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Identifying the molecular origins by which new morphological structures evolve is one of the long standing problems in evolutionary biology. To date, vanishingly few examples provide a compelling account of how new morphologies were initially formed, thereby limiting our understanding of how diverse forms of life derived their complex features. Here, we provide evidence that the large projections on the Drosophila eugracilis phallus that are implicated in sexual conflict have evolved through co-option of the trichome genetic network. These unicellular apical projections on the phallus postgonal sheath are reminiscent of trichomes that cover the Drosophila body but are up to 20-fold larger in size. During their development, they express the transcription factor Shavenbaby, the master regulator of the trichome network. Consistent with the co-option of the Shavenbaby network during the evolution of the D. eugracilis projections, somatic mosaic CRISPR/Cas9 mutagenesis shows that shavenbaby is necessary for their proper length. Moreover, mis-expression of Shavenbaby in the sheath of D. melanogaster , a naïve species that lacks these extensions, is sufficient to induce small trichomes. These induced extensions rely on a genetic network that is shared to a large extent with the D. eugracilis projections, indicating its co-option but also some genetic rewiring. Thus, by leveraging a genetically tractable evolutionarily novelty, our work shows that the trichome-forming network is flexible enough that it can be co-opted in a new context, and subsequently refined to produce unique apical projections that are barely recognizable compared to their simpler ancestral beginnings.
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Congenital heart disease often arises from perturbations of transcription factors (TFs) that guide cardiac development. ISLET1 (ISL1) is a TF that influences early cardiac cell fate, as well as differentiation of other cell types including motor neuron progenitors (MNPs) and pancreatic islet cells. While lineage specificity of ISL1 function is likely achieved through combinatorial interactions, its essential cardiac interacting partners are unknown. By assaying ISL1 genomic occupancy in human induced pluripotent stem cell-derived cardiac progenitors (CPs) or MNPs and leveraging the deep learning approach BPNet, we identified motifs of other TFs that predicted ISL1 occupancy in each lineage, with NKX2.5 and GATA motifs being most closely associated to ISL1 in CPs. Experimentally, nearly two-thirds of ISL1-bound loci were co-occupied by NKX2.5 and/or GATA4. Removal of NKX2.5 from CPs led to widespread ISL1 redistribution, and overexpression of NKX2.5 in MNPs led to ISL1 occupancy of CP-specific loci. These results reveal how ISL1 guides lineage choices through a combinatorial code that dictates genomic occupancy and transcription.
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Células Madre Pluripotentes Inducidas , Factores de Transcripción , Humanos , Factores de Transcripción/metabolismo , Miocitos Cardíacos , Proteínas con Homeodominio LIM/genética , Proteínas con Homeodominio LIM/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Diferenciación Celular/genética , Proteína Homeótica Nkx-2.5/genética , Proteína Homeótica Nkx-2.5/metabolismo , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismoRESUMEN
Short tandem repeats (STRs) are enriched in eukaryotic cis-regulatory elements and alter gene expression, yet how they regulate transcription remains unknown. We found that STRs modulate transcription factor (TF)-DNA affinities and apparent on-rates by about 70-fold by directly binding TF DNA-binding domains, with energetic impacts exceeding many consensus motif mutations. STRs maximize the number of weakly preferred microstates near target sites, thereby increasing TF density, with impacts well predicted by statistical mechanics. Confirming that STRs also affect TF binding in cells, neural networks trained only on in vivo occupancies predicted effects identical to those observed in vitro. Approximately 90% of TFs preferentially bound STRs that need not resemble known motifs, providing a cis-regulatory mechanism to target TFs to genomic sites.
