dynamics, histone chaperone NAP1 coordinates cell cycle dependent cohesin release. These results reveal a novel molecular pathway for sister chromatid resolution and emphasizes a role for histone chaperones in control of eukaryotic genome replication and transmission. Genome-wide NAP1 and Cohesin ChIP-chip profiling in Drosophila S2 cells. The supplementary bed file S2_cohesin_sites.bed contains cohesin binding sites obtained by intersecting the sets of significant ChIP-chip peaks for SA (a cohesin subunit; stromalin) and SMC1.
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Project description:The fidelity of chromosome duplication through cell divisions requires timely binding and release of the cohesin. Cohesin is a ring-shaped protein complex linking newly replicated sister chromatids to ensure their appropriate transmission through mitosis. Upon commencement of mitosis cohesin is removed from DNA in two steps: first, from chromosome arms resulting in sister chromatid resolution, and, second, from centromers leading to sister chromatid segregation. As DNA of eukaryotic chromosomes is assembled into chromatin, regulation of sister chromatid cohesion-segregation may involve chromatin modifying machinery, but this link is not well understood. Here we report that H2A-H2B histone chaperone NAP1, a factor, which is primarily implicated in chromatin assembly, is required for cohesin release from mitotic chromosome arms. NAP1 and cohesin protein complex interact directly and share multiple binding sites on chromatin. Depletion of the NAP1 hinders cohesin removal during mitosis resulting in accumulation of unresolved sister chromatids. Thus, in addition to its well established functions in chromatin dynamics, histone chaperone NAP1 coordinates cell cycle dependent cohesin release. These results reveal a novel molecular pathway for sister chromatid resolution and emphasizes a role for histone chaperones in control of eukaryotic genome replication and transmission.
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Project description:Segregation of homologous maternal and paternal centromeres to opposite poles during meiosis I depends on post-replicative crossing over between homologous non-sister chromatids, which creates chiasmata and therefore bivalent chromosomes. Destruction of sister chromatid cohesion along chromosome arms due to proteolytic cleavage of cohesin's Rec8 subunit by separase resolves chiasmata and thereby triggers the first meiotic division. This produces univalent chromosomes, the chromatids of which are held together by centromeric cohesin that has been protected from separase by shugoshin (Sgo1/MEI-S332) proteins. Here we show in both fission and budding yeast that Sgo1 recruits to centromeres a specific form of protein phosphatase 2A (PP2A). Its inactivation causes loss of centromeric cohesin at anaphase I and random segregation of sister centromeres at the second meiotic division. Artificial recruitment of PP2A to chromosome arms prevents Rec8 phosphorylation and hinders resolution of chiasmata. Our data are consistent with the notion that efficient cleavage of Rec8 requires phosphorylation of cohesin and that this is blocked by PP2A at meiosis I centromeres. Keywords: ChIP-chip, Mitosis, Meiosis, Cell cycle, Saccharomyces cerevisiae, Chromosome VI tiling array, Sgo1, Pp2A, Cse4, Ndc10, Rts1, Rec8 â?¢ Experimental factors Distribution of Cse4, Ndc10, Rts1 in mitotic G2 phase and meiosis I (S. cerevisiae). Distribution of Rec8 in meiosis I (S.cerevisiae). All experiments were performed in cells with the same genetic background (Saccharomyces cerevisiae SK1). â?¢ Experimental design ChIP analysis: In all cases, hybridization data for ChIP fraction was compared with SUP (supernatant) fraction. SUP fraction is same as WCE fraction. â?¢ Quality control steps taken Duplication, confirmation using different tags, and different subunits of the same complex. Confirmation of protein distribution using deletion mutant strain. Confirmation of several loci by q-PCR. Checking of the ChIP fraction by Western blotting. Checking of the ChIP fraction by swapping SUP fraction. Samples used, extract preparation and labelling: â?¢ The origin of each biological sample Saccharomyces cerevisiae (SK1) . â?¢ Manipulation of biological samples and protocols used Chromatin immunoprecipitation (ChIP) and hybridization to Affimetrix high-density oligonucleotide arrays of S. cerevisiae chromosome VI was performed essentially as previously described (Katou et al., 2003, nature) (Lengronne et al., 2004, nature). â?¢ Technical protocols for preparing the hybridization extract The chromatin-immunprecipates were eluted and incubated over night at 65ºC to reverse the cross-link. Immunoprecipitated genomic DNA was incubated with proteinase K, extracted 2 times with phenol/chloroform/isoamylalcohol, precipitated, resuspended in TE and incubated with RnaseA. The DNA was then purified using the Qiagen PCR purification kit, and concentrated by ethanol precipitation. The DNA was amplified by PCR after random priming. 10 ug of amplified DNA was digested with Dnase I to a mean size of 100 bp. After Dnase I inactivation at 95ºC. DNA fragments were end-labeled by addition of 25 U of Terminal Transferase and 1 nmol Biotin-N6ddATP (NEN) for 1 hour at 37ºC as previously described by Winzeler et al. (Science. 281, 1194-1197, 1998). The entire sample was used for hybridization. â?¢ Hybridization procedures and parameters: Hybridization, blocking and washing were carried out as previously described (http://everythingchromosomevi.gsc.riken.go.jp). Each sample was hybridized to the array in 150 ul containing 6xSSPE; 0.005% TritonX-100; 15 ug fragmented denatured salmon sperm DNA (Gibco-BRL); 1 nmole 3â??biotin labelled control oligonucleotide (oligo B2, Affymetrix). Samples were denatured at 100ºC for 10 minutes, and then put on ice before being hybridized for 16 hours at 42ºC in a hybridization oven (GeneChip Hybridization Oven 640, Affymetrix). Washing and scanning protocol provided by Affymetrix was performed automatically on a fluidics station (GeneChip fluidics station 450, Affymetrix). â?¢ Measurement data and specifications: Arrays were scanned using the Genechip Scanner3000 7G following the library array description. All the raw data files can be downloaded from GEO database. The primary analysis of tiling chip data was performed following exactly the statistical algorithm used for Affymetrix GeneChip Operating Software (GCOS). The detailed information for the algorithm used can be downloaded from the Affymetrix web site at http://www.affymetrix.com/support/technical/technotes/statistical_reference_guide.pdf. The analysis is available on request. For the ChrVI array, one unit for analysis (locus) was set to 300bp. Fold change value, change p-value, and detection p-value for each locus were obtained by primary analysis. For the discrimination of positive and negative signals for the binding, we used three criteria as follows. First, the reliability of the signal strength was judged by detection p-value of each locus (p-valueâ?¤0.025 for cerevisiae). Secondly, reliability of binding ratio was judged by change p-value (p-valueâ?¤0.025 for cerevisiae). 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