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

Abstract

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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Figure 1: Crystal structure of the (CENP-A–H4) 2 heterotetramer.
Figure 2: The residues involved in the rotated CENP-A–CENP-A interface are essential for centromere targeting.
Figure 3: Surface and internal structural features unique to CENP-A-containing complexes.
Figure 4: The (CENP-A–H4) 2 heterotetramer assembles with H2A–H2B dimers into an octameric nucleosome with conventional handedness of DNA wrapping.

Accession codes

Primary accessions

Protein Data Bank

Data deposits

The structures of (CENP-A–H4)2 heterotetramers have been deposited in the Protein Data Bank under accession numbers 3NQJ and 3NQU.

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Acknowledgements

We thank D. Cleveland for plasmids and steadfast encouragement to pursue a physical understanding of the centromere; S. Wood for generating cleaved histone H2A; K. Gupta for help with collecting data and technical suggestions; and K. Ferguson, G. Van Duyne, M. Lemmon, J. Shorter, L. Jansen, D. Foltz, J. Shah, D. Alvarado, K. Moravcevic and T. Panchenko for discussions and comments on the manuscript. This work was supported by the NIH research grant GM82989, a Career Award in the Biomedical Sciences from the Burroughs Wellcome Fund, and a Rita Allen Foundation Scholar Award to B.E.B. N.S. is supported by a postdoctoral fellowship from the American Cancer Society and E.A.B. has been supported by the Penn Structural Biology Training Grant (NIH GM08275) and a predoctoral fellowship from the American Heart Association.

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N.S. designed and performed experiments, solved and refined the structures, analysed data and wrote the manuscript; E.A.B. and D.J.R. performed experiments and analysed data; and B.E.B. directed the project, designed and performed experiments, analysed data and wrote the manuscript.

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Correspondence to Ben E. Black.

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The authors declare no competing financial interests.

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Sekulic, N., Bassett, E., Rogers, D. et al. The structure of (CENP-A–H4)2 reveals physical features that mark centromeres. Nature 467, 347–351 (2010). https://doi.org/10.1038/nature09323

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