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In vitro centromere and kinetochore assembly on defined chromatin templates


During cell division, chromosomes are segregated to nascent daughter cells by attaching to the microtubules of the mitotic spindle through the kinetochore. Kinetochores are assembled on a specialized chromatin domain called the centromere, which is characterized by the replacement of nucleosomal histone H3 with the histone H3 variant centromere protein A (CENP-A). CENP-A is essential for centromere and kinetochore formation in all eukaryotes but it is unknown how CENP-A chromatin directs centromere and kinetochore assembly1. Here we generate synthetic CENP-A chromatin that recapitulates essential steps of centromere and kinetochore assembly in vitro. We show that reconstituted CENP-A chromatin when added to cell-free extracts is sufficient for the assembly of centromere and kinetochore proteins, microtubule binding and stabilization, and mitotic checkpoint function. Using chromatin assembled from histone H3/CENP-A chimaeras, we demonstrate that the conserved carboxy terminus of CENP-A is necessary and sufficient for centromere and kinetochore protein recruitment and function but that the CENP-A targeting domain—required for new CENP-A histone assembly2—is not. These data show that two of the primary requirements for accurate chromosome segregation, the assembly of the kinetochore and the propagation of CENP-A chromatin, are specified by different elements in the CENP-A histone. Our unique cell-free system enables complete control and manipulation of the chromatin substrate and thus presents a powerful tool to study centromere and kinetochore assembly.

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Figure 1: Reconstituted CENP-A chromatin supports centromere assembly in Xenopus egg extracts.
Figure 2: CENP-A chromatin specifically recruits kinetochore proteins as a response to a mimic of kinetochore detachment from microtubules.
Figure 3: Kinetochores assembled on reconstituted CENP-A chromatin bind microtubules and generate a mitotic checkpoint signal.
Figure 4: The CENP-A C terminus is required for centromere and kinetochore assembly in Xenopus egg extract.

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  1. Cheeseman, I. M. & Desai, A. Molecular architecture of the kinetochore-microtubule interface. Nature Rev. Mol. Cell Biol. 9, 33–46 (2008)

    CAS  Article  Google Scholar 

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

    ADS  CAS  Article  Google Scholar 

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

    CAS  Article  Google Scholar 

  4. 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)

    ADS  CAS  Article  Google Scholar 

  5. 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)

    CAS  Article  Google Scholar 

  6. 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)

    CAS  Article  Google Scholar 

  7. Dunleavy, E. M. et al. HJURP is a cell-cycle-dependent maintenance and deposition factor of CENP-A at centromeres. Cell 137, 485–497 (2009)

    CAS  Article  Google Scholar 

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

    CAS  Article  Google Scholar 

  9. Hu, H. et al. Structure of a CENP-A-histone H4 heterodimer in complex with chaperone HJURP. Genes Dev. 25, 901–906 (2011)

    CAS  Article  Google Scholar 

  10. Tachiwana, H. et al. Crystal structure of the human centromeric nucleosome containing CENP-A. Nature 10.1038/nature10258 (10 July, 2011)

  11. Blower, M. D., Sullivan, B. A. & Karpen, G. H. Conserved organization of centromeric chromatin in flies and humans. Dev. Cell 2, 319–330 (2002)

    CAS  Article  Google Scholar 

  12. Ribeiro, S. et al. A super-resolution map of the vertebrate kinetochore. Proc. Natl Acad. Sci. USA 107, 10484–10489 (2010)

    ADS  CAS  Article  Google Scholar 

  13. Zinkowski, R. P., Meyne, J. & Brinkley, B. R. The centromere-kinetochore complex: a repeat subunit model. J. Cell Biol. 113, 1091–1110 (1991)

    CAS  Article  Google Scholar 

  14. Huynh, V., Robinson, P. & Rhodes, D. A method for the in vitro reconstitution of a defined “30 nm” chromatin fibre containing stoichiometric amounts of the linker histone. J. Mol. Biol. 345, 957–968 (2005)

    CAS  Article  Google Scholar 

  15. Luger, K., Rechsteiner, T. J., Flaus, A. J., Waye, M. M. & Richmond, T. J. Characterization of nucleosome core particles containing histone proteins made in bacteria. J. Mol. Biol. 272, 301–311 (1997)

    CAS  Article  Google Scholar 

  16. Desai, A., Murray, A., Mitchison, T. J. & Walczak, C. E. The use of Xenopus egg extracts to study mitotic spindle assembly and function in vitro . Methods Cell Biol. 61, 385–412 (1999)

    CAS  Article  Google Scholar 

  17. Hori, T. et al. CCAN makes multiple contacts with centromeric DNA to provide distinct pathways to the outer kinetochore. Cell 135, 1039–1052 (2008)

    CAS  Article  Google Scholar 

  18. Kawashima, S. A., Yamagishi, Y., Honda, T., Ishiguro, K.-i. & Watanabe, Y. Phosphorylation of H2A by Bub1 prevents chromosomal instability through localizing shugoshin. Science 327, 172–177 (2010)

    ADS  CAS  Article  Google Scholar 

  19. Kelly, A. E. et al. Survivin reads phosphorylated histone H3 threonine 3 to activate the mitotic kinase Aurora B. Science 330, 235–239 (2010)

