The CENP-A chaperone complex spatially organizes centromeres.

Centromeres, defined by CENP-A-containing nucleosomes, direct kinetochore assembly for spindle attachment. In mitosis, CENP-A and the constitutive centromere-associated network (CCAN) of the inner kinetochore are arranged into bipartite subdomains within clearings of chromatin. However, less is known about their architecture during interphase. We report here an unrecognized structural role for the CENP-A chaperone machinery in establishing interphase centromere architecture. In interphase, CENP-A and the CCAN assemble conserved shell-like structures that enclose a chromatin-poor central cavity. This cavity is occupied by the interphase-specific CENP-A chaperone complex, which promotes CENP-A assembly once per cell cycle. The presence of the chaperone complex, but not its CENP-A-incorporating activity, is required to generate both the shell architecture and chromatin clearing. The CCAN scaffold CENP-C exhibits radial organization throughout the structure and is essential for its formation, primarily by recruiting the HJURP chaperone. Our findings broaden the role of the CENP-A chaperone machinery to include the structural organization of interphase vertebrate centromeres, independent of CENP-A deposition.