Silencing of genomic domains requires a complex series of interactions between inactivation centers called silencers and numerous repressor proteins. The silencers recruit repressor protein complexes composed of the Sir proteins that interact with histones in nucleosomes to form a chromatin domain that is inaccessible Our studies will focus on (1) the mechanisms by which silent domains are established and the role of histone variants in silencing (2) the biochemical and molecular characterization of the structure of the silent domain and its reconstitution in vitro. (3) The mechanism by which a silenced domain is restricted to a specific region of the genome. The establishment of silencing by Sir1p at the mating type loci was examined by analyzing mutations in Sir1p that are unable to establish silent state even after recruitment of Sir1 to the silencer. These mutants were employed to isolate suppressors . The relationship of such suppressors with Sir1p mutants were determined. The isolation of a histone H2A variant suggests a role for this protein in silencing and is being investigated. The mechanism by which the silenced chromatin domain is restricted to a specific region of the genome is being investigated. Specific DNA sequence elements that mediate boundary function were sought. Once such identified was a specific t-RNA gene. This as well as other results suggest that barriers to silencing function by forming a stable complex that probably remodels chromatin and blocks the spread of the silenced chromatin Genetic studies have revealed that multiple loci are transcriptionally repressed by distinct combinations of the Sir protein complexes. Studies were performed to purify Sir protein complexes from yeast cells followed by the identification and characterization of the individual components. Further studies will aim at identifying associated enzymatic activities as well as determining the molecular structure of the complexes. An important index of our current understanding of transcriptional silencing is whether or not it is possible to reconstitute the silenced state in vitro. Methods will be developed to deposit the Sir proteins onto reconstituted nucleosomes and the assembled chromatin will be tested for its ability to mimic the silenced state using numerous biochemical assays. Such a system will allow a biochemical dissection of the process of silencing and will yield new insights into the mechanisms by which genes are silenced.
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