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• W2041590024 abstract "Jun, Fos, and Ets proteins belong to distinct families of transcription factors that target specific DNA elements often found jointly in gene promoters. Physical and functional interactions between these families play important roles in modulating gene expression. Previous studies have demonstrated a direct interaction between the DNA-binding domains of the two partners. However, the molecular details of the interactions have not been investigated so far. Here we used the known three-dimensional structures of the ETS DNA-binding domain and Jun/Fos heterodimer to model an ETS-Jun/Fos-DNA ternary complex. Docking procedures suggested that certain ETS domain residues in the DNA recognition helix α3 interact with the N-terminal basic domain of Jun. To support the model, different Erg ETS domain mutants were obtained by deletion or by single amino acid substitutions and were tested for their ability to mediate DNA binding, Erg-Jun/Fos complex formation, and transcriptional activation. We identified point mutations that affect both the DNA binding properties of Erg and its physical interaction with Jun (R367K), as well as mutations that essentially prevent transcriptional synergy with the Jun/Fos heterodimer (Y371V). These results provide a framework of the ETS/bZIP interaction linked to the manifestation of functional activity in gene regulation. Jun, Fos, and Ets proteins belong to distinct families of transcription factors that target specific DNA elements often found jointly in gene promoters. Physical and functional interactions between these families play important roles in modulating gene expression. Previous studies have demonstrated a direct interaction between the DNA-binding domains of the two partners. However, the molecular details of the interactions have not been investigated so far. Here we used the known three-dimensional structures of the ETS DNA-binding domain and Jun/Fos heterodimer to model an ETS-Jun/Fos-DNA ternary complex. Docking procedures suggested that certain ETS domain residues in the DNA recognition helix α3 interact with the N-terminal basic domain of Jun. To support the model, different Erg ETS domain mutants were obtained by deletion or by single amino acid substitutions and were tested for their ability to mediate DNA binding, Erg-Jun/Fos complex formation, and transcriptional activation. We identified point mutations that affect both the DNA binding properties of Erg and its physical interaction with Jun (R367K), as well as mutations that essentially prevent transcriptional synergy with the Jun/Fos heterodimer (Y371V). These results provide a framework of the ETS/bZIP interaction linked to the manifestation of functional activity in gene regulation. basic zipper glutathione S-transferase DNA-binding domain ETS-binding site polyomavirus enhancer phosphate-buffered saline In eukaryotes, gene expression appears to be regulated by the assembly of various combinations of transcription factors at promoters and enhancers. The ability of transcription factors to interact specifically with one another, resulting in the formation of hetero-oligomeric complexes, enables the generation of diverse inducible and developmentally regulated programs of gene expression. Biochemical and functional characterization of transcription factor complexes has shown that the structural information necessary for their assembly is provided by both protein-protein and protein-DNA contacts (1Wolberger C. Annu. Rev. Biophys. Biomol. Struct. 