ETV4 Is Necessary for Estrogen Signaling and Growth in Endometrial Cancer Cells.

Estrogen signaling through estrogen receptor alpha (ER) plays a major role in endometrial cancer risk and progression, however, the molecular mechanisms underlying ER's regulatory role in endometrial cancer are poorly understood. In breast cancer cells, ER genomic binding is enabled by FOXA1 and GATA3, but the transcription factors that control ER genomic binding in endometrial cancer cells remain unknown. We previously identified ETV4 as a candidate factor controlling ER genomic binding in endometrial cancer cells, and here we explore the functional importance of ETV4. Homozygous deletion of ETV4, using CRISPR/Cas9, led to greatly reduced ER binding at the majority of loci normally bound by ER. Consistent with the dramatic loss of ER binding, the gene expression response to estradiol was dampened for most genes. ETV4 contributes to estrogen signaling in two distinct ways. ETV4 loss affects chromatin accessibility at some ER bound loci and impairs ER nuclear translocation. The diminished estrogen signaling upon ETV4 deletion led to decreased growth, particularly in 3D culture, where hollow organoids were formed and in vivo in the context of estrogen-dependent growth. These results show that ETV4 plays an important role in estrogen signaling in endometrial cancer cells. SIGNIFICANCE: Estrogen receptor alpha (ER) is a key oncogene in endometrial cancer. This study uncovers ETV4 as an important factor in controlling the activity of ER and the growth of endometrial cancer cells. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/6/1234/F1.large.jpg.

The Biophysical Basis for Phosphorylation-Enhanced DNA-Binding Autoinhibition of the ETS1 Transcription Factor.

The eukaryotic transcription factor ETS1 is regulated by an intrinsically disordered serine-rich region (SRR) that transiently associates with the adjacent ETS domain to inhibit DNA binding. In this study, we further elucidated the physicochemical basis for ETS1 autoinhibition by characterizing the interaction of its ETS domain with a series of synthetic peptides corresponding to the SRR. Binding is driven by the hydrophobic effect and enhanced electrostatically by phosphorylation of serines adjacent to aromatic residues in the amphipathic SRR. Structural characterization of the dynamic peptide/protein complex by NMR spectroscopy and X-ray crystallography revealed multiple modes of binding that lead to autoinhibition by synergistically blocking the DNA-binding interface of the ETS domain and stabilizing an appended helical inhibitory module against allosterically induced unfolding. Consistent with these conclusions, the SRR peptide does not interact with DNA-bound ETS1. In addition, we found that the ETS1 SRR phosphopeptide binds to distantly related PU.1 in vitro, indicating that autoinhibition exploits features of the ETS domain that are conserved across this family of transcription factors.

Development of High-Throughput Screening Assays for Inhibitors of ETS Transcription Factors.

ETS transcription factors from the ERG and ETV1/4/5 subfamilies are overexpressed in the majority of prostate cancer patients and contribute to disease progression. Here, we have developed two in vitro assays for the interaction of ETS transcription factors with DNA that are amenable to high-throughput screening. Using ETS1 as a model, we applied these assays to screen 110 compounds derived from a high-throughput virtual screen. We found that the use of lower-affinity DNA binding sequences, similar to those that ERG and ETV1 bind to in prostate cells, allowed for higher inhibition from many of these test compounds. Further pilot experiments demonstrated that the in vitro assays are robust for ERG, ETV1, and ETV5, three of the ETS transcription factors that are overexpressed in prostate cancer.

Electrostatic repulsion causes anticooperative DNA binding between tumor suppressor ETS transcription factors and JUN-FOS at composite DNA sites.

Many different transcription factors (TFs) regulate gene expression in a combinatorial fashion, often by binding in close proximity to each other on composite cis-regulatory DNA elements. Here, we investigated how ETS TFs bind with the AP1 TFs JUN-FOS at composite DNA-binding sites. DNA-binding ability with JUN-FOS correlated with the phenotype of ETS proteins in prostate cancer. We found that the oncogenic ETS-related gene (ERG) and ETS variant (ETV) 1/4/5 subfamilies co-occupy ETS-AP1 sites with JUN-FOS in vitro, whereas JUN-FOS robustly inhibited DNA binding by the tumor suppressors ETS homologous factor (EHF) and SAM pointed domain-containing ETS TF (SPDEF). EHF bound ETS-AP1 DNA with tighter affinity than ERG in the absence of JUN-FOS, possibly enabling EHF to compete with ERG and JUN-FOS for binding to ETS-AP1 sites. Genome-wide mapping of EHF- and ERG-binding sites in prostate epithelial cells revealed that EHF is preferentially excluded from closely spaced ETS-AP1 DNA sequences. Structural modeling and mutational analyses indicated that adjacent positively charged surfaces from EHF and JUN-FOS use electrostatic repulsion to disfavor simultaneous DNA binding. Conservation of positive residues on the JUN-FOS interface identified E74-like ETS TF 1 (ELF1) as an additional ETS TF exhibiting anticooperative DNA binding with JUN-FOS, and we found that ELF1 is frequently down-regulated in prostate cancer. In summary, divergent electrostatic features of ETS TFs at their JUN-FOS interface enable distinct binding events at ETS-AP1 DNA sites, which may drive specific targeting of ETS TFs to facilitate distinct transcriptional programs.

