Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Letter
  • Published:

The structure of (CENP-A–H4)2 reveals physical features that mark centromeres

Abstract

Centromeres are specified epigenetically, and the histone H3 variant CENP-A is assembled into the chromatin of all active centromeres1. Divergence from H3 raises the possibility that CENP-A generates unique chromatin features to mark physically centromere location. Here we report the crystal structure of a subnucleosomal heterotetramer, human (CENP-A–H4)2, that reveals three distinguishing properties encoded by the residues that comprise the CENP-A targeting domain (CATD; ref. 2): (1) a CENP-A–CENP-A interface that is substantially rotated relative to the H3–H3 interface; (2) a protruding loop L1 of the opposite charge as that on H3; and (3) strong hydrophobic contacts that rigidify the CENP-A–H4 interface. Residues involved in the CENP-A–CENP-A rotation are required for efficient incorporation into centromeric chromatin, indicating specificity for an unconventional nucleosome shape. DNA topological analysis indicates that CENP-A-containing nucleosomes are octameric with conventional left-handed DNA wrapping, in contrast to other recent proposals3,4,5,6. Our results indicate that CENP-A marks centromere location by restructuring the nucleosome from within its folded histone core.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Crystal structure of the (CENP-A–H4) 2 heterotetramer.
Figure 2: The residues involved in the rotated CENP-A–CENP-A interface are essential for centromere targeting.
Figure 3: Surface and internal structural features unique to CENP-A-containing complexes.
Figure 4: The (CENP-A–H4) 2 heterotetramer assembles with H2A–H2B dimers into an octameric nucleosome with conventional handedness of DNA wrapping.

Similar content being viewed by others

Accession codes

Primary accessions

Protein Data Bank

Data deposits

The structures of (CENP-A–H4)2 heterotetramers have been deposited in the Protein Data Bank under accession numbers 3NQJ and 3NQU.

References

  1. Black, B. E. & Bassett, E. A. The histone variant CENP-A and centromere specification. Curr. Opin. Cell Biol. 20, 91–100 (2008)

    Article  CAS  Google Scholar 

  2. Black, B. E. et al. Structural determinants for generating centromeric chromatin. Nature 430, 578–582 (2004)

    Article  ADS  CAS  Google Scholar 

  3. Mizuguchi, G., Xiao, H., Wisniewski, J., Smith, M. M. & Wu, C. Nonhistone Scm3 and histones CenH3–H4 assemble the core of centromere-specific nucleosomes. Cell 129, 1153–1164 (2007)

    Article  CAS  Google Scholar 

  4. Williams, J. S., Hayashi, T., Yanagida, M. & Russell, P. Fission yeast Scm3 mediates stable assembly of Cnp1/CENP-A into centromeric chromatin. Mol. Cell 33, 287–298 (2009)

    Article  CAS  Google Scholar 

  5. Dalal, Y., Wang, H., Lindsay, S. & Henikoff, S. Tetrameric structure of centromeric nucleosomes in interphase Drosophila cells. PLoS Biol. 5, e218 (2007)

    Article  Google Scholar 

  6. Furuyama, T. & Henikoff, S. Centromeric nucleosomes induce positive DNA supercoils. Cell 138, 104–113 (2009)

    Article  CAS  Google Scholar 

  7. Smith, M. M. Centromeres and variant histones: what, where, when and why? Curr. Opin. Cell Biol. 14, 279–285 (2002)

    Article  CAS  Google Scholar 

  8. Henikoff, S., Ahmad, K. & Malik, H. S. The centromere paradox: stable inheritance with rapidly evolving DNA. Science 293, 1098–1102 (2001)

    Article  CAS  Google Scholar 

  9. Black, B. E. et al. Centromere identity maintained by nucleosomes assembled with histone H3 containing the CENP-A targeting domain. Mol. Cell 25, 309–322 (2007)

    Article  CAS  Google Scholar 

  10. Erhardt, S. et al. Genome-wide analysis reveals a cell cycle-dependent mechanism controlling centromere propagation. J. Cell Biol. 183, 805–818 (2008)

    Article  CAS  Google Scholar 

  11. Shelby, R. D., Vafa, O. & Sullivan, K. F. Assembly of CENP-A into centromeric chromatin requires a cooperative array of nucleosomal DNA contact sites. J. Cell Biol. 136, 501–513 (1997)

