Genetic Analysis of Cell Cycle Dependent Transcription

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DESCRIPTION (Provided by applicant): The long-term goal is to understand the role of chromatin dynamics in transcription and the cell cycle. Chromatin undergoes global changes in folding during the cell cycle and local changes in folding during transcription, DNA repair, and recombination. These changes are intimately linked to normal cell physiology because multiple observations have linked alterations in chromatin remodeling factors to human diseases and cancer. The proposed studies will test the hypothesis that two factors - the cell cycle-regulated transcriptional corepressors Hir1p and Hir2p and a ubiquitinated species of histone H2B - affect transcription because of their direct roles in aspects of chromatin folding. In the first specific aim, yeast genes directly regulated by Hir1p and Hir2p will be identified by combining DNA microarray hybridization analysis with chromatin immunoprecipitation. The second specific aim will distinguish between chromatin-dependent and chromatin-independent mechanisms of Hir repression by analyzing transcription of the histone HTA I gene in mutants that are defective in RNA polymerase II holoenzyme components or histone deacetylases. The ability of Hir proteins to assemble nucleosomes in vitro and to organize DNA into topologically distinct domains in vivo will be tested. The third specific aim will examine the regulation of Hir protein function in the cell cycle. Chromatin immunoprecipitation will be used to identify the cell cycle signals that control the Hir-dependent recruitment of the Swi/Snf nucleosome remodeling factor to the HTA 1 gene, and nondenaturing chromatin preparations will be used to ask if Hir proteins and Swi/Snf cycle on and off chromatin. The hypothesis that Swi/Snf recruits mitotic Clb cyclins to the HTA1 promoter in late G 1 will be tested by chromatin immunoprecipitation. In the fourth specific aim, the transcriptional role of ubiquitinated H2B (uH2B) will be examined by testing the hypothesis that uH2B recruits nucleosome remodeling factors to specific genes. Genes that are regulated by uH2B and the genomic localization of uH2B will be identified by DNA microarray hybridization coupled with chromatin immunoprecipitation.