The overall objective of our research is to determine how chromosome structure influences nuclear processes, and one of our general strategies is to identify and characterize the factors that facilitate the folding of yeast nucleosomal arrays into specialized structures with unique biological functions. Our recent studies suggest than an HMG1-like protein, Sin1p, and the SIN domain of the histone octamer comprised of residues from both histones H3 and H4, play crucial roles in chromatin- mediated transcriptional repression during mitosis. Our working hypothesis is that interactions of Sin1p with chromatin requires an intact octamer SIN domain and that Sin1p regulates mitotic transcription by influencing compaction of nucleosomal arrays. We also propose that the yeast linker H1 homologue, Hhno1p, and the histone variants, Cse4p and Htz1p, create specialized, folded domains of nucleosomal arrays that contribute to the regulation of gene expression, DNA repair, recombination, or chromosome segregation. Over the next budget period we will continue to exploit the power genetic and biochemical opportunities available in yeast to directly test these hypotheses.
The first aim will investigate the role of Sin1p in mitosis and will test the hypothesis that Sin1p interacts with the histone octamer SIN domain and influences nucleosomal array folding. These studies will be addressed by indirect immunofluorescence, chromatin association, and chromatin immunoprecipitation assays to monitor in vivo interactions of Sin1p with chromatin as a function of cell cycle progression. We will also use analytical ultracentrifugation to characterize the binding of SIN1P to nucleosomal arrays reconstituted with wildtype and sin-histone octamers. The second objective will use both a candidate gene approach and genetic screens to identify genes that encode key components of mitotic chromatin.
The third aim will investigate the role of yeast histone H1 (Hho1p) in chromatin structure and function. In this aim we will use chromatin immunoprecipitation to test whether Hho1p is targeted to only a few loci or if Hho1p is uniformly distributed through yeast chromatin.
This aim will use analytical centrifugation to investigate the ability of Hho1p to condense nucleosomal arrays reconstituted with yeast histone octamers. The fourth objective will use analytical ultracentrifugation to analyze the folding dynamics of nucleosomal arrays that contain Cse4p- or Htz1p-containing nucleosomes. These studies will test the hypothesis that these histone variants create specialized chromatin structures.
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