Independence of centromeric and pericentromeric chromatin stability on CCAN components.

Abstract

The chromatin of the centromere provides the assembly site for the mitotic kinetochore that couples microtubule attachment and force production to chromosome movement in mitosis. The chromatin of the centromere is specified by nucleosomes containing the histone H3 variant, CENP-A. The constitutive centromeric-associated network (CCAN) and kinetochore are assembled on CENP-A chromatin to enable chromosome separation. CENP-A chromatin is surrounded by pericentromeric heterochromatin, which itself is bound by the sequence specific binding protein, CENP-B. We performed mechanical experiments on mitotic chromosomes while tracking CENP-A and CENP-B to observe the centromere's stiffness and the role of the CCAN. We degraded CENP-C and CENP-N containing auxin-inducible degrons, which we verified compromises the CCAN via observation of CENP-T loss. Chromosome stretching revealed that the CENP-A domain does not visibly stretch, even in the absence of CENP-C and/or CENP-N. Pericentromeric chromatin deforms upon force application, stretching ∼3-fold less than the entire chromosome. CENP-C and/or CENP-N loss has no impact on pericentromere stretching. Chromosome-disconnecting nuclease treatments showed no structural effects on CENP-A. Our experiments show that the core-centromeric chromatin is more resilient and likely mechanically disconnected from the underlying pericentromeric chromatin, while the pericentric chromatin is deformable yet stiffer than the chromosome arms.