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Regulación de la Expresión Génica , Repeticiones de Microsatélite , Factores de Transcripción , Células Eucariotas , Factores de Transcripción/química , Factores de Transcripción/genética , Unión Proteica , Humanos , Animales , Saccharomyces cerevisiae , Dominios Proteicos , Conformación ProteicaRESUMEN
While the accessibility of enhancers is dynamically regulated during development, promoters tend to be constitutively accessible and poised for activation by paused Pol II. By studying Lola-I, a Drosophila zinc finger transcription factor, we show here that the promoter state can also be subject to developmental regulation independently of gene activation. Lola-I is ubiquitously expressed at the end of embryogenesis and causes its target promoters to become accessible and acquire paused Pol II throughout the embryo. This promoter transition is required but not sufficient for tissue-specific target gene activation. Lola-I mediates this function by depleting promoter nucleosomes, similar to the action of pioneer factors at enhancers. These results uncover a level of regulation for promoters that is normally found at enhancers and reveal a mechanism for the de novo establishment of paused Pol II at promoters.
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Drosophila , Embrión de Mamíferos , Animales , Regiones Promotoras Genéticas/genética , Drosophila/genética , Desarrollo Embrionario , Nucleosomas/genética , ARN Polimerasa II/genéticaRESUMEN
Chromatin accessibility is integral to the process by which transcription factors (TFs) read out cis-regulatory DNA sequences, but it is difficult to differentiate between TFs that drive accessibility and those that do not. Deep learning models that learn complex sequence rules provide an unprecedented opportunity to dissect this problem. Using zygotic genome activation in Drosophila as a model, we analyzed high-resolution TF binding and chromatin accessibility data with interpretable deep learning and performed genetic validation experiments. We identify a hierarchical relationship between the pioneer TF Zelda and the TFs involved in axis patterning. Zelda consistently pioneers chromatin accessibility proportional to motif affinity, whereas patterning TFs augment chromatin accessibility in sequence contexts where they mediate enhancer activation. We conclude that chromatin accessibility occurs in two tiers: one through pioneering, which makes enhancers accessible but not necessarily active, and the second when the correct combination of TFs leads to enhancer activation.
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Transcription factors (TF) are proteins that bind DNA in a sequence-specific manner to regulate gene transcription. Despite their unique intrinsic sequence preferences, in vivo genomic occupancy profiles of TFs differ across cellular contexts. Hence, deciphering the sequence determinants of TF binding, both intrinsic and context-specific, is essential to understand gene regulation and the impact of regulatory, non-coding genetic variation. Biophysical models trained on in vitro TF binding assays can estimate intrinsic affinity landscapes and predict occupancy based on TF concentration and affinity. However, these models cannot adequately explain context-specific, in vivo binding profiles. Conversely, deep learning models, trained on in vivo TF binding assays, effectively predict and explain genomic occupancy profiles as a function of complex regulatory sequence syntax, albeit without a clear biophysical interpretation. To reconcile these complementary models of in vitro and in vivo TF binding, we developed Affinity Distillation (AD), a method that extracts thermodynamic affinities de-novo from deep learning models of TF chromatin immunoprecipitation (ChIP) experiments by marginalizing away the influence of genomic sequence context. Applied to neural networks modeling diverse classes of yeast and mammalian TFs, AD predicts energetic impacts of sequence variation within and surrounding motifs on TF binding as measured by diverse in vitro assays with superior dynamic range and accuracy compared to motif-based methods. Furthermore, AD can accurately discern affinities of TF paralogs. Our results highlight thermodynamic affinity as a key determinant of in vivo binding, suggest that deep learning models of in vivo binding implicitly learn high-resolution affinity landscapes, and show that these affinities can be successfully distilled using AD. This new biophysical interpretation of deep learning models enables high-throughput in silico experiments to explore the influence of sequence context and variation on both intrinsic affinity and in vivo occupancy.
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What new questions can we ask about transcriptional regulation given recent developments in large-scale approaches?
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Regulación de la Expresión Génica , Regulación de la Expresión Génica/genéticaRESUMEN
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.