    Article  CAS  Google Scholar 

  12. Camahort, R. et al. Cse4 is part of an octameric nucleosome in budding yeast. Mol. Cell 35, 794–805 (2009)

    Article  CAS  Google Scholar 

  13. Black, B. E., Brock, M. A., Bédard, S., Woods, V. L. & Cleveland, D. W. An epigenetic mark generated by the incorporation of CENP-A into centromeric nucleosomes. Proc. Natl Acad. Sci. USA 104, 5008–5013 (2007)

    Article  ADS  CAS  Google Scholar 

  14. Luger, K., Mäder, A. W., Richmond, R. K., Sargent, D. F. & Richmond, T. J. Crystal structure of the nucleosome core particle at 2.8 Å resolution. Nature 389, 251–260 (1997)

    Article  ADS  CAS  Google Scholar 

  15. Davey, C. A., Sargent, D. F., Luger, K., Maeder, A. W. & Richmond, T. J. Solvent mediated interactions in the structure of the nucleosome core particle at 1.9 Å resolution. J. Mol. Biol. 319, 1097–1113 (2002)

    Article  CAS  Google Scholar 

  16. Altaf, M. et al. Interplay of chromatin modifiers on a short basic patch of histone H4 tail defines the boundary of telomeric heterochromatin. Mol. Cell 28, 1002–1014 (2007)

    Article  CAS  Google Scholar 

  17. Lu, X. et al. The effect of H3K79 dimethylation and H4K20 trimethylation on nucleosome and chromatin structure. Nature Struct. Mol. Biol. 15, 1122–1124 (2008)

    Article  CAS  Google Scholar 

  18. Englander, S. W. Hydrogen exchange and mass spectrometry: a historical perspective. J. Am. Soc. Mass Spectrom. 17, 1481–1489 (2006)

    Article  CAS  Google Scholar 

  19. Lusser, A. & Kadonaga, J. T. Strategies for the reconstitution of chromatin. Nature Methods 1, 19–26 (2004)

    Article  CAS  Google Scholar 

  20. Carruthers, L. M., Tse, C., Walker, K. P. & Hansen, J. C. Assembly of defined nucleosomal and chromatin arrays from pure components. Methods Enzymol. 304, 19–35 (1999)

    Article  CAS  Google Scholar 

  21. Conde e Silva, N. et al. CENP-A-containing nucleosomes: easier disassembly versus exclusive centromeric localization. J. Mol. Biol. 370, 555–573 (2007)

    Article  CAS  Google Scholar 

  22. Simpson, R. T., Thoma, F. & Brubaker, J. M. Chromatin reconstituted from tandemly repeated cloned DNA fragments and core histones: a model system for study of higher order structure. Cell 42, 799–808 (1985)

    Article  CAS  Google Scholar 

  23. Esposito, F. & Sinden, R. R. Supercoiling in prokaryotic and eukaryotic DNA: changes in response to topological perturbation of plasmids in E. coli and SV40 in vitro, in nuclei and in CV-1 cells. Nucleic Acids Res. 15, 5105–5124 (1987)

    Article  CAS  Google Scholar 

  24. Shuaib, M., Ouararhni, K., Dimitrov, S. & Hamiche, A. HJURP binds CENP-A via a highly conserved N-terminal domain and mediates its deposition at centromeres. Proc. Natl Acad. Sci. USA 107, 1349–1354 (2010)

    Article  ADS  CAS  Google Scholar 

  25. Bina-Stein, M. & Simpson, R. T. Specific folding and contraction of DNA by histones H3 and H4. Cell 11, 609–618 (1977)

    Article  CAS  Google Scholar 

  26. Bassett, E. A. et al. Epigenetic centromere specification directs Aurora B accumulation but is insufficient to efficiently correct mitotic errors. J. Cell Biol. 190, 177–185 (2010)

    Article  CAS  Google Scholar 

  27. Vermaak, D., Hayden, H. S. & Henikoff, S. Centromere targeting element within the histone fold domain of Cid. Mol. Cell. Biol. 22, 7553–7561 (2002)

    Article  CAS  Google Scholar 

  28. Foltz, D. R. et al. Centromere-specific assembly of CENP-A nucleosomes is mediated by HJURP. Cell 137, 472–484 (2009)

