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In vitro centromere and kinetochore assembly on defined chromatin templates

Nature volume 477, pages 354–358 (2011)Cite this article

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Abstract

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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Acknowledgements

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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  1. Department of Biochemistry, Stanford Medical School, Beckman 409A, Stanford, California 94305-5307, USA,

    Annika Guse, Christopher W. Carroll, Ben Moree, Colin J. Fuller & Aaron F. Straight

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  1. Annika Guse
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Contributions

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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Guse, A., Carroll, C., Moree, B. et al. In vitro centromere and kinetochore assembly on defined chromatin templates. Nature 477, 354–358 (2011). https://doi.org/10.1038/nature10379

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