Organization of Chromatin by Global Regulators in Yeast

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DESCRIPTION (provided by applicant): Chromatin remodeling is now recognized as a key step in gene regulation. The long-term goal of our research is to understand how changes in chromatin structure are brought about, and how these changes enhance or repress transcription. Experiments in this proposal will address these fundamental questions via an analysis of the molecular mechanisms underlying repression mediated by a model corepressor complex in yeast, Tup1-Ssn6. We demonstrated previously that Tup1 interacts directly with underacetylated isoforms of histones H3 and H4. We also showed that Tup1-Ssn6 mediated repression is abrogated upon simultaneous disruption of 3 HDAC genes in yeast, RPD3, HOS1, and HOS2. Mutations in histones that weaken interactions with Tup1 in vitro compromise repression in vivo by destabilizing Tup1 interactions at target promoters. These findings led us to a model wherein Tup1-Ssn6 complexes are recruited to target genes via sequence-specific repressers, and in turn, recruit one or more histone modifying activities to create a self-reinforcing, repressive chromatin state. However, we do not know whether the corepressor uses the same tools and establishes the same kinds of structures at different types of target genes. We also do not know the order of events in the repression mechanism, or whether other types of chromatin remodeling activities also contribute to repression. We recently discovered that Ssn6 might have some separate functions in gene regulation, in addition to functions shared with Tup1. However, we have little information regarding these independent Ssn6 functions. To address these questions, we will 1) Define HDAC interaction domains within the Tup1-Ssn6 complex and determine whether multiple HDACs are recruited simultaneously by the corepressor; 2) Determine whether Ssn6 functions independently of Tup1 in the regulation of specific genes; 3) Define and compare the kinetics of Tup1-Ssn6 recruitment, HDAC recruitment, histone deacetylation, etc. at specific target genes and 4) Establish an in vitro system to further define the mechanism of repression. Tup1 shares structural and functional features with the TLE/groucho and the TBL1/TBLR1 corepressors that are important in controlling gene expression during embryonic development and in response to specific signal transduction pathways in higher organisms. Our studies of Tup1-Ssn6 in yeast will further our understanding of corepressor functions in general and will provide new insights to the functions of TLE/groucho and TBL family members both in normal cells and in disease states.