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

Retinoblastoma tumor suppressor proteins (Rb) are highly conserved metazoan transcriptional corepressors involved in regulating the expression of thousands of genes. The vertebrate lineage and the Drosophila genus independently experienced an Rb gene duplication event, leading to the expression of several Rb paralogs whose unique and redundant roles in gene regulation remain to be fully explored. Here, we used a novel CRISPRi system in Drosophila to identify the significance of paralogy in the Rb family. We engineered dCas9 fusions to the fly Rbf1 and Rbf2 paralogs and deployed them to gene promoters in vivo, studying them in their native chromatin context. By directly querying the in vivo response of dozens of genes to Rbf1 and Rbf2 targeting, using both transcriptional as well as sensitive developmental readouts, we find that Rb paralogs function as "soft repressors" and have highly context-specific activities. Our comparison of targeting endogenous genes to reporter genes in cell culture identified striking differences in activity, underlining the importance of using CRISPRi effectors in a physiologically relevant context to identify paralog-specific activities. Our study uncovers the complexity of Rb-mediated transcriptional regulation in a living organism, and serves as a stepping stone for future CRISPRi development in Drosophila.

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.

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.

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.

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.

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.

Genome-Wide Analysis of Drosophila RBf2 Protein Highlights the Diversity of RB Family Targets and Possible Role in Regulation of Ribosome Biosynthesis.

RBf2 is a recently evolved retinoblastoma family member in Drosophila that differs from RBf1, especially in the C-terminus. To investigate whether the unique features of RBf2 contribute to diverse roles in gene regulation, we performed chromatin immunoprecipitation sequencing for both RBf2 and RBf1 in embryos. A previous model for RB-E2F interactions suggested that RBf1 binds dE2F1 or dE2F2, whereas RBf2 is restricted to binding to dE2F2; however, we found that RBf2 targets approximately twice as many genes as RBf1. Highly enriched among the RBf2 targets were ribosomal protein genes. We tested the functional significance of this finding by assessing RBf activity on ribosomal protein promoters and the endogenous genes. RBf1 and RBf2 significantly repressed expression of some ribosomal protein genes, although not all bound genes showed transcriptional effects. Interestingly, many ribosomal protein genes are similarly targeted in human cells, indicating that these interactions may be relevant for control of ribosome biosynthesis and growth. We carried out bioinformatic analysis to investigate the basis for differential targeting by these two proteins and found that RBf2-specific promoters have distinct sequence motifs, suggesting unique targeting mechanisms. Association of RBf2 with these promoters appears to be independent of dE2F2/dDP, although promoters bound by both RBf1 and RBf2 require dE2F2/dDP. The presence of unique RBf2 targets suggest that evolutionary appearance of this corepressor represents the acquisition of potentially novel roles in gene regulation for the RB family.

The Evolutionarily Conserved C-terminal Domains in the Mammalian Retinoblastoma Tumor Suppressor Family Serve as Dual Regulators of Protein Stability and Transcriptional Potency.

The retinoblastoma (RB) tumor suppressor and related family of proteins play critical roles in development through their regulation of genes involved in cell fate. Multiple regulatory pathways impact RB function, including the ubiquitin-proteasome system with deregulated RB destruction frequently associated with pathogenesis. With the current study we explored the mechanisms connecting proteasome-mediated turnover of the RB family to the regulation of repressor activity. We find that steady state levels of all RB family members, RB, p107, and p130, were diminished during embryonic stem cell differentiation concomitant with their target gene acquisition. Proteasome-dependent turnover of the RB family is mediated by distinct and autonomously acting instability elements (IE) located in their C-terminal regulatory domains in a process that is sensitive to cyclin-dependent kinase (CDK4) perturbation. The IE regions include motifs that contribute to E2F-DP transcription factor interaction, and consistently, p107 and p130 repressor potency was reduced by IE deletion. The juxtaposition of degron sequences and E2F interaction motifs appears to be a conserved feature across the RB family, suggesting the potential for repressor ubiquitination and specific target gene regulation. These findings establish a mechanistic link between regulation of RB family repressor potency and the ubiquitin-proteasome system.

Role of Drosophila retinoblastoma protein instability element in cell growth and proliferation.

