The control of gene expression is a fundamental process underlying the development and maintenance of different cell types in eukaryotes. Because abnormalities in transcriptional regulation can lead to particular disease states, including cancer, studies of basic mechanisms of gene regulation may have a significant impact on medicine. Control of gene expression in animals results from a complex interplay of positive and negative regulators. The long-term goal of this work is an understanding of basic mechanisms of coordinate repression of regions or 'domains' of chromosomes. The 'silencing' of genes may play a fundamental role in determining the overall tissue-specificity of gene expression, and may underlie phenomena such as X-chromosome inactivation in mammals. The goal of the proposed research is a detailed molecular description of the silencing of mating-type genes in the yeast Saccharomyces cerevisiae, a system amenable to sophisticated genetic and biochemical analysis. Mating type (a or a) is determined by a locus called MAT. Additional copies of mating-type genes are present at other loci (HMLa and HMRa), but are repressed by flanking sequences, called 'silencers', located >lkb from their promoters. Surprisingly, one protein that binds to the silencers, called RAP1, also binds upstream of a large number of genes, where it appears to be involved in transcriptional activation, and to the Poly(Cl-3A) repeats at telomeres, where its role is less clear. A genetic analysis of RAP1 has shown that the protein plays a role at all three types of chromosomal sites to which it binds: silencers, activators, and telomeres. It seems likely that auxiliary proteins, each specific to a particular regulatory process, interact with RAP1 at these different sites and determine the ultimate regulatory outcome.
The aims of this project are to test this model and begin to describe the mechanism of silencing in molecular detail. We will utilize a number of both genetic and biochemical methods to analyze the function of RAP1 at the silent loci with the general aim of identifying and characterizing the protein-protein and protein-DNA interactions at the silencers responsible for initiating repression. Specifically, we propose to (1) isolate new alleles of rapl affecting silencing and use these mutants to analyze the involvement of RAP1 in the transmission of the repressed state during cell division; (2) identify RAPl-interacting factors at silencers through the isolation of suppressors of silencing-defective rapl mutants, and by a novel genetic screen for protein-protein interactions and (3) generate a non-essential, altered DNA-binding mutant of RAP1 that can be used to undertake a complete genetic study of parts of RAP1 involved in silencing.
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