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COVID-19 , Aprendizaje Profundo , Humanos , Genómica , Redes Neurales de la Computación , Expresión GénicaRESUMEN
The Spt/Ada-Gcn5 Acetyltransferase (SAGA) coactivator complex has multiple modules with different enzymatic and non-enzymatic functions. How each module contributes to gene expression is not well understood. During Drosophila oogenesis, the enzymatic functions are not equally required, which may indicate that different genes require different enzymatic functions. An analogy for this phenomenon is the handyman principle: while a handyman has many tools, which tool he uses depends on what requires maintenance. Here we analyzed the role of the non-enzymatic core module during Drosophila oogenesis, which interacts with TBP. We show that depletion of SAGA-specific core subunits blocked egg chamber development at earlier stages than depletion of enzymatic subunits. These results, as well as additional genetic analyses, point to an interaction with TBP and suggest a differential role of SAGA modules at different promoter types. However, SAGA subunits co-occupied all promoter types of active genes in ChIP-seq and ChIP-nexus experiments, and the complex was not specifically associated with distinct promoter types in the ovary. The high-resolution genomic binding profiles were congruent with SAGA recruitment by activators upstream of the start site, and retention on chromatin by interactions with modified histones downstream of the start site. Our data illustrate that a distinct genetic requirement for specific components may conceal the fact that the entire complex is physically present and suggests that the biological context defines which module functions are critical.
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Proteínas de Drosophila/metabolismo , Drosophila melanogaster/fisiología , Histona Acetiltransferasas/metabolismo , Oogénesis/fisiología , Regiones Promotoras Genéticas , Animales , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Histona Acetiltransferasas/genética , Oogénesis/genéticaRESUMEN
Core promoter types differ in the extent to which RNA polymerase II (Pol II) pauses after initiation, but how this affects their tissue-specific gene expression characteristics is not well understood. While promoters with Pol II pausing elements are active throughout development, TATA promoters are highly active in differentiated tissues. We therefore used a genomics approach on late-stage Drosophila embryos to analyze the properties of promoter types. Using tissue-specific Pol II ChIP-seq, we found that paused promoters have high levels of paused Pol II throughout the embryo, even in tissues where the gene is not expressed, while TATA promoters only show Pol II occupancy when the gene is active. The promoter types are associated with different chromatin accessibility in ATAC-seq data and have different expression characteristics in single-cell RNA-seq data. The two promoter types may therefore be optimized for different properties: paused promoters show more consistent expression when active, while TATA promoters have lower background expression when inactive. We propose that tissue-specific genes have evolved to use two different strategies for their differential expression across tissues.
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Drosophila melanogaster/embriología , Perfilación de la Expresión Génica/métodos , Regiones Promotoras Genéticas , ARN Polimerasa II/metabolismo , Animales , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Regulación del Desarrollo de la Expresión Génica , Especificidad de Órganos , Análisis de Secuencia de ARN , Análisis de la Célula Individual , TATA BoxRESUMEN
The arrangement (syntax) of transcription factor (TF) binding motifs is an important part of the cis-regulatory code, yet remains elusive. We introduce a deep learning model, BPNet, that uses DNA sequence to predict base-resolution chromatin immunoprecipitation (ChIP)-nexus binding profiles of pluripotency TFs. We develop interpretation tools to learn predictive motif representations and identify soft syntax rules for cooperative TF binding interactions. Strikingly, Nanog preferentially binds with helical periodicity, and TFs often cooperate in a directional manner, which we validate using clustered regularly interspaced short palindromic repeat (CRISPR)-induced point mutations. Our model represents a powerful general approach to uncover the motifs and syntax of cis-regulatory sequences in genomics data.