    ADS  CAS  Article  Google Scholar 

  20. Wang, F. et al. Histone H3 Thr-3 phosphorylation by Haspin positions Aurora B at centromeres in mitosis. Science 330, 231–235 (2010)

    ADS  CAS  Article  Google Scholar 

  21. Budde, P. P., Kumagai, A., Dunphy, W. G. & Heald, R. Regulation of Op18 during spindle assembly in Xenopus egg extracts. J. Cell Biol. 153, 149–158 (2001)

    CAS  Article  Google Scholar 

  22. Blow, J. J. & Laskey, R. A. Initiation of DNA replication in nuclei and purified DNA by a cell-free extract of Xenopus eggs. Cell 47, 577–587 (1986)

    CAS  Article  Google Scholar 

  23. Minshull, J., Sun, H., Tonks, N. K. & Murray, A. W. A MAP kinase-dependent spindle assembly checkpoint in Xenopus egg extracts. Cell 79, 475–486 (1994)

    CAS  Article  Google Scholar 

  24. Sawin, K. E. & Mitchison, T. J. Mitotic spindle assembly by two different pathways in vitro . J. Cell Biol. 112, 925–940 (1991)

    CAS  Article  Google Scholar 

  25. Heald, R. et al. Self-organization of microtubules into bipolar spindles around artificial chromosomes in Xenopus egg extracts. Nature 382, 420–425 (1996)

    ADS  CAS  Article  Google Scholar 

  26. Nicklas, R. B., Ward, S. C. & Gorbsky, G. J. Kinetochore chemistry is sensitive to tension and may link mitotic forces to a cell cycle checkpoint. J. Cell Biol. 130, 929–939 (1995)

    CAS  Article  Google Scholar 

  27. Rieder, C. L., Cole, R. W., Khodjakov, A. & Sluder, G. The checkpoint delaying anaphase in response to chromosome monoorientation is mediated by an inhibitory signal produced by unattached kinetochores. J. Cell Biol. 130, 941–948 (1995)

    CAS  Article  Google Scholar 

  28. Milks, K. J., Moree, B. & Straight, A. F. Dissection of CENP-C-directed centromere and kinetochore assembly. Mol. Biol. Cell 20, 4246–4255 (2009)

    CAS  Article  Google Scholar 

  29. Foltz, D. R. et al. The human CENP-A centromeric nucleosome-associated complex. Nature Cell Biol. 8, 458–469 (2006)

    CAS  Article  Google Scholar 

  30. McClelland, S. E. et al. The CENP-A NAC/CAD kinetochore complex controls chromosome congression and spindle bipolarity. EMBO J. 26, 5033–5047 (2007)

    CAS  Article  Google Scholar 

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

    CAS  Article  Google Scholar 

  32. 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)

    CAS  Article  Google Scholar 

  33. Murray, A. W. Cell cycle extracts. Methods Cell Biol. 36, 581–605 (1991)

    CAS  Article  Google Scholar 

  34. Kim, S., Song, E., Lee, K. & Ferrell, J., Jr Multisite M-phase phosphorylation of Xenopus Wee1A. Mol. Cell. Biol. 25, 10580–10590 (2005)

    CAS  Article  Google Scholar 

  35. Hannak, E. & Heald, R. Investigating mitotic spindle assembly and function in vitro using Xenopus laevis egg extracts. Nature Protocols 1, 2305–2314 (2006)

    CAS  Article  Google Scholar 

  36. Field, C. M., Oegema, K., Zheng, Y., Mitchison, T. J. & Walczak, C. E. Purification of cytoskeletal proteins using peptide antibodies. Methods Enzymol. 298, 525–541 (1998)

    CAS  Article  Google Scholar 

  37. Osborne, J. W. Best Practices in Quantitative Methods (Sage Publications, 2008)

    Book  Google Scholar 

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The authors would like to thank A.F.S. laboratory members for support and comments, J. E. Ferrell, A. Murray, R.-H. Chen, G. Kops and P. T. Stukenberg for providing antibodies. D. Rhodes, P. Robinson, K. Luger, J. Hansen, G. Narlikar and J. Yang for providing reagents and advice. A.G. was supported by a postdoctoral fellowship from the German Research Foundation (DFG). C.W.C. was supported by a postdoctoral fellowship from the Helen Hay Whitney Foundation and the American Heart Association (AHA). B.M. was supported by T32GM007276, C.J.F. was supported by a Stanford Graduate Fellowship and this work was supported by National Institutes of Health (NIH) R01GM074728 to A.F.S.

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Authors and Affiliations



A.G. and A.F.S. designed the experiments and wrote the manuscript. A.G. performed all the experiments. C.W.C. purified the CENP-A/H3 chimaeras and assembled arrays containing chimaeric proteins, analysed Xenopus cenp-n binding to human CENP-A mononucleosomes and provided advice. B.M. generated Xenopus centromere protein antibodies and C.J.F. designed and wrote the image analysis software for quantitative analysis.

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Correspondence to Aaron F. Straight.

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The authors declare no competing financial interests.

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Guse, A., Carroll, C., Moree, B. et al. In vitro centromere and kinetochore assembly on defined chromatin templates. Nature 477, 354–358 (2011).

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