1999; 28: 29-56Crossref PubMed Scopus (99) Google Scholar). Particularly, Ets and Jun/Fos family members commonly function as part of the integrated regulatory complexes. For example, complex formation of the Ets protein Elk-1/SAP-1 with the serum responsive factor is critical for the regulation of the c-fos promoter (2Dalton S. Treisman R. Cell. 1992; 68: 597-612Abstract Full Text PDF PubMed Scopus (534) Google Scholar); the Ets-related protein GAPBα forms heterotetramers with GABPβ to activate the immediate early promoters of HSV-1 (3LaMarco K. Thompson C.C. Byers B.P. Walton E.M. McKnight S.L. Science. 1991; 253: 789-792Crossref PubMed Scopus (259) Google Scholar, 4Thompson C.C. Brown T.A. McKnight S.L. Science. 1991; 253: 762-768Crossref PubMed Scopus (320) Google Scholar, 5Batchelor A.H. Piper D.E. de la Brousse F.C. McKnight S.L. Wolberger C. Science. 1998; 279: 1037-1041Crossref PubMed Scopus (266) Google Scholar); the association of Pu-1 with NF-EM5/Pip is important for the regulation of immunoglobulin light chain enhancers (6Eisenbeis C.F. Singh H. Storb U. Genes Dev. 1995; 9: 1377-1387Crossref PubMed Scopus (415) Google Scholar, 7Brass A.L. Zhu A.Q. Singh H. EMBO J. 1999; 18: 977-991Crossref PubMed Scopus (149) Google Scholar); and the binding of a Jun/Fos heterodimer along with the nuclear factor of activated T cells to composite elements is critical for the regulation of genes involved in T cell activation (8Chen L. Glover J.N. Hogan P.G. Rao A. Harrison S.C. Nature. 1998; 392: 42-48Crossref PubMed Scopus (413) Google Scholar). The Ets family proteins share a highly conserved 85-amino acid winged helix-turn-helix DNA-binding domain (ETS domain) that is able to bind the consensus DNA core sequence 5′-GGA(A/T)-3′ and to engage in protein-protein interactions (9Crépieux P. Coll J. Stéhelin D. Crit. Rev. Oncol. 1995; 5: 615-638Google Scholar, 10Graves B.J. Petersen J.M. Adv. Cancer Res. 1998; 75: 1-55Crossref PubMed Google Scholar, 11Li R. Pei H. Watson D.K. Oncogene. 2000; 19: 6514-6523Crossref PubMed Scopus (189) Google Scholar). The Jun and Fos families consist of related bZIP1proteins that are able to heterodimerize and bind the consensus DNA sequence 5′-TGACTCA-3′ (12Karin M. Liu Z. Zandi E. Curr. Opin. Cell Biol. 1997; 9: 240-246Crossref PubMed Scopus (2297) Google Scholar). Ets proteins act synergistically with a variety of other transcription factors to regulate many cellular and viral promoters and enhancers (9Crépieux P. Coll J. Stéhelin D. Crit. Rev. Oncol. 1995; 5: 615-638Google Scholar, 10Graves B.J. Petersen J.M. Adv. Cancer Res. 1998; 75: 1-55Crossref PubMed Google Scholar, 13Sementchenko V.I. Watson D.W. Oncogene. 2000; 19: 6533-6548Crossref PubMed Scopus (315) Google Scholar). The Jun/Fos heterodimer is one of the well characterized partners of Ets proteins (10Graves B.J. Petersen J.M. Adv. Cancer Res. 1998; 75: 1-55Crossref PubMed Google Scholar, 13Sementchenko V.I. Watson D.W. Oncogene. 2000; 19: 6533-6548Crossref PubMed Scopus (315) Google Scholar).In vitro binding studies have demonstrated a direct interaction of various Ets proteins like Ets-1, Elf-1, Pu-1, Fli-1, Ets-2, and Erg with the Jun protein moiety but never with Fos alone (14Bassuk A.G. Leiden J.M. Immunity. 1995; 3: 223-237Abstract Full Text PDF PubMed Scopus (175) Google Scholar, 15Butticè G. Duterque-Coquillaud M. Basuyaux J.P. Carrère S. Kurkinen M. Stéhelin D. Oncogene. 1996; 13: 2297-2306PubMed Google Scholar, 16Basuyaux J.P. Ferreira E. Stéhelin D. Butticè G. J. Biol. Chem. 1997; 272: 26188-26195Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 17Carrère S. Verger A. Flourens A. Stéhelin D. Duterque-Coquillaud M. Oncogene. 1998; 16: 3261-3268Crossref PubMed Scopus (96) Google Scholar). These direct protein interactions have been shown to involve the DNA-binding domain of the two partners. However, the amino acid residues critical for these highly intricate interactions have not been precisely mapped so far. Here we studied the molecular contacts between the human Erg protein and the Jun/Fos heterodimer to gain insights into the general mechanisms by which these two families of transcription factors interact to regulate gene expression. In this respect, we used molecular modeling techniques and the available crystal structures of the ETS domain and the Jun/Fos heterodimer complexed to DNA to predict individual residues involved in the Erg-Jun/Fos-DNA ternary complex formation. The selected ETS domain residues were thus mutated and then tested for their ability to support Jun/Fos recruitment in vitro and in vivo. Interestingly two conserved amino acids in the ETS domain (residues Arg367 and Tyr371 in Erg) are required for efficient recruitment of the Jun/Fos heterodimer. Whereas the R367K substitution abolished DNA binding, interaction with Jun, and consequently transcriptional cooperation, the Y371V mutation abrogated interaction with Jun and synergy without abolishing DNA binding. Therefore the structural determinants of Erg that are important for ternary complex assembly are also required for transcriptional synergy. We thus propose that interdependent protein-protein and protein-DNA contacts regulate Erg-Jun/Fos-DNA complex assembly. The functional changes induced by these mutations define the location of a putative conserved Jun binding interface in the ETS domain. The proposed ETS-Jun/Fos-DNA complex model assumes that the recognition consensus DNA sequence 5′-GGAA-3′ of the ETS domain is located upstream from the DNA binding site 5′-TGACTCA-3′ of the Jun/Fos heterodimer, as observed in the polyomavirus enhancer. The aim of this model is to predict protein-protein interactions when the Jun/Fos heterodimer and the ETS domain of Erg are simultaneously bound to their respective DNA targets, as observed on individual crystal structures. This model is based on the x-ray crystallographic coordinates of the Jun/Fos-DNA (Ref. 18Glover J.N. Harrison S.C. Nature. 1995; 373: 257-261Crossref PubMed Scopus (669) Google Scholar; Protein Data Bank code 1fos) and Elk-1-DNA (Ref. 19Mo Y. Vaessen B. Johnston K. Marmorstein R. Nat. Struct. Biol. 2000; 7: 292-297Crossref PubMed Scopus (82) Google Scholar; Protein Data Bank code 1dux) complexes and was built using the molecular modeling software Insight II (Molecular Simulations Inc.). The ETS domains of the Elk-1 and Erg proteins are indeed highly similar, with an overall sequence identity of 55%. The DNA fragment in the Elk-1-DNA complex x-ray structure (19Mo Y. Vaessen B. Johnston K. Marmorstein R. Nat. Struct. Biol. 2000; 7: 292-297Crossref PubMed Scopus (82) Google Scholar) displays an important bending centered on7G; only the part of DNA extending from G at position 7 to T at position 13 (5′-GAAGTGT-3′) and its complementary strand were then considered. Because important conformational rearrangements can occur upon ternary complex formation that remain unpredictable by molecular modeling means, we deliberately used a simple rigid docking approach. First the Jun/Fos heterodimer was replaced at the center of a 30-nucleotide DNA structure adopting a standard double-stranded B-conformation. Then to investigate individual residues that could be involved at the protein-protein interface when Elk-1 and Jun/Fos are simultaneously bound to their DNA targets, we computed all possible overlapping superimpositions of the selected Elk-1-DNA fragment (5′-GAAGTGT-3′) along the 30-nucleotide standard DNA structure with the Jun/Fos heterodimer remaining fixed at its center. The set of atoms used for DNA structure superimpositions were deoxyribose atoms C1′, C2′, C3′, C4′, and O4′. The root mean square values provided were around 1.5 Å. The ETS domain of the Elk-1-DNA