Genome-wide mapping of Ccp1 association sites in fission yeast
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ABSTRACT:
ORGANISM(S): Schizosaccharomyces pombe
PROVIDER: GSE95046 | GEO | 2018/06/13
REPOSITORIES: GEO
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Project description:At Schizosaccharomyces pombe centromeres, heterochromatin formation is required for de novo incorporation of the histone H3 variant CENP-A/Cnp1, which in turn directs kinetochore assembly and ultimately chromosome segregation during mitosis. Noncoding RNAs (ncRNAs) transcribed by RNA polymerase II (Pol II) directs heterochromatin formation via the RNAi machinery, but also through RNAiindependent RNA processing factors. Control of centromeric ncRNA transcription is therefore a key factor for proper centromere function. We here use transcriptional profiling, gene inactivation experiments, and chromatin immunoprecipitation analyses to demonstrate that the Mediator complex directs ncRNA transcription and regulates centromeric heterochromatin formation in fission yeast. Mediator co-localizes with Pol II at centromeres and loss of the Mediator subunit Med20 causes a dramatic increase in pericentromeric transcription and desilencing of the core centromere. As a consequence, heterochromatin formation is impaired both via the RNAi dependent and independent pathways, resulting in loss of CENP-A/Cnp1 from the core centromere, defect kinetochore function, and a severe chromosome segregation defect. Interestingly, the increased centromeric transcription observed in med20Δ appears to directly block CENP-A/Cnp1 incorporation and inhibition of Pol II transcription can suppress the observed phenotypes. Our data thus identify Mediator as a crucial regulator of ncRNA transcription at fission yeast centromeres and add another crucial layer of regulation to centromere function.
2012-11-06 | GSE35524 | GEO
Project description:A key element for defining the centromere identity is the incorporation of a specific histone H3, CENP-A, known as Cnp1p in S. pombe. Previous studies have suggested that functional S. pombe centromeres lack nucleosome arrays and may involve chromatin remodeling as a key step of kinetochore assembly. We used tiling microarrays to show that nucleosomes are in fact positioned in regular intervals in the core of centromere 2, providing the first high resolution map of regional centromere chromatin. Nucleosome locations are not disrupted by mutations in kinetochore proteins cnp1, mis18, mis12, nuf2, mal2, overexpression of Cnp1p, or deletion of ams2. Bioinformatic analysis of the centromere sequence indicates certain enriched motifs in linker regions between nucleosomes and reveals a sequence-bias in nucleosome positioning. We conclude that centromeric nucleosome positions are stable and may be derived from the underlying DNA sequence. In addition, sequence analysis of nucleosome-free regions identifies novel binding sites for the GATA-like protein Ams2p, which participates in CENP-A incorporation. Keywords: Nucleosome Mapping Study Entire cnt regions and histone-related genes were tiled at 1-5 bp spacing using 60-mer probes.
2008-05-30 | E-GEOD-10742 | biostudies-arrayexpress
Project description:In most eukaryotes, the centromere is epigenetically defined by nucleosomes that contain the histone H3 variant centromere protein A (CENP-A). Specific targeting of the CENP-A-loading chaperone to the centromere is vital for stable centromere propagation; however, the existence of ectopic centromeres (neocentromeres) indicates that this chaperone can function in different chromatin environments. The mechanism responsible for accommodating the CENP-A chaperone at novel chromatin regions is poorly understood. Here, we report the identification of transient, immature neocentromeres in Schizosaccharomyces pombe, which show reduced association with the CENP-A chaperone Scm3 attributable to persistence of the histone H2A variant H2A.Z. Following acquisition of adjacent heterochromatin or relocation of the immature neocentromeres to subtelomeric regions, H2A.Z was depleted and Scm3 was replenished, leading to subsequent stabilization of the neocentromeres. These findings provide novel insights into histone variant-mediated epigenetic control of neocentromere establishment.
