Abstract
CENP-A is an evolutionarily conserved, centromere-specific variant of histone H3 that is thought to play a central role in directing kinetochore assembly and in centromere function. Here, we have analyzed the consequences of disrupting the CENP-A gene in the chicken DT40 cell line. In CENP-A-depleted cells, kinetochore protein assembly is impaired, as indicated by mislocalization of the inner kinetochore proteins CENP-I, CENP-H, and CENP-C as well as the outer components Nuf2/Hec1, Mad2, and CENP-E. However, BubR1 and the inner centromere protein INCENP are efficiently recruited to kinetochores. Following CENP-A depletion, chromosomes are deficient in proper congression on the mitotic spindle and there is a transient delay in prometaphase. CENP-A-depleted cells further proceed through anaphase and cytokinesis with unequal chromosome segregation, suggesting that some kinetochore function remains following substantial depletion of CENP-A. We furthermore demonstrate that CENP-A-depleted cells exhibit a specific defect in maintaining kinetochore localization of the checkpoint protein BubR1 under conditions of checkpoint activation. Our data thus point to a specific role for CENP-A in assembly of kinetochores competent in the maintenance of mitotic checkpoint signaling.
ACKNOWLEDGMENTS
We are grateful to A. Abrieu for providing the anti-Xenopus CENP-E antibody and Gabriele Reinkensmeier for technical assistance with FACS analysis. We thank François-Xavier Barre, Chris-Tyler Smith, and Ed Southern for discussions and comments.
This work was supported by grants from Cancer Research UK, BBSRC, and the Wellcome Trust. V.R. was supported by fellowships from European Molecular Biology Organization and Ligue Nationale contre le Cancer, and W.C.E. is a Wellcome Trust Principal Research Fellow. The FACS machine was provided by the Wellcome trust.