A regulatory role for the unstructured C-terminal domain of the CtBP transcriptional corepressor

The C-terminal Binding Protein (CtBP) is a transcriptional corepressor that plays critical roles in development, tumorigenesis, and cell fate. CtBP proteins are structurally similar to alpha hydroxyacid dehydrogenases and feature a prominent intrinsically disordered region in the C-terminus. In the mammalian system, CtBP proteins lacking the C-terminal Domain (CTD) are able to function as transcriptional regulators and oligomerize, putting into question the significance of this unstructured domain for gene regulation. Yet, the presence of an unstructured CTD of ~100 residues, including some short motifs, is conserved across Bilateria, indicating the importance of maintaining this domain over evolutionary time. To uncover the significance of the CtBP CTD, we functionally tested naturally occurring Drosophila isoforms of CtBP that possess or lack the CTD, namely CtBP(L) and CtBP(S). We used the CRISPRi system to recruit dCas9-CtBP(L) and dCas9-CtBP(S) to endogenous promoters to directly compare their transcriptional impacts in vivo. Interestingly, CtBP(S) was able to significantly repress transcription of the Mpp6 promoter, while CtBP(L) was much weaker, suggesting that the long CTD may modulate CtBP’s repression activity. In contrast, in cell culture, the isoforms behaved similarly on a transfected Mpp6 reporter gene. The context-specific differences in activity of these two developmentally-regulated isoforms suggests that the CTD may help provide a spectrum of repression activity suitable for developmental programs.