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Biología Computacional/métodos , Motivos de Nucleótidos , Factores de Transcripción/metabolismo , Animales , Sitios de Unión , Inmunoprecipitación de Cromatina , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Aprendizaje Profundo , Ratones , Células Madre Embrionarias de Ratones/fisiología , Proteína Homeótica Nanog/metabolismo , Redes Neurales de la Computación , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Reproducibilidad de los Resultados , Factores de Transcripción SOXB1/metabolismoRESUMEN
Genomics data are now being generated at large quantities, of exquisite high resolution and from single cells. They offer a unique opportunity to develop powerful machine learning algorithms, including neural networks, to uncover the rules of the cis-regulatory code. However, current modeling assumptions are often not based on state-of-the-art knowledge of the cis-regulatory code from transcription, developmental genetics, imaging and structural studies. Here I aim to fill this gap by giving a brief historical overview of the field, describing common misconceptions and providing knowledge that might help to guide computational approaches. I will describe the principles and mechanisms involved in the combinatorial requirement of transcription factor binding motifs for enhancer activity, including the role of chromatin accessibility, repressors and low-affinity motifs in the cis-regulatory code. Deciphering the cis-regulatory code would unlock an enormous amount of regulatory information in the genome and would allow us to locate cis-regulatory genetic variants involved in development and disease.
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Mouse embryonic stem cells (ESCs) cultured in defined medium resemble the pre-implantation epiblast in the ground state, with full developmental capacity including the germline. ß-Catenin is required to maintain ground state pluripotency in mouse ESCs, but its exact role is controversial. Here, we reveal a Tcf3-independent role of ß-catenin in restraining germline and somatic lineage differentiation genes. We show that ß-catenin binds target genes with E2F6 and forms a complex with E2F6 and HMGA2 or E2F6 and HP1γ. Our data indicate that these complexes help ß-catenin restrain and fine-tune germ cell and neural developmental potential. Overall, our data reveal a previously unappreciated role of ß-catenin in preserving lineage differentiation integrity in ground state ESCs.
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Diferenciación Celular , Linaje de la Célula , Células Madre Embrionarias de Ratones/citología , Células Madre Embrionarias de Ratones/metabolismo , beta Catenina/metabolismo , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Diferenciación Celular/genética , Linaje de la Célula/genética , Regulación hacia Abajo/genética , Células Germinativas/citología , Células Germinativas/metabolismo , Ratones , Células Madre Pluripotentes/metabolismo , Unión Proteica , Factores de Transcripción/metabolismoRESUMEN
During development, transcription factors and signaling molecules govern gene regulatory networks to direct the formation of unique morphologies. As changes in gene regulatory networks are often implicated in morphological evolution, mapping transcription factor landscapes is important, especially in tissues that undergo rapid evolutionary change. The terminalia (genital and anal structures) of Drosophila melanogaster and its close relatives exhibit dramatic changes in morphology between species. While previous studies have identified network components important for patterning the larval genital disc, the networks governing adult structures during pupal development have remained uncharted. Here, we performed RNA-seq in whole Drosophila melanogaster male terminalia followed by in situ hybridization for 100 highly expressed transcription factors during pupal development. We find that the male terminalia are highly patterned during pupal stages and that specific transcription factors mark separate structures and substructures. Our results are housed online in a searchable database (https://flyterminalia.pitt.edu/) as a resource for the community. This work lays a foundation for future investigations into the gene regulatory networks governing the development and evolution of Drosophila terminalia.
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Drosophila melanogaster/anatomía & histología , Drosophila melanogaster/genética , Regulación del Desarrollo de la Expresión Génica , Factores de Transcripción/genética , Animales , Masculino , Pupa/anatomía & histología , Pupa/genética , Factores de Transcripción/metabolismoRESUMEN
FACT (facilitates chromatin transcription) is an evolutionarily conserved histone chaperone that was initially identified as an activity capable of promoting RNA polymerase II (Pol II) transcription through nucleosomes in vitro. In this report, we describe a global analysis of FACT function in Pol II transcription in Drosophila. We present evidence that loss of FACT has a dramatic impact on Pol II elongation-coupled processes including histone H3 lysine 4 (H3K4) and H3K36 methylation, consistent with a role for FACT in coordinating histone modification and chromatin architecture during Pol II transcription. Importantly, we identify a role for FACT in the maintenance of promoter-proximal Pol II pausing, a key step in transcription activation in higher eukaryotes. These findings bring to light a broader role for FACT in the regulation of Pol II transcription.