complex was thus progressively advanced toward the Jun/Fos heterodimer. From all of the built ETS-Jun/Fos-DNA complexes, only one displays intermolecular contacts without steric clashes between protein backbones (see Fig. 2). Finally, facing the simplicity of the followed strategy, the structures of DNA and proteins being kept rigid, energy minimization was not appropriate in this context. ROS 17.2/8 (rat osteosarcoma) cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. The Erg deletion mutant expression vectors (constructs Erg (1) and Erg (307)) have been described previously (17Carrère S. Verger A. Flourens A. Stéhelin D. Duterque-Coquillaud M. Oncogene. 1998; 16: 3261-3268Crossref PubMed Scopus (96) Google Scholar). The amino acid substitutions and disrupted ETS domain derivatives were constructed by polymerase chain reaction using appropriate primers flanked by convenient restriction sites. Full details and primer sequences are available on request. Briefly, deletion mutants were obtained by polymerase chain reaction amplification and subcloned into pCRII (Invitrogen). Constructs were then cloned via EcoRI/BglII sites into pSG5 vector for further transcription and translation in vitro. To construct recombinant pGEX-2TK (Amersham Pharmacia Biotech), the DNA fragments encoding Jun, DBD, DBD-R367K, DBD-Y371V, and DBD-D374V were first generated by using polymerase chain reaction amplification of pSG5 hu-Jun and pSG5 hu-Ergp55, respectively, and subcloned into pCRII vector. The BamHI/EcoRI (Jun) or EcoRI (DBD) fragments were cloned in-frame into the cloning sites present within the pGEX-2TK polylinker. The amino acid junctions of the GST vector with the Jun or Erg sequences (in bold type) are: GST-Jun, SVGSMTAK; GST-DBD, GRPVYLGSGQ; GST-DBD-R367K, IRPVYLGSGQ; GST-DBD-Y371V, GRPVYLGSGQ; and GST-DBD-D374V, GRPVYLGSGQ. All constructs were verified by DNA sequencing. The day before transfection, ROS 17 2/8 cells were plated at 50–60% confluence in 6-well plates. For transfection, cells were incubated with 1.0 μg of plasmid DNA and 4 μl of polyethyleneimine (Euromedex, Souffelweyersheim, France) for 6 h in 1 ml of OptiMEM and then in fresh complete medium. When necessary, pSG5 plasmid was used as a carrier. For reporter assays, detailed transfection conditions are indicated in the relevant figure legend. Cells were lysed 24 h after transfection and assayed for luciferase activity with a Berthold (Nashua, NH) chemioluminometer. Results presented are the means of at least five transfections. In vitro translated proteins were generated with a rabbit reticulocyte in vitro transcription/translation system (TNT/Promega) and labeling with 50 μCi of [35S]methionine/50 μl of reticulocyte lysate. The translation products were visualized by SDS-polyacrylamide gel electrophoresis and quantified by PhosphorImager (Molecular Dynamics). The bacterial expression of GST constructs and the purification of GST fusion proteins were performed as described (17Carrère S. Verger A. Flourens A. Stéhelin D. Duterque-Coquillaud M. Oncogene. 1998; 16: 3261-3268Crossref PubMed Scopus (96) Google Scholar). Expression levels of the various GST fusion proteins were confirmed by Coomassie staining (data not shown) and by Western blot (see Fig. 5 B). For pull-down assays, 50 μl of glutathione-Sepharose beads (Amersham Pharmacia Biotech) were incubated with 1 μg of GST, GST-Jun, GST-DBD, GST-DBD-R367K, GST-DBD-Y371V, or GST-DBD-D374V fusion proteins in NETN (20 mm Tris-HCl, pH 8, 200 mm NaCl, 1 mm EDTA, 0, 5% Nonidet P-40) for 1 h at 4 °C. Beads were washed three times with incubation buffer (12 mmHEPES, pH 7.9, 4 mm Tris-HCl, pH 7.9, 50 mmNaCl, 10 mm KCl, 1 mm EDTA) and resuspended in 30 μl of a mixture containing 35S-labeled protein