Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Mediator promotes CENP-A incorporation at fission yeast centromeres [ChIP-seq]

ABSTRACT: 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.

ORGANISM(S): Schizosaccharomyces pombe

SUBMITTER: Claes Gustafsson

PROVIDER: E-GEOD-35524 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Mediator promotes CENP-a incorporation at fission yeast centromeres.

Carlsten Jonas O JO Szilagyi Zsolt Z Liu Beidong B Lopez Marcela Davila MD Szászi Erzsébet E Djupedal Ingela I Nyström Thomas T Ekwall Karl K Gustafsson Claes M CM Zhu Xuefeng X

Molecular and cellular biology 20120730 19

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 through not only the RNA interference (RNAi) machinery but also RNAi-independent RNA processing factors. Control of centromeric ncRNA transcription is theref ...[more]

PMID: 22851695

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Project description:Specialized chromatin containing CENP-A nucleosomes instead of H3 nucleosomes is found at all centromeres. However, the mechanisms that specify the locations at which CENP-A chromatin is assembled remain elusive in organisms with regional, epigenetically regulated centromeres. It is known that normal centromeric DNA is transcribed in several systems including the fission yeast, Schizosaccharomyces pombe. Here, we show that factors which preserve stable histone H3 chromatin during transcription also play a role in preventing promiscuous CENP-A(Cnp1) deposition in fission yeast. Mutations in the histone chaperone FACT impair the maintenance of H3 chromatin on transcribed regions and promote widespread CENP-A(Cnp1) incorporation at non-centromeric sites. FACT has little or no effect on CENP-A(Cnp1) assembly at endogenous centromeres where CENP-A(Cnp1) is normally assembled. In contrast, Clr6 complex II (Clr6-CII; equivalent to Rpd3S) histone deacetylase function has a more subtle impact on the stability of transcribed H3 chromatin and acts to prevent the ectopic accumulation of CENP-A(Cnp1) at specific loci, including subtelomeric regions, where CENP-A(Cnp1) is preferentially assembled. Moreover, defective Clr6-CII function allows the de novo assembly of CENP-A(Cnp1) chromatin on centromeric DNA, bypassing the normal requirement for heterochromatin. Thus, our analyses show that alterations in the process of chromatin assembly during transcription can destabilize H3 nucleosomes and thereby allow CENP-A(Cnp1) to assemble in its place. We propose that normal centromeres provide a specific chromatin context that limits reassembly of H3 chromatin during transcription and thereby promotes the establishment of CENP-A(Cnp1) chromatin and associated kinetochores. These findings have important implications for genetic and epigenetic processes involved in centromere specification. In total, 24 samples: 22 ChIP DNA files (10 different conditions), 2 Input files.

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. For transcription: Total RNA from top3-105 mutant and WT control cells after 8 hours at 36C in biological duplicates. For Top3-myc chromatin immunoprecipitation: DNA immunoprecipitated with mouse anti-Myc using chromatin extracts from cells expressing Top3-Myc from the endogenous locus at 30C in biological duplicates normalized to input DNA from wild type cells at 30C in biological duplicates. For CENP-A/Cnp1 chromatin immunoprecipitation: DNA immunoprecipitated with anti-Cnp1 serum using chromatin extracts from top3-105 mutant and wild type control cells after 8 hours at 36C in in biological duplicates normalized to input DNA from each strain.

Dataset Information

Mediator promotes CENP-A incorporation at fission yeast centromeres [ChIP-seq]

Publications

Mediator promotes CENP-a incorporation at fission yeast centromeres.

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