SMART MODE:

NORMAL GENOMIC

• Gilliland DG
• The diverse role of the ETS family of transcription factors in cancer.
• Clin Cancer Res. 2001; 7: 451-3

• Sato Y
• Role of ETS family transcription factors in vascular development and angiogenesis.
• Cell Struct Funct. 2001; 26: 19-24
• Display abstract

• The ETS family of transcription factors is defined by a conserved DNA-binding ETS domain that forms a winged helix-turn-helix structural motif. This family of transcription factors is involved in a diverse array of biological functions including cellular growth and differentiation, as well as organ development. Among the members of this family, ETS-1, ERG, Fli-1, TEL, and NERF-2 are expressed in endothelial cells and their progenitors. This review will summarize the role of ETS family transcription factors in vascular development and angiogenesis.

• Anderson MK, Sun X, Miracle AL, Litman GW, Rothenberg EV
• Evolution of hematopoiesis: Three members of the PU.1 transcription factor family in a cartilaginous fish, Raja eglanteria.
• Proc Natl Acad Sci U S A. 2001; 98: 553-8
• Display abstract

• T lymphocytes and B lymphocytes are present in jawed vertebrates, including cartilaginous fishes, but not in jawless vertebrates or invertebrates. The origins of these lineages may be understood in terms of evolutionary changes in the structure and regulation of transcription factors that control lymphocyte development, such as PU.1. The identification and characterization of three members of the PU.1 family of transcription factors in a cartilaginous fish, Raja eglanteria, are described here. Two of these genes are orthologs of mammalian PU.1 and Spi-C, respectively, whereas the third gene, Spi-D, is a different family member. In addition, a PU.1-like gene has been identified in a jawless vertebrate, Petromyzon marinus (sea lamprey). Both DNA-binding and transactivation domains are highly conserved between mammalian and skate PU.1, in marked contrast to lamprey Spi, in which similarity is evident only in the DNA-binding domain. Phylogenetic analysis of sequence data suggests that the appearance of Spi-C may predate the divergence of the jawed and jawless vertebrates and that Spi-D arose before the divergence of the cartilaginous fish from the lineage leading to the mammals. The tissue-specific expression patterns of skate PU.1 and Spi-C suggest that these genes share regulatory as well as structural properties with their mammalian orthologs.

• Sementchenko VI, Watson DK
• Ets target genes: past, present and future.
• Oncogene. 2000; 19: 6533-48
• Display abstract

• Ets is a family of transcription factors present in species ranging from sponges to human. All family members contain an approximately 85 amino acid DNA binding domain, designated the Ets domain. Ets proteins bind to specific purine-rich DNA sequences with a core motif of GGAA/T, and transcriptionally regulate a number of viral and cellular genes. Thus, Ets proteins are an important family of transcription factors that control the expression of genes that are critical for several biological processes, including cellular proliferation, differentiation, development, transformation, and apoptosis. Here, we tabulate genes that are regulated by Ets factors and describe past, present and future strategies for the identification and validation of Ets target genes. Through definition of authentic target genes, we will begin to understand the mechanisms by which Ets factors control normal and abnormal cellular processes.

• Bartel FO, Higuchi T, Spyropoulos DD
• Mouse models in the study of the Ets family of transcription factors.
• Oncogene. 2000; 19: 6443-54
• Display abstract

• The Ets family of transcription factors is one of a growing number of master regulators of development. This family was originally defined by the presence of a conserved DNA binding domain, the Ets domain. To date, nearly 30 members of this family have been identified and implicated in a wide range of physiological and pathological processes. Despite the likely importance of Ets-family members, each of their precise roles has not been delineated. Herein, we describe the elucidation of essential functions of a few of these family members in vivo using knockout mouse models. Of the knockouts generated to date, the majority shows important functions in hematopoiesis, ranging from PU.1, a principle regulator of myelo-lymphopoiesis, to Spi-B which regulates the proper function of terminally differentiated cells. Ets1 was shown to be of intermediate importance as a regulator of pan-lymphoid development. Other Ets family members such as Fli1 and TEL1 display distinct and/or overlapping functions in vasculo/angiogenesis, hemostasis and hematopoiesis. The remaining knockouts generated, Ets2 and Er81, show non-hematopoietic defects related to extraembryonic development and neurogenesis, respectively. The pioneering group of knockout models described reveals only the most distinct functions of each of these Ets family members. A better understanding of the roles and hierarchies of Ets family members in cellular differentiation will come with the generation of new null alleles in previously untargeted family members, more mutant alleles in members already disrupted, double knockouts, ES cell differentiation and chimera rescue experiments, and tissue-specific inducible knockouts.

• Yordy JS, Muise-Helmericks RC
• Signal transduction and the Ets family of transcription factors.
• Oncogene. 2000; 19: 6503-13
• Display abstract

• Cellular responses to environmental stimuli are controlled by a series of signaling cascades that transduce extracellular signals from ligand-activated cell surface receptors to the nucleus. Although most pathways were initially thought to be linear, it has become apparent that there is a dynamic interplay between signaling pathways that result in the complex pattern of cell-type specific responses required for proliferation, differentiation and survival. One group of nuclear effectors of these signaling pathways are the Ets family of transcription factors, directing cytoplasmic signals to the control of gene expression. This family is defined by a highly conserved DNA binding domain that binds the core consensus sequence GGAA/T. Signaling pathways such as the MAP kinases, Erk1 and 2, p38 and JNK, the PI3 kinases and Ca2+-specific signals activated by growth factors or cellular stresses, converge on the Ets family of factors, controlling their activity, protein partnerships and specification of downstream target genes. Interestingly, Ets family members can act as both upstream and downstream effectors of signaling pathways. As downstream effectors their activities are directly controlled by specific phosphorylations, resulting in their ability to activate or repress specific target genes. As upstream effectors they are responsible for the spacial and temporal expression or numerous growth factor receptors. This review provides a brief survey of what is known to date about how this family of transcription factors is regulated by cellular signaling with a special focus on Ras responsive elements (RREs), the MAP kinases (Erks, p38 and JNK) and Ca2+-specific pathways and includes a description of the multiple roles of Ets family members in the lymphoid system. Finally, we will discuss other potential mechanisms and pathways involved in the regulation of this important family of transcription factors.

• Li R, Pei H, Watson DK
• Regulation of Ets function by protein - protein interactions.
• Oncogene. 2000; 19: 6514-23
• Display abstract

• Ets proteins are a family of transcription factors that share an 85 amino acid conserved DNA binding domain, the ETS domain. Over 25 mammalian Ets family members control important biological processes, including cellular proliferation, differentiation, lymphocyte development and activation, transformation and apoptosis by recognizing the GGA core motif in the promoter or enhancer of their target genes. Protein - protein interactions regulates DNA binding, subcellular localization, target gene selection and transcriptional activity of Ets proteins. Combinatorial control is a characteristic property of Ets family members, involving interaction between Ets and other key transcriptional factors such as AP-1, NFkappaB and Pax family members. Specific domains of Ets proteins interact with many protein motifs such as bHLH, bZipper and Paired domain. Such interactions coordinate cellular processes in response to diverse signals including cytokines, growth factors, antigen and cellular stresses.

• Mo Y, Vaessen B, Johnston K, Marmorstein R
• Structure of the elk-1-DNA complex reveals how DNA-distal residues affect ETS domain recognition of DNA.
• Nat Struct Biol. 2000; 7: 292-7
• Display abstract

• SAP-1 and Elk-1 are members of a large group of eukaryotic transcription factors that contain a conserved ETS DNA binding domain and that cooperate with the serum response factor (SRF) to activate transcription of the c-fos protooncogene. Despite the high degree of sequence similarity, which includes an identical amino acid sequence for the DNA recognition helix within the ETS domain of these proteins, they exhibit different DNA binding properties. Here we report the 2.1 ? crystal structure of the ETS domain of Elk-1 bound to a high affinity E74 DNA (E74DNA) site and compare it to a SAP-1-E74DNA complex. This comparison reveals that the differential DNA binding properties of these proteins are mediated by non-conserved residues distal to the DNA binding surface that function to orient conserved residues in the DNA recognition helix for protein-specific DNA contacts. As a result, nearly one-third of the interactions between the protein recognition helix and the DNA are different between the SAP-1 and Elk-1 DNA complexes. Taken together, these studies reveal a novel mechanism for the modulation of DNA binding specificity within a conserved DNA binding domain, and have implications for how highly homologous ETS proteins exhibit differential DNA-binding properties.

• Szymczyna BR, Arrowsmith CH
• DNA binding specificity studies of four ETS proteins support an indirect read-out mechanism of protein-DNA recognition.
• J Biol Chem. 2000; 275: 28363-70
• Display abstract

• Members of the ETS family of transcription factors are involved in several developmental and physiological processes, and, when overexpressed or misexpressed, can contribute to a variety of cancers. Each family member has a conserved DNA-binding domain that recognizes DNA sequences containing a G-G-A trinucleotide. Discrimination between potential ETS-binding sites appears to be governed by both the nucleotides flanking the G-G-A sequence and protein-protein interactions. We have used an adaptation of the "length-encoded multiplex" approach (Desjarlais, J. R., and Berg, J. M. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 11099-11103) to define DNA binding specificities for four ETS proteins: Fli-1, SAP-1, PU.1, and TEL. Our results support a model in which cooperative effects among neighboring bases flanking the central G-G-A site contribute to the formation of stable ETS/DNA complexes. These results are consistent with a mechanism for specific DNA binding that is partially governed by an indirect read-out of the DNA sequence, in which a sequence-specific DNA conformation is sensed or induced.

