The structure of (CENP-A–H4)2 reveals physical features that mark centromeres

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Abstract
The hallmark of all eukaryotic centromeres is the presence of a histone H3 variant, CENP-A, but the nature of the epigenetic centromere mark is the subject of great debate. The crystal structure of the human CENP-A–H4 complex has now been determined. It reveals the structural basis for the rigidity that CENP-A imparts to nucleosomes, as well as several surprising global and local structural alterations that have major implications for epigenetic centromere marking. CENP-A seems to alter the chromatin into which it assembles — not by switching the handedness of DNA wrapping or disrupting the octameric histone stoichiometry, but by altering nucleosome structure. The centromeres of chromosomes are specified epigenetically, and the histone H3 variant CENP-A is assembled into the chromatin of all active centromeres. Here, the crystal structure of CENP-A in a tetrameric complex with histone H4 reveals the physical features of centromeric chromatin. CENP-A seems to mark the centromere by altering nucleosome structure from within its folded histone core. 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 mark physically centromere location. Here we report the crystal structure of a subnucleosomal heterotetramer, human (CENP-A–H4)2, that reveals three distinguishing properties encoded by the residues that comprise the CENP-A targeting domain (CATD; ref. 2): (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 that 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, indicating specificity for an unconventional nucleosome shape. DNA topological analysis indicates that CENP-A-containing nucleosomes are octameric with conventional left-handed DNA wrapping, in contrast to other recent proposals3,4,5,6. Our results indicate that CENP-A marks centromere location by restructuring the nucleosome from within its folded histone core.

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