Octameric nucleosomes are present within the inner kinetochore of human centromeres

The centromere is a vital locus on each chromosome which 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, using a combination of biophysical and biochemical analyses we provide evidence for the existence of two populations of structurally distinct CENP-A nucleosomes that co-exist at human centromeres. These two populations display unique sedimentation patterns in a glycerol gradient, and CENP-A nucleosomes that are physically associated with the inner kinetochore appear stabilized in an octameric conformation, with restricted access to the nucleosomal DNA by DNase I. In contrast, the bulk population of CENP-A nucleosomes have diminished heights and weakened DNA interactions. These data suggest that, in vivo, a reserve pool of immature CENP-A nucleosomes exist which wrap DNA loosely, whereas an active inner kinetochore complex associates with stabilized CENP-A nucleosomes. Our data have implications for a function for CENP-A that may be independent of its role in mitotis. Overall design: ChIP-seq