The objective of this proposal is to determine how distinct domains of gene expression are established and propagated in eukaryotes. Data from several laboratories have identified some of the proteins that are required for silencing such as Orc, Rap1 and the Sir proteins. These studies have also revealed an intimate association between components of chromatin and silencing thus setting the stage for a detailed investigation of the mechanism of silencing by the Sir proteins in the context of chromatin. Our studies will focus on (1) the mechanisms by which silent domains are established and faithfully propagated through generations using genetical and biochemical means and (2) the structure of the silent domain and its analysis in vitro. The establishment of silencing by Sir1p at the mating type loci will be examined by analyzing mutations in Sir1p that are unable to establish and inherit stable silent domains. These mutants will be subsequently employed to isolate additional mutations that can suppress the Sir1p defect in silencing. The relationship of such suppressors with Sir1p mutants will be determined biochemically. The mechanism by which the silenced chromatin domain is restricted to a specific region of the genome will be investigated. Specific DNA sequence elements that mediate boundary function will be sought. Once such elements are identified, proteins that bind these elements to prevent the spread of heterochromatin into neighboring euchromatin will then be isolated and characterized. Genetic studies have revealed that distinct combinations of the Sir proteins transcriptionally repress multiple loci. While Sir2p, Sir3p and Sir4p mediate silencing at HML, HMR and at the telomeres, only Sir2p is involved in silencing at the rDNA locus. It is therefore of interest to determine whether there are multiple Sir protein complexes in the cell and what the composition of these complexes might be. Studies will aim at purifying the Sir protein complexes from yeast cells followed by the identification and characterization of the individual components. The complexes will also be analyzed for various enzyme activities and will be uilized for further in vitro studies. This research has broad relevance with respect to understanding how the function of genes is influenced by their position in the genome. This research has implications in gene replacement therapy, how chromosome rearrangements can lead to diseases, how X-inactivation and parental imprinting of genes form heritable states of gene expression as well as in understanding oncogenesis and aging. - Yeast, Silencing, Heterochromatin, Sir Proteins, Genetics, Biochemistry, Boundary Elements
