In addition to gene specific mechanisms of regulation, gene expression is known to be modulated by more global changes in chromsomal structure as evidenced by such phenomenon as position effect variegation, imprinting, and silencing. Silencing of telomeric gene expression and of the HML and HMR mating type loci in S. cerevisiae represents one of the best defined systems in which this phenomenon has been studied. Work from several laboratories has resulted in the identification of a number of the critical components of this system, including RAP1, SIR1-4, RIF1, and histones H3 and H4. The cis acting sequences at the E mating type silencer are the A, E, and B domains. These bind different trans-acting factors: the ORC replication complex, RAP1 a protein which has been identified as a context dependent repressor or activator, and ABF (ARS binding factor), respectively. Silencing requires two of three of these domains and the aforementioned transacting factors. Of the identified factors, RAP1 is the only one with DNA binding activity. It has been shown to interact with SIR3 and its role is presumed to involve docking other proteins to the DNA. The Shore lab recently showed that SIR1 if targeted to the DNA by a heterologous fusion domain bypassed the requirement for RAP1. Silencing propagated inward from the telomeres and requires the same factors with the exception of SIR1 and is less stable. This grant is highly focused on defining the initiating signals for silencing and on understanding the interactions among the requisite protein factors. There are three specific aims: 1) Characterization of the protein-protein interactions between RAP1 and SIR3 and SIR4; 2) studying the interactions between pairs of SIR proteins in order to identify functional domains involved in initiation and propagation of repressed chromatin; and 3) definition of the molecular role of SIR1 in the establishment of silencing at HM loci.
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