Groucho (Gro) is a transcriptional corepressor that plays a critical role in Drosophila embryonic development. It contains ordered C- and N-terminal domains required, respectively, for repressor binding and tetramerization in addition to a disordered, but essential, central region. While many mechanisms of Gro mediated repression have been proposed, none fully account for the functional importance of the disordered central domains. In this thesis, I attempt to elucidate the function of this region.
The unstructured Gro central domains may mediate a wide range of protein-protein interactions. To identify Gro interacting proteins, I performed affinity purification followed by mass spectrometry analysis. I recovered over 160 potential interacting proteins that function in chromatin organization, developmental processes, and mRNA splicing. In addition, a co-IP experiment was performed to confirm that one of the interacting complexes, the U1 snRNP complex, physically interacts with Gro. I also employed a high throughput RNAi screen involving Gal4-Gro-responsive luciferase reporter to systematically test the functional relevance of the 160 potential interacting proteins. Two of the U1 specific proteins were shown to be positive regulators of Gro function. As the affinity purification and reporter assay results imply regulation of Gro activity by the U1 snRNP complex, I analyzed the expression profile of cells subjected to knockdown of Gro or snRNP U1C. The two data sets revealed a significant overlap between genes regulated by these two factors. Furthermore, comparison of our RNA-seq data with Gro and RNA polymerase II ChIP data led to number of insights, including the finding that Gro-repressed genes are enriched for promoter-proximal RNA polymerase II. In conclusion, these findings suggest roles for spliceosomal components and paused Pol II in Gro mediated repression.