The ratio between centromeric proteins CENP-A and CENP-C maintains homeostasis of human centromeres

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.