The RB tumor suppressor, a regulator of the cell cycle, apoptosis, senescence, and differentiation, is frequently mutated in human cancers. We recently described an evolutionarily conserved C-terminal "instability element" (IE) of the Drosophila Rbf1 retinoblastoma protein that regulates its turnover. Misexpression of wild-type or non-phosphorylatable forms of the Rbf1 protein leads to repression of cell cycle genes. In contrast, overexpression of a defective form of Rbf1 lacking the IE (ΔIE), a stabilized but transcriptionally less active form of the protein, induced ectopic S phase in cell culture. To determine how mutations in the Rbf1 IE may induce dominant effects in a developmental context, we assessed the impact of in vivo expression of mutant Rbf1 proteins on wing development. ΔIE expression resulted in overgrowth of larval wing imaginal discs and larger adult wings containing larger cells. In contrast, a point mutation in a conserved lysine of the IE (K774A) generated severely disrupted, reduced wings. These contrasting effects appear to correlate with control of apoptosis; expression of the pro-apoptotic reaper gene and DNA fragmentation measured by acridine orange stain increased in flies expressing the K774A isoform and was suppressed by expression of Rbf1ΔIE. Intriguingly, cancer associated mutations affecting RB homologs p130 and p107 may similarly induce dominant phenotypes.

Integrated stability and activity control of the Drosophila Rbf1 retinoblastoma protein.

The retinoblastoma (RB) family transcriptional corepressors regulate diverse cellular events including cell cycle, senescence, and differentiation. The activity and stability of these proteins are mediated by post-translational modifications; however, we lack a general understanding of how distinct modifications coordinately impact both of these properties. Previously, we showed that protein turnover and activity are tightly linked through an evolutionarily conserved C-terminal instability element (IE) in the Drosophila RB-related protein Rbf1; surprisingly, mutant proteins with enhanced stability were less, not more active. To better understand how activity and turnover are controlled in this model RB protein, we assessed the impact of Cyclin-Cdk kinase regulation on Rbf1. An evolutionarily conserved N-terminal threonine residue is required for Cyclin-Cdk response and showed a dominant impact on turnover and activity; however, specific residues in the C-terminal IE differentially impacted Rbf1 activity and turnover, indicating an additional level of regulation. Strikingly, specific IE mutations that impaired turnover but not activity induced dramatic developmental phenotypes in the Drosophila eye. Mutation of the highly conserved Lys-774 residue induced hypermorphic phenotypes that mimicked the loss of phosphorylation control; mutation of the corresponding codon of the human RBL2 gene has been reported in lung tumors. Our data support a model in which closely intermingled residues within the conserved IE govern protein turnover, presumably through interactions with E3 ligases, and protein activity via contacts with E2F transcription partners. Such functional relationships are likely to similarly impact mammalian RB family proteins, with important implications for development and disease.

Evidence for autoregulation and cell signaling pathway regulation from genome-wide binding of the Drosophila retinoblastoma protein.

The retinoblastoma (RB) tumor suppressor protein is a transcriptional cofactor with essential roles in cell cycle and development. Physical and functional targets of RB and its paralogs p107/p130 have been studied largely in cultured cells, but the full biological context of this family of proteins' activities will likely be revealed only in whole organismal studies. To identify direct targets of the major Drosophila RB counterpart in a developmental context, we carried out ChIP-Seq analysis of Rbf1 in the embryo. The association of the protein with promoters is developmentally controlled; early promoter access is globally inhibited, whereas later in development Rbf1 is found to associate with promoter-proximal regions of approximately 2000 genes. In addition to conserved cell-cycle-related genes, a wholly unexpected finding was that Rbf1 targets many components of the insulin, Hippo, JAK/STAT, Notch, and other conserved signaling pathways. Rbf1 may thus directly affect output of these essential growth-control and differentiation pathways by regulation of expression of receptors, kinases and downstream effectors. Rbf1 was also found to target multiple levels of its own regulatory hierarchy. Bioinformatic analysis indicates that different classes of genes exhibit distinct constellations of motifs associated with the Rbf1-bound regions, suggesting that the context of Rbf1 recruitment may vary within the Rbf1 regulon. Many of these targeted genes are bound by Rbf1 homologs in human cells, indicating that a conserved role of RB proteins may be to adjust the set point of interlinked signaling networks essential for growth and development.

Ubiquitination of retinoblastoma family protein 1 potentiates gene-specific repression function.

The retinoblastoma (RB) tumor suppressor family functions as a regulatory node governing cell cycle progression, differentiation, and apoptosis. Post-translational modifications play a critical role in modulating RB activity, but additional levels of control, including protein turnover, are also essential for proper function. The Drosophila RB homolog Rbf1 is subjected to developmentally cued proteolysis mediated by an instability element (IE) present in the C terminus of this protein. Paradoxically, instability mediated by the IE is also linked to Rbf1 repression potency, suggesting that proteolytic machinery may also be directly involved in transcriptional repression. We show that the Rbf1 IE is an autonomous degron that stimulates both Rbf1 ubiquitination and repression potency. Importantly, Rbf1 IE function is promoter-specific, contributing to repression of cell cycle responsive genes but not to repression of cell signaling genes. The multifunctional IE domain thus provides Rbf1 flexibility for discrimination between target genes embedded in divergent cellular processes.