ETV4 and AP1 Transcription Factors Form Multivalent Interactions with three Sites on the MED25 Activator-Interacting Domain.

The recruitment of transcriptional cofactors by sequence-specific transcription factors challenges the basis of high affinity and selective interactions. Extending previous studies that the N-terminal activation domain (AD) of ETV5 interacts with Mediator subunit 25 (MED25), we establish that similar, aromatic-rich motifs located both in the AD and in the DNA-binding domain (DBD) of the related ETS factor ETV4 interact with MED25. These ETV4 regions bind MED25 independently, display distinct kinetics, and combine to contribute to a high-affinity interaction of full-length ETV4 with MED25. High-affinity interactions with MED25 are specific for the ETV1/4/5 subfamily as other ETS factors display weaker binding. The AD binds to a single site on MED25 and the DBD interacts with three MED25 sites, allowing for simultaneous binding of both domains in full-length ETV4. MED25 also stimulates the in vitro DNA binding activity of ETV4 by relieving autoinhibition. ETV1/4/5 factors are often overexpressed in prostate cancer and genome-wide studies in a prostate cancer cell line indicate that ETV4 and MED25 occupy enhancers that are enriched for ETS-binding sequences and are both functionally important for the transcription of genes regulated by these enhancers. AP1-motifs, which bind JUN and FOS transcription factor families, were observed in MED25-occupied regions and JUN/FOS also contact MED25; FOS strongly binds to the same MED25 site as ETV4 AD and JUN interacts with the other two MED25 sites. In summary, we describe features of the multivalent ETV4- and AP1-MED25 interactions, thereby implicating these factors in the recruitment of MED25 to transcriptional control elements.

Structured and disordered regions cooperatively mediate DNA-binding autoinhibition of ETS factors ETV1, ETV4 and ETV5.

Autoinhibition enables spatial and temporal regulation of cellular processes by coupling protein activity to surrounding conditions, often via protein partnerships or signaling pathways. We report the molecular basis of DNA-binding autoinhibition of ETS transcription factors ETV1, ETV4 and ETV5, which are often overexpressed in prostate cancer. Inhibitory elements that cooperate to repress DNA binding were identified in regions N- and C-terminal of the ETS domain. Crystal structures of these three factors revealed an α-helix in the C-terminal inhibitory domain that packs against the ETS domain and perturbs the conformation of its DNA-recognition helix. Nuclear magnetic resonance spectroscopy demonstrated that the N-terminal inhibitory domain (NID) is intrinsically disordered, yet utilizes transient intramolecular interactions with the DNA-recognition helix of the ETS domain to mediate autoinhibition. Acetylation of selected lysines within the NID activates DNA binding. This investigation revealed a distinctive mechanism for DNA-binding autoinhibition in the ETV1/4/5 subfamily involving a network of intramolecular interactions not present in other ETS factors. These distinguishing inhibitory elements provide a platform through which cellular triggers, such as protein-protein interactions or post-translational modifications, may specifically regulate the function of these oncogenic proteins.

Conformational Dynamics and the Binding of Specific and Nonspecific DNA by the Autoinhibited Transcription Factor Ets-1.

The affinity of the Ets-1 transcription factor for DNA is autoinhibited by an intrinsically disordered serine-rich region (SRR) and a helical inhibitory module (IM) appended to its winged helix-turn-helix ETS domain. Using NMR spectroscopy, we investigated how Ets-1 recognizes specific versus nonspecific DNA, with a focus on the roles of protein dynamics and autoinhibition in these processes. Upon binding either DNA, the two marginally stable N-terminal helices of the IM predominantly unfold, but still sample partially ordered conformations. Also, on the basis of amide chemical shift perturbation mapping, Ets-1 associates with both specific and nonspecific DNA through the same canonical ETS domain interface. These interactions are structurally independent of the SRR, and thus autoinhibition does not impart DNA-binding specificity. However, relative to the pronounced NMR spectroscopic changes in Ets-1 resulting from specific DNA binding, the spectra of the nonspecific DNA complexes showed conformational exchange broadening and lacked several diagnostic amide and indole signals attributable to hydrogen bonding interactions seen in reported X-ray crystallographic structures of this transcription factor with its cognate DNA sequences. Such differences are highlighted by the chemical shift and relaxation properties of several interfacial lysine and arginine side chains. Collectively, these data support a general model in which Ets-1 interacts with nonspecific DNA via dynamic electrostatic interactions, whereas hydrogen bonding drives the formation of well-ordered complexes with specific DNA.

Autoinhibition of ETV6 DNA Binding Is Established by the Stability of Its Inhibitory Helix.

The ETS transcriptional repressor ETV6 (or TEL) is autoinhibited by an α-helix that sterically blocks its DNA-binding ETS domain. The inhibitory helix is marginally stable and unfolds when ETV6 binds to either specific or non-specific DNA. Using NMR spectroscopy, we show that folding of the inhibitory helix requires a buried charge-dipole interaction with helix H1 of the ETS domain. This interaction also contributes directly to autoinhibition by precluding a highly conserved dipole-enhanced hydrogen bond between the phosphodiester backbone of bound DNA and the N terminus of helix H1. To probe further the thermodynamic basis of autoinhibition, ETV6 variants were generated with amino acid substitutions introduced along the solvent exposed surface of the inhibitory helix. These changes were designed to increase the intrinsic helical propensity of the inhibitory helix without perturbing its packing interactions with the ETS domain. NMR-monitored amide hydrogen exchange measurements confirmed that the stability of the folded inhibitory helix increases progressively with added helix-promoting substitutions. This also results in progressively reinforced autoinhibition and decreased DNA-binding affinity. Surprisingly, locking the inhibitory helix onto the ETS domain by a disulfide bridge severely impairs, but does not abolish DNA binding. Weak interactions still occur via an interface displaced from the canonical ETS domain DNA-binding surface. Collectively, these studies establish a direct thermodynamic linkage between inhibitory helix stability and ETV6 autoinhibition, and demonstrate that helix unfolding does not strictly precede DNA binding. Modulating inhibitory helix stability provides a potential route for the in vivo regulation of ETV6 activity.

Interaction of the Jhd2 Histone H3 Lys-4 Demethylase with Chromatin Is Controlled by Histone H2A Surfaces and Restricted by H2B Ubiquitination.

Histone H3 lysine 4 (H3K4) methylation is a dynamic modification. In budding yeast, H3K4 methylation is catalyzed by the Set1-COMPASS methyltransferase complex and is removed by Jhd2, a JMJC domain family demethylase. The catalytic JmjC and JmjN domains of Jhd2 have the ability to remove all three degrees (mono-, di-, and tri-) of H3K4 methylation. Jhd2 also contains a plant homeodomain (PHD) finger required for its chromatin association and H3K4 demethylase functions. The Jhd2 PHD finger associates with chromatin independent of H3K4 methylation and the H3 N-terminal tail. Therefore, how Jhd2 associates with chromatin to perform H3K4 demethylation has remained unknown. We report a novel interaction between the Jhd2 PHD finger and histone H2A. Two residues in H2A (Phe-26 and Glu-57) serve as a binding site for Jhd2 in vitro and mediate its chromatin association and H3K4 demethylase functions in vivo. Using RNA sequencing, we have identified the functional target genes for Jhd2 and the H2A Phe-26 and Glu-57 residues. We demonstrate that H2A Phe-26 and Glu-57 residues control chromatin association and H3K4 demethylase functions of Jhd2 during positive or negative regulation of transcription at target genes. Importantly, we show that H2B Lys-123 ubiquitination blocks Jhd2 from accessing its binding site on chromatin, and thereby, we have uncovered a second mechanism by which H2B ubiquitination contributes to the trans-histone regulation of H3K4 methylation. Overall, our study provides novel insights into the chromatin binding dynamics and H3K4 demethylase functions of Jhd2.

Synergy of aromatic residues and phosphoserines within the intrinsically disordered DNA-binding inhibitory elements of the Ets-1 transcription factor.

