Silent Chromatin: Mechanisms of Transcriptional Repression

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Chromatin plays a critical role in regulating the expression of eukaryotic genes. To a first approximation, it exists in two forms: as an unfolded, 10-nm-like filament, comprised of arrays of nucleosomes in a beads-on-a-string configuration, or as a compacted 30-nm-like fiber. The former is characteristic of transcriptionally active euchromatin, while the latter is characteristic of silent chromatin (heterochromatin), which represses transcription in a regional rather than promoter-specific fashion. How silent chromatin inhibits gene expression is not well understood, although the traditional view is that it does so by sterically hindering access of positive regulators of transcription to the underlying DNA. In the budding yeast, Saccharomyces cerevisiae, silent chromatin is formed by Silent Information Regulator (SIR) proteins. Previous work has revealed, very unexpectedly, that silent chromatin is permissive to the binding of both gene-specific activators and general transcription factors (GTFs). Indeed, an initiation-competent form of RNA polymerase II (Pol II) is present at hyperrepressed promoters. By contrast, occupancy of downstream factors, including enzymes that cap the nascent mRNA and factors that facilitate Pol II elongation, is virtually abolished, resulting in Pol II stalling at the gene's 5' end. How this differential accessibility is established is unknown, but is central to understanding the mechanism of transcriptional silencing. In this project, two important issues concerning the fundamental mechanisms by which silent chromatin regulates gene transcription will be investigated: 1. How do Sir proteins silence basal gene transcription? This question will be addressed by (i) Testing the hypothesis that differential factor accessibility in silent chromatin reflects the temporal order of factor recruitment to a gene during the transcription cycle; (ii) Characterizing the role of key downstream factors in triggering silencing through mutational and functional analyses; and (iii) Evaluating the role of factor deacetylation in triggering silencing. 2. How does gene activation take place in silent chromatin? This will be investigated by (i) Characterizing the dynamic alterations of nucleosomes during gene activation in silent chromatin; (ii) Determining the extent of spread of Sir proteins at transgenes subject to different degrees of silencing; and (iii) Comparing the coactivator requirements of euchromatic and heterochromatic genes. Benefits of this project, beyond the anticipated scientific discoveries, include the training of two full-time Ph.D. graduate students and two undergraduates.