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bioRxiv · 10.1101/604223

CENP-C stabilizes the conformation of CENP-A nucleosomes within the inner kinetochore at human centromere

Melters, D. P.·Rakshit, T.·Grigoryev, S. A.·Sturgill, D.·Dalal, Y.
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Abstract

The centromere is the chromosomal locus that seeds the kinetochore, allowing for a physical connection between the chromosome and the mitotic spindle. At the heart of the centromere is the centromere-specific histone H3 variant CENP-A/CENH3. Throughout the cell cycle the constitutive centromere associated network is bound to CENP-A chromatin, but how this protein network modifies CENP-A nucleosome dynamics in vivo is unknown. Here, we purify kinetochore associated native centromeric chromatin and analyze its biochemical features using a combinatorial approach. We report that kinetochore bound chromatin has strongly reduced DNA accessibility and a distinct stabilized nucleosomal configuration. Disrupting the balance between CENP-A and CENP-C result in reduced centromeric occupancy of RNA polymerase 2 and impaired de novo CENP-A loading on the centromeric chromatin fiber, correlating with significant mitotic defects. CENP-A mutants that restore the ratio rescue the mitotic defects. These data support a model in which CENP-C bound centromeric nucleosomes behave as a barrier to the transcriptional machinery and suggest that maintaining the correct ratio between CENP-A and CENP-C levels is critical for centromere homeostasis.

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Melters, D. P., Rakshit, T., Grigoryev, S. A., Sturgill, D., Dalal, Y.. 2019-04-10. CENP-C stabilizes the conformation of CENP-A nucleosomes within the inner kinetochore at human centromere. https://doi.org/10.1101/604223

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