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Proteínas Portadoras/metabolismo , Proteínas de Drosophila/metabolismo , Histonas/metabolismo , Procesamiento Proteico-Postraduccional , ARN Polimerasa II/metabolismo , Elongación de la Transcripción Genética , Animales , Proteínas Portadoras/genética , Proteínas de Drosophila/genética , Drosophila melanogaster , Histonas/genética , ARN Polimerasa II/genéticaRESUMEN
RNA polymerase II (Pol II) pausing is a general regulatory step in transcription, yet the stability of paused Pol II varies widely between genes. Although paused Pol II stability correlates with core promoter elements, the contribution of individual sequences remains unclear, in part because no rapid assay is available for measuring the changes in Pol II pausing as a result of altered promoter sequences. Here, we overcome this hurdle by showing that ChIP-nexus captures the endogenous Pol II pausing on transfected plasmids. Using this reporter-ChIP-nexus assay in Drosophila cells, we show that the pausing stability is influenced by downstream promoter sequences, but that the strongest contribution to Pol II pausing comes from the initiator sequence, in which a single nucleotide, a G at the +2 position, is critical for stable Pol II pausing. These results establish reporter-ChIP-nexus as a valuable tool to analyze Pol II pausing.
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Estabilidad de Enzimas , Biología Molecular/métodos , ARN Polimerasa II/química , ARN Polimerasa II/metabolismo , ARN Mensajero/química , ARN Mensajero/metabolismo , Transcripción Genética , Animales , Línea Celular , Drosophila , Genes Reporteros , Plásmidos , TransfecciónRESUMEN
The Drosophila genus is a unique group containing a wide range of species that occupy diverse ecosystems. In addition to the most widely studied species, Drosophila melanogaster, many other members in this genus also possess a well-developed set of genetic tools. Indeed, high-quality genomes exist for several species within the genus, facilitating studies of the function and evolution of cis-regulatory regions and proteins by allowing comparisons across at least 50 million years of evolution. Yet, the available genomes still fail to capture much of the substantial genetic diversity within the Drosophila genus. We have therefore tested protocols to rapidly and inexpensively sequence and assemble the genome from any Drosophila species using single-molecule sequencing technology from Oxford Nanopore. Here, we use this technology to present highly contiguous genome assemblies of 15 Drosophila species: 10 of the 12 originally sequenced Drosophila species (ananassae, erecta, mojavensis, persimilis, pseudoobscura, sechellia, simulans, virilis, willistoni, and yakuba), four additional species that had previously reported assemblies (biarmipes, bipectinata, eugracilis, and mauritiana), and one novel assembly (triauraria). Genomes were generated from an average of 29x depth-of-coverage data that after assembly resulted in an average contig N50 of 4.4 Mb. Subsequent alignment of contigs from the published reference genomes demonstrates that our assemblies could be used to close over 60% of the gaps present in the currently published reference genomes. Importantly, the materials and reagents cost for each genome was approximately $1,000 (USD). This study demonstrates the power and cost-effectiveness of long-read sequencing for genome assembly in Drosophila and provides a framework for the affordable sequencing and assembly of additional Drosophila genomes.
