Crystal structure of the human centromeric nucleosome containing CENP-A

Abstract

In eukaryotes, accurate chromosome segregation during mitosis and meiosis is coordinated by kinetochores, which are unique chromosomal sites for microtubule attachment1,2. Centromeres specify the kinetochore formation sites on individual chromosomes, and are epigenetically marked by the assembly of nucleosomes containing the centromere-specific histone H3 variant, CENP-A3,4,5,6,7,8,9,10,11,12. Although the underlying mechanism is unclear, centromere inheritance is probably dictated by the architecture of the centromeric nucleosome. Here we report the crystal structure of the human centromeric nucleosome containing CENP-A and its cognate α-satellite DNA derivative (147 base pairs). In the human CENP-A nucleosome, the DNA is wrapped around the histone octamer, consisting of two each of histones H2A, H2B, H4 and CENP-A, in a left-handed orientation. However, unlike the canonical H3 nucleosome, only the central 121 base pairs of the DNA are visible. The thirteen base pairs from both ends of the DNA are invisible in the crystal structure, and the αN helix of CENP-A is shorter than that of H3, which is known to be important for the orientation of the DNA ends in the canonical H3 nucleosome13. A structural comparison of the CENP-A and H3 nucleosomes revealed that CENP-A contains two extra amino acid residues (Arg 80 and Gly 81) in the loop 1 region, which is completely exposed to the solvent. Mutations of the CENP-A loop 1 residues reduced CENP-A retention at the centromeres in human cells. Therefore, the CENP-A loop 1 may function in stabilizing the centromeric chromatin containing CENP-A, possibly by providing a binding site for trans-acting factors. The structure provides the first atomic-resolution picture of the centromere-specific nucleosome.

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Figure 1: Crystal structure of the human CENP-A nucleosome.
Figure 2: Structure of the DNA entrance and exit of the human CENP-A nucleosome.
Figure 3: Structural differences in the loop 1 regions between CENP-A and H3.
Figure 4: Less stable association of CENP-A(del) with the centromere.

Accession codes

Primary accessions

Protein Data Bank

Data deposits

The atomic coordinates of the CENP-A nucleosome have been deposited in the Protein Data Bank, under the accession code 3AN2.

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Acknowledgements

We thank the beamline scientists, N. Shimizu, Y. Kawano, M. Makino and T. Hikima, for their assistance with data collection at the BL41XU and BL45XU beamlines of SPring-8. We also thank R. Matsumoto for technical assistance, K. Yoda for anti-CENP-C, and T. Fukagawa and Y. Hiraoka for discussions. This work was supported in part by Grants-in-Aid from the Japanese Society for the Promotion of Science (JSPS), and the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan. H.Ku. was also supported by the Waseda Research Institute for Science and Engineering.

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H.T., T.S., A.O. and Y.M. purified the histones and CENP-A, crystallized the CENP-A nucleosome, and performed biochemical analyses. H.T., W.K., K.S. and T.S. collected X-ray diffraction data, and H.T., W.K., and S.-Y.P. performed the structural analysis of the CENP-A nucleosome. H.T., A.O., Y.H.-T. and H.Ki. performed the cell biological experiments. T.O., H.T., W.K. and M.S. performed SAXS analysis. H.Ku. conceived, designed and supervised all of the work, and H.Ku., W.K. and H.T. wrote the paper. All of the authors discussed the results and commented on the manuscript.

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Correspondence to Hitoshi Kurumizaka.

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Tachiwana, H., Kagawa, W., Shiga, T. et al. Crystal structure of the human centromeric nucleosome containing CENP-A. Nature 476, 232–235 (2011). https://doi.org/10.1038/nature10258

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