Elucidation of core biological processes requires an understanding of how transcription is regulated. Transcriptional regulation is critical for fine tuning gene expression to achieve precise temporal and spatial expression patterns. Key components of transcriptional regulation are repressors and corepressors—proteins that are recruited to DNA to reduce or turn off gene expression. Corepressors such as Rb and CtBP are essential metazoan transcription factors that regulate the expression of a diversity of genes. Retinoblastoma (Rb) was the first identified human tumor suppressor protein. It represses gene expression by binding to E2F transcriptional activators and recruiting histone modifiers, and has been shown to regulate genes through preferential interactions with promoter-proximal sequences. The C-terminal binding protein (CtBP) can function from both promoters and enhancers, and also regulates gene expression through recruitment of chromatin-modifying factors. CtBP is homologous to alpha-hydroxy acid dehydrogenases, and requires NADH binding and oligomerization to function. It has an unstructured C-terminal domain whose function in gene regulation is unknown. Both Rb and CtBP have diversified over evolutionary time through gene duplications in specific lineages, and CtBP has evolved unique protein isoforms through alternative splicing. The gene encoding Rb has undergone duplication in vertebrates and in the Drosophila lineage, and the gene encoding CtBP has duplicated multiple times in vertebrates, with alternative splice isoforms found across Metazoa, including in Drosophila. Such evolutionary variation is undoubtedly significant for Rb and CtBP gene regulatory functions, as many of the paralogs and certain alternative splice forms are highly conserved. The specific activities and positive selection for multiple Rb paralogs and CtBP isoforms is not well understood, however. To uncover differences between Rb paralogs and CtBP isoforms in the Drosophila system, I made use of a modified CRISPR/Cas9 technology, called CRISPRi. In CRISPRi, a nuclease-dead Cas9 is fused to transcription factors and epigenetic modifiers to recruit them to genomic sequences via gene-specific guide RNAs for gene regulation. I adapted this technology to recruit Rb and CtBP proteins to gene promoters to characterize their repression properties and identify possible intrinsic differences between paralogs and isoforms. This direct, comparative approach to characterizing corepressor action in vivo takes advantage of powerful molecular genetic tools to shed light on how evolutionary forces have sculpted the activity of these conserved regulatory proteins. Additionally, through studies of Rb, we identified a form of gene regulation that we term “soft repression”, in which a repressor functions to subtly, but precisely, modulate gene expression, rather than acting as an on/off switch. In a separate but related study that stemmed from structure-function questions about the CtBP intrinsically disordered C-terminal domain, I, along with a team of junior researchers, took a comparative phylogenetic approach to uncover the history of this protein’s evolution. Through analysis of extant ‘omic data, phylogenetic analyses, and precise molecular biology tools outlined here, we furthered our understanding of how these two key corepressors function in the Drosophila system, which we expect will inform future studies that have biomedical relevance.
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