The E26 transformation-specific (Ets-1) transcription factor is autoinhibited by a conformationally disordered serine-rich region (SRR) that transiently interacts with its DNA-binding ETS domain. In response to calcium signaling, autoinhibition is reinforced by calmodulin-dependent kinase II phosphorylation of serines within the SRR. Using mutagenesis and quantitative DNA-binding measurements, we demonstrate that phosphorylation-enhanced autoinhibition requires the presence of phenylalanine or tyrosine (ϕ) residues adjacent to the SRR phosphoacceptor serines. The introduction of additional phosphorylated Ser-ϕ-Asp, but not Ser-Ala-Asp, repeats within the SRR dramatically reinforces autoinhibition. NMR spectroscopic studies of phosphorylated and mutated SRR variants, both within their native context and as separate trans-acting peptides, confirmed that the aromatic residues and phosphoserines contribute to the formation of a dynamic complex with the ETS domain. Complementary NMR studies also identified the SRR-interacting surface of the ETS domain, which encompasses its positively charged DNA-recognition interface and an adjacent region of neutral polar and nonpolar residues. Collectively, these studies highlight the role of aromatic residues and their synergy with phosphoserines in an intrinsically disordered regulatory sequence that integrates cellular signaling and gene expression.

Dual views of SRF: a genomic exposure.

Esnault and colleagues (pp. 943-958) take a genomics approach to investigate the role of SRF (serum response factor) in the serum response of fibroblasts. The well-established dual role of SRF with alternative cofactors and responsiveness to two signaling pathways is illustrated at the genome-wide level, yet new insight comes from this global picture.

Steric mechanism of auto-inhibitory regulation of specific and non-specific DNA binding by the ETS transcriptional repressor ETV6.

DNA binding by the ETS transcriptional repressor ETV6 (or TEL) is auto-inhibited ~50-fold due to an α-helix that sterically blocks its ETS domain binding interface. Using NMR spectroscopy, we demonstrate that this marginally stable helix is unfolded, and not displaced to a non-inhibitory position, when ETV6 is bound to DNA containing a consensus (5')GGAA(3') recognition site. Although significantly lower in affinity, binding to non-specific DNA is auto-inhibited ~5-fold and is also accompanied by helix unfolding. Based on NMR chemical shift perturbations, both specific and non-specific DNA are bound via the same canonical ETS domain interface. However, spectral perturbations are smaller for the non-specific complex, suggesting weaker and less well-defined interactions than in the specific complex. In parallel, the crystal structure of ETV6 bound to a specific DNA duplex was determined. The structure of this complex reveals that a non-conserved histidine residue in the ETS domain recognition helix helps establish the specificity of ETV6 for DNA-binding sites containing (5')GGAA(3')versus(5')GGAT(3'). These studies provide a unified steric mechanism for attenuating ETV6 binding to both specific and non-specific DNA and expand the repertoire of characterized auto-inhibitory strategies utilized to regulate ETS factors.

Autoinhibition of ETV6 (TEL) DNA binding: appended helices sterically block the ETS domain.

ETV6 (or TEL), a transcriptional repressor belonging to the ETS family, is frequently involved in chromosomal translocations linked with human cancers. It displays a DNA-binding mode distinct from other ETS proteins due to the presence of a self-associating PNT domain. In this study, we used NMR spectroscopy to dissect the structural and dynamic bases for the autoinhibition of ETV6 DNA binding by sequences C-terminal to its ETS domain. The C-terminal inhibitory domain (CID) contains two helices, H4 and H5, which sterically block the DNA-binding interface of the ETS domain. Importantly, these appended helices are only marginally stable as revealed by amide hydrogen exchange and (15)N relaxation measurements. The CID is thus poised to undergo a facile conformational change as required for DNA binding. The CID also dampens millisecond timescale motions of the ETS domain hypothesized to be critical for the recognition of specific ETS target sequences. This work illustrates the use of appended sequences on conserved structural domains to generate biological diversity and complements previous studies of the allosteric mechanism of ETS1 autoinhibition to reveal both common and divergent features underlying the regulation of DNA binding by ETS transcription factors.

Gene deregulation and chronic activation in natural killer cells deficient in the transcription factor ETS1.

