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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM040094-07
Application #
2180172
Study Section
Genetics Study Section (GEN)
Project Start
1988-04-01
Project End
1996-01-21
Budget Start
1994-12-01
Budget End
1996-01-21
Support Year
7
Fiscal Year
1995
Total Cost
Indirect Cost
Name
Columbia University (N.Y.)
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
167204994
City
New York
State
NY
Country
United States
Zip Code
10032
Moretti, P; Shore, D (2001) Multiple interactions in Sir protein recruitment by Rap1p at silencers and telomeres in yeast. Mol Cell Biol 21:8082-94
Vannier, D; Damay, P; Shore, D (2001) A role for Sds3p, a component of the Rpd3p/Sin3p deacetylase complex, in maintaining cellular integrity in Saccharomyces cerevisiae. Mol Genet Genomics 265:560-8
Marcand, S; Wotton, D; Gilson, E et al. (1997) Rap1p and telomere length regulation in yeast. Ciba Found Symp 211:76-93; discussion 93-103
Marcand, S; Gilson, E; Shore, D (1997) A protein-counting mechanism for telomere length regulation in yeast. Science 275:986-90
Shore, D (1997) Telomere length regulation: getting the measure of chromosome ends. Biol Chem 378:591-7
Wotton, D; Shore, D (1997) A novel Rap1p-interacting factor, Rif2p, cooperates with Rif1p to regulate telomere length in Saccharomyces cerevisiae. Genes Dev 11:748-60
Shore, D (1997) Telomerase and telomere-binding proteins: controlling the endgame. Trends Biochem Sci 22:233-5
Marcand, S; Buck, S W; Moretti, P et al. (1996) Silencing of genes at nontelomeric sites in yeast is controlled by sequestration of silencing factors at telomeres by Rap 1 protein. Genes Dev 10:1297-309
Chi, M H; Shore, D (1996) SUM1-1, a dominant suppressor of SIR mutations in Saccharomyces cerevisiae, increases transcriptional silencing at telomeres and HM mating-type loci and decreases chromosome stability. Mol Cell Biol 16:4281-94
Wotton, D; Freeman, K; Shore, D (1996) Multimerization of Hsp42p, a novel heat shock protein of Saccharomyces cerevisiae, is dependent on a conserved carboxyl-terminal sequence. J Biol Chem 271:2717-23

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