    Article  CAS  Google Scholar 

  29. Carroll, C. W., Silva, M. C. C., Godek, K. M., Jansen, L. E. T. & Straight, A. F. Centromere assembly requires the direct recognition of CENP-A nucleosomes by CENP-N. Nature Cell Biol. 11, 896–902 (2009)

    Article  CAS  Google Scholar 

  30. Kabsch, W. Automatic processing of rotation diffraction data from crystals of initially unknown symmetry and cell constants. J. Appl. Crystallogr. 26, 795–800 (1993)

    Article  CAS  Google Scholar 

  31. McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007)

    Article  CAS  Google Scholar 

  32. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D 60, 2126–2132 (2004)

    Article  Google Scholar 

  33. Dodson, E. J., Winn, M. & Ralph, A. Collaborative Computational Project, number 4: providing programs for protein crystallography. Methods Enzymol. 277, 620–633 (1997)

    Article  CAS  Google Scholar 

  34. Langer, G., Cohen, S. X., Lamzin, V. S. & Perrakis, A. Automated macromolecular model building for X-ray crystallography using ARP/wARP version 7. Nature Protocols 3, 1171–1179 (2008)

    Article  CAS  Google Scholar 

  35. Konarev, P. V., Volkov, V. V., Sokolova, A. V., Koch, M. H. & Svergun, D. I. PRIMUS: a Windows PC-based system for small-angle scattering data analysis. J. Appl. Crystallogr. 36, 1277–1282 (2003)

    Article  CAS  Google Scholar 

  36. Svergun, D. I. Determination of the regularization parameter in indirect-transform methods using perceptual criteria. J. Appl. Crystallogr. 25, 495–503 (1992)

    Article  CAS  Google Scholar 

  37. Svergun, D., Barberato, C. & Koch, M. H. J. CRYSOL—a program to evaluate X-ray solution scattering of biological macromolecules from atomic coordinates. J. Appl. Crystallogr. 28, 768–773 (1995)

    Article  CAS  Google Scholar 

  38. Wriggers, W. Using Situs for the integration of multi-resolution structures. Biophys. Rev. 2, 21–27 (2010)

    Article  Google Scholar 

  39. Volkov, V. V. & Svergun, D. I. Uniqueness of ab initio shape determination in small-angle scattering. J. Appl. Crystallogr. 36, 860–864 (2003)

    Article  CAS  Google Scholar 

  40. Pettersen, E. F. et al. UCSF Chimera—A visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004)

    Article  CAS  Google Scholar 

  41. Fyodorov, D. V. & Kadonaga, J. T. Chromatin assembly in vitro with purified recombinant ACF and NAP-1. Methods Enzymol. 371, 499–515 (2003)

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank D. Cleveland for plasmids and steadfast encouragement to pursue a physical understanding of the centromere; S. Wood for generating cleaved histone H2A; K. Gupta for help with collecting data and technical suggestions; and K. Ferguson, G. Van Duyne, M. Lemmon, J. Shorter, L. Jansen, D. Foltz, J. Shah, D. Alvarado, K. Moravcevic and T. Panchenko for discussions and comments on the manuscript. This work was supported by the NIH research grant GM82989, a Career Award in the Biomedical Sciences from the Burroughs Wellcome Fund, and a Rita Allen Foundation Scholar Award to B.E.B. N.S. is supported by a postdoctoral fellowship from the American Cancer Society and E.A.B. has been supported by the Penn Structural Biology Training Grant (NIH GM08275) and a predoctoral fellowship from the American Heart Association.

Author information

Authors and Affiliations

Authors

Contributions

N.S. designed and performed experiments, solved and refined the structures, analysed data and wrote the manuscript; E.A.B. and D.J.R. performed experiments and analysed data; and B.E.B. directed the project, designed and performed experiments, analysed data and wrote the manuscript.

Corresponding author

Correspondence to Ben E. Black.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains Supplementary Table 1, Supplementary Figures 1-12 with legends and additional References. (PDF 5830 kb)

PowerPoint slides

Rights and permissions

Reprints and permissions

About this article

Cite this article

Sekulic, N., Bassett, E., Rogers, D. et al. The structure of (CENP-A–H4)2 reveals physical features that mark centromeres. Nature 467, 347–351 (2010). https://doi.org/10.1038/nature09323

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature09323

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing