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Physical Interaction and Functional Synergy between Glucocorticoid Receptor and Ets2 Proteins for Transcription Activation of the Rat Cytochrome P-450c27 Promoter*

  • Jayati Mullick
    Affiliations
    Department of Animal Biology and Mari Lowe Center for Comparative Oncology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104,
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  • Hindupur K. Anandatheerthavarada
    Affiliations
    Department of Animal Biology and Mari Lowe Center for Comparative Oncology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104,
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  • Govindasamy Amuthan
    Affiliations
    Department of Animal Biology and Mari Lowe Center for Comparative Oncology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104,
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  • Shripad V. Bhagwat
    Footnotes
    Affiliations
    Department of Animal Biology and Mari Lowe Center for Comparative Oncology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104,
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  • Gopa Biswas
    Affiliations
    Department of Animal Biology and Mari Lowe Center for Comparative Oncology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104,
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  • Vijayasarathy Camasamudram
    Affiliations
    Department of Animal Biology and Mari Lowe Center for Comparative Oncology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104,
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  • Narayan K. Bhat
    Affiliations
    Science Applications International Corporation, Frederick Cancer Research and Development Center, Frederick, Maryland 21702, and the
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  • Shyam E.P. Reddy
    Affiliations
    Program in Cancer Genetics, Cancer Center, Medical College of Pennsylvania and Hahnemann University, Philadelphia, Pennsylvania 19102
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  • Veena Rao
    Affiliations
    Program in Cancer Genetics, Cancer Center, Medical College of Pennsylvania and Hahnemann University, Philadelphia, Pennsylvania 19102
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  • Narayan G. Avadhani
    Correspondence
    To whom correspondence should be addressed:
    Affiliations
    Department of Animal Biology and Mari Lowe Center for Comparative Oncology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104,
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  • Author Footnotes
    * This work was supported in part by National Institutes of Health Grants GM34883 and CA22762.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
    § Present address: Dept. of Experimental Oncology, St. Jude Children's Research Hospital, 332 N. Lauderdale, Memphis, TN 38105.
Open AccessPublished:May 25, 2001DOI:https://doi.org/10.1074/jbc.M100671200
      We demonstrate that dexamethasone-mediated transcription activation of the cytochrome P-450c27 promoter involves a physical interaction and functional synergy between glucocorticoid receptor (GR) and Ets2 factor. Ets2 protein binding to a “weak” Ets-like site of the promoter is dependent on GR bound to the adjacent cryptic glucocorticoid response element. Coimmunoprecipitation and chemical cross-linking experiments show physical interaction between GR and Ets2 proteins. Mutational analyses show synergistic effects of Ets2 and GR in dexamethasone-mediated activation of the cytochrome P-450c27 promoter. The DNA-binding domain of GR, lacking the transcription activation and ligand-binding domains, was fully active in synergistic activation of the promoter with intact Ets2. The DNA-binding domain of Ets2 lacking the transcription activation domain showed a dominant negative effect on the transcription activity. Finally, a fusion protein consisting of the GR DNA-binding domain and the transcription activation domain of Ets2 fully supported the transcription activity, suggesting a novel synergy between the two proteins, which does not require the transactivation domain of GR. Our results also provide new insights on the role of putative weak consensus Ets sites in transcription activation, possibly through synergistic interaction with other gene-specific transcription activators.
      CYP27
      cytochrome P-450c27
      GR
      glucocorticoid receptor
      Dx
      dexamethasone
      GRE
      glucocorticoid response element
      tEts2
      DNA-binding domain of Ets2 protein
      GRDBD
      DNA-binding domain of glucocorticoid receptor protein
      BME
      β-mercaptoethanol
      EMSA
      electrophoretic mobility shift analysis for DNA-protein binding
      kb
      kilobase(s)
      PCR
      polymerase chain reaction
      CMV
      cytomegalovirus
      β-gal
      β-galactosidase
      CAT
      chloramphenicol acetyltransferase
      bp
      base pair(s)
      DSP
      dithiobis[succinimidylpropionate]
      sulfo-MBS
      m-maleimidobenzyl-N-hydroxysulfosuccinimide ester
      Adx
      adrenodoxin
      SREBP
      sterol response element-binding protein
      GBP
      GA-binding protein.
      Cytochrome P-450c27 (CYP27)1 is a multifunctional enzyme with major activities for the c27 hydroxylation of cholesterol and c25 hydroxylation of vitamin D3 (
      • Su P.
      • Rennert H.
      • Shayiq R.M.
      • Yamamoto R.
      • Zhwng Y.M.
      • Addya S.
      • Strauss J.F.
      • Avadhani N.G.
      ). Cholesterol c27 hydroxylation in the liver mitochondria is a rate-limiting step in the acidic bile acid pathway and also in the feedback regulation of cholesterol biosynthesis (
      • Bjorkhem I.
      ,
      • Russell D.W.
      • Setchell K.D.
      ,
      • Stravitz R.T.
      • Vlahcevic Z.R.
      • Russell T.L.
      • Heizer M.L.
      • Avadhani N.G.
      • Hylemon P.B.
      ). Vitamin D3 25 hydroxylation in liver mitochondria, on the other hand, represents an important first step toward the conversion of inactive vitamin into the active hormonal form. Thus, the enzyme plays important roles in different pathways of cholesterol homeostasis, bile acid metabolism, and Ca2+ homeostasis (
      • Su P.
      • Rennert H.
      • Shayiq R.M.
      • Yamamoto R.
      • Zhwng Y.M.
      • Addya S.
      • Strauss J.F.
      • Avadhani N.G.
      ,
      • Bjorkhem I.
      ,
      • Stravitz R.T.
      • Vlahcevic Z.R.
      • Russell T.L.
      • Heizer M.L.
      • Avadhani N.G.
      • Hylemon P.B.
      ). In the rat, the CYP27 gene is expressed as a major species of 2-kb mRNA and a minor species of 5′-extended 2.3-kb mRNA (
      • Shayiq R.M.
      • Avadhani N.G.
      ). The physiological function of the latter species remains unclear. Previous studies from our and other laboratories (
      • Shayiq R.M.
      • Avadhani N.G.
      ,
      • Mullick J.
      • Addya S.
      • Sucharov C.
      • Avadhani N.G.
      ,
      • Vlahcevic Z.R.
      • Jairath S.K.
      • Heuman D.M.
      • Stravitz R.T.
      • Hylemon P.B.
      • Avadhani N.G.
      • Pandak W.M.
      ) showed that CYP27 gene expression, particularly the transcription of the 2.0-kb mRNA, is under the control of pituitary growth hormone, glucocorticoids, and also bile acid-dependent diurnal regulation (
      • Rao Y.P.
      • Vlahcevic Z.R.
      • Stravitz R.T.
      • Mallonee D.H.
      • Mullick J.
      • Avadhani N.G.
      • Hylemon P.B.
      ). We have characterized the rat CYP27 gene structure and found that the transcription initiation site and the basal promoter elements required for transcription of the major 2-kb mRNA are localized within the 5′ end of the second exon (
      • Mullick J.
      • Addya S.
      • Sucharov C.
      • Avadhani N.G.
      ). In the present study, we have investigated mechanisms of glucocorticoid-mediated transcription activation of the 2-kb mRNA and found that the essential elements needed for hormone-mediated activation are localized in the 5′-proximal, putative intron region, upstream of the 2-kb mRNA start site. Throughout this study, this downstream promoter will be referred to as the CYP27 promoter.
      GR is a steroid receptor family zinc finger protein that activates transcription of many liver-specific genes (). Binding of Dx, a synthetic glucocorticoid hormone, and related ligands to GR induces structural changes in the receptor resulting in the transcription activation of target genes (). Variant forms of GREs with different affinities for GR binding and hence various activation potentials have been reported (
      • Pearce D.
      • Matsui W.
      • Miner J.N.
      • Yamamoto K.R.
      ,
      • Chen J.D.
      • Evans R.M.
      ). In most cases, the 15-nucleotide-long GRE consists of two half-sites, with consensus sequence (T/G)GTACAXXXTGTTCT (). One molecule of dimeric GR binds to each of the half-sites, TGTACA and TGTTCT, in a cooperative manner (
      • Tsai S.Y.
      • Carlstedt-Duke J.
      • Weigel N.L.
      • Dahlman K.
      • Gustafsson J.A.
      • Tsai M.J.
      • O'Malley B.W.
      ,
      • Espinas M.L.
      • Roux J.
      • Pictet R.
      • Grange T.
      ). A duplicated GRE motif is a more potent transcription activator when compared with a single GRE motif (
      • Schule R.
      • Muller M.
      • Otsuka-Murakami H.
      • Renkawitz R.
      ,
      • Strahle U.
      • Schmid W.
      • Schutz G.
      ).
      GR has been reported to regulate various signaling events by interacting with other transcription factors, such as members of the Ap-1 family, Jun and Fos (
      • Jonat C.
      • Rahmsdorf H.J.
      • Park K.K.
      • Cato A.C.
      • Gebel S.
      • Ponta H.
      • Herrlich P.
      ,
      • Schule R.
      • Rangarajan P.
      • Kliewer S.
      • Ransone L.J.
      • Bolado J.
      • Yang N.
      • Verma I.M.
      • Evans R.M.
      ,
      • Grange T.
      • Roux J.
      • Rigaud G.
      • Pictet R.
      ,
      • Yang-Yen H.F.
      • Chambard J.C.
      • Sun Y.L.
      • Smeal T.
      • Schmidt T.J.
      • Drouin J.
      • Karin M.
      ), RelA protein of the NFκB pathway (
      • Caldenhoven E.
      • Liden J.
      • Wissink S.
      • Van de Stolpe A.
      • Raaijmakers J.
      • Koenderman L.
      • Okret S.
      • Gustafsson J.A.
      • Van der Saag P.T.
      ,
      • Ray A.
      • Prefontaine K.E.
      ,
      • Zhang Z.
      • Jones S.
      • Hagood J.S.
      • Fuentes N.L.
      • Fuller G.M.
      ), liver-specific factor HNF3 (
      • Rigaud G.
      • Roux J.
      • Pictet R.
      • Grange T.
      ,
      • Roux J.
      • Pictet R.
      • Grange T.
      ), and Stat-5 (
      • Stocklin E.
      • Wissler M.
      • Gouilleux F.
      • Groner B.
      ). GR also modulates transcription activity through receptor-receptor interaction between proteins bound to tandem GRE sites (
      • Strahle U.
      • Schmid W.
      • Schutz G.
      ,
      • Grange T.
      • Roux J.
      • Rigaud G.
      • Pictet R.
      ). Recently Zhang et al. (
      • Zhang Z.
      • Jones S.
      • Hagood J.S.
      • Fuentes N.L.
      • Fuller G.M.
      ) demonstrated a complex pattern of interaction between interleukin-6 response element-bound protein and GR bound to an adjacent GRE site. This interaction was suggested to help synergize transcriptional effects of the interleukin-6 response element and GRE sites through Stat-3, which acts as a potent coactivator of GRE-mediated transcription.
      Some studies suggest that transcription activation by GR is also subject to regulation by Ets family transcription factors. Transient transfection studies showed that PU.1 and GR reciprocally modulate the activity of each factor, resulting in transcriptional repression (
      • Crepieux P.
      • Coll J.
      • Stehelin D.
      ,
      • Gauthier J.M.
      • Bourachot B.
      • Doucas V.
      • Yaniv M.
      • Moreau-Gachelin F.
      ). The precise mechanisms of repression, however, remain unclear. Another study showed that mutations targeted to the Ets1-binding site, overlapping the GR binding motif of the tyrosine aminotransferase promoter, resulted in a 2-fold reduction in transcription activity, suggesting synergistic effects of the two factors (
      • Espinas M.L.
      • Roux J.
      • Ghysdale J.
      • Pictet R.
      • Grange T.
      ). However, the precise mode of physical interaction between the factors was not investigated. In the present study we show that Dx-mediated activation of CYP27 gene expression involves GR binding to a variant GRE site and its synergistic interaction with factors binding to an adjacent “weak” consensus Ets site, referred to in this study as an Ets-like site. Interestingly, the activation appears to require a novel functional and physical interaction between the DNA-binding domain of GR and the transcription activation domain of the ubiquitously expressed Ets2 transcription factor.

      DISCUSSION

      Transcription regulation of mRNA coding genes in mammalian cells requires a complex cooperative interaction of gene-specific transcription activators and coactivators with the basal transcription machinery, which includes the TFIID complex and polymerase II holoenzyme complex (,
      • Roeder R.G.
      ). In this paper we demonstrate that Dx-mediated transcription activation of the rat CYP27 gene involves a cooperative interaction between GR and Ets2 factors. Functional synergies between gene/tissue-specific and ubiquitous housekeeping varieties of sequence-specific DNA-binding transcription factors can be classified into the following categories. The first type involves ligand or physiological pathway-specific transcription factors and ubiquitous constitutive factors binding to the same sequence motif as heteromeric complexes. Examples of heteromeric factors include myogenic factors MyoD and E2A (
      • Lassar A.B.
      • Davis R.L.
      • Wright W.E.
      • Kadesch T.
      • Murre C.
      • Voronova A.
      • Baltimore D.
      • Weintraub H.
      ), proteins belonging to the Ap1/Ap2/CAAT family (Jun/Fos, NFATc and CEBP) (
      • Basuyaux J.P.
      • Ferreira E.
      • Stehelin D.
      • Buttice G.
      ,
      • MacDougald O.A.
      • Lane M.D.
      ,
      • McCaffrey P.G.
      • Jain J.
      • Jamieson C.
      • Sen R.
      • Rao R.
      ), and ligand-specific factors belonging to the steroid superfamily nuclear receptors (
      • Tsai M.J.
      • O'Malley B.W.
      ). The second type involves cooperativity between the same class or homologous factors bound to tandemly duplicated DNA-binding sites leading to functional synergy. Transcription activation of promoter sites with duplicated binding sites by GR (
      • Tsai M.J.
      • O'Malley B.W.
      ,
      • Schmid W.
      • Strahle U.
      • Schutz G.
      • Schmitt J.
      • Stunnenberg H.
      ) and Ets family GABP factors (
      • Carter R.S.
      • Avadhani N.G.
      ,

      LaMarco, K., Thompson, C. C., Byers, B. P., Walton, E. M., and McKnight, S. L. Science 253,789–792.

      ) belong to this class. The third type involves synergy between transcription factors bound to spatially separated DNA sites possibly through interaction with common coactivator proteins. Synergistic activation of the low density lipoprotein receptor promoter by sterol response element-binding protein (SREBP) and the ubiquitous factor Sp1 through interaction with coactivator cAMP-response element-binding protein-binding protein is a classical example of this type (
      • Schmid W.
      • Strahle U.
      • Schutz G.
      • Schmitt J.
      • Stunnenberg H.
      ,
      • Naar A.M.
      • Beaurang P.A.
      • Robinson K.M.
      • Oliner J.D.
      • Avizonis D.
      • Scheek S.
      • Zwicker J.
      • Kadonaga J.T.
      • Tjian R.
      ). The fourth type of functional synergy is seen in the recruitment of factor Pu.1 interacting protein by DNA-bound PU.1 or E2A in the activation of immunoglobulin κ 3′ enhancer (
      • Nagulapalli S.
      • Atchison M.L.
      ,
      • Pongubala J.M.
      • Nagulapalli S.
      • Klemsz M.J.
      • McKercher S.R.
      • Maki R.A.
      • Atchison M.L.
      ). The association of Pu.1 interacting protein with the promoter sites appears to involve both protein-protein interaction with DNA-bound PU.1 or E2A (
      • Nagulapalli S.
      • Atchison M.L.
      ,
      • Pongubala J.M.
      • Nagulapalli S.
      • Klemsz M.J.
      • McKercher S.R.
      • Maki R.A.
      • Atchison M.L.
      ) and also direct interaction with DNA. Thus, the functional synergy between the GR and Ets2 proteins observed in this study shares similarities with the last two mechanisms described above.
      Physical and functional analyses of the Dx-responsive region of the promoter by site-directed mutations (Figs. 1 and 3) suggest that the cryptic GRE motif and a downstream Ets-like motif are important for Dx-mediated transcription activation of the promoter. Cotransfection with GR and Ets2 cDNAs resulting in synergistic activation of CAT activity further substantiated the role of these cis-DNA elements (Figs. 1, 3, and 4). In support of these functional data, the cryptic GRE motif (TGCTGT) bound to proteins from nuclear extracts, which cross-reacted with GR-specific antibody as seen by a supershift in EMSA (Fig. 2 A). Additionally, bacterially expressed purified GRDBD also bound to this motif (Fig. 2 B). However, the Ets-like motif by itself failed to form a detectable complex with both purified Ets2 protein (Fig. 2 C) and 3T3 nuclear extract (not shown). These results are in full support of general consensus in the field that the core sequences (G/T)GGAA(T/A) are weak motifs for binding to ubiquitously expressed Ets2 and GABP (variably called NRF2) as well as tissue-specific Ets1 and other members of Ets family proteins (
      • Seth A.
      • Ascione R.
      • Fisher R.J.
      • Mavrothalassitis G.J.
      • Bhat N.K.
      • Papas T.S.
      ,
      • Sharrocks A.D.
