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.

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.

Retinoblastoma protein activity revealed by CRISPRi study of divergent Rbf1 and Rbf2 paralogs.

Retinoblastoma tumor suppressor proteins regulate the key transition from G1 to S phase of the cell cycle. The mammalian Rb family comprises Rb, p107, and p130, with overlapping and unique roles in gene regulation. Drosophila experienced an independent gene duplication event, leading to the Rbf1 and Rbf2 paralogs. To uncover the significance of paralogy in the Rb family, we used CRISPRi. We engineered dCas9 fusions to Rbf1 and Rbf2, and deployed them to gene promoters in developing Drosophila tissue to study their relative impacts on gene expression. On some genes, both Rbf1 and Rbf2 mediate potent repression, in a highly distance-dependent manner. In other cases, the two proteins have different effects on phenotype and gene expression, indicating different functional potential. In a direct comparison of Rb activity on endogenous genes and transiently transfected reporters, we found that only qualitative, but not key quantitative aspects of repression were conserved, indicating that the native chromatin environment generates context-specific effects of Rb activity. Our study uncovers the complexity of Rb-mediated transcriptional regulation in a living organism, which is clearly impacted by the different promoter landscapes and the evolution of the Rb proteins themselves.

The Cynosure of CtBP: Evolution of a Bilaterian 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.

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

After a COVID-related hiatus, the fifth biennial symposium on Evolution and Core Processes in Gene Regulation met at the Stowers Institute in Kansas City, Missouri July 21 to 24, 2022. This symposium, sponsored by the American Society for Biochemistry and Molecular Biology (ASBMB), featured experts in gene regulation and evolutionary biology. Topic areas covered enhancer evolution, the cis-regulatory code, and regulatory variation, with an overall focus on bringing the power of deep learning (DL) to decipher DNA sequence information. DL is a machine learning method that uses neural networks to learn complex rules that make predictions about diverse types of data. When DL models are trained to predict genomic data from DNA sequence information, their high prediction accuracy allows the identification of impactful genetic variants within and across species. In addition, the learned sequence rules can be extracted from the model and provide important clues about the mechanistic underpinnings of the cis-regulatory code.

Tête-à-tête with CtBP dimers.

Jecrois et al. (2020) use cryoelectron microscopy to illuminate the tetrameric conformation of the CtBP2 transcriptional corepressor, a protein frequently overexpressed in human cancers. The in vivo functional characterization of tetramer-destabilizing mutants indicates that tetramerization is a physiologically important process, critical for CtBP control of gene regulation and cell migration.

Soft repression: Subtle transcriptional regulation with global impact.

Pleiotropically acting eukaryotic corepressors such as retinoblastoma and SIN3 have been found to physically interact with many widely expressed "housekeeping" genes. Evidence suggests that their roles at these loci are not to provide binary on/off switches, as is observed at many highly cell-type specific genes, but rather to serve as governors, directly modulating expression within certain bounds, while not shutting down gene expression. This sort of regulation is challenging to study, as the differential expression levels can be small. We hypothesize that depending on context, corepressors mediate "soft repression," attenuating expression in a less dramatic but physiologically appropriate manner. Emerging data indicate that such regulation is a pervasive characteristic of most eukaryotic systems, and may reflect the mechanistic differences between repressor action at promoter and enhancer locations. Soft repression may represent an essential component of the cybernetic systems underlying metabolic adaptations, enabling modest but critical adjustments on a continual basis.

Feasibility and acceptability of an online response inhibition cognitive training program for youth with Williams syndrome.

Williams syndrome (WS) is a genetic neurodevelopmental disorder often accompanied by inhibitory difficulties. Online cognitive training programs show promise for improving cognitive functions. No such interventions have been developed for individuals with WS, but to explore the practicality of large-scale online cognitive training for this population, we must first investigate whether families of those with WS find these programs feasible and acceptable. Twenty individuals aged 10-17 years with WS, along with parents, participated in a pilot online cognitive training program supervised in real time using videoconference software. We evaluated the feasibility and acceptability of this response inhibition training using three parent questionnaires. Descriptive data are reported for the measures of feasibility and acceptability. Overall, the online procedures received a positive reaction from families. Parents were likely to recommend the study to others. They indicated training was ethical and acceptable despite feeling neutral about effectiveness. The frequency and duration of sessions were acceptable to families (two 20-to-30-min sessions per week; 10 sessions total). Families provided feedback and offered suggestions for improvement, such as more flexibility in scheduling and decreasing time spent in review of procedures.

Transcriptional Regulation of INSR, the Insulin Receptor Gene.

The insulin receptor gene encodes an evolutionarily conserved signaling protein with a wide spectrum of functions in metazoan development. The insulin signaling pathway plays key roles in processes such as metabolic regulation, growth control, and neuronal function. Misregulation of the pathway features in diabetes, cancer, and neurodegenerative diseases, making it an important target for clinical interventions. While much attention has been focused on differential pathway activation through ligand availability, sensitization of overall signaling may also be mediated by differential expression of the insulin receptor itself. Although first characterized as a "housekeeping" gene with stable expression, comparative studies have shown that expression levels of the human INSR mRNA differ by tissue and in response to environmental signals. Our recent analysis of the transcriptional controls affecting expression of the Drosophila insulin receptor gene indicates that a remarkable amount of DNA is dedicated to encoding sophisticated feedback and feed forward signals. The human INSR gene is likely to contain a similar level of transcriptional complexity; here, we summarize over three decades of molecular biology and genetic research that points to a still incompletely understood regulatory control system. Further elucidation of transcriptional controls of INSR will provide the basis for understanding human genetic variation that underlies population-level physiological differences and disease.