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.

Soft repression and chromatin modification by conserved transcriptional corepressors.

Transcriptional regulation in eukaryotic cells involves the activity of multifarious DNA-binding transcription factors and recruited corepressor complexes. Together, these complexes interact with the core transcriptional machinery, chromatin, and nuclear environment to effect complex patterns of gene regulation. Much focus has been paid to the action of master regulatory switches that are key to developmental and environmental responses, as these genetic elements have important phenotypic effects. The regulation of widely-expressed metabolic control genes has been less well studied, particularly in cases in which physically-interacting repressors and corepressors have subtle influences on steady-state expression. This latter phenomenon, termed "soft repression" is a topic of increasing interest as genomic approaches provide ever more powerful tools to uncover the significance of this level of control. This review provides an oversight of classic and current approaches to the study of transcriptional repression in eukaryotic systems, with a specific focus on opportunities and challenges that lie ahead in the study of soft repression.

Long-range repression by ecdysone receptor on complex enhancers of the insulin receptor gene.

The insulin signalling pathway is evolutionarily conserved throughout metazoans, playing key roles in development, growth, and metabolism. Misregulation of this pathway is associated with a multitude of disease states including diabetes, cancer, and neurodegeneration. The human insulin receptor gene (INSR) is widely expressed throughout development and was previously described as a 'housekeeping' gene. Yet, there is abundant evidence that this gene is expressed in a cell-type specific manner, with dynamic regulation in response to environmental signals. The Drosophila insulin-like receptor gene (InR) is homologous to the human INSR gene and was previously shown to be regulated by multiple transcriptional elements located primarily within the introns of the gene. These elements were roughly defined in ~1.5 kbp segments, but we lack an understanding of the potential detailed mechanisms of their regulation. We characterized the substructure of these cis-regulatory elements in Drosophila S2 cells, focusing on regulation through the ecdysone receptor (EcR) and the dFOXO transcription factor. By identifying specific locations of activators and repressors within 300 bp subelements, we show that some previously identified enhancers consist of relatively compact clusters of activators, while others have a distributed architecture not amenable to further reduction. In addition, these assays uncovered a long-range repressive action of unliganded EcR. The complex transcriptional circuitry likely endows InR with a highly flexible and tissue-specific response to tune insulin signalling. Further studies will provide insights to demonstrate the impact of natural variation in this gene's regulation, applicable to human genetic studies.

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.

Long-range repression by ecdysone receptor on complex enhancers of the insulin receptor gene.

The insulin signaling pathway is evolutionarily conserved throughout metazoans, playing key roles in development, growth, and metabolism. Misregulation of this pathway is associated with a multitude of disease states including diabetes, cancer, and neurodegeneration. Genome-wide association studies indicate that natural variants in putative intronic regulatory elements of the human insulin receptor gene ( INSR) are associated with metabolic conditions, however, this gene's transcriptional regulation remains incompletely studied. INSR is widely expressed throughout development and was previously described as a 'housekeeping' gene. Yet, there is abundant evidence that this gene is expressed in a cell-type specific manner, with dynamic regulation in response to environmental signals. The Drosophila insulin-like receptor gene ( InR ) is homologous to the human INSR gene and was previously shown to be regulated by multiple transcriptional elements located primarily within the introns of the gene. These elements were roughly defined in ∼1.5 kbp segments, but we lack an understanding of the potential detailed mechanisms of their regulation, as well as the integrative output of the battery of enhancers in the entire locus. Using luciferase assays, we characterized the substructure of these cis-regulatory elements in Drosophila S2 cells, focusing on regulation through the ecdysone receptor (EcR) and the dFOXO transcription factor. The direct action of EcR on Enhancer 2 reveals a bimodal form of regulation, with active repression in the absence of the ligand, and positive activation in the presence of 20E. By identifying the location of activators of this enhancer, we characterized a long-range of repression acting over at least 475 bp, similar to the action of long-range repressors found in the embryo. dFOXO and 20E have contrasting effects on some of the individual regulatory elements, and for the adjacent enhancers 2 and 3, their influence was/was not found to be additive, indicating that enhancer action on this locus can/cannot be characterized in part by additive models. Other characterized enhancers from within this locus exhibited "distributed" or "localized" modes of action, suggesting that predicting the joint functional output of multiple regulatory regions will require a deeper experimental characterization. The noncoding intronic regions of InR have demonstrated dynamic regulation of expression and cell type specificity. This complex transcriptional circuitry goes beyond the simple conception of a 'housekeeping' gene. Further studies are aimed at identifying how these elements work together in vivo to generate finely tuned expression in tissue- and temporal-specific manners, to provide a guide to understanding the impact of natural variation in this gene's regulation, applicable to human genetic studies.

