Animal development and homeostasis critically depend on the accurate regulation of gene transcription, which includes the silencing of genes that should not be expressed. Repression is mediated by a specific class of transcription factors (TFs) termed repressors that, via the recruitment of co-repressors (CoRs), can dominantly prevent transcription, even in the presence of activating cues. However, the relationship between specific CoRs and enhancers has remained unclear. Here, we used functional genomics to uncover regulatory specificities between CoRs and enhancers. We show that enhancers can typically be repressed by only a subset of CoRs. Enhancers classified by CoR sensitivity also show distinct biological functions and endogenous chromatin features. Moreover, enhancers that are sensitive or resistant to silencing by specific CoRs differ in TF motif content, and their sensitivity to CoRs can be predicted based on TF motif content. Finally, we identified and validated specific TF motifs that have a direct impact on enhancers sensitivity or resistance towards specific CoRs, using large scale motif mutagenesis and addition experiments. This study reveals the existence of TF motif-based regulatory rules that coordinate CoRs-enhancer compatibilities. These specificities between repressors and activators not only suggest that repression occurs via distinct mechanisms, but also provide an additional layer in transcriptional regulation that allows for differential repression at close genomic distances and offers multiple ways for de-repression.

Widespread regulatory specificities between transcriptional corepressors and enhancers in Drosophila

http://www.jbc.org/article/S0021925822012030/pdf

Off the deep end: What can deep learning do for the gene expression field?

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 additionally feature an unstructured C-terminal domain (CTD). The role of a possible dehydrogenase activity has been postulated for the corepressor, although in vivo substrates are unknown, but the functional significance of the CTD is unclear. In the mammalian system, CtBP proteins lacking the CTD are able to function as transcriptional regulators and oligomerize, putting into question the significance of the CTD 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 this domain. To study the in vivo functional significance of the CTD, we turned to the Drosophila melanogaster system, which naturally expresses isoforms with the CTD (CtBP(L)), and isoforms lacking the CTD (CtBP(S)). We used the CRISPRi system to test dCas9-CtBP(S) and dCas9-CtBP(L) on diverse endogenous genes, to directly compare their transcriptional impacts in vivo. Interestingly, CtBP(S) was able to significantly repress transcription of the E2F2 and Mpp6 genes, while CtBP(L) had minimal impact, suggesting that the long CTD modulates CtBP’s repression activity. In contrast, in cell culture, the isoforms behaved similarly on a transfected Mpp6 reporter. Thus, we have identified context-specific effects of these two developmentally-regulated isoforms, and propose that differential expression of CtBP(S) and CtBP(L) may provide a spectrum of repression activity suitable for developmental programs.

/pdf/a-regulatory-role-for-the-unstructured-c-terminal-domain-of-11a2hub5.pdf

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

Evolution of sequence-specific transcription factors clearly drives lineage-specific innovations, but less is known about how changes in the central transcriptional machinery may contribute to evolutionary transformations. In particular, transcriptional regulators are rich in intrinsically disordered regions that appear to be magnets for evolutionary innovation. The C-terminal Binding Protein (CtBP) is a transcriptional corepressor derived from an ancestral lineage of alpha hydroxyacid dehydrogenases; it is found in mammals and invertebrates, and features a core NAD-binding domain as well as an unstructured C-terminus (CTD) of unknown function. CtBP can act on promoters and enhancers to repress transcription through chromatin-linked mechanisms. Our comparative phylogenetic study shows that CtBP is a bilaterian innovation whose CTD of about 100 residues is present in almost all orthologs. CtBP CTDs contain conserved blocks of residues and retain a predicted disordered property, despite having variations in the primary sequence. Interestingly, the structure of the C-terminus has undergone radical transformation independently in certain lineages including flatworms and nematodes. Also contributing to CTD diversity is the production of myriad alternative RNA splicing products, including the production of “short” tailless forms of CtBP in Drosophila. Additional diversity stems from multiple gene duplications in vertebrates, where up to five CtBP orthologs have been observed. Vertebrate lineages show fewer major modifications in the unstructured CTD, possibly because gene regulatory constraints of the vertebrate body plan place specific constraints on this domain. Our study highlights the rich regulatory potential of this previously unstudied domain of a central transcriptional regulator.

/pdf/the-cynosure-of-ctbp-evolution-of-a-bilaterian-3snombkl.pdf

Akshay Seenivasan

Papers

The Cynosure of CtBP: Evolution of a Bilaterian Transcriptional Corepressor