84 Oral - Gene Regulation
Friday April 08, 11:30 AM - 11:45 AM

Authors:
Ana-Maria Raicu ¹; Patricia Castanheira ²; David Arnosti ²

Affiliations:
1) Cell and Molecular Biology Program, Michigan State University, East Lansing, MI; 2) Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI

Keywords:
d. repressors/corepressors; g. CRISPR/Cas9

CRISPR activation and interference (CRISPRa/i) have revolutionized gene expression studies, allowing us to precisely regulate genes through targeting activator and repressor domains across the genome. With CRISPRa/i, we can turn genes on and off with high precision and perform large-scale LOF and GOF screens in many systems. Yet, this modified CRISPR system is not often used for studying the mechanisms of gene regulation by transcription factors. Here, we have adapted the CRISPRi system for targeting transcriptional co-repressors to gene promoters to uncover their mechanisms of repression. We engineered dCas9 fusions to the Retinoblastoma family proteins (Rb) and the C-terminal binding protein (CtBP), which are highly conserved co-repressors across Metazoa. In D. melanogaster, the Rb family consists of the Rbf1 and Rbf2 paralogs, while a single CtBP gene encodes the CtBP-long and CtBP-short isoforms, which differ by about 100 residues in the CTD. We expressed the dCas9-Rb and dCas9-CtBP chimeras in the fly wing for in vivo targeting to specific promoters, and tested each protein’s effect on target gene expression and their mechanism of repression. We found that on some genes, both Rbf1 and Rbf2 are able to mediate potent repression, but that this happens in a distance-dependent manner. In contrast, we found examples where Rb proteins modestly activate the target gene, suggesting that they do not always function to turn off transcription. Notably, an Rbf1 CTD mutant lacking the Instability Element (IE), which we previously identified as being necessary for activity, was just as good a repressor as the wild type protein. This suggests that the IE is chiefly involved in recruiting and is not necessary for repression. Between the CtBP isoforms, CtBP-short was a much more potent repressor than CtBP-long, which indicates that the long CTD extension may have an inhibitory role in transcription. Our novel adaptation of a well-established CRISPR tool has allowed us to probe mechanisms of repression in vivo in the fly and compare factors in the same contexts with great precision. This molecular analysis of promoter-specific regulation by Rb and CtBP proteins will enhance our understanding of these conserved regulatory proteins in development and disease.