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.

Cell cycle expression of polarity genes features Rb targeting of Vang.

Specification of cellular polarity is vital to normal tissue development and function. Pioneering studies in Drosophila and C. elegans have elucidated the composition and dynamics of protein complexes critical for establishment of cell polarity, which is manifest in processes such as cell migration and asymmetric cell division. Conserved throughout metazoans, planar cell polarity (PCP) genes are implicated in disease, including neural tube closure defects associated with mutations in VANGL1/2. PCP protein regulation is well studied; however, relatively little is known about transcriptional regulation of these genes. Our earlier study revealed an unexpected role for the fly Rbf1 retinoblastoma corepressor protein, a regulator of cell cycle genes, in transcriptional regulation of polarity genes. Here we analyze the physiological relevance of the role of E2F/Rbf proteins in the transcription of the key core polarity gene Vang. Targeted mutations to the E2F site within the Vang promoter disrupts binding of E2F/Rbf proteins in vivo, leading to polarity defects in wing hairs. E2F regulation of Vang is supported by the requirement for this motif in a reporter gene. Interestingly, the promoter is repressed by overexpression of E2F1, a transcription factor generally identified as an activator. Consistent with the regulation of this polarity gene by E2F and Rbf factors, expression of Vang and other polarity genes is found to peak in G2/M phase in cells of the embryo and wing imaginal disc, suggesting that cell cycle signals may play a role in regulation of these genes. These findings suggest that the E2F/Rbf complex mechanistically links cell proliferation and polarity.

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.

Transcriptional Enhancers in Drosophila.

Key discoveries in Drosophila have shaped our understanding of cellular "enhancers." With a special focus on the fly, this chapter surveys properties of these adaptable cis-regulatory elements, whose actions are critical for the complex spatial/temporal transcriptional regulation of gene expression in metazoa. The powerful combination of genetics, molecular biology, and genomics available in Drosophila has provided an arena in which the developmental role of enhancers can be explored. Enhancers are characterized by diverse low- or high-throughput assays, which are challenging to interpret, as not all of these methods of identifying enhancers produce concordant results. As a model metazoan, the fly offers important advantages to comprehensive analysis of the central functions that enhancers play in gene expression, and their critical role in mediating the production of phenotypes from genotype and environmental inputs. A major challenge moving forward will be obtaining a quantitative understanding of how these cis-regulatory elements operate in development and disease.

Selective repression of the Drosophila cyclin B promoter by retinoblastoma and E2F proteins.

The Cyclin B1 gene encodes a G2/M cyclin that is deregulated in various human cancers, however, the transcriptional regulation of this gene is incompletely understood. The E2F and retinoblastoma family of proteins are involved in this gene's regulation, but there is disagreement on which of the E2F and retinoblastoma proteins interact with the promoter to regulate this gene. Here, we dissect the promoter region of the Drosophila CycB gene, and study the role of Rbf and E2F factors in its regulation. This gene exhibits remarkable features that distinguish it from G1/S regulated promoters, such as PCNA. The promoter is comprised of modular elements with dedicated repressor and activator functions, including a segment spanning the first intron that interferes with a 5' activator element. A highly active minimal promoter (-464, +100) is repressed by the Rbf1 retinoblastoma protein, but much more potently repressed by the Rbf2 protein, which has been linked in other studies to control of cell growth genes. Unlike many other cell-cycle genes, which are activated by E2F1 and repressed by E2F2, CycB is potently activated by E2F2, and repressed by E2F1. Although the bulk of Rbf binding is associated with a region 5' of the core promoter, E2F and retinoblastoma proteins functionally interact with the basal promoter region, in part through a conserved E2F site at -80 bp. The specific regulatory requirements of this late cell cycle promoter appear to be linked to the unique activities of E2F and retinoblastoma family members acting on a complex cis-regulatory circuit.

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.

Diversification of Retinoblastoma Protein Function Associated with Cis and Trans Adaptations.

