Box Repressor Research Articles

REST Is Not Resting: REST/NRSF in Health and Disease.

Chromatin modifications play a crucial role in the regulation of gene expression. The repressor element-1 (RE1) silencing transcription factor (REST), also known as neuron-restrictive silencer factor (NRSF) and X2 box repressor (XBR), was found to regulate gene transcription by binding to chromatin and recruiting chromatin-modifying enzymes. Earlier studies revealed that REST plays an important role in the development and disease of the nervous system, mainly by repressing the transcription of neuron-specific genes. Subsequently, REST was found to be critical in other tissues, such as the heart, pancreas, skin, eye, and vascular. Dysregulation of REST was also found in nervous and non-nervous system cancers. In parallel, multiple strategies to target REST have been developed. In this paper, we provide a comprehensive summary of the research progress made over the past 28 years since the discovery of REST, encompassing both physiological and pathological aspects. These insights into the effects and mechanisms of REST contribute to an in-depth understanding of the transcriptional regulatory mechanisms of genes and their roles in the development and progression of disease, with a view to discovering potential therapeutic targets and intervention strategies for various related diseases.

Roles of the Neuron-Restrictive Silencer Factor in the Pathophysiological Process of the Central Nervous System.

The neuron-restrictive silencer factor (NRSF), also known as repressor element 1 (RE-1) silencing transcription factor (REST) or X2 box repressor (XBR), is a zinc finger transcription factor that is widely expressed in neuronal and non-neuronal cells. It is a master regulator of the nervous system, and the function of NRSF is the basis of neuronal differentiation, diversity, plasticity, and survival. NRSF can bind to the neuron-restrictive silencer element (NRSE), recruit some co-repressors, and then inhibit transcription of NRSE downstream genes through epigenetic mechanisms. In neurogenesis, NRSF functions not only as a transcriptional silencer that can mediate the transcriptional inhibition of neuron-specific genes in non-neuronal cells and thus give neuron cells specificity, but also as a transcriptional activator to induce neuronal differentiation. Many studies have confirmed the association between NRSF and brain disorders, such as brain injury and neurodegenerative diseases. Overexpression, underexpression, or mutation may lead to neurological disorders. In tumorigenesis, NRSF functions as an oncogene in neuronal tumors, such as neuroblastomas, medulloblastomas, and pheochromocytomas, stimulating their proliferation, which results in poor prognosis. Additionally, NRSF-mediated selective targets gene repression plays an important role in the development and maintenance of neuropathic pain caused by nerve injury, cancer, and diabetes. At present, several compounds that target NRSF or its co-repressors, such as REST-VP16 and X5050, have been shown to be clinically effective against many brain diseases, such as seizures, implying that NRSF and its co-repressors may be potential and promising therapeutic targets for neural disorders. In the present review, we introduced the biological characteristics of NRSF; reviewed the progress to date in understanding the roles of NRSF in the pathophysiological processes of the nervous system, such as neurogenesis, brain disorders, neural tumorigenesis, and neuropathic pain; and suggested new therapeutic approaches to such brain diseases.

Abstract 3056: Development of an integrated CRISPR interference system targeting ΔNp63 to treat lung and esophageal squamous cell carcinoma

Abstract TP63 encodes two different transcripts that have opposite functions in transcriptional control. One transcript encodes TAp63 that is functionally similar to the tumor suppressor TP53. The other transcript, ΔNp63, lacks the transcription-activating domain of TAp63 and has been proposed to function as a potent oncogene in squamous cell carcinomas. In this study, we have developed an integrated CRISPR interference system to selectively suppress ΔNp63 and we have termed this system CRISPRiΔNp63. We engineered the CRISPRi using tandemized guide RNA expression cassettes to target the 50 to 100 bp downstream region of the transcription start site of ΔNp63 and we used inactivated Cas9 linked to the transcription repression module Krüppel-associated box repressor domain. The plasmid vector harboring CRISPRiΔNp63 repressed ΔNp63 transcriptional activity in lung and esophageal squamous cell carcinoma cells that express ΔNp63. Moreover, an all-in-one adenoviral vector containing tandemized gRNAs and the dCas9/KRAB expression cassette, Ad-CRISPRiΔNp63, suppressed ΔNp63 expression in squamous cell carcinomas cells. Ad-CRISPRiΔNp63 effectively decreased cell proliferation and colony formation and induced apoptosis in lung and esophageal squamous cell carcinoma cells in vitro. Moreover, the all-in-one vector significantly inhibited tumor growth in a lung squamous cell carcinoma xenograft mouse model in vivo. These results indicate that ΔNp63 suppression by CRISPRiΔNp63 might be an effective strategy for the treatment of lung and esophageal squamous cell carcinoma. Citation Format: Masakazu Yoshida, Etsuko Yokota, Tetsushi Sakuma, Nagio Takigawa, Toshikazu Ushijima, Takashi Yamamoto, Yoshio Naomoto, Takuya Fukazawa, Tomoki Yamatsuji. Development of an integrated CRISPR interference system targeting ΔNp63 to treat lung and esophageal squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3056.

Development of an integrated CRISPRi targeting ΔNp63 for treatment of squamous cell carcinoma.

TP63 encodes TAp63, which is functionally similar to the tumor suppressor TP53, and ΔNp63, which lacks the transcription-activating domain of TAp63 and appears potently oncogenic in squamous cell carcinomas (SCCs). In this study, we developed an integrated CRISPR interference (CRISPRi) system to selectively suppress ΔNp63 (CRISPRiΔNp63). We engineered this CRISPRi using tandemized guide RNA expression cassettes that targeted the 50 to 100 bp downstream of the transcription start site of ΔNp63 in combination with inactivated Cas9 linked to the transcription repression module Krüppel-associated box repressor domain. The plasmid vector harboring CRISPRiΔNp63 repressed ΔNp63 transcription in lung and esophageal SCC cells. Likewise, Ad-CRISPRiΔNp63, an all-in-one adenoviral vector containing the tandemized gRNAs and dCas9/KRAB expression cassette suppressed ΔNp63 expression in SCC cells. Ad-CRISPRiΔNp63 also effectively decreased cell proliferation and colony formation and induced apoptosis in lung and esophageal SCC cells in vitro and significantly inhibited tumor growth in a mouse lung SCC xenograft model in vivo. These results indicate that ΔNp63 suppression using CRISPRiΔNp63 may be an effective strategy for treating lung and esophageal SCC.

Application of TALE-Based Approach for Dissecting Functional MicroRNA-302/367 in Cellular Reprogramming.

MicroRNAs are small 18-24nt single-stranded noncoding RNA molecules involved in many biological processes, including stemness maintenance and cellular reprogramming. Current methods used in loss-of-function studies of microRNAs have several limitations. Here, we describe a new approach for dissecting miR-302/367 functions by transcription activator-like effectors (TALEs), which are natural effector proteins secreted by Xanthomonas and Ralstonia bacteria. Knockdown of the miR-302/367 cluster uses the Kruppel-associated box repressor domain fused with specific TALEs designed to bind the miR-302/367 cluster promoter. Knockout of the miR-302/367 cluster uses two pairs of TALE nucleases (TALENs) to delete the miR-302/367 cluster in human primary cells. Together, both TALE-based transcriptional repressor and TALENs are two promising approaches for loss-of-function studies of microRNA cluster in human primary cells.

483. Epigenetic Silencing of Hepatitis B cccDNA In Vitro and In Vivo Using AAV-Delivered Engineered Repressor Transcription Activator-Like Effector

Hepatitis B virus (HBV) infection is hyper-endemic (>8 % chronic carriers) to parts of Asia and sub-Saharan Africa. Persistent HBV infection predisposes to liver diseases such as cirrhosis and hepatocellular carcinoma. Current anti-HBV therapies face several limitations. RNA interference-based therapies lack the robustness and specificity required for therapeutic effect while interferon-α and nucleotide/side analogs function post-transcriptionally and thus allow for the persistence of the covalently closed circular (cccDNA). cccDNA may persist indefinitely and enables re-initiation of HBV replication after withdrawal of treatment. Disabling the cccDNA is essential for the successful treatment of chronic HBV. Our group previously described effective anti-HBV transcription activator-like effector (TALE) nucleases (TALENs). Although potentially useful to counter HBV replication, one drawback is that viral sequences integrated into the host genome may be susceptible to digestion by the TALENs. Transcriptional silencing, rather than cleavage, of cccDNA may therefore be preferable to avoid causing undesirable mutations in the host. To this end, TALE binding domains designed to target the viral preS2 promoter and the basic core promoter/enhancer II regions were fused to a Kruppel-associated box repressor domain to generate repressor-TALEs (rTALEs). These rTALEs were shown to inhibit viral replication in vitro and in vivo without inducing significant toxicity. In an in vivo murine model using hydrodynamic transfection with an HBV replication-competent plasmid, a reduction in secreted HBV surface antigen (HBsAg) of 97% and 98% was seen at day 3 and 93% and 96% at day 5 for P1L and P1R respectively. To develop this approach as a feasible therapy, rTALE-encoding sequences were incorporated into recombinant adeno-associated viruses (rAAVs) and assessed in the HepG2.hNTCP-C4 cell line. These cells overexpress the HBV receptor, human sodium taurocholate co-transporting peptide (hNTCP). The HepG2.NTCP-C4 cell line is infectable with HBV and viral gene expression is dependent on formation of cccDNA. Measurement of viral markers of replication may thus be used as an indicator of inhibitory effects on cccDNA. The anti-HBV efficacy of the rTALEs and epigenetic modification of the targeted HBV DNA was characterized. Chromatin immunoprecipitation assays determined the binding of the rTALEs to the cccDNA and induction of epigenetic markers of viral gene suppression. Mobility shift assays confirmed specificity of rTALE binding. In vivo efficacy of rAAV-delivered rTALEs was evaluated in transgenic HBV mice. Our study provides valuable information on the potential therapeutic utility of rTALEs and demonstrates the feasibility of the approach for treatment of HBV.

Stable oncogenic silencing in vivo by programmable and targeted de novo DNA methylation in breast cancer.

With the recent comprehensive mapping of cancer genomes, there is now a need for functional approaches to edit the aberrant epigenetic state of key cancer drivers to reprogram the epi-pathology of the disease. In this study we utilized a programmable DNA-binding methyltransferase to induce targeted incorporation of DNA methylation (DNAme) in the SOX2 oncogene in breast cancer through a six zinc finger (ZF) protein linked to DNA methyltransferase 3A (ZF-DNMT3A). We demonstrated long-lasting oncogenic repression, which was maintained even after suppression of ZF-DNMT3A expression in tumor cells. The de novo DNAme was faithfully propagated and maintained through cell generations even after the suppression of the expression of the chimeric methyltransferase in the tumor cells. Xenograft studies in NUDE mice demonstrated stable SOX2 repression and long-term breast tumor growth inhibition, which lasted for >100 days post implantation of the tumor cells in mice. This was accompanied with a faithful maintenance of DNAme in the breast cancer implants. In contrast, downregulation of SOX2 by ZF domains engineered with the Krueppel-associated box repressor domain resulted in a transient and reversible suppression of oncogenic gene expression. Our results indicated that targeted de novo DNAme of the SOX2 oncogenic promoter was sufficient to induce long-lasting epigenetic silencing, which was not only maintained during cell division but also significantly delayed the tumorigenic phenotype of cancer cells in vivo, even in the absence of treatment. Here, we outline a genome-based targeting approach to long-lasting tumor growth inhibition with potential applicability to many other oncogenic drivers that are currently refractory to drug design.

The Krüppel-associated box repressor domain induces reversible and irreversible regulation of endogenous mouse genes by mediating different chromatin states.

The Krüppel-associated box (KRAB) domain is a transcription repression module from the largest family of transcriptional regulators encoded by higher vertebrates. We developed a drug-controllable regulation system based on an artificial KRAB-containing repressor (tTS) that targets the endogenous Hprt gene to explore the regulatory mechanism and molecular basis of KRAB-containing regulators within the context of an endogenous gene in vivo. We show that KRAB can mediate irreversible and reversible regulation of endogenous genes in mouse that is dependent on embryonic developmental stage. KRAB-induced stable DNA methylation within the KRAB binding region during the early embryonic stage, resulting in irreversible gene repression. In later stages, KRAB mainly induced de-acetylation and methylation of histone, resulting in reversible gene repression. Thus, we have characterized the KRAB-mediated regulation system within the context of an endogenous gene and multiple spatiotemporal ranges, thereby providing a basis for identifying the function of KRAB-containing regulators and aiding development of novel KRAB-based gene regulation tools in vivo.

Dissecting the Roles of miR-302/367 Cluster in Cellular Reprogramming Using TALE-based Repressor and TALEN

SummaryMicroRNAs are important gene regulators involved in many biological processes, including stemness maintenance and cellular reprogramming. Current methods used in loss-of-function studies of microRNAs mainly include locked nucleic acid (LNA) oligonucleotides and miRZip inhibitors, which have several limitations. Due to their unique gene structures and small sizes, there is no efficient or simple strategy to knock down or knock out microRNAs or whole microRNA clusters. Here, we demonstrate knockdown of the miR-302/367 cluster by using the Kruppel-associated box repressor domain fused with specific transcription activator-like effectors (TALEs) designed to bind the miR-302/367 cluster promoter. We also designed two pairs of TALE nucleases (TALENs) to efficiently delete the miR-302/367 cluster in primary human fibroblasts and determined that knockout of the miR-302/367 cluster completely blocked induced pluripotent stem cell (iPSC) generation. Together, our results demonstrate that TALE-based transcriptional repressor and TALENs are two promising approaches for loss-of-function studies of microRNA clusters in somatic cells and pluripotent stem cells.

The Krüppel-associated Box Repressor Domain Can Induce Reversible Heterochromatization of a Mouse Locus in Vivo

The study of chromatin and its regulators is key to understanding and manipulating transcription. We previously exploited the Krüppel-associated box (KRAB) transcriptional repressor domain, present in hundreds of vertebrate-specific zinc finger proteins, to assess the effect of its binding to gene bodies. These experiments revealed that the ectopic and doxycycline (dox)-controlled tet repressor KRAB fusion protein (tTRKRAB) can induce reversible and long-range silencing of cellular promoters. Here, we extend this system to in vivo applications and use tTRKRAB to achieve externally controllable repression of an endogenous mouse locus. We employed lentiviral-mediated transgenesis with promoterless TetO-containing gene traps to engineer a mouse line where the endogenous kinesin family member 2A (Kif2A) promoter drives a YFP reporter gene. When these mice were crossed to animals expressing the TetO-binding tTRKRAB repressor, this regulator was recruited to the Kif2A locus, and YFP expression was reduced. This effect was reversed when dox was given to embryos or adult mice, demonstrating that the cellular Kif2A promoter was only silenced upon repressor binding. Molecular analyses confirmed that tTRKRAB induced transcriptional repression through the spread of H3K9me3-containing heterochromatin, without DNA methylation of the trapped Kif2A promoter. Therefore, we demonstrate that targeting of tTRKRAB to a gene body in vivo results in reversible transcriptional repression through the spreading of facultative heterochromatin. This finding not only sheds light on KRAB-mediated transcriptional processes, but also suggests approaches for the externally controllable and reversible modulation of chromatin and transcription in vivo.

Transcription‐dependence of histone H3 lysine 27 trimethylation at the Arabidopsis polycomb target gene FLC

The FLC gene encodes a MADS box repressor of flowering that is the main cause of the late-flowering phenotype of many Arabidopsis ecotypes. Expression of FLC is repressed by vernalization; maintenance of this repression is associated with the deposition of histone 3 K27 trimethylation (H3K27me3) at the FLC locus. However, whether this increased H3K27me3 is a consequence of reduced FLC transcription or the cause of transcriptional repression is not well defined. In this study we investigate the effect of changes in transcription rate on the abundance of H3K27me3 in the FLC gene body, a chromatin region that includes sequences required to maintain FLC repression following vernalization. We show that H3K27me3 is inversely correlated with transcription across the FLC gene body in a range of ecotypes and mutants with different flowering times. We demonstrate that the FLC gene body becomes marked with H3K27me3 in the absence of transcription. When transcription of the gene body is directed by an inducible promoter, H3K27me3 is removed following activation of transcription and H3K27me3 is added after transcription is decreased. The rate of addition of H3K27me3 to the FLC transgene following inactivation of transcription is similar to that observed in the FLC gene body following vernalization. Our data suggest that reduction of FLC transcription during vernalization leads to an increase of H3K27me3 levels in the FLC gene body that in turn maintains FLC repression.

Transcriptional regulation and expression of guanylyl cyclase/natriuretic peptide receptor‐A gene by δEF1:Role of TGF‐β1 signaling

The binding of atrial and brain natriuretic peptides (ANP and BNP) to guanylyl cyclase‐A/natriuretic peptide receptor‐A (GC‐A/NPR‐A) produces second messenger cGMP, which plays a critical role in various intracellular signaling pathways including; hypertension, cardiovascular events, and cancer. Transcription factor δEF1, an E‐2 box repressor has been predicted to have binding sites in the 5′ flanking region of the Npr1(coding for GC‐A/NPRA) gene promoter. The objective of this study was to determine the repressive effect of δEF1 on transcriptional regulation of murine Npr1 gene promoter in mouse mesangial cells (MMCs) and rat thoracic aortic vascular smooth muscle cells (RTASMCs). MMCs were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) and ITS (insulin, transferrin, and selenium). RTASMCs were cultured in DMEM containing 10% FBS. Co‐transfection of δEF1 expression plasmid with the Npr1 gene promoter‐luciferase constructs showed 70–75% reduction in the luciferase activity in MMCs and RTASMCs. Also, Npr1 gene promoter‐luciferase constructs embodying δEF1 sites showed up to 90 % reduction in luciferase activity after treatment with TGF‐β1 in a time‐ and dose‐ dependent manner. Deletion of δEF1 sites eliminated the repression of luciferase activity induced by TGF‐β1. Electrophoretic mobility shift assay as well as supershift assay confirmed the binding of δEF1 to the specific sites of Npr1 gene promoter. The findings of this study suggest that δEF1 exerts a repressive effect on Npr1 gene transcription and expression, which plays a critical role in hypertension and cardiovascular regulation. This work is supported by National Institutes of Health Grants (HL57531 and HL 62147).

The Krüppel-associated Box Repressor Domain Can Trigger de Novo Promoter Methylation during Mouse Early Embryogenesis

The Krüppel-associated box (KRAB) domain is a transcriptional repression module responsible for the DNA binding-dependent gene silencing activity of hundreds of vertebrate zinc finger proteins. We previously exploited KRAB-mediated repression within the context of a tet repressor-KRAB fusion protein and of lentiviral vectors to create a method of external gene control. We demonstrated that with this system transcriptional silencing was fully reversible in cell culture as well as in vivo. Here we reveal that, in sharp contrast, KRAB-mediated repression results in irreversible gene silencing through promoter DNA methylation if it acts during the first few days of mouse development.

Phorbol Ester-induced Transcription of a Fibroblast Growth Factor-binding Protein Is Modulated by a Complex Interplay of Positive and Negative Regulatory Promoter Elements

Earlier studies from our laboratory showed that a secreted binding protein for fibroblast growth factors (FGF-BP) is expressed at high levels in squamous cell carcinoma (SCC) cell lines. Overexpression studies or conversely reduced expression of FGF-BP by ribozyme targeting have elucidated a direct role of this protein in angiogenesis during tumor development. We have also observed a significant up-regulation of FGF-BP during TPA (12-O-tetradecanoylphorbol-13-acetate) promotion of skin cancer. Here we investigate the mechanism of TPA induction of FGF-BP gene expression in the human ME-180 SCC cell line. We found that TPA increased FGF-BP mRNA levels in a time- and dose-dependent manner mediated via the protein kinase C signal transduction pathway. Results from actinomycin D and cycloheximide experiments as well as nuclear transcription assays revealed that TPA up-regulated the steady-state levels of FGF-BP mRNA by increasing its rate of gene transcription independently of de novo protein synthesis. We isolated the human FGF-BP promoter and determined by deletion analysis that TPA regulatory elements were all contained in the first 118 base pairs upstream of the transcription start site. Further mutational analysis revealed that full TPA induction required interplay between several regulatory elements with homology to Ets, AP-1, and CAATT/enhancer binding protein C/EBP sites. In addition, deletion or mutation of a 10-base pair region juxtaposed to the AP-1 site dramatically increased TPA induced FGF-BP gene expression. This region represses the extent of the FGF-BP promoter response to TPA and contained sequences recognized by the family of E box helix-loop-helix transcription factors. Gel shift analysis showed specific and TPA-inducible protein binding to the Ets, AP-1, and C/EBP sites. Furthermore, distinct, specific, and TPA-inducible binding to the imperfect E box repressor element was also apparent. Overall, our data indicate that TPA effects on FGF-BP gene transcription are tightly controlled by a complex interplay of positive elements and a novel negative regulatory element.

A zinc finger protein that represses transcription of the human MHC class II gene, DPA.

