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Centromeres, Kinetochores, and Chromosome Dynamics in Human Cells

K. Monier, 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 on each chromosome), the structures 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. A unique process of chromatin assembly incorporates a centromere-specific histone, CENP-A, in place of normal histone H3 at centromeres and may provide a mechanistic basis for epigenetic specification of centromeres.

Previously, we found that CENP-A is synthesized in the G₂ phase of the cell cycle, long after its cognate DNA has been replicated in S phase. In experiments in which we used transfection to produce expression of CENP-A in the presence of inhibitors of DNA synthesis, we discovered that CENP-A can assemble into kinetochores in the complete absence of DNA replication. To determine the precise timing of CENP-A incorporation into centromeric chromatin, we microinjected plasmids expressing CENP-A tagged with green fluorescent protein into synchronized Hela cells.

Fluorescent CENP-A was detectable within 1-2 hours as a faint nucleoplasmic staining in nucleoli and/or as brighter foci that did not correspond to centromeres. However, 4 hours after injection, fluorescent CENP-A was detected in centromeres irrespective of the phase of the cell cycle. The time lag between CENP-A synthesis and assembly suggests that CENP-A must be modified before it accumulates at centromeres or that the centromeres must be made accessible for CENP-A.

The constitutive presence of the machinery for CENP-A assembly into chromatin suggests that assembly of CENP-A may be mediated by common chromatin assembly or remodeling factors. We are identifying those factors to determine how CENP-A is incorporated uniquely at centromeres in a normal cell cycle.

Posttranslational modification of histone subunits in chromatin is a major mechanism for regulating a variety of chromosomal functions. We are studying the distinctive modifications that occur in centromeric chromatin and in the surrounding pericentric heterochromatin that flanks the centromere on most chromosomes. In preparation for mitosis, histone H3 is phosphorylated. In the past year, we showed that CENP-A is also phosphorylated as cells enter mitosis, at a site analogous to the major site of mitotic phosphorylation of histone 3. This phosphorylation of CENP-A most likely is mediated by Aurora B kinase, also thought to be the major histone H3 kinase in metazoan cells.

Mutational analysis of the mitotic CENP-A phosphorylation site revealed a surprising role for this modification at the last step in mitosis, cytokinesis, which occurs after kinetochore-dependent segregation of chromosomes. In cells with mutations in the phosphorylation site, Aurora B kinase and its putative partner phosphatase PP1g are not localized properly during telophase and cytokinesis. Thus, CENP-A chromatin plays a role in directing the subcellular localization of enzymes required for completion of cytokinesis.

PUBLICATIONS

Sadoni, N., Sullivan, K.F., Weinzierl, P., Stelzer, E.H., Zink, D. Large-scale chromatin fibers of living cells display a discontinuous functional organization. Chromosoma 110:39, 2001.

Van Hooser, A.A., Ouspenski, I.I., Gregson, H.C., Starr, D.A., Yen, T.J., Goldberg, M.L., Yokomori, K., Earnshaw, W.C., Sullivan, K.F., Brinkley, B.R. Specification of kinetochore-forming chromatin by the histone H3 variant CENP-A. J. Cell Sci. 114:3529, 2001.

Zeitlin, S.G., Monier, K., Sullivan, K.F. Phospho-histone antibody immunofluorescence for analysis of cell cycle progression in G₂ and prophase. Chemtracts 14:557, 2001.

Zeitlin, S.G., Shelby, R.D., Sullivan, K.F. CENP-A is phosphorylated by Aurora B kinase and plays an unexpected role in completion of cytokinesis. J. Cell Biol. 155:1147, 2001.