2013-11-03 | GSE37094 | GEO
Project description:At Schizosaccharomyces pombe centromeres, heterochromatin formation is required for de novo incorporation of the histone H3 variant CENP-A/Cnp1, which in turn directs kinetochore assembly and ultimately chromosome segregation during mitosis. Noncoding RNAs (ncRNAs) transcribed by RNA polymerase II (Pol II) directs heterochromatin formation via the RNAi machinery, but also through RNAiindependent RNA processing factors. Control of centromeric ncRNA transcription is therefore a key factor for proper centromere function. We here use transcriptional profiling, gene inactivation experiments, and chromatin immunoprecipitation analyses to demonstrate that the Mediator complex directs ncRNA transcription and regulates centromeric heterochromatin formation in fission yeast. Mediator co-localizes with Pol II at centromeres and loss of the Mediator subunit Med20 causes a dramatic increase in pericentromeric transcription and desilencing of the core centromere. As a consequence, heterochromatin formation is impaired both via the RNAi dependent and independent pathways, resulting in loss of CENP-A/Cnp1 from the core centromere, defect kinetochore function, and a severe chromosome segregation defect. Interestingly, the increased centromeric transcription observed in med20Δ appears to directly block CENP-A/Cnp1 incorporation and inhibition of Pol II transcription can suppress the observed phenotypes. Our data thus identify Mediator as a crucial regulator of ncRNA transcription at fission yeast centromeres and add another crucial layer of regulation to centromere function. 3 samples examined: wild type chromatin incubated with beads as the non antibody control, wild type chromatin incubated with RNA Polymerase II CTD domain antibody and Protein G beads, and TAP-Med7 cells chromatin incubated with IgG beads.
2012-11-06 | E-GEOD-35524 | biostudies-arrayexpress
Project description:In most eukaryotes, the centromere is epigenetically defined by nucleosomes that contain the histone H3 variant centromere protein A (CENP-A). Specific targeting of the CENP-A-loading chaperone to the centromere is vital for stable centromere propagation; however, the existence of ectopic centromeres (neocentromeres) indicates that this chaperone can function in different chromatin environments. The mechanism responsible for accommodating the CENP-A chaperone at novel chromatin regions is poorly understood. Here, we report the identification of transient, immature neocentromeres in Schizosaccharomyces pombe, which show reduced association with the CENP-A chaperone Scm3 attributable to persistence of the histone H2A variant H2A.Z. Following acquisition of adjacent heterochromatin or relocation of the immature neocentromeres to subtelomeric regions, H2A.Z was depleted and Scm3 was replenished, leading to subsequent stabilization of the neocentromeres. These findings provide novel insights into histone variant-mediated epigenetic control of neocentromere establishment. Comparison of chromosomal distributions of centromeric proteins and heterochromatin proteins between the NC survivors and their derivatives.
2013-11-03 | E-GEOD-37094 | biostudies-arrayexpress
Project description:Centromeres are specialized chromatin regions marked by the presence of nucleosomes containing the centromere-specific histone H3 variant CENP-A, which is essential for chromosome segregation. Assembly and disassembly of nucleosomes is intimately linked to DNA topology and DNA topoisomerases have previously been implicated in the dynamics of canonical H3 nucleosomes. Here we show that Schizosaccharomyces pombe Top3 and its partner Rqh1 are involved in controlling the levels of CENP-ACnp1 at centromeres. Both top3 and rqh1 mutants display defects in chromosome segregation. Using chromatin immunoprecipitation and tiling microarrays we show that Top3 unlike Top1 and Top2 is highly enriched at centromeric central domains, demonstrating that Top3 is the major topoisomerase in this region. Moreover, centromeric Top3 occupancy positively correlates with CENP-ACnp1 occupancy. Intriguingly, both top3 and rqh1 mutants display increased relative enrichment of CENP-ACnp1 at centromeric central domains. Thus, Top3 and Rqh1 normally limit the levels of CENP-ACnp1 in this region. This new role is independent of the established function of Top3 and Rqh1 in homologous recombination downstream of Rad51. Therefore, we hypothesize that the Top3-Rqh1 complex has an important role in controlling centromere DNA topology which in turn affects the dynamics of CENP-ACnp1 nucleosomes.
