Epigenetic silencing, the heritable repression of transcription within chromatin domains, is critical in eukaryotic gene regulation. Maintaining homeostasis requires a delicate balance of gene regulatory events involving transcription factors. Deregulation of silencing patterns ushers growth of aberrant cells, and development and proliferation of cancer. However, the molecular mechanism of epigenetic silencing remains unknown. My previous data indicate that silencing is initiated downstream of activator binding but upstream of TFIIB recruitment and preinitiation-complex assembly suggesting silencing acts through posttranslationally modifying histones, to govern TFIIB recruitment to promoters.
This project aims to identify transcription factors and molecular mechanisms responsible for transcriptional silencing. We will determine the essential regulators of silenced genes by backtracking from TFIIB binding to activator binding and quantify the relative occupancy of these crucial regulators at silenced versus active genes. Utilizing the silenced mating system in yeast to examine the mechanism of silencing in a natural context, the extent of occupancy of key regulatory factors at the silenced/active mating loci in "a" and "alpha" cells, will be measured by chromatin immunoprecipitation and real-time polymerase chain reaction. We will test the hypothesis that these key regulators control silencing through ongoing, reversible, dynamic competition rather than through frozen, inert structure.