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Centromeres are marked by 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 conformations in vivo is unknown. Here, we purify endogenous centromeric chromatin associated with the CENP-C complex across the cell cycle and analyze the structures by single-molecule imaging and biochemical assays. CENP-C complex–bound chromatin was refractory to MNase digestion. The CENP-C complex increased in height throughout the cell cycle culminating in mitosis, and the smaller CENP-C complex corresponds to the dimensions of in vitro reconstituted constitutive centromere-associated network. In addition, we found two distinct CENP-A nucleosomal configurations; the taller variant was associated with the CENP-C complex. Finally, CENP-A mutants partially corrected CENP-C overexpression–induced centromeric transcription and mitotic defects. In all, our data support a working model in which CENP-C is critical in regulating centromere homeostasis by supporting a unique higher order structure of centromeric chromatin and altering the accessibility of the centromeric chromatin fiber for transcriptional machinery. 

Histone variants fine-tune transcription, replication, DNA damage repair, and faithful chromosome segregation. Whether and how nucleosome variants encode unique mechanical properties to their cognate chromatin structures remains elusive. Here, using in silico and in vitro nanoindentation methods, extending to in vivo dissections, we report that histone variant nucleosomes are intrinsically more elastic than their canonical counterparts. Furthermore, binding proteins, which discriminate between histone variant nucleosomes, suppress this innate elasticity and also compact chromatin. Interestingly, when we overexpress the binding proteins in vivo, we also observe increased compaction of chromatin enriched for histone variant nucleosomes, correlating with diminished access. Taken together, these data suggest a plausible link between innate mechanical properties possessed by histone variant nucleosomes, the adaptability of chromatin states in vivo, and the epigenetic plasticity of the underlying locus.