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Centromere assembly requires the direct recognition of CENP-A nucleosomes by CENP-N

Nature Cell Biology volume 11, pages 896–902 (2009)Cite this article

Abstract

Centromeres are specialized chromosomal domains that direct kinetochore assembly during mitosis. CENP-A (centromere protein A), a histone H3-variant present exclusively in centromeric nucleosomes, is thought to function as an epigenetic mark that specifies centromere identity. Here we identify the essential centromere protein CENP-N as the first protein to selectively bind CENP-A nucleosomes but not H3 nucleosomes. CENP-N bound CENP-A nucleosomes in a DNA sequence-independent manner, but did not bind soluble CENP-A–H4 tetramers. Mutations in CENP-N that reduced its affinity for CENP-A nucleosomes caused defects in CENP-N localization and had dominant effects on the recruitment of CENP-H, CENP-I and CENP-K to centromeres. Depletion of CENP-N using siRNA (short interfering RNA) led to similar centromere assembly defects and resulted in reduced assembly of nascent CENP-A into centromeric chromatin. These data suggest that CENP-N interprets the information encoded within CENP-A nucleosomes and recruits other proteins to centromeric chromatin that are required for centromere function and propagation.

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Figure 1: CENP-N binds CENP-A nucleosomes.
Figure 2: Identification and characterization of CENP-N mutants defective in CENP-A-nucleosome binding.
Figure 3: CENP-N mutants show centromere assembly defects.
Figure 4: Depletion of CENP-N affects centromere assembly.

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Acknowledgements

The authors would like to thank members of the Straight Lab for helpful comments and support, J. Minshull and DNA2.0 (CA, USA) for gene synthesis, S. H. Hanissian for the CENP-U(50) cDNA, J. Yang, G. Narlikar, M. Resch, K. Luger and J. Hansen for reagents and help with nucleosome reconstitution, S.-T. Liu for the CENP-H and CENP-I antibodies and D. Herschlag for advice. C.W.C. was supported by a postdoctoral fellowship from the Helen Hay Whitney Foundation. K.G. was supported by a predoctoral fellowship from the National Science Foundation and by a NIH grant (T32GM007276). A.F.S. is a Gordon Family Scholar supported by the Damon Runyon Cancer Research Foundation, and this work was supported by a NIH grant (R01GM074728). M.C.C.S. is supported by the Fundação para a Ciência e a Tecnologia (FCT; SFRH/BD33219/2007). LETJ is supported by the FCT, Fundação Calouste Gulbenkian and the EU Seventh Framework Programme.

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Authors and Affiliations

  1. Department of Biochemistry, Stanford University School of Medicine, Beckman Center, Room 409, 279 Campus Drive, Palo Alto, 94503-5307, CA, USA

    Christopher W. Carroll, Kristina M. Godek & Aaron F. Straight

  2. Laboratory for Epigenetic Mechanisms, Instituto Gulbenkian de Ciência, Rua da Quinta Grande, 6, Oeiras, 2780-156, Portugal

    Mariana C.C. Silva & Lars E.T. Jansen

  3. Department of Biochemistry, Stanford University School of Medicine, Beckman Center, Room 409, 279 Campus Drive, Palo Alto, 94503-5307, CA, USA

    Aaron F. Straight

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  1. Christopher W. Carroll
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  2. Mariana C.C. Silva
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  3. Kristina M. Godek
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  4. Lars E.T. Jansen
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Contributions

C.W.C. and A.F.S. designed the experiments and wrote the manuscript; C.W.C. performed all the experiments except those presented in Figure 4c, which were performed by M.C.C.S. and L.E.T.J.; and K.G. purified histones and helped with nucleosome assembly.

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Correspondence to Aaron F. Straight.

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Carroll, C., Silva, M., Godek, K. et al. Centromere assembly requires the direct recognition of CENP-A nucleosomes by CENP-N. Nat Cell Biol 11, 896–902 (2009). https://doi.org/10.1038/ncb1899

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