expressed in reticulocyte lysate and incubation buffer. After 1 h at 4 °C, beads were washed six times with NETN and mixed with SDS sample buffer. The bound proteins were analyzed by SDS-polyacrylamide gel electrophoresis followed by autoradiography. The DNA binding reaction was performed at room temperature for 30 min in a total volume of 20 μl containing 20 mm Tris, pH 7.5, 80 mm NaCl, 2 mm dithiothreitol, 0.1% Triton X-100, 5% glycerol, and 5 μg/ml poly(dI-dC). As a probe, we used a double-stranded oligonucleotide corresponding to the polyomavirus enhancer (Py) 5′-GATCTTTAAGCAGGAAGTGACTAACTGACCGCAGGTGGATC-3′ or to an ETS consensus (EBS) 5′-GATCTTCGAAACGGAAGTTCGAG-3′ and labeled with [γ-32P]dATP at a concentration of 10,000 cpm/reaction. Protein-DNA complexes were resolved by 5–10% polyacrylamide gel containing 2% glycerol in TBE buffer. Autoradiography was performed on dry gels using an extra film to quench radioactivity arising from the 35S-labeled proteins. Before electrophoretic mobility shift assay, GST fusion proteins were visualized and quantified by Western blotting. Proteins were separated by SDS-polyacrylamide gel electrophoresis and then transferred to a Hybond-C Extra membrane (Amersham Pharmacia Biotech) with a Bio-Rad dry blotter using 25 mm Tris-base, 192 mm glycine, and 20% (v/v) methanol as the transfer buffer. Membrane was blocked in PBS with 5% (w/v) dry milk for 1.5 h and stained with a rabbit antibody against the ETS domain of the human Erg protein (20Dhordain P. Dewitte F. Desbiens X. Stéhelin D. Duterque- Coquillaud M. Mech. Dev. 1995; 50: 17-28Crossref PubMed Scopus (79) Google Scholar) and a secondary goat anti rabbit/peroxidase antibody (Amersham Pharmacia Biotech). Antibody incubations were performed for 1.5 h in PBS with 5% (w/v) dry milk followed by four 15-min washes in PBS with 0.1% Nonidet P-40. For detection we used the ECL chemiluminescent peroxidase substrate kit from Amersham Pharmacia Biotech. For transient transfection experiments, cell extracts were prepared from confluent 6-well plates. Cells were washed in cold PBS and harvested with Laemmli SDS sample buffer. Extracts were boiled for 5 min, and samples were resolved on 12% SDS-polyacrylamide gels and transferred to a Hybond-C Extra membrane (Amersham Pharmacia Biotech) as for GST fusion detection. In our previous study (17Carrère S. Verger A. Flourens A. Stéhelin D. Duterque-Coquillaud M. Oncogene. 1998; 16: 3261-3268Crossref PubMed Scopus (96) Google Scholar), we defined functional domains of the Erg transcription factor (Fig.1 A) and showed that Erg sequences 253–472, including the ETS domain, are necessary for Jun/Fos recruitment. To better characterize the Erg sequences important for Jun recruitment and Erg-Jun/Fos-DNA ternary complex assembly, we prepared a recombinant protein, named GST-DBD, comprising the ETS domain of Erg (amino acids 307–392 fused to GST) and Jun/Fos proteins (full-length proteins translated in vitro using rabbit reticulocyte lysates) to perform GST pull-down assay. As shown in Fig.1 B, 35S-labeled Jun (lane 5) and the Jun/Fos heterodimer (lane 6), but not Fos alone (lane 4), bind specifically to immobilized intact GST-DBD but not to GST alone (lanes 7–9). Thus these results suggest that the ETS domain of Erg alone includes minimal sequences for recruiting the Jun/Fos heterodimer in vitro. We then performed electrophoretic mobility shift assays with the Py, known to contain adjacent EBS and AP-1 binding sites and to bind Ets and Jun/Fos proteins (21Wasylyk B. Wasylyk C. Flores P. Begue A. Leprince D. Stehelin D. Nature. 