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.

  • Article
  • Published:

The octamer is the major form of CENP-A nucleosomes at human centromeres

Abstract

The centromere is the chromosomal locus that ensures fidelity in genome transmission at cell division. Centromere protein A (CENP-A) is a histone H3 variant that specifies centromere location independently of DNA sequence. Conflicting evidence has emerged regarding the histone composition and stoichiometry of CENP-A nucleosomes. Here we show that the predominant form of the CENP-A particle at human centromeres is an octameric nucleosome. CENP-A nucleosomes are very highly phased on α-satellite 171-base-pair monomers at normal centromeres and also display strong positioning at neocentromeres. At either type of functional centromere, CENP-A nucleosomes exhibit similar DNA-wrapping behavior, as do octameric CENP-A nucleosomes reconstituted with recombinant components, having looser DNA termini than those on conventional nucleosomes containing canonical histone H3. Thus, the fundamental unit of the chromatin that epigenetically specifies centromere location in mammals is an octameric nucleosome with loose termini.

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: Structure-based predictions for MNase protection and experimental outcomes with CENP-A–containing particles assembled with recombinant components.
Figure 2: Nuclease digestion of native CENP-A–containing particles resembles that of octameric nucleosomes with loose termini.
Figure 3: The three size classes of CENP-A nucleosomes localize to the same prominent positions on neocentromeres.
Figure 4: CENP-A nucleosomes on the repetitive α-satellite DNA of normal centromeres have a tripartite distribution of nuclease-protected DNA fragments.
Figure 5: Terminally unwrapped CENP-A nucleosomes and their conventional counterparts with wrapped termini are similarly phased at normal centromeres.
Figure 6: Phasing of CENP-A nucleosomes at annotated regions of α-satellite DNA from the X and Y chromosomes.
Figure 7: CENP-A nucleosomes are less phased and gain symmetric MNase digestion on the Y-chromosome centromere that lacks functional CENP-B boxes.

Similar content being viewed by others

Accession codes

Primary accessions

Gene Expression Omnibus

Referenced accessions

Protein Data Bank

References

  1. Cleveland, D.W., Mao, Y. & Sullivan, K.F. Centromeres and kinetochores: from epigenetics to mitotic checkpoint signaling. Cell 112, 407–421 (2003).

    Article  CAS  Google Scholar 

  2. Mendiburo, M.J., Padeken, J., Fülöp, S., Schepers, A. & Heun, P. Drosophila CENH3 is sufficient for centromere formation. Science 334, 686–690 (2011).

    Article  CAS  Google Scholar 

  3. Barnhart, M.C. et al. HJURP is a CENP-A chromatin assembly factor sufficient to form a functional de novo kinetochore. J. Cell Biol. 194, 229–243 (2011).

    Article  CAS  Google Scholar 

  4. Guse, A., Carroll, C.W., Moree, B., Fuller, C.J. & Straight, A.F. In vitro centromere and kinetochore assembly on defined chromatin templates. Nature 477, 354–358 (2011).

    Article  CAS  Google Scholar 

  5. Warburton, P.E. Chromosomal dynamics of human neocentromere formation. Chromosome Res. 12, 617–626 (2004).

    Article  CAS  Google Scholar 

  6. Amor, D.J. et al. Human centromere repositioning 'in progress'. Proc. Natl. Acad. Sci. USA 101, 6542–6547 (2004).

    Article  CAS  Google Scholar 

  7. 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 

  8. Alonso, A. et al. Co-localization of CENP-C and CENP-H to discontinuous domains of CENP-A chromatin at human neocentromeres. Genome Biol. 8, R148 (2007).

    Article  Google Scholar 

  9. Alonso, A. et al. Genomic microarray analysis reveals distinct locations for the CENP-A binding domains in three human chromosome 13q32 neocentromeres. Hum. Mol. Genet. 12, 2711–2721 (2003).

    Article  CAS  Google Scholar 

  10. Hasson, D. et al. Formation of novel CENP-A domains on tandem repetitive DNA and across chromosome breakpoints on human chromosome 8q21 neocentromeres. Chromosoma 120, 621–632 (2011).

    Article  Google Scholar 

  11. Dechassa, M.L. et al. Structure and Scm3-mediated assembly of budding yeast centromeric nucleosomes. Nat. Commun. 2, 313 (2011).

    Article  Google Scholar 

  12. Kingston, I.J., Yung, J.S.Y. & Singleton, M.R. Biophysical characterization of the centromere-specific nucleosome from budding yeast. J. Biol. Chem. 286, 4021–4026 (2011).

    Article  CAS  Google Scholar 

  13. Panchenko, T. et al. Replacement of histone H3 with CENP-A directs global nucleosome array condensation and loosening of nucleosome superhelical termini. Proc. Natl. Acad. Sci. USA 108, 16588–16593 (2011).

