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In vitro centromere and kinetochore assembly on defined chromatin templates

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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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.

Corresponding author

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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