1990; 346: 191-193Crossref PubMed Scopus (415) Google Scholar). Thus we incubated a 32P-labeled Py probe with purified GST recombinant proteins, corresponding to the Erg DNA-binding domain (GST-DBD), and with Jun/Fos proteins cotranslated in vitro. As shown in Fig. 1 C, the Py probe formed, in the presence of both Erg-DBD and Jun/Fos proteins, a specific ternary complex that migrated more slowly than the secondary Jun/Fos-Py complex alone (compare lanes 2 and 4). A similar ternary complex was observed with full-length Erg (data not shown). Assembly of the ternary complex required both intact EBS and AP-1 binding sites because mutation of these sequences prevented ternary complex formation (data not shown). To verify that the ternary complex contained both Erg-DBD and the Jun/Fos heterodimer, we incubated the binding reactions with specific antibodies against GST fusion protein or Jun (αGST-IgG and αJun-IgG, respectively). The addition of αJun-IgG abolished formation of the Jun/Fos-Py and Erg-DBD-Jun/Fos-Py complexes but not the Erg-DBD-Py complex (Fig.1 C, lane 5). Conversely αGST-IgG hindered formation of the Erg-DBD-Py and Erg-DBD-Jun/Fos-Py complexes but not the Jun/Fos-Py complex (lane 6). The functional consequences of these physical interactions were then analyzed by transient transfections with a reporter plasmid containing four copies of the polyomavirus enhancer (Fig. 1 D). As expected, cotransfection of Erg and Jun/Fos proteins resulted in a 10-fold transactivation, which revealed a cooperative effect between the two partners. Together these results indicate that Erg and the Jun/Fos heterodimer can form a ternary complex on the adjacent binding sites of the polyomavirus enhancer and that physical interactions between the two proteins contribute to ternary complex assembly. Although we identified the ETS DNA-binding domain as an instrumental scaffold for interaction with the Jun/Fos heterodimer and for transcriptional cooperation, we did not identify amino acids that participate in this mechanism. To make predictions about residues that could be involved in interactions between Erg and the Jun/Fos heterodimer, we modeled an ETS-Jun/Fos-DNA ternary complex (Fig.2) using the available crystal coordinates of the Jun/Fos-DNA (18Glover J.N. Harrison S.C. Nature. 1995; 373: 257-261Crossref PubMed Scopus (669) Google Scholar) and Elk-1-DNA complexes (19Mo Y. Vaessen B. Johnston K. Marmorstein R. Nat. Struct. Biol. 2000; 7: 292-297Crossref PubMed Scopus (82) Google Scholar). The use of Elk-1-DNA instead of Erg-DNA complexes for which no crystallographic data are as yet available seemed appropriate; there is close similarity in the overall scaffold of the ETS domains because the winged helix-turn-helix motif and DNA-contacting residues are strongly conserved (19Mo Y. Vaessen B. Johnston K. Marmorstein R. Nat. Struct. Biol. 2000; 7: 292-297Crossref PubMed Scopus (82) Google Scholar, 22Pio F. Kodandapani R. Ni C.Z. Shepard W. Klemsz M. McKercher S.R. Maki R.A. Ely K.R. J. Biol. Chem. 1996; 271: 23329-23337Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). The rigid docking strategy followed to build the ETS-Jun/Fos-DNA ternary complex model is described under “Experimental Procedures.” In our ETS-Jun/Fos-DNA model (Fig. 2), the two protein partners bind to the major groove on opposite sides of the DNA helix, positioning the helix α3 of the ETS domain and the N-terminal part of the Jun basic domain in close proximity. Principally we located Jun residue Lys267 and Elk-1 residues Arg62, Tyr66, and Asp69 conserved in Erg (Arg367, Tyr371, and Asp374) at the protein-protein interface. As observed in the different available crystal structures of ETS-DNA complexes (5Batchelor A.H. Piper D.E. de la Brousse F.C. McKnight S.L. Wolberger C. Science. 