    Article  CAS  Google Scholar 

  14. Sekulic, N., Bassett, E.A., Rogers, D.J. & Black, B.E. The structure of (CENP-A-H4)2 reveals physical features that mark centromeres. Nature 467, 347–351 (2010).

    Article  CAS  Google Scholar 

  15. Tachiwana, H. et al. Crystal structure of the human centromeric nucleosome containing CENP-A. Nature 476, 232–235 (2011).

    Article  CAS  Google Scholar 

  16. 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 

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

    Article  CAS  Google Scholar 

  18. Black, B.E. & Cleveland, D.W. Epigenetic centromere propagation and the nature of CENP-A nucleosomes. Cell 144, 471–479 (2011).

    Article  CAS  Google Scholar 

  19. 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 

  20. 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 

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

    Article  CAS  Google Scholar 

  22. Bui, M. et al. Cell-cycle-dependent structural transitions in the human CENP-A nucleosome in vivo. Cell 150, 317–326 (2012).

    Article  CAS  Google Scholar 

  23. Shivaraju, M. et al. Cell-cycle-coupled structural oscillation of centromeric nucleosomes in yeast. Cell 150, 304–316 (2012).

    Article  CAS  Google Scholar 

  24. 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 

  25. 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. Nat. Cell Biol. 11, 896–902 (2009).

    Article  CAS  Google Scholar 

  26. Carroll, C.W., Milks, K.J. & Straight, A.F. Dual recognition of CENP-A nucleosomes is required for centromere assembly. J. Cell Biol. 189, 1143–1155 (2010).

    Article  CAS  Google Scholar 

  27. 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  CAS  Google Scholar 

  28. Shaw, B.R., Herman, T.M., Kovacic, R.T., Beaudreau, G.S. & Van Holde, K.E. Analysis of subunit organization in chicken erythrocyte chromatin. Proc. Natl. Acad. Sci. USA 73, 505–509 (1976).

    Article  CAS  Google Scholar 

  29. Axel, R. Cleavage of DNA in nuclei and chromatin with staphylococcal nuclease. Biochemistry 14, 2921–2925 (1975).

    Article  CAS  Google Scholar 

  30. Decanniere, K., Babu, A.M., Sandman, K., Reeve, J.N. & Heinemann, U. Crystal structures of recombinant histones HMfA and HMfB from the hyperthermophilic archaeon Methanothermus fervidus. J. Mol. Biol. 303, 35–47 (2000).

    Article  CAS  Google Scholar 

  31. Dong, F. & Van Holde, K.E. Nucleosome positioning is determined by the (H3–H4)2 tetramer. Proc. Natl. Acad. Sci. USA 88, 10596–10600 (1991).

    Article  CAS  Google Scholar 

  32. Read, C.M., Baldwin, J.P. & Crane-Robinson, C. Structure of subnucleosomal particles. tetrameric (H3/H4)2 146 base pair DNA and hexameric (H3/H4)2(H2A/H2B)1 146 base pair DNA complexes. Biochemistry 24, 4435–4450 (1985).

    Article  CAS  Google Scholar 

  33. Park, P.J. ChIP-seq: advantages and challenges of a maturing technology. Nat. Rev. Genet. 10, 669–680 (2009).

    Article  CAS  Google Scholar 

  34. Zhang, Z. & Pugh, B.F. High-resolution genome-wide mapping of the primary structure of chromatin. Cell 144, 175–186 (2011).

    Article  CAS  Google Scholar 

  35. Willard, H.F. & Waye, J.S. Hierarchical order in chromosome-specific human alpha satellite DNA. Trends Genet. 3, 192–198 (1987).

    Article  CAS  Google Scholar 

  36. Vafa, O. & Sullivan, K.F. Chromatin containing CENP-A and α-satellite DNA is a major component of the inner kinetochore plate. Curr. Biol. 7, 897–900 (1997).

    Article  CAS  Google Scholar 

  37. Masumoto, H., Masukata, H., Muro, Y., Nozaki, N. & Okazaki, T. A human centromere antigen (CENP-B) interacts with a short specific sequence in alphoid DNA, a human centromeric satellite. J. Cell Biol. 109, 1963–1973 (1989).

    Article  CAS  Google Scholar 

  38. Rudd, M.K. & Willard, H.F. Analysis of the centromeric regions of the human genome assembly. Trends Genet. 20, 529–533 (2004).

    Article  CAS  Google Scholar 

  39. Waye, J.S. & Willard, H.F. Chromosome-specific alpha satellite DNA: nucleotide sequence analysis of the 2.0 kilobasepair repeat from the human X chromosome. Nucleic Acids Res. 13, 2731–2743 (1985).

    Article  CAS  Google Scholar 

  40. Warburton, P.E. Epigenetic analysis of kinetochore assembly on variant human centromeres. Trends Genet. 17, 243–247 (2001).

