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The CENP-H–I complex is required for the efficient incorporation of newly synthesized CENP-A into centromeres

Abstract

In vertebrates, centromeres lack defined sequences and are thought to be propagated by epigenetic mechanisms involving the incorporation of specialized nucleosomes containing the histone H3 variant centromere protein (CENP)-A. However, the precise mechanisms that target CENP-A to centromeres remain poorly understood. Here, we isolated a multi-subunit complex, which includes the established inner kinetochore components CENP-H and CENP-I, and nine other proteins, from both human and chicken cells. Our analysis of these proteins demonstrates that the CENP-H–I complex can be divided into three functional sub-complexes, each of which is required for faithful chromosome segregation. Interestingly, newly expressed CENP-A is not efficiently incorporated into centromeres in knockout mutants of a subclass of CENP-H–I complex proteins, indicating that the CENP-H–I complex may function, in part, as a marker directing CENP-A deposition to centromeres.

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Figure 1: The chicken CENP-H–I complex localizes constitutively to centromeres.
Figure 2: Purification of the CENP-H–I complex from human cells.
Figure 3: The CENP-H–I complex is essential for normal cell-cycle progression and faithful chromosome segregation.
Figure 4: Localization analyses define three distinct subgroups within the CENP-H–I complex.
Figure 5: Level of Hec1 in mutants for CENP-H–I complex associated proteins.
Figure 6: Endogenous CENP-A in mutants of the CENP-H–I complex associated proteins.
Figure 7: Newly synthesized CENP-A is not efficiently incorporated into centromeres in mutants for CENP-H–I complex associated proteins.
Figure 8: Model of a mechanism for newly synthesized CENP-A loading into centromeres.

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Acknowledgements

The authors are very grateful to M. Takahashi, K. Suzuki and K. Kita for technical assistance. We thank Y. Mikami and M. Kwon for providing materials and H. Kimura for histone H3–GFP plasmid. This work was supported by Grants-in-Aid for Scientific Research on Priority Areas 'Cancer Cell Biology' and 'Nuclear Dynamics' from the Ministry of Education, Science, Sports and Culture of Japan. This work was also supported by funding from the Ludwig Institute for Cancer Research to A.D., a National Institutes of Health grant to A.D. (R01GM074215–01). I.M.C. is a fellow of the Jane Coffin Childs Memorial Fund for Medical Research. A.D. is a Damon Runyon Scholar supported by the Damon Runyon Cancer Research Foundation (DRS 38–04).

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Correspondence to Tatsuo Fukagawa.

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Supplementary Figures S1, S2, S3, S4, S5, S6, S7, S8, S9 and S10 (PDF 4173 kb)

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Okada, M., Cheeseman, I., Hori, T. et al. The CENP-H–I complex is required for the efficient incorporation of newly synthesized CENP-A into centromeres. Nat Cell Biol 8, 446–457 (2006). https://doi.org/10.1038/ncb1396

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