• Mattot V, Pourtier-Manzanedo A, Vercamer C, Soncin F, Vandenbunder B
• [Experimental angiogenesis : strategy for the functional study of the transcription factors of the Ets family during morphogenesis of the vascular tree]
• Ann Endocrinol (Paris). 2000; 61: 61-9
• Display abstract

• During morphogenesis of the vascular tree, the massive outgrowth of primitive capillaries is followed by the development and the maturation of some capillary branches whereas others regress. The direct observation and the manipulation of in vivo models, including a series of recent knock-out experiments, allow to delineate the mechanisms controlling this process, and to identify factors involved in the formation of a mature capillary, surrounded with a basal lamina and pericytes. The expression of several members of the Ets family of transcription factors, Ets1, Erg and Fli, correlates with the occurrence of invasive processes, such as angiogenesis during normal and pathological development. The description of the phenotype of cultured endothelial cells expressing the DNA binding domain of Ets1 suggests that members of the Ets family take part in the morphogenesis of the -vascular tree. Although transient transfection experiments allowed the identification of putative targets genes for Ets1 during angiogenesis, deciphering the Ets1 regulation networks remains a major goal for the future.

• Nishimura Y
• [DNA-recognition by helix-turn-helix variants in DNA-binding domains]
• Tanpakushitsu Kakusan Koso. 2000; 45: 1683-93

• Cowley DO, Graves BJ
• Phosphorylation represses Ets-1 DNA binding by reinforcing autoinhibition.
• Genes Dev. 2000; 14: 366-76
• Display abstract

• Phosphorylation of transcription factors is a key link between cell signaling and the control of gene expression. Here we report that phosphorylation regulates DNA binding of the Ets-1 transcription factor by reinforcing an autoinhibitory mechanism. Quantitative DNA-binding assays show that calcium-dependent phosphorylation inhibits Ets-1 DNA binding 50-fold. The four serines that mediate this inhibitory effect are distant from the DNA-binding domain but near structural elements required for autoinhibition. Mutational analyses demonstrate that an intact inhibitory module is required for phosphorylation-dependent regulation. Partial proteolysis studies indicate that phosphorylation stabilizes an inhibitory conformation. These findings provide a structural mechanism for phosphorylation-dependent inhibition of Ets-1 DNA binding and demonstrate a new function for inhibitory modules as structural mediators of negative signaling events.

• Mimeault M
• Structure-function studies of ETS transcription factors.
• Crit Rev Oncog. 2000; 11: 227-53
• Display abstract

• The functional characterization of ETS transcription factors have allowed the association of numerous physiological and pathological roles to these proteins in the regulation of gene expression during the maturation of hematopoietic cell lineages and in tumor cell growth, invasion, and metastasis. Moreover, structural investigations have allowed the determination of certain amino acid domains of ETS proteins that are essential for the recognition and transcriptional activation or repression of gene promoters or enhancers. It has been observed that the mechanisms of action of ETS proteins can be ruled in part by their intermolecular interactions with other transcription factors and by their phosphorylation status. This review describes information about structure-function relationships of different ETS family members in order to establish the structural differences that are important for their affinities and intrinsic activities to DNA binding sites.

• Wheat W et al.
• The highly conserved beta-hairpin of the paired DNA-binding domain is required for assembly of Pax-Ets ternary complexes.
• Mol Cell Biol. 1999; 19: 2231-41
• Display abstract

• Pax family transcription factors bind DNA through the paired domain. This domain, which is comprised of two helix-turn-helix motifs and a beta-hairpin structure, is a target of mutations in congenital disorders of mice and humans. Previously, we showed that Pax-5 (B-cell-specific activator protein) recruits proteins of the Ets proto-oncogene family to bind a composite DNA site that is essential for efficient transcription of the early-B-cell-specific mb-1 promoter. Here, evidence is provided for specific interactions between Ets-1 and the amino-terminal subdomains of Pax proteins. By tethering deletion fragments of Pax-5 to a heterologous DNA-binding domain, we show that 73 amino acids (amino acids 12 to 84) of its amino-terminal subdomain can recruit the ETS domain of Ets-1 to bind the composite site. Furthermore, an amino acid (Gln22) within the highly conserved beta-hairpin motif of Pax-5 is essential for efficient recruitment of Ets-1. The ability to recruit Ets proteins to bind DNA is a shared property of Pax proteins, as demonstrated by cooperative DNA binding of Ets-1 with sequences derived from the paired domains of Pax-2 and Pax-3. The strict conservation of sequences required for recruitment of Ets proteins suggests that Pax-Ets interactions are important for regulating transcription in diverse tissues during cellular differentiation.

• Grutz G, Forster A, Rabbitts TH
• Identification of the LMO4 gene encoding an interaction partner of the LIM-binding protein LDB1/NLI1: a candidate for displacement by LMO proteins in T cell acute leukaemia.
• Oncogene. 1998; 17: 2799-803
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• The T cell oncogenes LMO1 and LMO2 are activated by distinct chromosomal translocations in childhood T cell acute leukaemias. Transgenic mouse models of this disease demonstrate that enforced expression of Lmo1 and Lmo2 cause T cell leukaemias with long latency and that Lmo2 expression leads to an inhibition of the T cell differentiation programme, prior to overt disease. These functions appear to be partly mediated by interaction of LMO1 or LMO2 with the LIM-binding protein LDB1/ NLI1. We have now identified a new member of the Lmo family, designated Lmo4, via its interaction with Ldb1. Lmo4 is widely expressed in mouse tissues, including adult thymus (mainly CD4, CD8-double positive T cells) and embryonic thymus (mainly CD4, CD8-double negative T cells). These characteristics imply that Ldb1-Lmo4 interaction may function in the T cell developmental programme and that enforced expression of LMO1 or LMO2 by chromosomal translocations or transgenesis may displace Lmo4 from this complex and thereby influence T cell differentiation prior to T cell tumour occurrence.

• Panitz F, Krain B, Hollemann T, Nordheim A, Pieler T
• The Spemann organizer-expressed zinc finger gene Xegr-1 responds to the MAP kinase/Ets-SRF signal transduction pathway.
• EMBO J. 1998; 17: 4414-25
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• The transcriptional activity of a set of genes, which are all expressed in overlapping spatial and temporal patterns within the Spemann organizer of Xenopus embryos, can be modulated by peptide growth factors. We identify Xegr-1, a zinc finger protein-encoding gene, as a novel member of this group of genes. The spatial expression characteristics of Xegr-1 during gastrulation are most similar to those of Xbra. Making use of animal cap explants, analysis of the regulatory events that govern induction of Xegr-1 gene activity reveals that, in sharp contrast to transcriptional regulation of Xbra, activation of Ets-serum response factor (SRF) transcription factor complexes is required and sufficient for Xegr-1 gene expression. This finding provides the first indication for Ets-SRF complexes bound to serum response elements to be activated during gastrulation. MAP kinase signalling cascades can induce and sustain expression of both Xegr-1 and Xbra. Ectopic Xbra can induce Xegr-1 transcription by an indirect mechanism that appears to operate via primary activation of fibroblast growth factor secretion. These findings define a cascade of events that links Xbra activity to the signal-regulated control of Xegr-1 transcription in the context of early mesoderm induction in Xenopus laevis.

• Chinenov Y, Schmidt T, Yang XY, Martin ME
• Identification of redox-sensitive cysteines in GA-binding protein-alpha that regulate DNA binding and heterodimerization.
• J Biol Chem. 1998; 273: 6203-9
• Display abstract

• The transcription factor GA-binding protein (GABP) is composed of two subunits, GABPalpha and GABPbeta. The DNA-binding subunit, GABPalpha, is a member of the Ets family of transcription factors, characterized by the conserved Ets-domain that mediates DNA binding and associates with GABPbeta, which lacks a discernible DNA binding domain, through ankyrin repeats in the NH2 terminus of GABPbeta. We previously demonstrated that GABP is subject to redox regulation in vitro and in vivo through four COOH-terminal cysteines in GABPalpha. To determine the roles of individual cysteines in GABP redox regulation, we generated a series of serine substitution mutants by site-directed mutagenesis and identified three redox-sensitive cysteine residues in GABPalpha (Cys388, Cys401, and Cys421). Sulfhydryl modification of Cys388 and Cys401 inhibits DNA binding by GABPalpha, whereas, modification of Cys421 has no effect on GABPalpha DNA binding but inhibits dimerization with GABPbeta. The positions of Cys388 and Cys401 within the known Ets-domain structure suggest two very different mechanisms for redox regulation of DNA binding. Sulfhydryl modification of Cys388 could directly interfere with DNA binding or might alter the positioning of the DNA-binding helix 3. Modification of Cys401 may inhibit DNA binding through stabilization of an inhibitory helix similar to that described in the Ets-1 protein. Thus, GABP is regulated through at least two redox-sensitive activities, DNA binding and heterodimerization.

