News and Publications

Centromeres, Kinetochores, and Chromosome Dynamics in Human Cells

K. Monier, R. Shelby, A. Visser, S. Zeitlin, K. Sullivan

During each cell cycle, the genome is packaged for transport on the mitotic spindle, the replicated chromosomes are segregated, and the nucleus is reformed to resume its normal program of gene expression. We are interested in how chromosome segregation is accomplished in mitosis and in how the large-scale organization of the nucleus is set up in postmitotic cells.

Chromosome segregation during cell division is mediated by centromeres (one centromere per chromosome), the structures on chromosomes that direct formation of the microtubule-binding kinetochore at the surface of the chromosomes. In metazoan chromosomes, the position and molecular composition of centromeres appear to be determined largely through epigenetic mechanisms. We identified a unique process of chromatin assembly that is essential for replication of centromeres and that may provide a mechanistic basis for epigenetic specification of centromeres.

The chromatin that forms the molecular foundation for the assembly of kinetochores contains a unique type of nucleosome in which histone H3 is replaced by a centromere-specific H3 homolog, CENP-A. Normally, histone synthesis is tightly coupled to DNA replication during the S phase of the cell cycle. We used chromatin immunoprecipitation to determine the timing of replication of CENP-A--associated DNA during S phase. We found that kinetochores replicate in mid-to-late S phase, along with the bulk of centromeric DNA. CENP-A itself, however, is not synthesized until phase G₂, approximately 3 hours after peak rates of kinetochore DNA synthesis. In addition, experiments indicated that CENP-A can assemble into kinetochores in the complete absence of DNA replication. This unique pathway leads to the hypothesis that distinctive chromatin assembly factors are essential for the mechanism of epigenetic propagation of centromeres.

In preparation for mitosis, histone H3 is phosphorylated. In the past year, we showed that CENP-A is also phosphorylated as cells enter mitosis. Using immunocytochemical analysis, we showed that phosphorylation of CENP-A is a discrete biochemical step that occurs after phosphorylation of histone H3 is completed. Along with the high degree of spatial organization of H3 phosphorylation, these results indicate that a tightly regulated sequence of events leads to the complete mitotic phosphorylation of the H3 family of proteins.

Mutational analysis of the mitotic CENP-A phosphorylation site revealed a highly specific role for this modification. Surprisingly, this function is related to the last step in mitosis--cytokinesis--which occurs after kinetochore-dependent chromosome segregation. Characterization of the mechanism of the CENP-A--dependent cytokinesis defect is ongoing, but initial results suggest that CENP-A chromatin plays a role in directing the subcellular localization of enzymes required for completion of cytokinesis. These results indicate an unexpected connection between chromosome movement, kinetochore structure, and mitotic exit in human cells.

PUBLICATIONS

Lomonte, P., Sullivan, K.F., Everett, R.D. Degradation of nucleosome-associated centromeric histone H3-like protein CENP-A induced by herpes simplex virus type 1 protein ICP0. J. Biol. Chem. 276:5829, 2001.

Monier, K., Armas, J.C., Etteldorf, S., Ghazal, P., Sullivan, K.F. Annexation of the interchromosomal space during viral infection. Nat. Cell Biol. 2:661, 2000.

Shelby, R.D., Monier, K., Sullivan, K.F. Chromatin assembly at kinetochores is uncoupled from DNA replication. J. Cell Biol. 151:1113, 2000.

Sullivan, K.F. A solid foundation: Functional specialization of centromeric chromatin. Curr. Opin. Genet. Dev. 11:182, 2001.

Yao, X., Abrieu, A., Zheng, Y., Sullivan, K.F., Cleveland, D.W. CENP-E forms a link between attachment of spindle microtubules to kinetochores and the mitotic checkpoint. Nat. Cell Biol. 2:484, 2000.

Zeitlin, S.G., Barber, C.M., Allis, C.D., Sullivan, K. Differential regulation of CENP-A and histone H3 phosphorylation in G₂/M. J. Cell Sci. 114:653, 2001.

Zeitlin, S.G., Sullivan, K.F. Animal cytokinesis: Breaking up is hard to do. Curr. Biol. 11:R514, 2001.

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