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Telomeric repeats facilitate CENP-A incorporation via telomere binding proteins

ABSTRACT: The histone H3 variant, CENP-ACnp1, is normally assembled upon canonical centromeric sequences, but there is no apparent obligate coupling of sequence and assembly, suggesting that centromere location can be epigenetically determined. To explore the tolerances and constraints on CENP-ACnp1 deposition we investigated whether certain locations are favoured when additional CENP-ACnp1 is present in fission yeast cells. Our analyses show that additional CENP-ACnp1 accumulates within and close to heterochromatic centromeric outer repeats, and over regions adjacent to rDNA and telomeres. The use of minichromosome derivatives with unique DNA sequences internal to chromosome ends shows that telomeres are sufficient to direct CENP-ACnp1 deposition. However, chromosome ends are not required as CENP-ACnp1 deposition also occurs at telomere repeats inserted at an internal locus and correlates with the presence of H3K9 methylation near these repeats. The Ccq1 protein, which is known to bind telomere repeats and recruit telomerase, was found to be required to induce H3K9 methylation and thus promote the incorporation of CENP-A near telomere repeats. These analyses demonstrate that at non-centromeric chromosomal locations the presence of heterochromatin influences the sites at which CENP-A is incorporated into chromatin and thus, potentially the location of centromeres.

ORGANISM(S): Schizosaccharomyces pombe

PROVIDER: GSE46427 | GEO | 2013/08/22

SECONDARY ACCESSION(S): PRJNA200313

REPOSITORIES: GEO

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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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Telomeric repeats facilitate CENP-A incorporation via telomere binding proteins

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