      • Brown A.L.
      • Ling Y.
      • Yates P.R.
      ,
      • Sucharov C.
      • Basu A.
      • Carter R.S.
      • Avadhani N.G.
      ). EMSA with the nuclear extract and 135-bp hormone response region DNA probe also yielded a slow migrating complex, which was competed by both cryptic GRE and Ets consensus DNA (Fig. 2,A and C), suggesting that GR binding to the cryptic GRE site helps recruiting an Ets family protein. Use of bacterially expressed purified proteins and competition with site-specific DNA indeed supported this possibility (Fig.2 C). Direct evidence for the functional role of Ets2 factor and the physiological significance of its in vitrofunctional synergy with GR is presented by experiments showing the dominant negative effects of tEts2 (only the DNA-binding domain of Ets2) on the activity of the promoter construct and also the endogenous CYP27 gene expression in 3T3 fibroblasts.
      Ets2 factor binding to the hormone response region of the promoter is dependent on GR or GRDBD bound to the cryptic GRE motif as well as the downstream Ets-like sequence motif, the latter thought to be a weak consensus sequence for binding to Ets family proteins (
      • Seth A.
      • Ascione R.
      • Fisher R.J.
      • Mavrothalassitis G.J.
      • Bhat N.K.
      • Papas T.S.
      ,
      • Sharrocks A.D.
      • Brown A.L.
      • Ling Y.
      • Yates P.R.
      ). This conclusion is based on EMSA results showing that slow migrating complex a obtained with purified GRDBD and Ets2 proteins (Fig. 2, Cand D) was effectively competed by Ets-like DNA but not by a mutant form. A DNA probe carrying mutations at the Ets-like sequence of the GHR region probe formed only complex b but not the higher order complex a even in the presence of added GRDBD and Ets2 proteins (results not shown). Similarly, the intact Ets-like sequence motif is required for the synergistic activation of the promoter by cotransfection with GR (or GRDBD) and Ets2 cDNAs (Fig. 3). Results of cotransfection also show that only the Ets2 protein with an intact DNA-binding domain, but not the deletion mutant lacking this domain, is able to induce transcription activation (results not presented). These results suggest the need for DNA binding by Ets2 protein as an essential part of the functional synergy with the adjacently bound GR. Thus, a novel finding of this work relates to the role of the putative weak Ets consensus site in the transcription activation. The putative weak Ets consensus sites are widely distributed on many promoters. In view of our results, the roles of the ubiquitously expressed Ets2 protein and the weak consensus Ets-like sites, particularly those located in the close proximity of other sequence-specific factor binding sites, need to be further evaluated.
      It is known that Ets1 and Ets2 factors can interact with members of Jun/Fos and other AP-1 family proteins (
      • Jonat C.
      • Rahmsdorf H.J.
      • Park K.K.
      • Cato A.C.
      • Gebel S.
      • Ponta H.
      • Herrlich P.
      ,
      • Schule R.
      • Rangarajan P.
      • Kliewer S.
      • Ransone L.J.
      • Bolado J.
      • Yang N.
      • Verma I.M.
      • Evans R.M.
      ,
      • Grange T.
      • Roux J.
      • Rigaud G.
      • Pictet R.
      ,
      • Yang-Yen H.F.
      • Chambard J.C.
      • Sun Y.L.
      • Smeal T.
      • Schmidt T.J.
      • Drouin J.
      • Karin M.
      ,
      • Basuyaux J.P.
      • Ferreira E.
      • Stehelin D.
      • Buttice G.
      ) and also other leucine zipper proteins such as Myb (
      • Strahle U.
      • Schmid W.
      • Schutz G.
      ,
      • Dudek H.
      • Tantravathi R.V.
      • Rao V.N.
      • Reddy E.S.
      • Reddy E.P.
      ), modulating their transcription activity. Results of coimmunoprecipitation (Fig. 6) and chemical cross-linking experiments (Fig. 7) reported in this study for the first time demonstrate a direct physical interaction between GRDBD and Ets2 as well as Ets1 proteins. Furthermore, the interaction requires the DNA-binding domains of both proteins because the transcription activation domain of Ets2, lacking the DNA-binding domain, showed no detectable interaction with the GRDBD protein. Thus, it is likely that the two factors interact physically in their DNA-bound form on the hormone response region of the CYP27 promoter. It is known that GRDBD, which lacks the ligand-binding domain, has a constitutively activated DNA binding activity (
      • Lefstin J.A.