Multiple transcription factors guide the development of mature functional natural killer (NK) cells, yet little is known about their function. We used global gene expression and genome-wide binding analyses combined with developmental and functional studies to unveil three roles for the ETS1 transcription factor in NK cells. ETS1 functions at the earliest stages of NK cell development to promote expression of critical transcriptional regulators including T-BET and ID2, NK cell receptors (NKRs) including NKp46, Ly49H, and Ly49D, and signaling molecules essential for NKR function. As a consequence, Ets1(-/-) NK cells fail to degranulate after stimulation through activating NKRs. Nonetheless, these cells are hyperresponsive to cytokines and have characteristics of chronic stimulation including increased expression of inhibitory NKRs and multiple activation-associated genes. Therefore, ETS1 regulates a broad gene expression program in NK cells that promotes target cell recognition while limiting cytokine-driven activation.

Oncogenic ETS proteins mimic activated RAS/MAPK signaling in prostate cells.

The aberrant expression of an oncogenic ETS transcription factor is implicated in the progression of the majority of prostate cancers, 40% of melanomas, and most cases of gastrointestinal stromal tumor and Ewing's sarcoma. Chromosomal rearrangements in prostate cancer result in overexpression of any one of four ETS transcription factors. How these four oncogenic ETS genes differ from the numerous other ETS genes expressed in normal prostate and contribute to tumor progression is not understood. We report that these oncogenic ETS proteins, but not other ETS factors, enhance prostate cell migration. Genome-wide binding analysis matched this specific biological function to occupancy of a unique set of genomic sites highlighted by the presence of ETS- and AP-1-binding sequences. ETS/AP-1-binding sequences are prototypical RAS-responsive elements, but oncogenic ETS proteins activated a RAS/MAPK transcriptional program in the absence of MAPK activation. Thus, overexpression of oncogenic ETS proteins can replace RAS/MAPK pathway activation in prostate cells. The genomic description of this ETS/AP-1-regulated, RAS-responsive, gene expression program provides a resource for understanding the role of these ETS factors in both an oncogenic setting and the developmental processes where these genes normally function.

Genomic and biochemical insights into the specificity of ETS transcription factors.

ETS proteins are a group of evolutionarily related, DNA-binding transcriptional factors. These proteins direct gene expression in diverse normal and disease states by binding to specific promoters and enhancers and facilitating assembly of other components of the transcriptional machinery. The highly conserved DNA-binding ETS domain defines the family and is responsible for specific recognition of a common sequence motif, 5'-GGA(A/T)-3'. Attaining specificity for biological regulation in such a family is thus a conundrum. We present the current knowledge of routes to functional diversity and DNA binding specificity, including divergent properties of the conserved ETS and PNT domains, the involvement of flanking structured and unstructured regions appended to these dynamic domains, posttranslational modifications, and protein partnerships with other DNA-binding proteins and coregulators. The review emphasizes recent advances from biochemical and biophysical approaches, as well as insights from genomic studies that detect ETS-factor occupancy in living cells.

The ETS gene ETV4 is required for anchorage-independent growth and a cell proliferation gene expression program in PC3 prostate cells.

Chromosomal abnormalities that give rise to elevated expression levels of the ETS genes ETV1, ETV4, ETV5, or ERG are prevalent in prostate cancer, but the function of these transcription factors in carcinogenesis is not clear. Previous work in cell lines implicates ERG, ETV1, and ETV5 as regulators of invasive growth but not transformation. Here we show that the PC3 prostate cancer cell line provides a model system to study the over-expression of ETV4. Migration assays, anchorage independent growth assays, and microarray analysis indicate that high ETV4 expression contributes to both transformation and cellular motility in PC3 cells. ETV4 directly bound the 5' and 3' MYC enhancers and modulated expression of both MYC and other cell proliferation genes, demonstrating a potential role in cell growth control. Despite this novel role for ETV4 in anchorage independent growth, ETV4 over-expression in normal prostate-derived RWPE-1 cells showed effects similar to ETV1 over-expression - increased cellular motility, and an up-regulation of genes encoding extracellular proteins as well as ones important for development, inflammation, and wound healing. Because ETV1 and ETV4 have similar roles when introduced to the same cellular background, we suggest that the requirement of high ETV4 expression for maintenance of the anchorage-independent growth in PC3 cells is due to a specific characteristic of this cell line rather than a function of ETV4 that is distinct from the other oncogenic ETS genes. Thus, the function of ETS genes in prostate cancer may differ based on other genetic alterations in a tumor.

ETS-4 is a transcriptional regulator of life span in Caenorhabditis elegans.

