Telomere Structure and Function in Yeast

Tulane University, New Orleans LA

Investigators

Abstract

Telomeres, the protein-DNA structures present at the ends of eukaryotic chromosomes, serve functions necessary for maintaining chromosome integrity. These functions include a) a mode of replication that solves a problem with the replication of ends, b) the capping of the chromosomal DNA to prevent terminal degradation and instability, and c) the ability of telomeres to quiet gene expression from genes near the ends of the chromosomes. The long-term goal of this project is to understand the mechanism and inheritance of telomeric silencing using the yeast Saccharomyces cerevisiae. Three specific aims include: First, continuation of previous work aimed at understanding the function of one of the central regulators of silencing, Sir3p. Several key steps of silencing require Sir3p. These include a) the initiation of silencing, b) subtelomeric/telomeric associations, c) the propagation (or spreading) of silencing, and d) the ability to interact with key silencing factors. Sir3p may represent a functional paradigm for silencing regulators. Specific Aim 1 involves defining the functionally critical amino acids in the Sir3p C-terminal domain (CTD) minimal silencer, the proteins that interact with the CTD, and the sir3p domains involved in events downstream of the initiation events. Cellular memory, the ability of a cell to perpetuate a specific transcription state through multiple generations, is a central characteristic of position effect, of variegation and of differentiation in most eukaryotes. Specific Aim 2 centers specifically on the relationship between telomeric chromatin state and heritability The telomeric chromatin state will be determined by the degree of accessibility of artificially introduced telomeric restriction sites to exogenous probes. Specific Aim 3 investigates the relationship between heritability and subtelomeric structure using the chromatin immunoprecipitation assay system. This technique permits the mapping of specific chromatin factors that are in close proximity to defined segments of subtelomeric DNA. Several issues are being explored, including the relationship between a)telomeric length and the formation of unique subtelomeric chromatin structures, b) heritability and formation of specific subtelomeric structures, and c)telomeric and subtelomeric states. In addition the unique role of yeast chromatin factor-1 in the propagation of silencing at the structural level is being examined. Understanding how chromatin structure can regulate gene expression is very important as this type of regulation takes place in imprinting and during development. It is also a major issue in the generation of transgenic organisms. Yeast is the most tractable system in which to study this phenomenon.