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Structural determinants for generating centromeric chromatin


Mammalian centromeres are not defined by a consensus DNA sequence. In all eukaryotes a hallmark of functional centromeres—both normal ones and those formed aberrantly at atypical loci—is the accumulation of centromere protein A (CENP-A), a histone variant that replaces H3 in centromeric nucleosomes1,2,3,4,5,6,7. Here we show using deuterium exchange/mass spectrometry coupled with hydrodynamic measures that CENP-A and histone H4 form sub-nucleosomal tetramers that are more compact and conformationally more rigid than the corresponding tetramers of histones H3 and H4. Substitution into histone H3 of the domain of CENP-A responsible for compaction is sufficient to direct it to centromeres. Thus, the centromere-targeting domain of CENP-A confers a unique structural rigidity to the nucleosomes into which it assembles, and is likely to have a role in maintaining centromere identity.

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Figure 1: The CENP-A–H4 complex is a compact heterotetramer.
Figure 2: Unique structural features at the CENP-A–H4 interface.
Figure 3: Converting H3 to a centromeric histone.

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We thank J. Kahana, J. Shah and K. Sullivan for reagents, and I. Cheeseman, B. Cottrell, P. Dyer, C. Gessner, F. Gordon, J. Kim, S. McBryant, D. Pantazatos and S. W. Englander for advice and technical assistance over the course of this study. We also thank J. Shah for comments on the manuscript. This research was supported by grants from the NIH to D.W.C and V.L.W., BioStar and Life Sciences Informatics grants from the University of California and ExSar Corporation to V.L.W., and by postdoctoral fellowships from the American Cancer Society (B.E.B.) and the NIH (D.R.F.). Salary support for D.W.C. was provided by the Ludwig Institute for Cancer Research.

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Correspondence to Virgil L. Woods Jr or Don W. Cleveland.

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V.L.W. has an equity interest in ExSar Corporation.

Supplementary information

Supplementary Figure S1

Replacing particular CENP-A residues with the corresponding residues from histone H3 disrupts centromere targeting. (PDF 525 kb)

Supplementary Figure S2

A model for the epigenetic maintenance of centromere identity via specialized, CENP-A-containing chromatin. (PDF 159 kb)

Supplementary Figure Legends

Legends for Supplementary Figs S1 and S2. (PDF 47 kb)

Supplementary Methods

A detailed description of protein fragmentation mapping, deuterium exchange, and analysis of exchange profiles. (PDF 66 kb)

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Black, B., Foltz, D., Chakravarthy, S. et al. Structural determinants for generating centromeric chromatin. Nature 430, 578–582 (2004).

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