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

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, which permits purification of inner kinetochore bound CENP-A chromatin away from bulk CENP-A nucleosomes. The bulk population of CENP-A nucleosomes have diminished heights and weakened DNA interactions, whereas CENP-A nucleosomes robustly associated with the inner kinetochore are stabilized in an octameric conformation, with restricted access to nucleosomal DNA. Immuno-labeling coupled to atomic force microscopy of these complexes confirms their identity at the nanoscale resolution. These data provide a systematic and detailed description of inner-kinetochore bound CENP-A chromatin from human centromeres, with implications for the state of CENP-A chromatin that is actively engaged during mitosis.