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Vol. 20, Issue 19, 4194-4204, October 1, 2009

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Hierarchical Inactivation of a Synthetic Human Kinetochore by a Chromatin Modifier

Stefano Cardinale^*,,, Jan H. Bergmann^*,, David Kelly^*, Megumi Nakano^,||, Manuel M. Valdivia^¶, Hiroshi Kimura^#, Hiroshi Masumoto^||, Vladimir Larionov, and William C. Earnshaw^*

*Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh EH9 3JR, United Kingdom; Laboratory of Molecular Pharmacology, National Cancer Institute, Bethesda, MD 20892; ^¶Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias, Universidad de Cádiz, 11510 Puerto Real, Cádiz, Spain; ^#Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka 565-0871, Japan; ^||Laboratory of Cell Engineering, Department of Human Genome Research, Kazusa DNA Research Institute, Kisarazu, Chiba 292-0818, Japan

Submitted June 16, 2009; Revised July 15, 2009; Accepted July 23, 2009
Monitoring Editor: Yixian Zheng

We previously used a human artificial chromosome (HAC) with a synthetic kinetochore that could be targeted with chromatin modifiers fused to tetracycline repressor to show that targeting of the transcriptional repressor tTS within kinetochore chromatin disrupts kinetochore structure and function. Here we show that the transcriptional corepressor KAP1, a downstream effector of the tTS, can also inactivate the kinetochore. The disruption of kinetochore structure by KAP1 subdomains does not simply result from loss of centromeric CENP-A nucleosomes. Instead it reflects a hierarchical disruption of the outer kinetochore, with CENP-C levels falling before CENP-A levels and, in certain instances, CENP-H being lost more readily than CENP-C. These results suggest that this novel approach to kinetochore dissection may reveal new patterns of protein interactions within the kinetochore.

These authors contributed equally to this work.

Present address: Lawrence Berkeley National Laboratory, 1 Cyclotron Road, Mailstop Stanley 922, Berkeley, CA 94720.

Address correspondence to: William C. Earnshaw (bill.earnshaw{at}ed.ac.uk).