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Drosophila/genética , Genoma , Genómica , Secuenciación de Nucleótidos de Alto Rendimiento , Animales , Biología Computacional/métodos , Genómica/métodos , Genotipo , Secuenciación de Nucleótidos de Alto Rendimiento/economía , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Mutación INDEL , Nanoporos , Filogenia , Análisis de Secuencia de ADNRESUMEN
Transplantation of hematopoietic stem cells (HSCs) from human umbilical cord blood (hUCB) holds great promise for treating a broad spectrum of hematological disorders including cancer. However, the limited number of HSCs in a single hUCB unit restricts its widespread use. Although extensive efforts have led to multiple methods for ex vivo expansion of human HSCs by targeting single molecules or pathways, it remains unknown whether it is possible to simultaneously manipulate the large number of targets essential for stem cell self-renewal. Recent studies indicate that N6-methyladenosine (m6A) modulates the expression of a group of mRNAs critical for stem cell-fate determination by influencing their stability. Among several m6A readers, YTHDF2 is recognized as promoting targeted mRNA decay. However, the physiological functions of YTHDF2 in adult stem cells are unknown. Here we show that following the conditional knockout (KO) of mouse Ythdf2 the numbers of functional HSC were increased without skewing lineage differentiation or leading to hematopoietic malignancies. Furthermore, knockdown (KD) of human YTHDF2 led to more than a 10-fold increase in the ex vivo expansion of hUCB HSCs, a fivefold increase in colony-forming units (CFUs), and more than an eightfold increase in functional hUCB HSCs in the secondary serial of a limiting dilution transplantation assay. Mapping of m6A in RNAs from mouse hematopoietic stem and progenitor cells (HSPCs) as well as from hUCB HSCs revealed its enrichment in mRNAs encoding transcription factors critical for stem cell self-renewal. These m6A-marked mRNAs were recognized by Ythdf2 and underwent decay. In Ythdf2 KO HSPCs and YTHDF2 KD hUCB HSCs, these mRNAs were stabilized, facilitating HSC expansion. Knocking down one of YTHDF2's key targets, Tal1 mRNA, partially rescued the phenotype. Our study provides the first demonstration of the function of YTHDF2 in adult stem cell maintenance and identifies its important role in regulating HSC ex vivo expansion by regulating the stability of multiple mRNAs critical for HSC self-renewal, thus identifying potential for future clinical applications.
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Adenosina/análogos & derivados , Autorrenovación de las Células , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Proteínas de Unión al ARN/antagonistas & inhibidores , Proteínas de Unión al ARN/metabolismo , Adenosina/metabolismo , Animales , Células Madre Hematopoyéticas/patología , Ratones , Ratones NoqueadosRESUMEN
In the initial published version of this article, there was an inadvertent omission from the Acknowledgements that this work was supported by Stowers Institute for Medical Research (SIMR-1004) and NIH National Cancer Institute grant to University of Kansas Cancer Center (P30 CA168524). This omission does not affect the description of the results or the conclusions of this work.
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The HMG-box protein Capicua (Cic) is a conserved transcriptional repressor that functions downstream of receptor tyrosine kinase (RTK) signaling pathways in a relatively simple switch: In the absence of signaling, Cic represses RTK-responsive genes by binding to nearly invariant sites in DNA, whereas activation of RTK signaling down-regulates Cic activity, leading to derepression of its targets. This mechanism controls gene expression in both Drosophila and mammals, but whether Cic can also function via other regulatory mechanisms remains unknown. Here, we characterize an RTK-independent role of Cic in regulating spatially restricted expression of Toll/IL-1 signaling targets in Drosophila embryogenesis. We show that Cic represses those targets by binding to suboptimal DNA sites of lower affinity than its known consensus sites. This binding depends on Dorsal/NF-κB, which translocates into the nucleus upon Toll activation and binds next to the Cic sites. As a result, Cic binds to and represses Toll targets only in regions with nuclear Dorsal. These results reveal a mode of Cic regulation unrelated to the well-established RTK/Cic depression axis and implicate cooperative binding in conjunction with low-affinity binding sites as an important mechanism of enhancer regulation. Given that Cic plays a role in many developmental and pathological processes in mammals, our results raise the possibility that some of these Cic functions are independent of RTK regulation and may depend on cofactor-assisted DNA binding.