1998; 279: 1037-1041Crossref PubMed Scopus (266) Google Scholar, 19Mo Y. Vaessen B. Johnston K. Marmorstein R. Nat. Struct. Biol. 2000; 7: 292-297Crossref PubMed Scopus (82) Google Scholar, 23Kodandapani R. Pio F. Ni C.Z. Piccialli G. Klemsz M. McKercher S. Maki R.A. Ely K.R. Nature. 1996; 380: 456-460Crossref PubMed Scopus (267) Google Scholar, 24Mo Y. Vaessen B. Johnston K. Marmorstein R. Mol. Cell. 1998; 2: 201-212Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar), the arginine residue Arg62 (Arg367 in Erg) is involved in hydrogen bonds with the DNA core sequence 5′-GGAA-3′ and seems to be unavailable for supplementary interactions. Moreover the positive charge of its guanidinium group should not allow favorable interactions with the N-terminal basic domain of Jun, which is also positively charged. On the other hand, the phenol ring of Tyr66(Tyr371 in Erg) involved in DNA contacts in the SAP-1-DNA complex structure (24Mo Y. Vaessen B. Johnston K. Marmorstein R. Mol. Cell. 1998; 2: 201-212Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar) but not in Elk-1-DNA (19Mo Y. Vaessen B. Johnston K. Marmorstein R. Nat. Struct. Biol. 2000; 7: 292-297Crossref PubMed Scopus (82) Google Scholar) and the carboxylate group of Asp69 (Asp374 in Erg), well accessible at the protein surface, could interact with the basic residue Lys267 of Jun (Fig. 2). However, if Tyr66 is highly conserved within the sequences of the ETS family and may thus be instrumental, Asp69 is frequently mutated (22Pio F. Kodandapani R. Ni C.Z. Shepard W. Klemsz M. McKercher S.R. Maki R.A. Ely K.R. J. Biol. Chem. 1996; 271: 23329-23337Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar) and thus may not play a major role in ETS-Jun/Fos complex formation (see below). Structural studies have revealed that the ETS domain forms a winged helix-turn-helix motif described to fold into a four-stranded anti-parallel β-sheet with three α-helices (Fig. 3 A), where helix α3 is the major DNA recognition component (19Mo Y. Vaessen B. Johnston K. Marmorstein R. Nat. Struct. Biol. 2000; 7: 292-297Crossref PubMed Scopus (82) Google Scholar, 22Pio F. Kodandapani R. Ni C.Z. Shepard W. Klemsz M. McKercher S.R. Maki R.A. Ely K.R. J. Biol. Chem. 1996; 271: 23329-23337Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 24Mo Y. Vaessen B. Johnston K. Marmorstein R. Mol. Cell. 1998; 2: 201-212Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). Our model (Fig. 2) suggests that this helix α3 should also include sequences allowing the recruitment of the Jun/Fos heterodimer. To test this hypothesis, we prepared a set of differentially truncated polypeptides in the ETS domain and expressed in rabbit reticulocyte lysates. We then generated different Erg protein mutants in which either the first helix α1, both helices α1 and α2, or both β3 and β4 β-sheets of the ETS domain were deleted (Fig. 4 A). As previously described (25Siddique H.R. Rao V.N. Lee L. Reddy E.S.P. Oncogene. 1993; 8: 1751-1755PubMed Google Scholar), the integrity of the 85-amino acid ETS domain is necessary to bind an EBS, whereas Erg proteins lacking N-terminal helix α1, helices α1 and α2, or β3 and β4 β-sheet failed to bind DNA (Fig. 4 B). Strikingly our pull-down assays using glutathione S-transferase (GST) fusion proteins indicated that the ETS domain retained the ability to interact with GST-Jun (Fig. 4 C, lanes 3, 6,9, 12, 15, and 18), even when the α1 and α2 helices or the β3 and β4 β-sheets had been deleted and the DNA binding had been abolished (Fig. 4, B andC). Interestingly, these results revealed that deletion of the helix α1 did not affect Erg-Jun/Fos interaction, excluding the possibility that the conserved LXXLL motif, a potential protein-protein interface (26Heery D.M. Kalkhoven E. Hoare S. Parker M.G. Nature. 1997; 387: 733-736Crossref PubMed Scopus (1768) Google Scholar, 27Torchia J. Rose D.W. Inostroza J. Kamei Y. Westin S. Glass C.K. Rosenfeld M.G. Nature. 