The human CENP-A centromeric nucleosome-associated complex

Abstract

The basic element for chromosome inheritance, the centromere, is epigenetically determined in mammals. The prime candidate for specifying centromere identity is the array of nucleosomes assembled with CENP-A, the centromere-specific histone H3 variant. Here, we show that CENP-A nucleosomes directly recruit a proximal CENP-A nucleosome associated complex (NAC) comprised of three new human centromere proteins (CENP-M, CENP-N and CENP-T), along with CENP-U(50), CENP-C and CENP-H. Assembly of the CENP-A NAC at centromeres is dependent on CENP-M, CENP-N and CENP-T. Facilitates chromatin transcription (FACT) and nucleophosmin-1 (previously implicated in transcriptional chromatin remodelling and as a multifunctional nuclear chaperone, respectively) are absent from histone H3-containing nucleosomes, but are stably recruited to CENP-A nucleosomes independent of CENP-A NAC. Seven new CENP-A-nucleosome distal (CAD) centromere components (CENP-K, CENP-L, CENP-O, CENP-P, CENP-Q, CENP-R and CENP-S) are identified as assembling on the CENP-A NAC. The CENP-A NAC is essential, as disruption of the complex causes errors of chromosome alignment and segregation that preclude cell survival despite continued centromere-derived mitotic checkpoint signalling.

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Figure 1: Purification of CENP-A nucleosomes.
Figure 2: CENP-A dependent centromere localization of four CENP-A nucleosome associated proteins.
Figure 3: Purification of the CENP-A NAC and identification of its associated CAD complex components.
Figure 4: Assembly of the CENP-A NAC requires CENP-M and CENP-N.
Figure 5: CENP-U(50) depletion causes mitotic errors without affecting the mitotic checkpoint.
Figure 6: Mitotic defects due to CENP-A NAC disruption by depletion of CENP-M, CENP-N or CENP-T.

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Acknowledgements

The authors thank T. Fukagawa, I. Cheeseman, J. Shah, P. Maddox, K. Weis, F. Furnari and K. Yoda for generously providing reagents and assistance; D. Young and the University of California at San Diego (UCSD) Cancer Center for flow cytometry and the Oegema and Desai laboratorys for use of spinning disk confocal and deconvolution microscopes. This work has been supported by grants from the National Institutes of Health (NIH) to D.W.C. (GM 29513) and J.R.Y. (RR11823). D.R.F. has been supported by a postdoctoral fellowship from the NIH and B.E.B. has been supported by a postdoctoral fellowship from the American Cancer Society and in part by a Career Award in the Biomedical Sciences from the Burroughs Welcome Fund. D.W.C. receives salary support from the Ludwig Institute for Cancer Research.

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Correspondence to Don W. Cleveland.

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