• Ogata K
• [Structure and dynamics of the transcription factor, Myb, in DNA-sequence recognition]
• Seikagaku. 1998; 70: 1233-50

• Sharrocks AD, Brown AL, Ling Y, Yates PR
• The ETS-domain transcription factor family.
• Int J Biochem Cell Biol. 1997; 29: 1371-87
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• During recent years, several significant discoveries have been made concerning the function of ETS-domain transcription factors. This family of transcription factors was originally defined on the basis of the conserved primary sequence of their DNA-binding domains. The ETS DNA-binding domain is also conserved at the structural level and is a divergent member of the winged helix-turn-helix superfamily of DNA binding proteins. This sequence conservation is reflected by their overlapping DNA-binding specificities based on the central GGAA/T motif. In addition to DNA-protein interactions, protein-protein interactions with partner proteins often play major roles in targeting ETS-domain proteins to specific promoters. Several such partner proteins have been identified. ETS-domain proteins function as either transcriptional activators or repressors and their activities are often regulated by signal transduction pathways, including the MAP kinase pathways. Specific links between such pathways and ETS-domain proteins have been established in several different experimental systems. ETS-domain transcription factors regulate a diverse array of biological functions including mammalian haematopoiesis and Drosophila eye development. In vertebrates, many ETS-domain proteins regulate embryonic and adult haematopoiesis. Deregulation of ETS-domain protein activity often leads to tumorigenesis. Future work will uncover further details of how these transcription factors work at the molecular level to regulate specific biological processes.

• Wasylyk C, Bradford AP, Gutierrez-Hartmann A, Wasylyk B
• Conserved mechanisms of Ras regulation of evolutionary related transcription factors, Ets1 and Pointed P2.
• Oncogene. 1997; 14: 899-913
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• Cell transformation by the Ras oncogene is mediated by members of the ets gene family. To analyse the mechanisms of regulation, we have studied activation of several ets factors by Ras expression. We show that expression of Ha-Ras strongly activates the Ets1 p68 and p54 isoforms and Ets2 in F9 EC cells. We have mapped the Ras responsive elements of Ets1 p68 to two domains, RI+II and RIII. Mutation of threonine 82 to alanine in RI+II abolishes both Ras activation and phosphorylation by MAP kinase. Threonine 82 is part of a sequence that is conserved in Drosophila Pointed P2, an ets protein that has been shown both genetically and biochemically to mediate Ras signalling in Drosophila cells. We extend the comparison of these evolutionary related proteins by showing that Pointed P2 is activated by Ras in mammalian cells and mutation of the homologous threonine abolishes activation. Furthermore, we show that Pointed P2 resembles Ets1, in that it has conserved sequences in a similar position adjacent to the ets DNA binding domain that negatively auto-regulates DNA binding. These results go towards showing that the Drosophila Pointed and vertebrate Ets1 are evolutionary related proteins that have remarkably conserved Ras regulatory mechanisms downstream from MAP kinase.

• Solano R, Fuertes A, Sanchez-Pulido L, Valencia A, Paz-Ares J
• A single residue substitution causes a switch from the dual DNA binding specificity of plant transcription factor MYB.Ph3 to the animal c-MYB specificity.
• J Biol Chem. 1997; 272: 2889-95
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• Transcription factor MYB.Ph3 from Petunia binds to two types of sequences, MBSI and MBSII, whereas murine c-MYB only binds to MBSI, and Am305 from Antirrhinum only binds to MBSII. DNA binding studies with hybrids of these proteins pointed to the N-terminal repeat (R2) as the most involved in determining binding to MBSI and/or MBSII, although some influence of the C-terminal repeat (R3) was also evident. Furthermore, a single residue substitution (Leu71 --> Glu) in MYB.Ph3 changed its specificity to that of c-MYB, and c-MYB with the reciprocal substitution (Glu132 --> Leu) essentially gained the MYB.Ph3 specificity. Molecular modeling and DNA binding studies with site-specific MYB.Ph3 mutants strongly supported the notion that the drastic changes in DNA binding specificity caused by the Leu --> Glu substitution reflect the fact that certain residues influence this property both directly, through base contacts, and indirectly, through interactions with other base-contacting residues, and that a single residue may establish alternative base contacts in different targets. Additionally, differential effects of mutations at non-base-contacting residues in MYB.Ph3 and c-MYB were observed, reflecting the importance of protein context on DNA binding properties of MYB proteins.

• Williams CE, Grotewold E
• Differences between plant and animal Myb domains are fundamental for DNA binding activity, and chimeric Myb domains have novel DNA binding specificities.
• J Biol Chem. 1997; 272: 563-71
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• Several Myb domain proteins have been identified in plants, in which they play important regulatory roles in specific cellular processes. Plant and animal Myb domains have significant differences, but how these differences are important for function is not yet understood. The P gene encodes a Myb domain protein that activates a subset of flavonoid biosynthetic genes in maize floral organs. P and v-Myb bind different DNA sequences in vitro. Here we show that the Myb domain is solely responsible for the sequence-specific DNA binding activity of P, which binds DNA only in the reduced state. Differences in the DNA binding domains of v-Myb and P, which are conserved among animal and plant Myb domains, are fundamental for the high affinity DNA binding activity of these proteins to the corresponding binding sites but are not sufficient for the distinct DNA binding specificities of P and v-Myb. We conclude that significant structural differences distinguish plant from animal Myb domains. A chimeric Myb domain with a novel DNA binding specificity was created by combining Myb repeats of P and v-Myb. This approach could be used to artificially create novel Myb domains and to target transcription factors to genes containing specific promoters or to modify Myb-mediated interactions with other cellular factors.

• Graves BJ, Gillespie ME, McIntosh LP
• DNA binding by the ETS domain.
• Nature. 1996; 384: 322-322

• Donaldson LW, Petersen JM, Graves BJ, McIntosh LP
• Solution structure of the ETS domain from murine Ets-1: a winged helix-turn-helix DNA binding motif.
• EMBO J. 1996; 15: 125-34
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• Ets-1 is the prototypic member of the ets family of transcription factors. This family is characterized by the conserved ETS domain that mediates specific DNA binding. Using NMR methods, we have determined the structure of a fragment of murine Ets-1 composed of the 85 residue ETS domain and a 25 amino acid extension that ends at its native C-terminus. The ETS domain folds into a helix-turn-helix motif on a four-stranded anti-parallel beta-sheet scaffold. This structure places Ets-1 in the winged helix-turn-helix (wHTH) family of DNA binding proteins and provides a model for interpreting the sequence conservation of the ETS domain and the specific interaction of Ets-1 with DNA. The C-terminal sequence of Ets-1, which is mutated in the v-Ets oncoprotein, forms an alpha-helix that packs anti-parallel to the N-terminal helix of the ETS domain. In this position, the C-terminal helix is poised to interact directly with an N-terminal inhibitory region in Ets-1 as well as the wHTH motif. This explains structurally the concerted role of residues flanking the ETS domain in the intramolecular inhibition of Ets-1 DNA binding.

• Dash AB, Orrico FC, Ness SA
• The EVES motif mediates both intermolecular and intramolecular regulation of c-Myb.
• Genes Dev. 1996; 10: 1858-69
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• The c-Myb transcription factor is a proto-oncoprotein whose latent transforming activity can be unmasked by truncation of either terminus. Because both ends of Myb are involved in negative regulation, we tested whether they could associate in a two-hybrid assay and identified a carboxy-terminal motif that interacts with the amino-terminal DNA-binding domain. The EVES motif is highly conserved in vertebrate c-Myb proteins and contains a known site of phosphorylation previously implicated in the negative regulation of c-Myb. Interestingly, a related EVES motif is present in p100, a ubiquitously expressed transcriptional coactivator found in diverse species. We show that p100 interacts with and influences the activity of c-Myb, implicating it in the regulation of c-Myb, differentiation, and cell growth. Our results suggest that Myb is regulated by a novel mechanism in which intramolecular interactions and conformational changes control the intermolecular associations among Myb, p100, and the transcriptional apparatus.

• Mann RS, Chan SK
• Extra specificity from extradenticle: the partnership between HOX and PBX/EXD homeodomain proteins.
• Trends Genet. 1996; 12: 258-62
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• For many DNA-binding transcription factors it is often difficult to reconcile their highly specific in vivo functions with their less specific in vitro DNA-binding properties. Cooperative DNA binding with cofactors often provides part of the answer to this paradox and recent studies have demonstrated this to be the case for the homeotic complex (HOX) family of transcription factors. However, the unique problem posed by these highly related and developmentally important transcription factors requires additional twists to the standard solution, which are beginning to become apparent from the characterization of the HOX cofactors encoded by the extradenticle and PBX genes.

• Emery P, Strubin M, Hofmann K, Bucher P, Mach B, Reith W
• A consensus motif in the RFX DNA binding domain and binding domain mutants with altered specificity.
• Mol Cell Biol. 1996; 16: 4486-94
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• The RFX DNA binding domain is a novel motif that has been conserved in a growing number of dimeric DNA-binding proteins, having diverse regulatory functions, in eukaryotic organisms ranging from yeasts to humans. To characterize this novel motif, we have performed a detailed dissection of the site-specific DNA binding activity of RFX1, a prototypical member of the RFX family. First, we have performed a site selection procedure to define the consensus binding site of RFX1. Second, we have developed a new mutagenesis-selection procedure to derive a precise consensus motif, and to test the accuracy of a secondary structure prediction, for the RFX domain. Third, a modification of this procedure has allowed us to isolate altered-specificity RFX1 mutants. These results should facilitate the identification both of additional candidate genes controlled by RFX1 and of new members of the RFX family. Moreover, the altered-specificity RFX1 mutants represent valuable tools that will permit the function of RFX1 to be analyzed in vivo without interference from the ubiquitously expressed endogenous protein. Finally, the simplicity, efficiency, and versatility of the selection procedure we have developed make it of general value for the determination of consensus motifs, and for the isolation of mutants exhibiting altered functional properties, for large protein domains involved in protein-DNA as well as protein-protein interactions.

• Shore P, Whitmarsh AJ, Bhaskaran R, Davis RJ, Waltho JP, Sharrocks AD
• Determinants of DNA-binding specificity of ETS-domain transcription factors.
• Mol Cell Biol. 1996; 16: 3338-49
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• Several mechanisms are employed by members of transcription factor families to achieve sequence-specific DNA recognition. In this study, we have investigated how members of the ETS-domain transcription factor family achieve such specificity. We have used the ternary complex factor (TCF) subfamily as an example. ERK2 mitogen-activated protein kinase stimulates serum response factor-dependent and autonomous DNA binding by the TCFs Elk-1 and SAP-la. Phosphorylated Elk-1 and SAP-la exhibit specificities of DNA binding similar to those of their isolated ETS domains. The ETS domains of Elk-1 and SAP-la and SAP-2 exhibit related but distinct DNA-binding specificities. A single residue, D-69 (Elk-1) or V-68 (SAP-1), has been identified as the critical determinant for the differential binding specificities of Elk-1 and SAP-1a, and an additional residue, D-38 (Elk-1) or Q-37 (SAP-1), further modulates their DNA binding. Creation of mutations D38Q and D69V is sufficient to confer SAP-la DNA-binding specificity upon Elk-1 and thereby allow it to bind to a greater spectrum of sites. Molecular modelling indicates that these two residues (D-38 and D-69) are located away from the DNA-binding interface of Elk-1. Our data suggest a mechanism in which these residues modulate DNA binding by influencing the interaction of other residues with DNA.

• Chytil M, Verdine GL
• The Rel family of eukaryotic transcription factors.
• Curr Opin Struct Biol. 1996; 6: 91-100
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• Members of the Rel transcription factor family mediate the response of eukaryotic cells to a broad range of environmental threats, in addition to serving an essential role in the development of certain vertebrate and insect cells. It is now apparent that there are two classes of Rel proteins, which differ in whether they bind DNA as monomers or dimers and which use markedly different mechanisms to transduce intracellular signals. Recent progress has been made towards understanding the structural basis for the fascinating biology of these proteins.

• Kanei-Ishii C et al.
• Structure and function of the proteins encoded by the myb gene family.
• Curr Top Microbiol Immunol. 1996; 211: 89-98

• Pio F et al.
• New insights on DNA recognition by ets proteins from the crystal structure of the PU.1 ETS domain-DNA complex.
• J Biol Chem. 1996; 271: 23329-37
• Display abstract

• Transcription factors belonging to the ets family regulate gene expression and share a conserved ETS DNA-binding domain that binds to the core sequence 5'-(C/A)GGA(A/T)-3'. The domain is similar to alpha+beta ("winged") helix-turn-helix DNA-binding proteins. The crystal structure of the PU.1 ETS domain complexed to a 16-base pair oligonucleotide revealed a pattern for DNA recognition from a novel loop-helix-loop architecture (Kodandapani, R., Pio, F., Ni. C.-Z., Piccialli, G., Klemsz, M., McKercher, S., Maki, R. A., and Ely, K. R. (1996) Nature 380, 456-460). Correlation of this model with mutational analyses and chemical shift data on other ets proteins confirms this complex as a paradigm for ets DNA recognition. The second helix in the helix-turn-helix motif lies deep in the major groove with specific contacts with bases in both strands in the core sequence made by conserved residues in alpha3. On either side of this helix, two loops contact the phosphate backbone. The DNA is bent (8 degrees) but uniformly curved without distinct kinks. ETS domains bind DNA as a monomer yet make extensive DNA contacts over 30 A. DNA bending likely results from phosphate neutralization of the phosphate backbone in the minor groove by both loops in the loop-helix-loop motif. Contacts from these loops stabilize DNA bending and may mediate specific base interactions by inducing a bend toward the protein.

• Sgouras DN, Athanasiou MA, Beal GJ Jr, Fisher RJ, Blair DG, Mavrothalassitis GJ
• ERF: an ETS domain protein with strong transcriptional repressor activity, can suppress ets-associated tumorigenesis and is regulated by phosphorylation during cell cycle and mitogenic stimulation.
• EMBO J. 1995; 14: 4781-93
• Display abstract

• ERF (ETS2 Repressor Factor) is a novel member of the ets family of genes, which was isolated by virtue of its interaction with the ets binding site (EBS) within the ETS2 promoter. The 2.7 kb ubiquitously expressed ERF mRNA encodes a 548 amino acid phosphoprotein that exhibits strong transcriptional repressor activity on promoters that contain an EBS. The localization of the DNA-binding domain of the protein at the N-terminus and th repression domain at the C-terminus is reminiscent of the organization of ELK1-like members of the ets family; however, there is no significant homology between ERF and ELK1 or any other ets member outside the DNA-binding domain. The repressor activity of ERF can antagonize the activity of other ets genes that are known transcriptional activators. Furthermore, ERF can suppress the ets-dependent transforming activity of the gag-myb-ets fusion oncogene of ME26 virus. Although ERF protein levels remain constant throughout the cell cycle, the phosphorylation level of the protein is altered as a function of the cell cycle and after mitogenic stimulation. The ERF protein is also hyperphosphorylated in cells transformed by the activated Ha-ras and v-src genes and the transcription repressor activity of ERF is decreased after co-transfection with activated Ha-ras or the kinase domain of the c-Raf-1 gene, indicating that ERF activity is probably regulated by the ras/MAPK pathway. Consistent with the in vivo phosphorylation and inactivation by ras, ERF is efficiently phosphorylated in vitro by Erk2 and cdc2/cyclin B kinases, at sites similar to those detected in vivo. Furthermore, a single mutation at position 526 results in the loss of a specific phosphopeptide both in in vivo and in vitro (by Erk2) labeling. Substitution of Thr526 for glutamic acid also decreases the repression ability of ERF. Our data suggest a model in which modulation of ERF activity is involved in the transcriptional regulation of genes activated during entry into G1 phase. Obstruction of the ERF repressor function by the transactivating members of the ets family of genes (i.e.gag-myb-ets) may be essential for the control of genes involved in cell proliferation and may also underlie their tumorigenic effects.

• Shore P, Sharrocks AD
• The ETS-domain transcription factors Elk-1 and SAP-1 exhibit differential DNA binding specificities.
• Nucleic Acids Res. 1995; 23: 4698-706
• Display abstract

• The ETS DNA-binding domain is conserved amongst many eukaryotic transcription factors. ETS-domains bind differentially to specific DNA sites containing a central GGA trinucleotide motif. The nucleotides flanking this motif define the binding specificity of individual proteins. In this study we have investigated binding specificity of the ETS-domains from two members of the ternary complex factor (TCF) subfamily, Elk-1 and SAP-1. The ETS DNA-binding domains of Elk-1 (Elk-93) and SAP-1 (SAP-92) select similar sites from random pools of double stranded oligonucleotides based on the consensus sequence ACCGGAAGTR. However, SAP-92 shows a more relaxed binding site selectivity and binds efficiently to a greater spectrum of sites than does Elk-93. This more relaxed DNA binding site selectivity is most pronounced in nucleotides located on the 3' side of the GGA motif. This differential DNA-binding specificity is also exhibited by longer TCF derivatives and, indeed by the full-length proteins. Our results suggest that the range of potential in vivo target sites for SAP-1 is likely to be greater than for Elk-1. We discuss our results in relation to other similar studies carried out with more divergent ETS-domains.

• Hovring I, Bostad A, Ording E, Myrset AH, Gabrielsen OS
• DNA-binding domain and recognition sequence of the yeast BAS1 protein, a divergent member of the Myb family of transcription factors.
• J Biol Chem. 1994; 269: 17663-9
• Display abstract

• The yeast BAS1 protein is a transcriptional activator with an amino-terminal domain homologous to the DNA-binding domain of the oncoprotein Myb containing three imperfect tryptophan-rich repeats. In contrast to Myb-related transcription factors from higher eukaryotes, where the second and third repeat constitutes a minimal independent DNA-binding domain, all three repeats of BAS1 were found to be necessary for sequence-specific DNA binding. Moreover, an active DNA-binding subdomain was obtained only if the first repeat was enlarged in the amino-terminal direction to include 3 tryptophans and a 23-amino acid insertion and if 55 amino acids carboxyl-terminal to the third repeat were included. The BAS1 DNA-binding site was analyzed in detail and found to cover 8-9 base pairs with no similarity to the Myb recognition element. The binding site included a conserved hexameric TGACTC motif, the methylation of which abolished BAS1 binding, as well as a 3-base pair extension that seemed to have a modulatory effect on BAS1 affinity and where binding was less affected by methylation.