1997; 387: 677-684Crossref PubMed Scopus (1105) Google Scholar), participates in the binding of Jun/Fos. Moreover Erg and Jun proteins could form a complex independently of DNA binding because specific interactions between the Jun/Fos heterodimer and the ETS domain of Erg were also observed in the presence of ethidium bromide (data not shown), which is known to disrupt DNA-protein interactions (28Lai J.S. Herr W. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6958-6962Crossref PubMed Scopus (397) Google Scholar). Thus although the ETS domain of Erg is the minimal Jun/Fos interaction domain, altering structural features such as the secondary structures can be without effect on the interaction with Jun. We have previously shown that deletion of the whole ETS domain abolishes Jun binding (17Carrère S. Verger A. Flourens A. Stéhelin D. Duterque-Coquillaud M. Oncogene. 1998; 16: 3261-3268Crossref PubMed Scopus (96) Google Scholar). Consequently all these results point out the DNA recognition helix α3 as the major ETS domain component for interaction with Jun protein.Figure 4Identification of Erg ETS domain regions essential for interaction with Jun/Fos. A, structure of the Erg deletion mutants. Amino acid numbers of encoded proteins are indicated for each construct. Interactions observed in B andC are summarized on the right. The minimal domain (helix α3) that is involved in interaction with Jun is indicated. B, band shift analysis of wild type and Erg protein mutants. 5–10 μl of in vitro translated proteins were incubated with the labeled ETS consensus oligonucleotide 5′-GATCTTCGAAACGGAAGTTCGAG-3′. The empty vector pSG5 utilized as a control is indicated. F, free probe. C, GST pull-down assays showing the interaction between GST-Jun and Erg, using the deletion constructs shown in A.View Large Image Figure ViewerDownload Hi-res image Download (PPT) We then attempted to locate in the helix α3 the amino acids more specifically involved in Erg-Jun/Fos interactions. Based on our molecular model of the ETS-Jun/Fos-DNA ternary complex, we identified essentially two Erg residues Tyr371 and Asp374 (Tyr66and Asp69 in Elk-1) (Fig. 2). In the x-ray structure of the SAP-1-DNA complex (24Mo Y. Vaessen B. Johnston K. Marmorstein R. Mol. Cell. 1998; 2: 201-212Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar), the tyrosine residue (Tyr371 in Erg) clearly participates in DNA recognition by contacting the thymine located on the complementary strand to the 5′-GGAA-3′ core sequence. However this residue seems not strictly required for DNA binding because its substitution does not always abolish DNA recognition (22Pio F. Kodandapani R. Ni C.Z. Shepard W. Klemsz M. McKercher S.R. Maki R.A. Ely K.R. J. Biol. Chem. 1996; 271: 23329-23337Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar,29Liang H. Mao X. Olejniczak E.T. Nettesheim D.G., Yu, L. Meadows R.P. Thompson C.B. Fesik S.W. Nat. Struct. Biol. 1994; 1: 871-875Crossref PubMed Scopus (102) Google Scholar). Note also that in the Elk-1-DNA complex crystal structure, no direct contacts of this residues are observed with DNA (19Mo Y. Vaessen B. Johnston K. Marmorstein R. Nat. Struct. Biol. 2000; 7: 292-297Crossref PubMed Scopus (82) Google Scholar). Aspartic acid 374 of Erg (Asp69 of E" @default.
• W2041590024 created "2016-06-24" @default.
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• W2041590024 date "2001-05-01" @default.
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• W2041590024 title "Identification of Amino Acid Residues in the ETS Transcription Factor Erg That Mediate Erg-Jun/Fos-DNA Ternary Complex Formation" @default.
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