    Article  CAS  Google Scholar 

  41. Earnshaw, W.C. et al. Molecular cloning of cDNA for CENP-B, the major human centromere autoantigen. J. Cell Biol. 104, 817–829 (1987).

    Article  CAS  Google Scholar 

  42. Zhang, W., Colmenares, S.U. & Karpen, G.H. Assembly of Drosophila centromeric nucleosomes requires CID dimerization. Mol. Cell 45, 263–269 (2012).

    Article  CAS  Google Scholar 

  43. Bassett, E.M. et al. HJURP uses distinct CENP-A surfaces to recognize and to stabilize CENP-A/histone H4 for centromere assembly. Dev. Cell 22, 749–762 (2012).

    Article  CAS  Google Scholar 

  44. Miell, M.A. et al. CENP-A confers a reduction in height on octameric nucleosomes. Nat. Struct. Mol. Biol. advance online publication, doi:10.1038/nsmb.2574 (5 May 2013).10.1038/nsmb.2574

  45. Tanaka, Y. et al. Crystal structure of the CENP-B protein-DNA complex: the DNA-binding domains of CENP-B induce kinks in the CENP-B box DNA. EMBO J. 20, 6612–6618 (2001).

    Article  CAS  Google Scholar 

  46. 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 

  47. Luger, K., Rechsteiner, T.J. & Richmond, T.J. Preparation of nucleosome core particle from recombinant histones. Methods Enzymol. 304, 3–19 (1999).

    Article  CAS  Google Scholar 

  48. Lowary, P.T. & Widom, J. New DNA sequence rules for high affinity binding to histone octamer and sequence-directed nucleosome positioning. J. Mol. Biol. 276, 19–42 (1998).

    Article  CAS  Google Scholar 

  49. Huynh, V.A.T., Robinson, P.J.J. & Rhodes, D. A method for the in vitro reconstitution of a defined '30nm' chromatin fibre containing stoichiometric amounts of the linker histone. J. Mol. Biol. 345, 957–968 (2005).

    Article  CAS  Google Scholar 

  50. Brünger, A.T. et al. Crystallography & NMR system: A new software suite for macromolecular structure determination. Acta Crystallogr. D Biol. Crystallogr. 54, 905–921 (1998).

    Article  Google Scholar 

  51. Brunger, A.T. Version 1.2 of the crystallography and NMR system. Nat. Protoc. 2, 2728–2733 (2007).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank O. Jabado (Icahn School of Medicine at Mount Sinai, New York, New York, USA) for help with Illumina sequencing, T. Patel (University of Pennsylvania, Philadelphia, Pennsylvania, USA (UPenn)) and B. Cole (UPenn) for advice on data analysis, D. Rogers (UPenn) and B. Gregory (UPenn) for advice, E. Bernstein (Icahn School of Medicine at Mount Sinai, New York, New York, USA) for mentoring and advising D.H., M. Lampson (UPenn) for comments on the manuscript, K. Luger (Colorado State University, Fort Collins, Colorado, USA), D. Cleveland (University of California, San Diego, La Jolla, California, USA), A. Straight (Stanford University, Stanford, California, USA) and D. Rhodes (Medical Research Council Laboratory of Molecular Biology, Cambridge, UK) for plasmids, A. Choo (Murdoch Children's Research Institute, Victoria, Australia) for the cell line containing the PD-NC4 chromosome and R. Allshire (University of Edinburgh, Edinburgh, Scotland, UK) for sharing results before publication. This work was supported by US National Institutes of Health research grant GM082989 (B.E.B.), a Career Award in the Biomedical Sciences from the Burroughs Wellcome Fund (B.E.B.), a Rita Allen Foundation Scholar Award (B.E.B.), a predoctoral fellowship from the American Heart Association (K.J.S.) and a postdoctoral fellowship from the American Cancer Society (N.S.). T.P. acknowledges support from US National Institutes of Health grant GM08275 (UPenn Structural Biology Training Grant).

Author information

Authors and Affiliations

Authors

Contributions

D.H. designed and performed experiments and analyzed data. K.J.S. and T.P. designed and performed experiments, developed new analytical tools, analyzed data and wrote the manuscript. M.U.S. developed new analytical tools. N.S. analyzed data and modeled nucleosomes. A.A. performed experiments and provided technical advice on ChIP experiments. P.E.W. directed the project, designed experiments and analyzed data. B.E.B. directed the project, designed experiments, analyzed 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 Text and Figures

Supplementary Figures 1–7 and Supplementary Tables 1 and 2 (PDF 6276 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Hasson, D., Panchenko, T., Salimian, K. et al. The octamer is the major form of CENP-A nucleosomes at human centromeres. Nat Struct Mol Biol 20, 687–695 (2013). https://doi.org/10.1038/nsmb.2562

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb.2562

This article is cited by

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