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 reconstitution 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 promote stable silent domains. These mutants will be subsequently employed to isolate additional mutations that can bypass the Sir1p defect in silencing. The relationship of such suppressors with Sir1p mutants will be determined biochemically. The chromatin structure of the silenced domain and the role of various proteins in maintaining the silenced domain will also be investigated. Silenced chromatin will be purified and its protein composition and chromatin structure studied. Once the chromatin structure has been characterized in vitro reconstitution of silenced chromatin will be performed, initially on templates that reduce the requirements for silencing. The assembled chromatin will be tested for its ability to mimic the silent state and this system will allow a biochemical dissection of the process of silencing and yield valuable new insights into the mechanisms by which genes are stably repressed. This research has broad medical relevance with respect to understanding how the function of genes is influenced by their position in the genome with implications in gene replacement therapy and how chromosome rearrangements lead to the diseased state, as well as how X inactivation and the parental imprinting of genes forms heritable states of gene expression.
