Elucidating the molecular mechanisms of chromosome segregation is the major interest of the Megee lab. DNA replication produces identical copies of each chromosome, which are referred to as replicated sister chromatids. The proper segregation of sister chromatids to opposite poles of the cell during mitosis paradoxically requires that sister chromatids remain tightly associated with one another from the time of DNA replication until anaphase onset. The cohesion of sister chromatids occurs at discrete sites along the length of the chromosome and is especially robust flanking the centromere, a specialized region of the chromosome that mediates its attachment to spindle microtubules. Sister chromatid cohesion plays an important role in promoting chromosome biorientation, the process whereby kinetochores on pairs of sister chromatids form attachments to microtubules from opposite poles. The failure to establish such attachments may lead to the transmission of abnormal chromosome numbers to daughter cells. This condition, known as aneuploidy, often results in genetic disease or miscarriage and is also a hallmark of tumor cells. Thus, mechanisms that promote chromosome biorientation and correct aberrant kinetochore-microtubule attachments are essential for the prevention of genomic instability.
Despite its fundamental importance for the fidelity of chromosome segregation, the process of sister chromatid cohesion remains poorly understood at the molecular level. In collaboration with Dr. Jennifer Gerton of the Stowers Institute, we have used chromatin immunoprecipitation of individual cohesin subunits followed by microarray analyses to provide the first genome-wide map of cohesin binding in any model organism (see figure).
DNA crosslinked to cohesin subunit Mcd1 and input DNA not subject to immunoprecipitation were competitively hybridized to arrays of the complete budding yeast genome. Profiles of Mcd1 binding were superimposed on a map of the 16 chromosomes. Regions in red are enriched for Mcd1 binding, with the brightness of the red indicating the intensity of Mcd1 binding. Centromeric regions, which have the highest magnitudes of Mcd1 binding, are marked by asterisks. Grey regions are not enriched for Mcd1 binding, and hybridization data are not available for the blue regions.
Using a combination of genetic, molecular, and cell biological approaches, our lab now focuses on understanding the mechanism(s) through which cohesin, the protein complex that mediates cohesion, is recruited to both centromere-proximal and distal locations.
Specific Projects:
Role of the kinetochore in cohesin recruitment: In higher eukaryotes, centromere-flanking heterochromatin has been shown to contribute to cohesin recruitment. Interestingly, budding yeast lack pericentric heterochromatin, but still have extensive domains flanking kinetochores that are highly enriched for cohesin binding. In previously published work, we have demonstrated that the budding yeast kinetochore is essential for the generation of the pericentric cohesin domains. We have now identified a number of conserved kinetochore proteins that are essential for pericentric cohesin recruitment. These observations suggest that both kinetochore-dependent and independent pathways for pericentric cohesin recruitment exist, a model that we are actively pursuing.
Generation of pericentric cohesin domains: We have shown previously that the kinetochore behaves as an enhancer of cohesin binding at both endogenous and ectopic centromere locations. Although the ability of the centromere/kinetochore complex to enhance cohesin binding operates over a rather large domain, its ability to affect cohesin binding is limited to a finite region of each chromosome. We are currently investigation how enhancer activity is regulated.
Biological functions of pericentric cohesin domains: We and others have provided evidence that the enrichment of cohesin in kinetochore-flanking regions is important for chromosome segregation during mitosis. It is likely, however, that these extensive cohesin domains have other functions important for chromosome biology. We are currently examining the role of these cohesin domains in other DNA metabolic processes such as transcription and recombination.
Selected Publications
Eckert, C. A., D. Gravdahl, and P. C. Megee. (2006). The enhancement of pericentric cohesin association by conserved kinetochore components promotes high fidelity chromosome segregation and is sensitive to microtubule-based tension. Manuscript in preparation.
Megee, P. C. (2006). Chromosome guardians on duty. Nature 441: 35-37.
Kiburz, B. M., D. B. Reynolds, P. C. Megee, A. L. Marston, B. H. Lee, T. I. Lee, S. S. Levine, R. A., Young, and A. Amon. (2005). The core centromere and Sgo1 establish a 50 kb cohesin-protected domain around centromeres during meiosis I. Genes Dev. 19: 3017-3030.
Weber, S.A., J. L. Gerton, J. E. Polancic, J. L. DeRisi, D. Koshland, and P. C. Megee. (2004). The kinetochore is an enhancer of pericentric cohesin binding. PLoS Biology 2: 1340-1353.
Glynn, E., P. C. Megee, H.-G. Yu, C. Mistrot, E. Unal, D. Koshland, J. L. DeRisi, and J. L. Gerton. (2004). Genome-wide mapping of the cohesin complex in the yeast Saccharomyces cerevisiae. PLoS Biology 2: 1325-1339.
Bird, A. W., D. Y. Yu, M. G. Pray-Grant, Q. Qiu, K. E. Harmon, P. C. Megee, P. A. Grant, M. M. Smith, and M. F. Christman. (2002). Histone H4 tail acetylation by Esa1p is required for DNA double strand break repair. Nature 419: 411-415.
Megee. P. C., C. Mistrot, V. Guacci, and D. Koshland. (1999). The centromeric sister chromatid cohesion site directs Mcd1p binding to adjacent sequences. Mol. Cell 4: 445-450.
Megee, P. C. and D. Koshland. (1999). A functional assay for centromere-associated sister chromatid cohesion. Science 285: 254-257.
Smith, M. M., P. Yang, M. S. Santestiban, P. W. Boone, A. T. Goldstein, and P. C. Megee. (1996). A novel histone H4 mutant defective in nuclear division and mitotic chromosome transmission. Mol. Cell. Biol. 16: 1017-1027.
Megee, P. C., B. A. Morgan, and M. M. Smith. (1995). Histone H4 and the maintenance of genome integrity. Genes Dev. 9: 1716-1727.
Megee, P. C., B. A. Morgan, B. A. Mittman, and M. M. Smith. (1990). Genetic analysis of histone H4: essential role of lysines subject to reversible acetylation. Science 247: 841-845.
Bird, A. W., D. Y. Yu, M. G.
Pray-Grant, Q. Qiu, K. E. Harmon, P. C. Megee, P. A.
Grant, M. M. Smith, and M. F. Christman. 2002.
Histone H4 tail acetylation by Esa1p is required for
DNA double strand break repair. Nature 419:
411-415.
Megee, P. C., C. Mistrot, V.
Guacci, and D. Koshland. 1999. The centromeric
sister chromatid cohesion site directs Mcd1p binding
to adjacent sequences. Molecular Cell 4: 445-450.
Megee, P. C. and D. Koshland.
1999. A functional assay for centromere-associated
sister chromatid cohesion. Science 285: 254-257.
Smith, M. M., P. Yang, M. S.
Santisteban, P. W. Boone, A. T. Goldstein, and P. C.
Megee. 1996. A novel histone H4 mutant defective
in nuclear division and mitotic chromosome
transmission. Mol. Cell. Biol. 16: 1017-1027.
Megee, P. C., B. A. Morgan, and
M. M. Smith. 1995. Histone H4 and the maintenance
of genome integrity. Genes Dev. 9: 1716-1727.
Megee, P. C., B. A. Morgan, B. A. Mittman, and M. M.
Smith. 1990. Genetic analysis of histone H4:
essential role of lysines subject to reversible
acetylation. Science 247: 841-845.
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