Induced ectopic kinetochore assembly bypasses the requirement for CENP-A nucleosomes

Cell. 2011 Apr 29;145(3):410-22. doi: 10.1016/j.cell.2011.03.031.

Karen E Gascoigne ¹, Kozo Takeuchi, Aussie Suzuki, Tetsuya Hori, Tatsuo Fukagawa, Iain M Cheeseman

Affiliations

Affiliation

1 Whitehead Institute for Biomedical Research, Department of Biology, Nine Cambridge Center, MA 02142, USA.

PMID: 21529714 DOI: 10.1016/j.cell.2011.03.031

Abstract

Accurate chromosome segregation requires assembly of the multiprotein kinetochore complex at centromeres. Although prior work identified the centromeric histone H3-variant CENP-A as the important upstream factor necessary for centromere specification, in human cells CENP-A is not sufficient for kinetochore assembly. Here, we demonstrate that two constitutive DNA-binding kinetochore components, CENP-C and CENP-T, function to direct kinetochore formation. Replacing the DNA-binding regions of CENP-C and CENP-T with alternate chromosome-targeting domains recruits these proteins to ectopic loci, resulting in CENP-A-independent kinetochore assembly. These ectopic kinetochore-like foci are functional based on the stoichiometric assembly of multiple kinetochore components, including the microtubule-binding KMN network, the presence of microtubule attachments, the microtubule-sensitive recruitment of the spindle checkpoint protein Mad2, and the segregation behavior of foci-containing chromosomes. We additionally find that CENP-T phosphorylation regulates the mitotic assembly of both endogenous and ectopic kinetochores. Thus, CENP-C and CENP-T form a critical regulated platform for vertebrate kinetochore assembly.

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