David M. Shore
Chromosome Structure and FunctionWe are interested in the general question of the relationship between chromosome structure and function in eukaryotic cells. Currently, our work is focused on two specific issues: the coordinate regulation of chromosomal domains that causes gene 'silencing', and the specialized structures at the ends of all chromosomes called 'telomeres'. We use the simple eukaryote Saccharomyces cerevisiae (bakers' yeast) as a model system.
Gene silencing was first identified in the mating-type system of yeast, but is now recognized to be a general phenomenon in eukaryotic cells. For example, the stable expression patterns of homeotic genes required for proper development in the fruitfly Drosophila depends upon a form of gene silencing. The silent copies of mating-type genes in yeast are absolutely required for the programmed developmental process that leads to mating-type switching, mating, and diploidization. Interestingly, gene silencing also occurs immediately adjacent to all telomeres in yeast, though the biological role of telomeric silencing (telomere position effect) is still unclear.
Our studies have been focused on an essential sequence-specific DNA-binding protein, called Rap1p, that is involved in both mating-type and telomeric silencing. Rap1p binds to both silencer elements at mating-type loci and to the simple repeat sequences (C1-3A) that comprise the extreme ends of all yeast telomeres. Strikingly, Rap1 also binds to the promoters of many genes, where it functions as an activator of transcription. We have shown that Rap1 works at silencers and telomeres by recruiting a complex of specialized repressors (Sir proteins) to these sites, where they then interact with nucleosomes to block transcription. We are currently trying to understand this process in molecular detail through a combination of genetic and biochemical analysis.
Telomere repeat tracts are variable in length, and are synthesized by a specialized reverse transcriptase called telomerase. All organisms appear to regulate telomere tract length to a species-specific average value. Significantly, telomere length regulation has been associated in mammalian cells with both aging and cancer. We have recently presented evidence that yeast cells regulate telomere length through a negative feedback mechanism that can discriminate the precise number of Rap1p molecules bound to the repeat tracts. This mechanism requires two Rap1-interacting factors (Rif1p and Rif2p) that we have identified using the two-hybrid system. The target of this regulatory system, which may be either telomerase itself of associated proteins, is currently under investigation.