Aging is a complex phenotype responsive to a plethora of environmental inputs; yet only a limited number of transcriptional regulators are known to influence life span. How the downstream expression programs mediated by these factors (or others) are coordinated into common or distinct set of aging effectors is an addressable question in model organisms, such as C. elegans. Here, we establish the transcription factor ETS-4, an ortholog of vertebrate SPDEF, as a longevity determinant. Adult worms with ets-4 mutations had a significant extension of mean life span. Restoring ETS-4 activity in the intestine, but not neurons, of ets-4 mutant worms rescued life span to wild-type levels. Using RNAi, we demonstrated that ets-4 is required post-developmentally to regulate adult life span; thus uncoupling the role of ETS-4 in aging from potential functions in worm intestinal development. Seventy ETS-4-regulated genes, identified by gene expression profiling of two distinct ets-4 alleles and analyzed by bioinformatics, were enriched for known longevity effectors that function in lipid transport, lipid metabolism, and innate immunity. Putative target genes were enriched for ones that change expression during normal aging, the majority of which are controlled by the GATA factors. Also, some ETS-4-regulated genes function downstream of the FOXO factor, DAF-16 and the insulin/IGF-1 signaling pathway. However, epistasis and phenotypic analyses indicate that ets-4 functioned in parallel to the insulin/IGF-1 receptor, daf-2 and akt-1/2 kinases. Furthermore, ets-4 required daf-16 to modulate aging, suggesting overlap in function at the level of common targets that affect life span. In conclusion, ETS-4 is a new transcriptional regulator of aging, which shares transcriptional targets with GATA and FOXO factors, suggesting that overlapping pathways direct common sets of lifespan-related genes.

Ras signaling requires dynamic properties of Ets1 for phosphorylation-enhanced binding to coactivator CBP.

Ras/MAPK signaling is often aberrantly activated in human cancers. The downstream effectors are transcription factors, including those encoded by the ETS gene family. Using cell-based assays and biophysical measurements, we have determined the mechanism by which Ras/MAPK signaling affects the function of Ets1 via phosphorylation of Thr38 and Ser41. These ERK2 phosphoacceptors lie within the unstructured N-terminal region of Ets1, immediately adjacent to the PNT domain. NMR spectroscopic analyses demonstrated that the PNT domain is a four-helix bundle (H2-H5), resembling the SAM domain, appended with two additional helices (H0-H1). Phosphorylation shifted a conformational equilibrium, displacing the dynamic helix H0 from the core bundle. The affinity of Ets1 for the TAZ1 (or CH1) domain of the coactivator CBP was enhanced 34-fold by phosphorylation, and this binding was sensitive to ionic strength. NMR-monitored titration experiments mapped the interaction surfaces of the TAZ1 domain and Ets1, the latter encompassing both the phosphoacceptors and PNT domain. Charge complementarity of these surfaces indicate that electrostatic forces act in concert with a conformational equilibrium to mediate phosphorylation effects. We conclude that the dynamic helical elements of Ets1, appended to a conserved structural core, constitute a phospho-switch that directs Ras/MAPK signaling to downstream changes in gene expression. This detailed structural and mechanistic information will guide strategies for targeting ETS proteins in human disease.

DNA binding by the ETS protein TEL (ETV6) is regulated by autoinhibition and self-association.

The ETS protein TEL, a transcriptional repressor, contains a PNT domain that, as an isolated fragment in vitro, self-associates to form a head-to-tail polymer. How such polymerization might affect the DNA-binding properties of full-length TEL is unclear. Here we report that monomeric TEL binds to a consensus ETS site with unusually low affinity (K(d) = 2.8 x 10(-8) M). A deletion analysis demonstrated that the low affinity was caused by a C-terminal inhibitory domain (CID) that attenuates DNA binding by approximately 10-fold. An NMR spectroscopically derived structure of a TEL fragment, deposited in the Protein Data Bank, revealed that the CID consists of two alpha-helices, one of which appears to block the DNA binding surface of the TEL ETS domain. Based on this structure, we substituted two conserved glutamic acids (Glu-431 and Glu-434) with alanines and found that this activated DNA binding and enhanced trypsin sensitivity in the CID. We propose that TEL displays a conformational equilibrium between inhibited and activated states and that electrostatic interactions involving these negatively charged residues play a role in stabilizing the inhibited conformation. Using a TEL dimer as a model polymer, we show that self-association facilitates cooperative binding to DNA. Cooperativity was observed on DNA duplexes containing tandem consensus ETS sites at variable spacing and orientations, suggesting flexibility in the region of TEL linking its self-associating PNT domain and DNA-binding ETS domain. We speculate that TEL compensates for the low affinity, which is caused by autoinhibition, by binding to DNA as a cooperative polymer.