Centromeres are specified epigenetically, and the histone H3 variant, CENP-A, is assembled into the chromatin of all active centromeres1. Divergence from H3 raises the possibility that CENP-A generates unique chromatin features to physically mark centromere location. The crystal structure of the sub-nucleosomal heterotetramer reported here reveals three distinguishing properties encoded by the residues that comprise the CENP-A Targeting Domain (CATD2): (1) a CENP-A/CENP-A interface that is substantially rotated relative to the H3/H3 interface, (2) a protruding loop L1 of the opposite charge as on H3, and (3) strong hydrophobic contacts that rigidify the CENP-A/H4 interface. Residues involved in the CENP-A/CENP-A rotation are required for efficient incorporation into centromeric chromatin suggesting specificity for an unconventional nucleosome shape. DNA topological analysis indicates that CENP-A-containing nucleosomes are nonetheless octameric with conventional left-handed DNA wrapping, in contrast to other recent proposals3-6. Our results indicate rather that CENP-A marks centromere location by restructuring the nucleosome from within its folded histone core.

Summary Centromeres are defined by the presence of chromatin containing the histone H3 variant, CENP-A, whose assembly into nucleosomes requires the chromatin assembly factor HJURP. We find that while surface-exposed residues in the CENP-A targeting domain (CATD) are the primary sequence determinants for HJURP recognition, buried CATD residues that generate rigidity with H4 are also required for efficient incorporation into centromeres. HJURP contact points adjacent to the CATD on the CENP-A surface are not used for binding specificity but rather to transmit stability broadly throughout the histone fold domains of both CENP-A and H4. Further, an intact CENP-A/CENP-A interface is a requirement for stable chromatin incorporation immediately upon HJURP-mediated assembly. These data offer insight into the mechanism by which HJURP discriminates CENP-A from bulk histone complexes and chaperones CENP-A/H4 for a substantial portion of the cell cycle prior to mediating chromatin assembly at the centromere.

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