Retinoblastoma proteins are eukaryotic transcriptional corepressors that play central roles in cell cycle control, among other functions. Although most metazoan genomes encode a single retinoblastoma protein, gene duplications have occurred at least twice: in the vertebrate lineage, leading to Rb, p107, and p130, and in Drosophila, an ancestral Rbf1 gene and a derived Rbf2 gene. Structurally, Rbf1 resembles p107 and p130, and mutation of the gene is lethal. Rbf2 is more divergent and mutation does not lead to lethality. However, the retention of Rbf2 >60 My in Drosophila points to essential functions, which prior cell-based assays have been unable to elucidate. Here, using genomic approaches, we provide new insights on the function of Rbf2. Strikingly, we show that Rbf2 regulates a set of cell growth-related genes and can antagonize Rbf1 on specific genes. These unique properties have important implications for the fly; Rbf2 mutants show reduced egg laying, and lifespan is reduced in females and males. Structural alterations in conserved regions of Rbf2 gene suggest that it was sub- or neofunctionalized to develop specific regulatory specificity and activity. We define cis-regulatory features of Rbf2 target genes that allow preferential repression by this protein, indicating that it is not a weaker version of Rbf1 as previously thought. The specialization of retinoblastoma function in Drosophila may reflect a parallel evolution found in vertebrates, and raises the possibility that cell growth control is equally important to cell cycle function for this conserved family of transcriptional corepressors.

Complex cis-regulatory landscape of the insulin receptor gene underlies the broad expression of a central signaling regulator.

Insulin signaling plays key roles in development, growth and metabolism through dynamic control of glucose uptake, global protein translation and transcriptional regulation. Altered levels of insulin signaling are known to play key roles in development and disease, yet the molecular basis of such differential signaling remains obscure. Expression of the insulin receptor (InR) gene itself appears to play an important role, but the nature of the molecular wiring controlling InR transcription has not been elucidated. We characterized the regulatory elements driving Drosophila InR expression and found that the generally broad expression of this gene is belied by complex individual switch elements, the dynamic regulation of which reflects direct and indirect contributions of FOXO, EcR, Rbf and additional transcription factors through redundant elements dispersed throughout ∼40 kb of non-coding regions. The control of InR transcription in response to nutritional and tissue-specific inputs represents an integration of multiple cis-regulatory elements, the structure and function of which may have been sculpted by evolutionary selection to provide a highly tailored set of signaling responses on developmental and tissue-specific levels.

Quantitative perturbation-based analysis of gene expression predicts enhancer activity in early Drosophila embryo.

Enhancers constitute one of the major components of regulatory machinery of metazoans. Although several genome-wide studies have focused on finding and locating enhancers in the genomes, the fundamental principles governing their internal architecture and cis-regulatory grammar remain elusive. Here, we describe an extensive, quantitative perturbation analysis targeting the dorsal-ventral patterning gene regulatory network (GRN) controlled by Drosophila NF-κB homolog Dorsal. To understand transcription factor interactions on enhancers, we employed an ensemble of mathematical models, testing effects of cooperativity, repression, and factor potency. Models trained on the dataset correctly predict activity of evolutionarily divergent regulatory regions, providing insights into spatial relationships between repressor and activator binding sites. Importantly, the collective predictions of sets of models were effective at novel enhancer identification and characterization. Our study demonstrates how experimental dataset and modeling can be effectively combined to provide quantitative insights into cis-regulatory information on a genome-wide scale.

Regulation of cell polarity determinants by the Retinoblastoma tumor suppressor protein.

In addition to their canonical roles in the cell cycle, RB family proteins regulate numerous developmental pathways, although the mechanisms remain obscure. We found that Drosophila Rbf1 associates with genes encoding components of the highly conserved apical-basal and planar cell polarity pathways, suggesting a possible regulatory role. Here, we show that depletion of Rbf1 in Drosophila tissues is indeed associated with polarity defects in the wing and eye. Key polarity genes aPKC, par6, vang, pk, and fmi are upregulated, and an aPKC mutation suppresses the Rbf1-induced phenotypes. RB control of cell polarity may be an evolutionarily conserved function, with important implications in cancer metastasis.

Cori meets Dobzhansky: Evolution and Gene Expression in St. Louis: A report on the "Evolution and Core Processes in Gene Regulation" meeting in St. Louis, June 25-28, 2015.

St. Louis and its famous Gateway Arch were the setting of the Special Symposium: Evolution and Core Processes in Gene Regulation, sponsored by the American Society for Biochemistry and Molecular Biology. Biochemists and evolutionary biologists highlighted growing connections between studies of biochemical mechanism and natural selection on gene expression.