ANALYSIS OF THE CHROMATIN-REMODELING COMPLEX RSC

University Of Utah, Salt Lake City UT

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Abstract

Our objective is to understand how ATP-dependent chromatin remodeling complexes (CRCs) contribute to the regulation of transcription and other vital cellular processes. Chromatin represses transcription, and all eukaryotes utilize a related set of CRCs to remove this repression. The yeast S. cerevisiae contains two related CRCs, SWI/SNF and RSC, which are highly similar in composition and in vitro activities to their metazoan counterparts. Yeast SWI/SNF is a rare, non-essential complex that remodels chromatin at many pol II promoters. In contrast, RSC is both abundant and essential for viability, but its functions are unknown. Human SWI/SNF is both abundant and essential for viability, and mutations can cause aggressive rhabdoid tumors. Our Specific Aims are to determine how RSC is targeted to particular genomic loci and to identify RSC- interacting proteins, with the goal of understanding more about how these conserved chromatin remodeling complexes contribute to the control of gene expression: 1) Identify the genomic targets for RSC action. The Rsc3 component of RSC is highly similar to known sequence-specific DNA-binding transcriptional activators. We will identify Rsc3 (and RSC) targets through DNA microarray analysis of rsc3 Ts- mutants. The Rsc3 binding site will be determined by site-selection studies, and chromatin immunoprecipitation experiments will be utilized to test candidate promoters for Rsc3 occupancy in vivo. 2) Determine the function of Rsc1, Rsc2, and the bromodomain (BD). Rsc1 and Rsc2 members that contain BDs and a BAH domain, two motifs of unknown function found in many important transcriptional regulators. We will perform a structure/function analysis of the Rsc2 BD using random mutagenesis, isolate conditional mutations, and utilize genetic suppression of conditional mutants to isolate BD- interactions proteins. 3) Examine the structure/function of a BAH region. We will perform a structure/function analysis of the Rsc2 BAH region using random mutagenesis, and utilize suppression of conditional mutants to isolate BAH-interacting proteins. 4) Ascertain the function of actin-related proteins (Arp) in RSC. We will purify RSC lacking Arp proteins and test for function, perform DNA microarray analysis with our arp Ts- mutants, and acquire genetic suppressors of arp mutants to identify cellular processes that require Arp function.