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Dbp7 and RNA exosome subunit Dis3 interacts to mediate CENP-A loading via CENP-A chaperone Sim3

ABSTRACT: Background: Centromeres are crucial for proper chromosomes segregation during cell division. Centromeres in most eukaryotes are epigenetically specified by the histone 3 (H3) variant, CENP-A. How CENP-A precisely targets to centromeres remains elusive. Centromeres are typically transcribed into non-coding RNAs, which have been implicated in centromere functions. But the exact role of centromeric RNA in CENP-A loading is still poorly understood. In addition, centromere transcription induce formation of R-loops consisting of a DNA:RNA hybrid and a displaced single-stranded DNA. Excessive R-loops lead to genome instability. Little is known about the mechanism used for safeguarding centromeres against R-loops. Results: Here we show that through a visual genetic screen, we identified a conserved RNA binding protein Dbp7 that plays a critical role in CENP-A loading to centromeres. Deletion of dbp7+ results in CENP-A mislocalization and defects in chromosome segregation and centromere silencing. We found that Dbp7 associates with centromere transcripts, and further showed that Dbp7 prevents accumulation of R-loops at centromeres. Our domain-deletion analysis demonstrated that the DEAD-box helicase domain and the C-terminal extension motif of Dbp7 contribute to its role in centromere function. In addition, Dbp7 interacts with the key exosome subunit Dis3. Importantly, we found that Dis3 promotes CENP-A centromere loading by interacting the CENP-A chaperone Sim3. Conclusions: Together, this study uncovered a previously unrecognized CENP-A loading mechanism, by which Dbp7 and Dis3 bind to centromeric transcripts and in turn mediate recruitment of CENP-A via recruiting Sim3. Our work also provides mechanistic insight into R-loop resolution at centromeres.

ORGANISM(S): Schizosaccharomyces pombe

PROVIDER: GSE285031 | GEO | 2025/01/15

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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Dbp7 and RNA exosome subunit Dis3 interacts to mediate CENP-A loading via CENP-A chaperone Sim3

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