• Donaldson LW, Petersen JM, Graves BJ, McIntosh LP
• Secondary structure of the ETS domain places murine Ets-1 in the superfamily of winged helix-turn-helix DNA-binding proteins.
• Biochemistry. 1994; 33: 13509-16
• Display abstract

• The members of the ets gene family of transcription factors are characterized by a conserved 85-residue DNA-binding region, termed the ETS domain, that lacks sequence homology to structurally characterized DNA-binding motifs. The secondary structure of the ETS domain of murine Ets-1 was determined on the basis of NMR chemical shifts, NOE and J-coupling constraints, amide hydrogen exchange, circular dichroism, and FT-IR spectroscopy. The ETS domain is composed of three alpha-helices (H) and four beta-strands (S) arranged in the order H1-S1-S2-H2-H3-S3-S4. The four-stranded antiparallel beta-sheet is the scaffold for a putative helix-turn-helix DNA recognition motif formed by helices 2 and 3. The 25 residues extending beyond the ETS domain to the native C-terminus of the truncated Ets-1 also contain a helical segment. On the basis of the similarity of this topology with that of catabolite activator protein (CAP), heat shock factor (HSF), and hepatocyte nuclear factor (HNF-3 gamma), we propose that ets proteins are members of the superfamily of winged helix-turn-helix DNA-binding proteins.

• Mavrothalassitis G, Fisher RJ, Smyth F, Watson DK, Papas TS
• Structural inferences of the ETS1 DNA-binding domain determined by mutational analysis.
• Oncogene. 1994; 9: 425-35
• Display abstract

• The ets family of transcription factors is characterized by a conserved region that harbors the DNA-binding activity. We performed extensive deletion and mutational analyses, as well as DNA-peptide interaction studies necessary to identify the determinants of the DNA-binding activity of the ETS1 oncoprotein. We found that amino acids beyond the 85 amino acid conserved region are required in order to afford maximum DNA-binding activity in a heterologous system. Mutation throughout the binding domain can have a detrimental effect on binding activity, indicating that proper folding of the entire domain is necessary for DNA binding. A peptide, as small as 37 residues (K37N), derived from the basic region of the ETS1 binding domain, is sufficient to exhibit sequence-specific DNA recognition. Total randomization of Lysine 379, Lysine 381 and Arginine 391 within this region fails to provide functional substitutions, indicating that these specific amino acids within the basic region are required for binding. Transactivation activity of the ETS1 proteins bearing mutations was consistent with their DNA-binding activity, indicating that the primary (if not only) function of this domain is to provide sequence-specific DNA recognition activity. Our mutational analysis, as well as modeling predictions, lead us to propose a helix-turn-helix structure for the basic region of the ETS1 binding domain that is able to interact directly with DNA. We also propose that the hydrophobic alpha-helical region, surrounding tryptophan 338, is fundamental for proper protein folding and functioning of the ets binding domain.

• Liu J, Sodeoka M, Lane WS, Verdine GL
• Evidence for a non-alpha-helical DNA-binding motif in the Rel homology region.
• Proc Natl Acad Sci U S A. 1994; 91: 908-12
• Display abstract

• The Rel family of transcription factors serve as terminal messengers in a variety of developmental and receptor-mediated signaling pathways. These proteins are related by a domain of approximately 280 amino acids, the Rel homology region, which mediates dimerization and sequence-specific binding to DNA. Here we report the use of photocrosslinking and site-directed mutagenesis to identify specific contact partners in a Rel protein-DNA interface. Within the Rel homology region of NF-kappa B p50 (also known as KBF1), two amino acid residues were identified by photocrosslinking to adjacent bases in a beta-interferon regulatory element. Secondary structure analysis suggests that the DNA-binding motif of the Rel homology region comprises a beta-turn-beta structure, in contrast to the alpha-helical motifs so commonly observed in transcription factors.

• Stehelin D
• Invasive carcinomas dismantle surrounding stroma induced to express the nuclear factor c-ets-1.
• Princess Takamatsu Symp. 1994; 24: 171-86

• Bosselut R, Levin J, Adjadj E, Ghysdael J
• A single amino-acid substitution in the Ets domain alters core DNA binding specificity of Ets1 to that of the related transcription factors Elf1 and E74.
• Nucleic Acids Res. 1993; 21: 5184-91
• Display abstract

• Ets proteins form a family of sequence specific DNA binding proteins which bind DNA through a 85 aminoacids conserved domain, the Ets domain, whose sequence is unrelated to any other characterized DNA binding domain. Unlike all other known Ets proteins, which bind specific DNA sequences centered over either GGAA or GGAT core motifs, E74 and Elf1 selectively bind to GGAA corecontaining sites. Elf1 and E74 differ from other Ets proteins in three residues located in an otherwise highly conserved region of the Ets domain, referred to as conserved region III (CRIII). We show that a restricted selectivity for GGAA core-containing sites could be conferred to Ets1 upon changing a single lysine residue within CRIII to the threonine found in Elf1 and E74 at this position. Conversely, the reciprocal mutation in Elf1 confers to this protein the ability to bind to GGAT core containing EBS. This, together with the fact that mutation of two invariant arginine residues in CRIII abolishes DNA binding, indicates that CRIII plays a key role in Ets domain recognition of the GGAA/T core motif and lead us to discuss a model of Ets proteins--core motif interaction.

• Fleischman LF, Pilaro AM, Murakami K, Kondoh A, Fisher RJ, Papas TS
• c-Ets-1 protein is hyperphosphorylated during mitosis.
• Oncogene. 1993; 8: 771-80
• Display abstract

• The ets-1 and ets-2 proto-oncogene products can serve as transcription factors and become phosphorylated in response to Ca(2+)-mediated signals. We have examined expression of Ets proteins during the cell cycle in cells synchronized by centrifugal elutriation or nocodazole-induced mitotic block. Both methods revealed the presence of a hyperphosphorylated isoform of Ets-1 during the mitotic phase. This isoform showed a characteristic mobility shift and was observed during mitosis in each of four cell lines (three human T-cell lines and a human astrocytoma) that express ets-1. In elutriated cells, only a small portion of the Ets-1 in cells from the G2/M fractions was hyperphosphorylated, while in nocodazole-arrested cells more of the Ets-1 was shifted. When cells were released from nocodazole arrest, this isoform disappeared within 1-2 h as cells completed mitosis and entered G1. This suggests that hyperphosphorylated Ets-1 is present transiently during early mitosis, before or around the time of the metaphase-anaphase transition. Exposure of unsynchronized cells to okadaic acid resulted in a dramatic hyperphosphorylation of virtually all Ets-1, suggesting that changes in cellular phosphatase activity are important for cell cycle regulation of Ets-1. Hyperphosphorylated Ets-1 appears to arise from multiple phosphorylations on serine in the exon 7-encoded domain of the protein and did not appear to alter sequence-specific DNA-binding activity. Although enhanced phosphorylation of Ets-2 was detected in nocodazole-arrested cells, no Ets-2 hyperphosphorylation was seen.

• Laudet V, Niel C, Duterque-Coquillaud M, Leprince D, Stehelin D
• Evolution of the ets gene family.
• Biochem Biophys Res Commun. 1993; 190: 8-14
• Display abstract

• Over the past few years a variety of genes have been described whose protein products share similarity with that of the c-ets-1 proto-oncogene, the cellular counterpart of the v-ets oncogene of the avian E26 retrovirus. This so-called "ets family" of transcription factors includes at least a dozen members present in several organisms. We have questioned the common evolutionary origin of these various gene products. By constructing phylogenetical trees with different methods, we show that the ets family is very ancient since the duplication of the various groups of ets related proteins occurred before the Arthropods/Vertebrates split (ca. 500 million years).

• Degnan BM, Degnan SM, Naganuma T, Morse DE
• The ets multigene family is conserved throughout the Metazoa.
• Nucleic Acids Res. 1993; 21: 3479-84
• Display abstract

• This study provides the first empirical evidence for the conservation of the ets proto-oncogene transcription factor family throughout the Metazoa. Using the polymerase chain reaction with degenerate primers corresponding to conserved sequences within the ETS DNA-binding domain, we have detected ets genes in a range of lower metazoans, including sponges, ctenophores, anemones, flatworms and nematodes, and in several higher invertebrate metazoans. Many of these sequences are significantly divergent from the original v-ets-1 oncogene, although most can be aligned with recently defined groups within the ets gene family. Multiple ETS domain sequences were detected in a number of the lower metazoan species, providing evidence for the existence of an ets multigene family at the earliest stages of metazoan evolution. In contrast, we were unable to detect any ETS sequences in fungal, plant or several protozoan DNAs. Our findings suggest that the duplication and divergence of ets proto-oncogenes responsible for generating the multigene family occurred concomitantly with the development of metazoan animals. In addition, these data corroborate other recent molecular evidence in providing strong support for the monophyletic origin of all multicellular animals, including sponges.

• Laget MP, Callebaut I, de Launoit Y, Stehelin D, Mornon JP
• Predicted common structural features of DNA-binding domains from Ets, Myb and HMG transcription factors.
• Nucleic Acids Res. 1993; 21: 5987-96
• Display abstract

• The Ets family of transcription factors shares a 85 amino acid domain, named the ETS domain, which appears responsible for their DNA binding activity. This domain did not show any clear similarity with already known DNA binding motifs. Hydrophobic Cluster Analysis (HCA), a sensitive method able to detect protein structural relationships even at low sequence identity, was chosen in order to compare the ETS domain with other conventional DNA binding motifs. HCA analysis combined with known three-dimensional NMR data, suggests that the ETS domain may be structurally related to the Myb DNA binding domain and possibly to the HMG one. Indeed, the ETS domain is likely to contain two helix-loop-helix motifs.