2013-03-14 | GSE44206 | GEO
Project description:CENP-A chromatin specifies mammalian centromere identity, and its chaperone HJURP replenishes CENP-A when recruited by the Mis18 complex (Mis18C) via M18BP/KNL2 to CENP-C at kinetochores during interphase. However, the Mis18C recruitment mechanism remains unresolved in species lacking M18BP1, such as fission yeast. Fission yeast centromeres cluster at G2 spindle pole bodies (SPBs) when CENP-ACnp1 is replenished and where Mis18C also localizes. We show that SPBs play an unexpected role in concentrating Mis18C near centromeres through the recruitment of Mis18 by direct binding to the major SPB LInker of Nucleoskeleton and Cytoskeleton (LINC) complex component Sad1. Mis18 recruitment by Sad1 is important for CENP-ACnp1 chromatin establishment and acts in parallel with a CENP-C-mediated Mis18C recruitment pathway to maintain centromeric CENP-ACnp1, but is independent of Sad1-mediated centromere clustering. SPBs therefore provide a non-chromosomal scaffold for both Mis18C recruitment and centromere clustering during G2. This centromere-independent Mis18-SPB recruitment provides a mechanism that governs de novo CENP-ACnp1 chromatin assembly by the proximity of appropriate sequences to SPBs and highlights how nuclear spatial organization influences centromere identity.
2023-09-18 | PXD045191 | Pride
Project description:We use high-resolution chemical cleavage mapping and both native and cross-linked chromatin immunoprecipitation with paired-end sequencing to elucidate the profile of nuceleosomes containing the centromere-specific variant of H3 (cenH3), known as CENP-A or Cnp1 in fission yeast. We find that in the central domain of fission yeast centromeres H3 nucleosomes are nearly absent and CENP-A nucleosomes are more widely spaced that nucleosomes elsewere. CENP-A (Cnp1), CENP-C (Cnp3), CENP-T (Cnp20) and CENP-I (Mis6) are highly enriched at every position in the central domain except at tRNA genes, with weak enrichment in the flanking heterochromatin where these proteins show no evidence of the positioning that has been seen in point centromeres and in the satellite-rich centromeres of plants and animals. Our findings suggest that classical regional centromeres are distinguished from other centromere classes by the absence of cenH3 nucleosome positioning.
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Project description:Centromeres exert vital cellular functions in mitosis and meiosis. A specialized histone and other chromatin-bound factors nucleate a dynamic protein assembly that is required for the proper segregation of sister chromatids. In several organisms including the fission yeast S. pombe, a RNAi-related pathway further strengthens the epigenetic maintenance of centromere location, primarily through repressing transcription near centromeres. Little is known how accessory factors such as histone chaperones and chromatin remodelling factors might be required to establish a functioning centromere. Here we show that the chaperone and remodelling complex FACT is required for transcriptional gene silencing at centromeres and for accurate chromosome segregation during mitosis. We show that Spt16 and Pob3 are two subunits of the S. pombe FACT complex. Surprisingly, yeast strains deleted for Pob3 are viable and show a marked loss of gene silencing at centromeric repeats and at the mating locus. Importantly, Pob3-null strains fail to undergo mitosis at high accuracy and phenocopy histone methylation and RNAi mutants. Processing of centromeric RNA transcripts into siRNAs is maintained in Pob3 mutants, but Swi6-association at the centromere is affected. Our studies provide the first experimental evidence for a role of the RNA polymerase II cofactor FACT in centromere function. Keywords: Expression profiling of pob3 vs wt
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Project description:Ribonucleoprotein immunoprecipitation microarray (RIp-chip) study using the Rng3p myosin-specific chaperones from the fission Schizosaccaromyces pombe. Rng3p associates with RNAs encoding myosins. To investigate if the association of Rng3p with these RNAs occurs on ribosomes, Rng3-purified fractions were immunoprecipitated with antibodies against a ribosomal RNA or an unrelated antibody. The RNAs present in the second immunoprecipitate were analysed using DNA microarrays
2008-12-23 | E-TABM-564 | biostudies-arrayexpress