The Context Dependent Function of Transcriptional Regulator Rap1 in Gene silencing and Activation

Compaction of the eukaryotic genome into regions of open euchromatin and dense heterochromatin serves as a fundamental regulator of gene expression in cells. In the budding yeast Saccharomyces cerevisiae, the Silent information regulator (Sir) protein complex regulates a heterochromatin-like structure at the silent mating type loci, HML and HMR, and at the telomeres. In addition to the Sir proteins, Repressor activator protein 1 (Rap1) functions in establishing and maintaining silent chromatin at HML and HMR by binding to nucleation sites known as silencers (silent enhancers) and recruiting silencing machinery. As its name suggests, Rap1 is simultaneously an essential transcription factor that activates hundreds of genes across the genome, including many ribosomal protein genes and the MATα1 and α2 genes whose expression determine the mating-type of α cells. Sequence identity between the mating-type locus MAT and the auxiliary HML allows a unique opportunity to study the role of Rap1 in both silent and expressed contexts, and how local chromatin state affects function. Chapter two focuses on discerning the context-dependent functions of Rap1. Using ChIP-seq I established that Rap1 accessed its binding site at the promoter of HML, even in the silent context. My findings supported and extended the view that pre-initiation complex machinery is unable to act in silent chromatin, thus narrowing the mechanism of silencing to a step between recruitment of the native activator, Rap1, and occlusion of the pre-initiation complex. Surprisingly, Rap1 enrichment at its three binding sites across the silent locus was enhanced by the presence of Sir proteins, despite binding of this transcription factor preceding Sir protein recruitment. As Rap1 was bound in the silent locus but was not functionally recruiting transcription machinery, I tested whether the presence of this enigmatic protein could instead be enhancing silencing. Utilizing a highly sensitive assay that monitors loss-of-silencing events, I established a novel and specific contribution of promoter-bound Rap1, which was previously seen as having potential only for activation, to silencing. Furthermore, I investigated the mechanism by which Rap1 promotes transcription when acting as an activator and have evidence that it may be aiding in the transition from transcription initiation to elongation. ChIP offers a static view of protein-DNA interactions rather than a dynamic readout. To assess whether in vivo dwell time of Rap1 might contribute to its function as an activator or repressor, I coupled a nuclear depletion strategy with a ChIP-seq time course to measure enrichment at and decay of Rap1 from its binding sites genome wide. The apparent Rap1 dwell time did not differ between silenced HML and expressed MAT. However, genome-wide Rap1 residence time correlated with transcriptional output and nucleosome positioning. These results point toward a model in which the duality of Rap1 function is mediated by local chromatin environment rather than binding-site availability. I have conducted a number of experiments attempting to pinpoint the mechanism by which Rap1 switches function, following up mainly on post-translational modifications. Some of this work is described in the appendix. While study of Sir silencing at the HM loci has been integral in understanding the establishment and maintenance of heterochromatin in eukaryotes, the effects of these same proteins at telomeres has been less clear. Chapter three focuses on an incomplete set of experiments designed to better understand the functional similarities and differences in Sir-mediated silencing at telomeres and the HM loci. Early studies of telomere position effect in yeast led to a hypothesis that distance from telomere dictated the amount of gene repression imparted by Sir proteins in a gradated manner. At the time I began working on the project, there was mounting evidence against this hypothesis. I designed and performed preliminary experiments testing whether silencing was a function of distance from telomere utilizing fluorescent reporters for high throughput analysis. Furthermore, I investigated whether telomere length affected silencing of HML and HMR by titrating available Sir proteins away from these loci. I also pioneered usage of a novel form of single molecule RNA FISH in our lab and I planned to use this technique to assess whether subtelomeric genes, which are enriched for metabolic function, were spatially co-regulated in response to environmental stimuli.

Advisor: Rine, Jasper D