• Schneikert J, Lutz Y, Wasylyk B
• Two independent activation domains in c-Ets-1 and c-Ets-2 located in non-conserved sequences of the ets gene family.
• Oncogene. 1992; 7: 249-56
• Display abstract

• The c-Ets-1 oncoprotein is a transcription activator that specifically binds to DNA. We show, using fusion proteins with heterologous DNA-binding domains, that chicken c-Ets-1 (p68) contains two independent activation domains. The N-terminal activation domain is absent in c-Ets-1 (p54) that is generated from an alternatively spliced mRNA. A closely related member of the ets gene family, c-Ets-2, also contains two separate activation domains. They lie in the regions of the protein that are least conserved with c-Ets-1, suggesting that the activating function will determine the different physiological roles of these two proteins. The activation domains of c-Ets-1 (p68) and -2 are separated by a moderately conserved region that does not activate on its own. These sequences appear to affect stimulation by the domains, suggesting that they regulate transcription activation. Competition experiments show that c-Ets-1 and -2 interact with a common limiting coactivator. These studies provide important clues about the physiological roles of closely related members of the ets gene family.

• Albagli O, Flourens A, Crepieux P, Begue A, Stehelin D, Leprince D
• Phylogeny of the p68c-ets-1 amino-terminal transactivating domain reveals some highly conserved structural features.
• Oncogene. 1992; 7: 1435-9
• Display abstract

• The chicken c-ets-1 locus gives rise to two distinct transcription factors differing only in their structurally and functionally unrelated N-termini. One of these transcription factors, p54c-ets-1, contains a specific, short (27 amino acids), hydrophilic N-terminus encoded by a single exon, I54, that is widely conserved among vertebrates. The other one, p68c-ets-1, the cellular counterpart of the viral ets oncogene product, differs in the replacement of the I54 by two exons, termed alpha and beta, encoding a larger (71 amino acids), hydrophobic N-terminus which, in contrast to I54, exhibits properties of a transactivating domain. To date the alpha and beta exons have only been found in chicken. Here, we demonstrate the existence of the alpha and beta exons in other avian species (quail and duck) and the existence of the alpha exon in reptiles (turtle). However, none of them could be detected in mammals. Our results strongly suggest that, in contrast to the phylogenetically well-conserved I54 exon, the alpha exon is restricted to reptilian species (birds and 'true' reptiles), whereas the beta exon is detectable so far only in birds. Comparison of their amino acid sequences reveals that the alpha exon and to a much greater extent the beta exon have diverged faster than the I54 exon. In addition, we show that the N- and C-terminal thirds of the alpha exon and the highly hydrophobic nature of the alpha beta-encoded sequence are heavily conserved features and thus likely to be required for function as a transactivating domain in p68c-ets-1 and possibly in the viral P135gag-myb-ets transforming protein.

• Leprince D, Crepieux P, Stehelin D
• c-ets-1 DNA binding to the PEA3 motif is differentially inhibited by all the mutations found in v-ets.
• Oncogene. 1992; 7: 9-17
• Display abstract

• The proto-oncogene c-ets-1, one of the two cellular sequences transduced by the avian retrovirus E26, encodes for two transcription factors that activate through a purine-rich motif. The v-ets oncogene differs from its cellular progenitor p68c-ets-1 (i) by its fusion to gag- and myb-derived sequences in the E26 P135gag-myb-ets fusion protein, (ii) by two point mutations, and (iii) by the replacement of the 13 C-terminal amino acids present in c-ets-1 by 16 unrelated residues in v-ets. A 35 kDa protein which binds to the purine-rich PEA3 motif in a sequence-specific manner has been obtained by expression in Escherichia coli of the 311 carboxy-terminal amino acids of c-ets-1. Using various v-/c-ets-1 chimeric 35 kDa proteins expressed in bacteria, we have shown that all the mutations found in v-ets, when introduced into this c-ets-1 protein, diminish or even abolish its sequence-specific DNA binding. These results demonstrate that, in addition to the previously defined 85 amino acids located near the carboxy terminus of the c-ets-1 protein (the ETS domain), other sequences are required for sequence-specific DNA binding. In addition, the c-ets-1 35 kDa polypeptide carrying the two point mutations and the viral-specific carboxy terminus, and thus similar to the v-ets-encoded domain of the E26 P135gag-myb-ets, does not bind to the PEA3 motif.

• Wasylyk C, Wasylyk B
• Oncogenic conversion alters the transcriptional properties of ets.
• Cell Growth Differ. 1992; 3: 617-25
• Display abstract

• The vEts oncoprotein and its progenitor cEts1(p68) belong to a growing family of transcription factors that are related by the conserved ets domain. We show here that the ets domain and adjacent COOH-terminal amino acids are required for DNA binding by cEts1(p68). vEts differs from cEts1(p68) in both the COOH-terminal sequence and an amino acid substitution in the ets domain. The change in the COOH-terminal sequence markedly decreases its affinity for specific DNA, and the ets domain mutation further diminishes binding. vEts does not trans-activate through the ets (PEA3) motif in vivo. Surprisingly, vEts still efficiently trans-activates the promoters of two genes, stromelysin and collagenase, that are found to be overexpressed in transformed cells. The AP1 motifs of both promoters are required for efficient activation. vEts does not bind to the AP1 motif, even in the presence of cJun and cFos. The DNA-binding domain of Ets1 is required for activation through the AP1 element. Activation is inhibited by the expression of the glucocorticoid and retinoic acid receptors, suggesting that activation by Ets does not involve reversal of negative regulators of AP1. We suggest that activation is by an indirect mechanism involving activation of endogenous genes. Our results show that vEts differs from its progenitor cEts1(p68) in its trans-activating properties. The findings suggest that activation of the Jun and Fos oncoprotein pathway is important for transformation by Ets.

• Wasylyk C, Kerckaert JP, Wasylyk B
• A novel modulator domain of Ets transcription factors.
• Genes Dev. 1992; 6: 965-74
• Display abstract

• The ets gene family is composed of several oncogenes and codes for transcription factors. The Ets proteins have a similar sequence called the ets domain and bind to the core motif A/CGGAA. We show here that several members of the ets family have different trans-activating properties. The ets domain of Ets-1 is required for DNA binding. Adjacent to this domain there is a novel element that inhibits DNA binding. It appears to alter the structure of the DNA-binding domain before it interacts with DNA. There is a similar sequence in Ets-2 that also inhibits DNA binding. This sequence is absent in alternative splice products of h-Ets-1. PU1, the most distantly related member of the ets gene family, lacks this novel element. It has a distinct DNA-binding specificity that is determined by DNA sequences outside the core motif. These results have important implications for both the oncogenic and normal functions of ets family members.

• Nye JA, Petersen JM, Gunther CV, Jonsen MD, Graves BJ
• Interaction of murine ets-1 with GGA-binding sites establishes the ETS domain as a new DNA-binding motif.
• Genes Dev. 1992; 6: 975-90
• Display abstract

• The proto-oncogene ets-1 is the founding member of a new family of eukaryotic transcriptional regulators. Using deletion mutants of murine ets-1 cDNA expressed in Escherichia coli, we show that the DNA-binding domain corresponds closely to the ETS domain, an 85-amino-acid region that is conserved among ets family members. To investigate the specificity of DNA binding of the ETS domain, we mapped the DNA contacts of a monomeric Ets-1 fragment by chemical protection and interference assays. DNA backbone interactions span a 20-nucleotide region and are localized on one face of the helix. Close phosphate and base contacts are restricted to 10 central nucleotides. Contacts map to the major groove in the center of the site. Flanking minor groove interactions also are predicted. To determine the sequence preference in the close contact zone, we selected a pool of high-affinity binding sites using a purified Ets-1 carboxy-terminal fragment. Our Ets-1-selected consensus, 5'-A/GCCGGAA/TGT/C-3', differs from the binding consensus for the Drosophila ETS domain protein E74A, suggesting that specificity of action of ets family members is mediated by the ETS domain. Compared to other well-characterized classes of DNA-binding proteins, Ets-1 produces a unique pattern of DNA contacts. These studies demonstrate that the ETS domain proteins bind DNA in a novel manner.

