Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Chromatin immunoprecipitation of fission yeast to identify topoisomerase I and II activity

ABSTRACT: Eukaryotic topoisomerase I and II relax DNA and are key components in the processes of DNA replication, transcription and genome stability. It is not clear, however, how their activity controls epigenetic states across an entire eukaryotic genome. Using the fission yeast model Schizosaccharomyces pombe, we investigate genome-wide how topoisomerases affect chromatin formation through nucleosome occupancy and regulate transcription. We show that topoisomerase activity is required for nucleosome turnover at promoter regions, affecting epigenetic gene regulatory states, and for effective termination of transcription.

ORGANISM(S): Schizosaccharomyces pombe

SUBMITTER: Mickael DURAND-DUBIEF

PROVIDER: E-MEXP-1850 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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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.

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Chromatin immunoprecipitation of fission yeast to identify topoisomerase I and II activity

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