Uncovering molecular mechanisms of active transcriptional repression.
Animal development and homeostasis critically depend on the accurate regulation of gene expression, which includes the silencing of genes that should not be active. Silencing or repression of transcription is mediated by a specific class of transcription factors termed repressors that, typically via the recruitment of co-repressors, can dominantly suppress transcription, even in the presence of activating cues. While the importance of such “active repression” is emphasized by severe developmental defects and diseases like cancer that can result when repressors are mutated, how repressors function is not well understood. In particular, how repression is achieved mechanistically and whether all repressors can repress all activators has remained elusive.   Here, I propose to study the functional properties of repressors and the mechanisms of active repression by an interdisciplinary approach that combines genome-wide experiments, targeted assays, and bioinformatics. Specifically, I will use high-throughput functional assays in combination with the Gal4/UAS system to systematically test whether transcriptional repressors can repress all active promoters and enhancers or only specific ones but not others. Further, I aim to uncover the mechanisms behind active repression by recruiting repressors to active promoters and enhancers in a rapidly inducible manner, using chemically-inducible-proximity, to then assess the changes to DNA accessibility, histone modifications, and Pol II activity. In addition, I will measure differential protein composition and PTMs at active genomic regions, before and during induced repression. These approaches should identify critical molecular events, proteins, or PTMs and allow me to test their causal involvement in repression.   This project has the potential to greatly improve our mechanistic understanding of transcriptional repression, which despite its importance for gene expression, development and disease has remained poorly understood.