• Wang CY, Petryniak B, Ho IC, Thompson CB, Leiden JM
• Evolutionarily conserved Ets family members display distinct DNA binding specificities.
• J Exp Med. 1992; 175: 1391-9
• Display abstract

• Members of the Ets family of proto-oncogenes encode sequence-specific transcription factors that bind to a purine-rich motif centered around a conserved GGA trinucleotide. Ets binding sites have been identified in the transcriptional regulatory regions of multiple T cell genes including the T cell receptor alpha and beta (TCR-alpha and -beta) enhancers and the IL-2 enhancer, as well as in the enhancers of several T cell-trophic viruses including Maloney sarcoma virus, human leukemia virus type 1, and human immunodeficiency virus-2. T cells express multiple members of the Ets gene family including Ets-1, Ets-2, GABP alpha, Elf-1, and Fli-1. The different patterns of expression and protein-protein interactions of these different Ets family members undoubtedly contribute to their ability to specifically regulate distinct sets of T cell genes. However, previous studies have suggested that different Ets family members might also display distinct DNA binding specificities. In this report, we have examined the DNA binding characteristics of two Ets family members, Ets-1 and Elf-1, that are highly expressed in T cells. The results demonstrate that the minimal DNA binding domain of these proteins consists of adjacent basic and putative alpha-helical regions that are conserved in all of the known Ets family members. Both regions are required for DNA binding activity. In vitro binding studies demonstrated that Ets-1 and Elf-1 display distinct DNA binding specificities, and, thereby interact preferentially with different naturally occurring Ets binding sites. A comparison of known Ets binding sites identified three nucleotides at the 3' end of these sequences that control the differential binding of the Ets-1 and Elf-1 proteins. These results are consistent with a model in which different Ets family members regulate the expression of different T cell genes by binding preferentially to purine-rich sequences that share a GGA core motif, but contain distinct flanking sequences.

• Chen T, Bunting M, Karim FD, Thummel CS
• Isolation and characterization of five Drosophila genes that encode an ets-related DNA binding domain.
• Dev Biol. 1992; 151: 176-91
• Display abstract

• The recent determination of the site-specific DNA binding properties of several proteins related to the ets oncoprotein has allowed the definition of a novel DNA binding domain, designated the ETS domain. In Drosophila, an ETS domain is present in the early ecdysone-induced E74A protein, which binds DNA in a site-specific manner and interacts with many ecdysone-induced polytene chromosome puffs at the onset of metamorphosis. As a first step toward determining the function of ETS-domain proteins during Drosophila development, we have used PCR amplification with degenerate oligonucleotides to isolate five other ets-related genes. Two of these genes, D-ets-2 and D-elg, have been previously identified. The proteins encoded by these genes are highly related to one another and to the seven identified vertebrate ETS-domain proteins, within the approximately 85-amino-acid DNA binding domain. In situ hybridization to polytene chromosomes revealed that these ets-related genes are not clustered in the genome and that only E74 corresponds to an ecdysone-inducible puff locus. These five ets-related genes are distinguished further from E74 in that they are transcribed through most of development, suggesting that they do not perform a stage-specific function. They are, however, expressed in a variety of patterns in early embryos, suggesting roles in the development of specific cell types. D-ets-2 is expressed in a complex pattern that changes dynamically during early embryogenesis. D-ets-3 and D-ets-6 are expressed in the ventral nervous system. The expression of D-ets-3 is higher in the three thoracic segments and lower in the abdominal segments. The high levels of expression in the thoracic segments are dependent on the presence of the bithorax complex. D-ets-4 and D-elg are expressed at their highest levels in the pole cells, suggesting a role in the development of the germline. This study represents the first effort in any organism to systematically isolate members of the ets gene family. The identification of six independent ets-related genes demonstrates that the ETS-domain proteins constitute a new family of potential transcriptional regulators encoded by the Drosophila genome.

• Bhat NK, Papas TS
• Characterization and uses of monoclonal antibody derived against DNA binding domain of the ets family of genes.
• Hybridoma. 1992; 11: 277-94
• Display abstract

• A monoclonal antibody recognizing ets proteins from a variety of species has been developed. This antibody recognizes ets1, ets2, erg, and other related proteins. It has a high affinity for the ets1 protein. The epitope for the pan ets mAb consists of about 13 amino acids. This antibody can be used to isolate and characterize new members of ets gene family derived from a c-DNA expression library, as well as to identify other "ets motif" binding proteins.

• Qi S, Chen ZQ, Papas TS, Lautenberger JA
• The sea urchin erg homolog defines a highly conserved erg-specific domain.
• DNA Seq. 1992; 3: 127-9
• Display abstract

• A genomic clone, isolated from a phage library prepared from the DNA of the sea urchin Lytechinus variegatus, was shown by sequence analysis to be a homolog of the ets family genes, ERG and Fli-1. It contains an open reading frame of which the coding region begins at a consensus 3' splice site and extends for 173 amino acid residues. The first 84 amino acids are homologous with all members of the ets gene family, while the remainder of the sequence is only homologous with the human ERG and murine Fli-1 genes. This latter region, designated R, represents a highly conserved erg-specific domain.

• Rushlow C, Warrior R
• The rel family of proteins.
• Bioessays. 1992; 14: 89-95
• Display abstract

• The rel family of proteins can be defined as a group of proteins that share sequence homology over a 300 amino acid region termed the rel domain. The rel family comprises important regulatory proteins from a wide variety of species and includes the Drosophila morphogen dorsal, the mammalian transcription factor NF-kappa B, the avian oncogene v-rel, and the cellular proto-oncogene c-rel. Over the last two years it has become apparent that these proteins function as DNA-binding transcription factors, and that their activity is regulated at the level of subcellular localization.

• Brown TA, McKnight SL
• Specificities of protein-protein and protein-DNA interaction of GABP alpha and two newly defined ets-related proteins.
• Genes Dev. 1992; 6: 2502-12
• Display abstract

• The ets-related protein GABP alpha interacts with the four ankyrin-type (ANK) repeats of GABP beta to form a high-affinity DNA-binding complex that recognizes a site important for herpes simplex virus type I immediate early gene activation. To investigate the selectivity and specificity of the GABP complex, we have isolated two new ETS family members, termed ER81 and ER71. ER81 and GABP alpha were present in most tissues of adult mice, whereas ER71 was restricted to testis. We have compared the DNA-binding specificities of these proteins by binding site selection. GABP alpha, ER71, and ER81 recognized the common pentanucleotide DNA sequence 5'-CGGAA/T-3'. Although subtle differences were observed for nucleotide preferences flanking this pentanucleotide core, the overall similarity of the selected sequences was most striking. Given the observation that GABP alpha interaction with GABP beta requires its intact ETS domain, we further compared the ability of GABP beta to interact with other ETS proteins. GABP beta did not augment the DNA-binding activity of the highly similar ETS domains of ER81, ER71, or Ets-1. Moreover, probing of total tissue extracts with radiolabeled GABP beta demonstrated its exceedingly stringent specificity for GABP alpha. Given that the DNA-binding specificities of these ETS proteins are similar and that the protein-protein interactions between GABP beta and GABP alpha are highly specific, we conclude that the protein interactions determine the target site selection by GABP alpha.

• Fisher RJ, Mavrothalassitis G, Kondoh A, Papas TS
• High-affinity DNA-protein interactions of the cellular ETS1 protein: the determination of the ETS binding motif.
• Oncogene. 1991; 6: 2249-54
• Display abstract

• ETS1 protein purified from CEM cells was used to select its optimum DNA-binding sequence (pu) G/CCaGGA-AGTc (py). The sequence CCGGAAGT (ETS1-3) was preferred 5:1 over CAGGAAGT (PEA3). Quantitative electrophoretic mobility-shift assays (EMSA) indicated that the purified ETS1 protein binds to either ETS1-3 or PEA3 oligonucleotide probes with high affinity (Ka = 0.5-4.0 x 10(10) M-1) and that the purified ETS1 has different binding capacities for ETS1-3 and PEA3 oligonucleotide probes. The ETS1 protein binds 2-5 times more ETS1-3 than PEA3. Competitive binding experiments showed that the ETS1-3 and PEA3 probes effectively compete for the binding of ETS1-3. However, changing the core DNA-binding sequence from GGAA to AGAA eliminates competition. Since the human ETS1 protein selected the same DNA sequence from a mixture of random oligonucleotides as did the Drosophila E74A protein (one of the most divergent members of the ETS family), this strongly suggests that all proteins containing the ETS 85 amino acid domain (sequences which define the ETS family) will bind to the same sequence.

• Gabrielsen OS, Sentenac A, Fromageot P
• Specific DNA binding by c-Myb: evidence for a double helix-turn-helix-related motif.
• Science. 1991; 253: 1140-3
• Display abstract

• The c-Myb protein is a sequence-specific DNA binding protein that activates transcription in hematopoietic cells. Three imperfect repeats (R1, R2, and R3) that contain regularly spaced tryptophan residues form the DNA binding domain of c-Myb. A fragment of c-Myb that contained the R2 and R3 regions bound specifically to a DNA sequence recognized by c-Myb plus ten additional base pairs at the 3' end of the element. The R2R3 fragment was predicted to contain two consecutive helix-turn-helix (HTH) motifs with unconventional turns. Mutagenesis of amino acids in R2R3 at positions that correspond to DNA-contacting amino acids in other HTH-containing proteins abolished specific DNA binding without affecting nonspecific DNA interactions.

• Thompson CC, Brown TA, McKnight SL
• Convergence of Ets- and notch-related structural motifs in a heteromeric DNA binding complex.
• Science. 1991; 253: 762-8
• Display abstract

• Analysis of the heteromeric DNA binding protein GABP has revealed the interaction of two distinct peptide sequence motifs normally associated with proteins located in different cellular compartments. The alpha subunit of GABP contains an 85-amino acid segment related to the Ets family of DNA binding proteins. The ETS domain of GABP alpha facilitates weak binding to DNA and, together with an adjacent segment of 37 amino acids, mediates stable interaction with GABP beta. The beta subunit of GABP contains four imperfect repeats of a sequence present in several transmembrane proteins including the product of the Notch gene of Drosophila melanogaster. These amino-terminal repeats of GABP beta mediate stable interaction with GABP alpha and, when complexed with GABP alpha, directly contact DNA. These observations provide evidence for a distinct biochemical role for the 33-amino acid repeats, and suggest that they may serve as a module for the generation of specific dimerization interfaces.