      • Thomas J.R.
      • Yamamoto K.R.
      ). It was, however, surprising that GRDBD lacking both the N- and C-terminal activation domains (
      • Hollenberg S.M.
      • Evans S.M.
      ,
      • Lees J.A.
      • Fawell S.E.
      • Parker M.G.
      ) and the ligand-binding domain was able to synergistically activate the promoter with Ets2 (Fig. 4 B). The possible requirement for physical interaction between GRDBD and Ets2 for functional synergy was further supported in experiments showing that a fusion protein containing GRDBD and the Ets2 transcription activation domain could efficiently activate the promoter. Interestingly, mutation of the weak Ets-like site did not affect the fusion protein-induced activity as long as the cryptic GRE site was intact. Notably, the GRDBD-Ets2 fusion construct with mutated zinc finger domains of GRDBDB showed vastly reduced transcription activation of the promoter. Based on these results, we hypothesize that the role of the GR or GRDBD bound to the cryptic GRE of the promoter is to help recruit the Ets2 protein to the complex. Interaction with GR may induce conformational change(s) in the Ets protein (
      • Petersen J.M.
      • Skalivky J.J.
      • Donaldson L.E.
      • McIntosh L.P.
      • Arber T.
      • Graves B.J.
      ) such that it can bind to the weak Ets consensus site. Apparently, binding of Ets2 factor to the weak Ets site alone in the absence of adjacently bound GR is not strong enough for resolution of the complex through the gel matrix in EMSA. Thus, the requirement for physical interaction between GR and Ets2 for functional synergy is uniquely different from the synergistic activation by SREBP and Sp1, which does not involve intermolecular protein-protein interaction (
      • Naar A.M.
      • Beaurang P.A.
      • Robinson K.M.
      • Oliner J.D.
      • Avizonis D.
      • Scheek S.
      • Zwicker J.
      • Kadonaga J.T.
      • Tjian R.
      ). The requirement for the transcription activation domain of only Ets2 but not that of GR for synergistic interaction is reminiscent of the synergy between Pu.1 and Pu.1 interacting protein, Fos, and Jun for the activation of immunoglobulin κ 3′ enhancer (
      • Pongubala J.M.
      • Atchison M.L.
      ).
      A current model on the mechanism of synergistic activation by SREBP and Sp1 suggests that the transactivation domains of both of the proteins are essential for transactivation (
      • McCaffrey P.G.
      • Jain J.
      • Jamieson C.
      • Sen R.
      • Rao R.
      ). It is proposed that the transcription activation domain of Sp1 may be involved in interaction with the basal transcription machinery, whereas that of SREBP might be involved in recruiting factors with HAT activity (
      • McCaffrey P.G.
      • Jain J.
      • Jamieson C.
      • Sen R.
      • Rao R.
      ). The synergy observed with GR and Ets2 factors, on the other hand, appears to be highly dependent on the transcription activation domain of the latter, without requiring the presence of the former. It should be noted that tissue-specific Ets1 factor also binds to the promoter DNA in a GRDBD-dependent manner (results not shown) and physically interacts with GRDBD as tested by coimmunoprecipitation and chemical cross-linking (Figs. 7 and 8). However, cotransfection with Ets1 and GR or GRDBD cDNAs failed to show any synergistic activation of the −329/+23CAT promoter. Similarly, cotransfection with GRDBD-Ets1 fusion protein failed to show significant transcription activation of the promoter. These results suggest that the transcription activation domain of Ets2 protein is critical for the synergistic activation. Although the DNA-binding domains of Ets1 and Ets2 proteins share over 90% sequence homology, the N-terminal 300-amino acid region comprising the transcription activation domains of the two proteins shares only about 38% sequence identity. Notably, the transcription activation domain of Ets2 protein contains higher hydrophobic helical content and higher glutamine contents. It is likely that the structural features of Ets2 protein may be essential for interaction with components of basal transcription machinery or with specific coactivator proteins. In summary, we describe a novel functional synergy between GR and Ets2 proteins in the glucocorticoid hormone-dependent activation of the P-450c27 promoter.

      Acknowledgments

      We are thankful to Dr. Keath Yamamoto for generously providing the GR and GRDBD cDNA constructs and purified GRDBD protein used in this study. Thanks are also due to Drs. Michael Atchison and Haider Raza for a critical review of the manuscript and to Dr. Marie-Anne Robin for helping with the preparation of the manuscript.

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