Analysis of SWI1, SWI2, amd SWI3 proteins

点击翻译按钮获取中文摘要

DESCRIPTION (provided by applicant): The overall objective of our research is to determine how chromosome structure affects gene expression and how the transcription machinery contends with this structure. Our general strategy is to focus on an evolutionarily conserved protein complex, SWI/SNF, which is required for expression of a subset of yeast genes and for the activity of several transcriptional activators. Genetic studies in yeast suggest that SWI/SNF functions by antagonizing chromatin-mediated transcriptional repression, and our recent studies suggest a global role for SWI/SNF and the Gcn5p histone acetyltransferase during late mitosis. In vitro studies indicate that the -2Mda SWI/SNF complex can use the energy of ATP hydrolysis to disrupt nucleosome structure and that SWI/SNF can be targeted to specific nucleosomes through direct interactions with a variety of transcriptional activators. Over the next budget period we will continue to exploit the powerful genetic and biochemical opportunities available in yeast to investigate the role of SWI/SNF in vivo and the biochemical mechanism by which SWI/SNF disrupts nucleosome structure in vitro.The first aim of this proposal will investigate the roles of SWI/SNF and the Gcn5p HAT during late mitosis. This aim is addressed by Affymetrix gene chip expression analyses with RNA isolated from synchronized cells, chromatin immunoprecipitations, and in vivo chromatin structural analyses. The second objective will investigate the role of histone H3 serine 10 phosphorylation in the regulation of remodeling activities in vivo and in vitro. Aim 3 will test the hypothesis that the Ashi p repressor functions by blocking the recruitment or remodeling activity of SWI/SNF. The fourth objective will use a battery of SWI2/SNF2 ATPase motif mutants and a photoaffinity crosslin king method to define the role of ATP binding and hydrolysis in SWI/SNF remodeling. This aim will also use photoaffinity crosslinking and fluorescence methods to test the hypothesis that SWI/SNF action causes movements of nucleosomal DNA. The fifth objective will probe the structure of SWI/SNF by electron microscopy methods. This aim also describes a conditional degron strategy to define the role(s) of individual SWI/SNF subunits for both in vivo and in vitro functions