The proximal promoters of all MHC class II genes contain a sequence element, the 19-bp X box, that is conserved in both sequence and position. Extensive analysis using a wide variety of approaches has demonstrated that the integrity of the X box is essential for transcription initiation from all class II genes studied. However, the X box is now recognized to contain two subregions, termed X1 and X2. Radiolabeled oligonucleotides corresponding to the X2 box of the MHC class II genes DPA and DQB were used to screen B cell and T cell expression libraries. A novel cDNA, termed XBR (X box repressor), encoding a putative zinc finger protein that binds specifically to the DPA X2 box was isolated from a human T cell line. The XBR gene encodes a 7-kb message that is ubiquitously transcribed, although at higher levels in tissues of the lymphocytic compartment. Southern blots indicate that this gene is single copy in primates and contains regions that are highly divergent in other species. Overexpression of XBR in a B cell line resulted in a dramatic reduction of transcription from a reporter gene construct driven by the DPA promoter, but not from similar constructs with mutations in the X2 box. Similarly, overexpression of XBR reduced induction of reporter gene activity driven from the DPA promoter in HeLa cells treated with IFN-gamma. XBR may, therefore, mediate transcriptional repression, thus preventing inappropriate MHC class II expression. XBR function may in part explain the dominant trans-acting repression of MHC class II expression reported in cell fusion experiments.

Transcriptional silencing of human immunodeficiency virus type 1 long terminal repeat-driven gene expression by the Krüppel-associated box repressor domain targeted to the transactivating response element.

The evolutionarily conserved protein domain, called the Krüppel-associated box (KRAB), present in the amino termini of a large number of Krüppel-type zinc finger proteins is a strong repressor domain. In order to develop novel strategies to control human immunodeficiency virus type 1 (HIV-1) gene expression, we constructed a series of expression vectors expressing the wild-type Tat or Tat transdominant negative mutants fused to the KRAB repressor domain. We found that the KRAB domain tethered to the transactivating response element is able to suppress both basal and Tat-mediated activity of HIV-1 long terminal repeat-driven gene expression. These results suggest that the KRAB repressor domain fused to the Tat transdominant negative mutants can be successfully employed to control HIV-1 gene expression.

The delta-crystallin enhancer-binding protein delta EF1 is a repressor of E2-box-mediated gene activation.

The repressor delta EF1 was discovered by its action on the DC5 fragment of the lens-specific delta 1-crystallin enhancer. C-proximal zinc fingers of delta EF1 were found responsible for binding to the DC5 fragment and had specificity to CACCT as revealed by selection of high-affinity binding sequences from a random oligonucleotide pool. CACCT is present not only in DC5 but also in the E2 box (CACCTG) elements which are the binding sites of various basic helix-loop-helix activators and also the target of an unidentified repressor, raising the possibility that delta EF1 accounts for the E2 box repressor activity. delta EF1 competed with E47 for binding to an E2 box sequence in vitro. In lymphoid cells, endogenous delta EF1 activity as a repressor was detectable, and exogenous delta EF1 repressed immunoglobulin kappa enhancer by binding to the kappa E2 site. Moreover, delta EF1 repressed MyoD-dependent activation of the muscle creatine kinase enhancer and MyoD-induced myogenesis of 10T1/2 cells. Thus, delta EF1 counteracts basic helix-loop-helix activators through binding site competition and fulfills the conditions of the E2 box repressor. In embryonic tissues, the most prominent site of delta EF1 expression is the myotome. Myotomal expression as well as the above results argues for a significant contribution of delta EF1 in regulation of embryonic myogenesis through the modulation of the actions of MyoD family proteins.

The δ-Crystallin Enhancer-Binding Protein δEF1 Is a Repressor of E2-Box-Mediated Gene Activation

The repressor delta EF1 was discovered by its action on the DC5 fragment of the lens-specific delta 1-crystallin enhancer. C-proximal zinc fingers of delta EF1 were found responsible for binding to the DC5 fragment and had specificity to CACCT as revealed by selection of high-affinity binding sequences from a random oligonucleotide pool. CACCT is present not only in DC5 but also in the E2 box (CACCTG) elements which are the binding sites of various basic helix-loop-helix activators and also the target of an unidentified repressor, raising the possibility that delta EF1 accounts for the E2 box repressor activity. delta EF1 competed with E47 for binding to an E2 box sequence in vitro. In lymphoid cells, endogenous delta EF1 activity as a repressor was detectable, and exogenous delta EF1 repressed immunoglobulin kappa enhancer by binding to the kappa E2 site. Moreover, delta EF1 repressed MyoD-dependent activation of the muscle creatine kinase enhancer and MyoD-induced myogenesis of 10T1/2 cells. Thus, delta EF1 counteracts basic helix-loop-helix activators through binding site competition and fulfills the conditions of the E2 box repressor. In embryonic tissues, the most prominent site of delta EF1 expression is the myotome. Myotomal expression as well as the above results argues for a significant contribution of delta EF1 in regulation of embryonic myogenesis through the modulation of the actions of MyoD family proteins.