Heterochromatin-Mediated Repression in Yeast

Lsu Health Sciences Center -Shreveport, Shreveport LA

Investigators

Abstract

0091898 David Gross Heterochromatin is a condensed form of chromatin found within all eukaryotic (nucleated) cells. It is responsible for X-chromosome inactivation in mammalian females, position-effect variegation in the fruit fly, and cell-type determination in budding yeast. While heterochromatin has long been associated with genetic inactivation, the precise mechanism by which it represses gene transcription is unclear. To investigate this, a powerful model system has been developed. In this system, a dynamically regulated, stress-inducible yeast gene (HSP82) has been placed under control of a heterochromatin-inducing protein complex termed SIR (Silent Information Regulator). This model system will be used to address the following questions. First, which proteins bind to the promoter DNA of the SIR-repressed gene? Using a powerful new technique, chromatin immunoprecipitation, the presence of both positive and negative regulatory proteins will be examined in living cells. These include the sequence-specific activator, heat shock factor (HSF), components of the basic transcriptional machinery (including RNA polymerase), and the heterochromatin-binding proteins Sir2p and Sir3p. Second, the question of whether silencing of HSP82 occurs in each cell in the population will be investigated. Is silencing stably inherited from parent to daughter cell, or is heterochromatin-mediated repression variegated (seen in some cells of a population but not in others), as is the case in fruit fly? Third, what are the collaborators of HSF which permit it, when activated by stress, to override the repressive heterochromatic structure established by SIR? A gene knock-out approach will be taken to answer this question. Finally, what is the mechanism of a second global repression system, mediated by the Ssn6-Tup1 protein complex? A yeast strain will be genetically engineered so that the HSP82 gene promoter is targeted by the Ssn6-Tup1 protein complex. Parallel expression, structural, and genetic analyses to those described above will then be carried out, allowing a direct comparison between the two repression systems. Given the degree to which transcriptional mechanisms are evolutionarily conserved, these studies have the potential to unravel the means by which heterochromatin works not only in yeast but also in other eukaryotes, including human.