• Ben-David Y, Giddens EB, Letwin K, Bernstein A
• Erythroleukemia induction by Friend murine leukemia virus: insertional activation of a new member of the ets gene family, Fli-1, closely linked to c-ets-1.
• Genes Dev. 1991; 5: 908-18
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• The retroviral integration site Fli-1 is rearranged in 75% of the erythroleukemia cell clones induced by Friend murine leukemia virus (F-MuLV), whereas Spi-1/PU.1, a member of the ets family of DNA-binding proteins, is rearranged in 95% of the erythroleukemias induced by Friend spleen focus-forming virus (SFFV). To determine the transcriptional domain defined by Fli-1, we have isolated a cDNA clone that is highly expressed only in erythroleukemia cell lines with Fli-1 rearrangements. The protein sequence of this cDNA is very similar to Erg2, another member of the ets gene family. The hydrophilic carboxy-terminal end of the Fli-1 cDNA shares significant sequence similarity to the DNA-binding ETS domain found in all members of the ets family. PFGE analysis localized Fli-1 within 240 kb of the ets-1 proto-oncogene on mouse chromosome 9 and human chromosome 11q23, suggesting that ets-1 and Fli-1 arose from a common ancestral gene by gene duplication. The involvement of the murine Fli-1, Spi-1, and avian v-ets genes in erythroleukemia induction suggests that activation of ets gene family members plays an important role in the progression of these multistage malignancies.

• Frampton J, Gibson TJ, Ness SA, Doderlein G, Graf T
• Proposed structure for the DNA-binding domain of the Myb oncoprotein based on model building and mutational analysis.
• Protein Eng. 1991; 4: 891-901
• Display abstract

• Myb-related proteins from plants to humans are characterized by a DNA-binding domain which contains two to three imperfect repeats of approximately 50 amino acids each. Based on the evolutionary conservation of specific residues, secondary structural predictions suggest an arrangement of alpha helices homologous to that seen in the homeodomains, members of the helix-turn-helix family of DNA-binding proteins. We have used molecular modelling in conjunction with site-directed mutagenesis to test the feasibility of this structure. We propose that each Myb repeat consists of three alpha helices packed over a hydrophobic core which is built around the three highly conserved tryptophan residues. The C-terminal helix forms part of the helix-turn-helix motif and can be positioned into the major groove of B-form DNA, allowing prediction of residues critical for specificity of interaction. Modelling also allowed positioning of adjacent repeats around the major groove over an 8 bp binding site.

• Watson DK, Ascione R, Papas TS
• Molecular analysis of the ets genes and their products.
• Crit Rev Oncog. 1990; 1: 409-36
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• Organisms from human to Drosophila have been found to contain cellular sequences and transcripts that are homologous to the ets region of the avian retrovirus, E26. Ets-related sequences are present on at least two distinct functional loci in chickens and mammals, and have been designated ets-1 and ets-2. The E26 virus transduced sequences from the chicken ets-1 locus, which encompasses over 60 kb of DNA. The ets genes characterized so far from sea urchin and Drosophila are most closely related to the 3' end of the known ets genes. The predicted viral and avian ets proteins are very similar, except at the termini. The similarity between the predicted ets proteins so far described is discussed. The ets proteins have been identified and localized by immunoprecipitation and immunofluorescence. While the ets-1 proteins are found in the nuclear and cytoplasmic fractions, the viral gag-myb-ets protein (p135) and the ets-2 proteins are nuclear. The ets-1 and ets-2 genes are differentially regulated in different cell types, probably reflecting unique controlling elements. Because chromosomal translocations have been associated with different human leukemias, studies addressing the possible association with the ETS1 (11q23) or ETS2 (21q22.3) loci are reviewed.

• Gunther CV, Nye JA, Bryner RS, Graves BJ
• Sequence-specific DNA binding of the proto-oncoprotein ets-1 defines a transcriptional activator sequence within the long terminal repeat of the Moloney murine sarcoma virus.
• Genes Dev. 1990; 4: 667-79
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• The ets proto-oncogene family is a group of sequence-related genes whose normal cellular function is unknown. In a study of cellular proteins involved in the transcriptional regulation of murine retroviruses in T lymphocytes, we have discovered that a member of the ets gene family encodes a sequence-specific DNA-binding protein. A mouse ets-1 cDNA clone was obtained by screening a mouse thymus cDNA expression library with a double-stranded oligonucleotide probe representing 20 bp of the Moloney murine sarcoma virus (MSV) long terminal repeat (LTR). The cDNA sequence has an 813-bp open reading frame (ORF) whose predicted amino acid sequence is 97.6% identical to the 272 carboxy-terminal amino acids of the human ets-1 protein. The ORF was expressed in bacteria, and the 30-kD protein product was shown to bind DNA in a sequence-specific manner by mobility-shift assays, Southwestern blot analysis, and methylation interference. A mutant LTR containing four base pair substitutions in the ets-1 binding site was constructed and was shown to have reduced binding in vitro. Transcriptional efficiency of the MSV LTR promoter containing this disrupted ets-1 binding site was compared to the activity of a wild-type promoter in mouse T lymphocytes in culture, and 15- to 20-fold reduction in expression of a reporter gene was observed. We propose that ets-1 functions as a transcriptional activator of mammalian type-C retroviruses and speculate that ets-related genes constitute a new group of eukaryotic DNA-binding proteins.

• Reddy ES, Rao VN
• Localization and modulation of the DNA-binding activity of the human c-ets-1 protooncogene.
• Cancer Res. 1990; 50: 5013-6
• Display abstract

• The avian acute leukemia virus (E26) induces a mixed erythroid-myeloid leukemia in chickens and carries two distinct oncogenes, v-myb and v-ets. The viral protein responsible for transformation is a gag-myb-ets fusion protein that is located in the nucleus of the transformed cells. The cellular homologue of v-ets (c-ets-1) is highly expressed in lymphoid cells and differs from the v-ets gene at its carboxy terminal region. Here, we show that both the c-ets-1 and v-ets gene products are DNA-binding proteins and their DNA-binding activity is located in the carboxy terminal (46 amino acid residues) region. It appears that this DNA-binding activity is modulated by the extreme carboxy terminal region. The amino acid sequences of the putative ets DNA-binding domain at its carboxy terminal region showed a helix-turn-helix secondary structure. Exchanging the nonhomologous extreme carboxy terminal regions of c-ets-1 with v-ets gene sequences showed differences in DNA-binding affinity, indicating that these differences may be partly responsible for the activation of the ets oncogene.

• Klemsz MJ, McKercher SR, Celada A, Van Beveren C, Maki RA
• The macrophage and B cell-specific transcription factor PU.1 is related to the ets oncogene.
• Cell. 1990; 61: 113-24
• Display abstract

• We have isolated a cDNA clone, PU.1, that codes for a new tissue-specific DNA binding protein. Analysis of the binding site by methylation interference and DNAase 1 protection revealed that the PU.1 protein recognized a purine-rich sequence, 5'-GAGGAA-3' (PU box). The PU.1 protein was shown to be a transcriptional activator that is expressed in macrophages and B cells. cDNA constructions used to generate proteins lacking portions of either the amino- or carboxy-terminal ends of the PU.1 protein placed the DNA binding domain in the highly basic carboxy-terminal domain of the protein. The amino acid sequence in the binding domain of PU.1 has considerable identity with proteins belonging to the ets oncogene family.

• Saikumar P, Murali R, Reddy EP
• Role of tryptophan repeats and flanking amino acids in Myb-DNA interactions.
• Proc Natl Acad Sci U S A. 1990; 87: 8452-6
• Display abstract

• The c-myb protooncogene codes for a sequence-specific DNA-binding protein that appears to act as a transcriptional regulator and is highly conserved through evolution. The DNA-binding domain of Myb has been shown to contain three imperfectly conserved repeats of 52 amino acids that constitute the amino-terminal end. Within each repeat, there are three tryptophans that are separated by 18 or 19 amino acids and are flanked by basic amino acids. To determine the role of tryptophans and the flanking basic amino acids in the DNA-binding activity of Myb proteins, we have selectively mutagenized individual tryptophans as well as some of the amino acid residues that flank these tryptophans. Replacement of these tryptophans with glycine, proline, or arginine abolished the DNA-binding activity whereas replacement with other aromatic amino acids or leucine or alanine did not appreciably affect this activity. On the other hand the replacement of two amino acids, asparagine and lysine, that flank the last tryptophan with acidic amino acids completely abolished their DNA-binding activity. These results are consistent with a model we present in which the tryptophans form a hydrophobic scaffold that plays a crucial role in maintaining the helix-turn-helix structure of the DNA binding domain. Basic and polar amino acids adjacent to these tryptophans seem to participate directly in DNA binding.

• Frampton J, Leutz A, Gibson T, Graf T
• DNA-binding domain ancestry.
• Nature. 1989; 342: 134-134

• Kingston RE
• Transcription control and differentiation: the HLH family, c-myc and C/EBP.
• Curr Opin Cell Biol. 1989; 1: 1081-7

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