MOLECULAR MECHANISMS OF GLOBAL REPRESSION IN YEAST

Harvard University (Medical School), Boston MA

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Abstract

DESCRIPTION (Verbatim from the applicant's abstract): The molecular mechanisms by which eukaryotes regulate gene expression are important for basic scientific knowledge that is necessary for understanding many complex biological phenomena including human diseases. A fundamental class of mechanisms involves transcriptional corepressors that are recruited to specific promoters via DNA-binding proteins. One such corepressor, the Sin3-Rpd3 histone deacetylase, represses transcription by creating a highly localized domain of histone deacetylation. As such it is a clear case in which transcriptional regulators function by recruiting chromatin modifying activities to promoters as well as a cause and effect relationship between histone acetylation and transcription. Another yeast corepressor, Cyc8-Tupl, represses transcription through a domain of Tupl, but the mechanism is poorly understood. The overall goals of this proposal are to determine in detailed molecular terms how a domain of histone deacetylation causes transcriptional repression, whether and how other histone deacetylases and acetylases are recruited to specific promoters, and how Cyc8-Tupl represses transcripuon. To do this, the investigator will combine molecular, genetic and biochemical approaches to carry out the following projects. First, using mechanistically defined situations involving the Sin3-Rpd3 histone deacetylase, he will determine how the location and extent of the domain of deacetylated histones affects the binding of activators, components of the Pol II machinery, other chromatin modifying activities, chromatin structure, and ultimately transcriptional repression. Second, using a combination of chromatin immunoprecipitation and genome-wide arrays, he will directly examine whether yeast histone deacetylases and acetylases are recruited to specific promoters; if so, he will use standard molecular analysis to bootstrap our way backwards to the transcriptional regulators that mediate the recruitment and how they affect transcriptional regulation and chromatin structure of the genes. Third, through genetic analysis, he will analyze the function of histone tails particularly with respect to the issue of which histone tails are important for specific biological functions and the requirement for histone tails to be modified by acetylation. Fourth, by analogy with aim 1, he will establish the 'rules' of Cyc8-Tupl repression to determine at which step Cyc8-Tupl blocks transcription. In addition, he will test whether Cyc8-Tupl can repress transcription in other organisms, will test specific models of Cyc8-Tupl repression, and will perform a genetic screen to identify new proteins that affect Cyc8-Tupl repression. As both Cyc8-Tupl and Sin3-Rpd3 are evolutionarily conserved and have very important biological functions, a detailed molecular understanding of their repression mechanisms should be of broad significance.