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Transcription Activation by the Orphan Nuclear Receptor, Chicken Ovalbumin Upstream Promoter-Transcription Factor I (COUP-TFI)

DEFINITION OF THE DOMAIN INVOLVED IN THE GLUCOCORTICOID RESPONSE OF THE PHOSPHOENOLPYRUVATE CARBOXYKINASE GENE*
  • Takashi Sugiyama
    Affiliations
    Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0615
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  • Jen-Chywan Wang
    Affiliations
    Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0615
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  • Donald K. Scott
    Affiliations
    Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0615
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  • Daryl K. Granner
    Correspondence
    To whom correspondence should be addressed. Tel.: 615-322-7004; Fax: 615-322-7236
    Affiliations
    Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0615
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  • Author Footnotes
    * This work was supported by National Institutes of Health Grant DK 35107 and the Vanderbilt Diabetes Research and Training Center Grant DK 20593).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.
Open AccessPublished:February 04, 2000DOI:https://doi.org/10.1074/jbc.275.5.3446
      Chicken ovalbumin upstream promoter-transcription factors (COUP-TFs), orphan members of the nuclear receptor superfamily, play a key role in the regulation of organogenesis, neurogenesis, and cellular differentiation during embryogenic development. COUP-TFs are also involved in the regulation of several genes that encode metabolic enzymes. Although COUP-TFs function as potent transcription repressors, there are at least three different molecular mechanisms of activation of gene expression by COUP-TFs. First, as we have previously shown, COUP-TF is required as an accessory factor for the complete induction of phosphoenolpyruvate carboxykinase gene transcription by glucocorticoids. This action is mediated by the binding of COUP-TF to the glucocorticoid accessory factor 1 (gAF1) and 3 (gAF3) elements in the phosphoenolpyruvate carboxykinase gene glucocorticoid response unit. In addition, COUP-TF1 binds to DNA elements in certain genes and transactivates directly. Finally, COUP-TF1 serves as a coactivator through DNA-bound hepatic nuclear factor 4. Here we show that the same region of COUP-TFI, located between amino acids 184 and 423, is involved in these three mechanisms of transactivation by COUP-TFI. Furthermore, we show that GRIP1 and SRC-1 potentiate the activity of COUP-TFI and that COUP-TFI associates with these coactivators in vivo using the same region required for transcription activation. Finally, overexpression of GRIP1 or SRC-1 does not convert COUP-TFI from a transcriptional repressor into a transcriptional activator in HeLa cells.
      COUP-TF
      chicken ovalbumin upstream promoter-transcription factor
      PEPCK
      phosphoenolpyruvate carboxykinase
      CYP7A
      cholesterol 7α-hydroxylase
      HNF
      hepatocyte nuclear factor
      SMRT
      silencing mediator for retinoic acid and thyroid hormone receptor
      GRU
      glucocorticoid response unit
      AF
      accessory factor
      gAF1
      gAF2, and gAF3, glucocorticoid AF binding sites
      GR
      glucocorticoid receptor
      GR1 and GR2
      GR binding sites
      GRIP1
      GR-interacting protein-1
      SRC-1
      steroid receptor coactivator-1
      CAT
      chloramphenicol acetyltransferase
      The nuclear hormone receptor superfamily is composed of many diverse subsets of transcriptional factors, including receptors for steroids, retinoids, and thyroid hormones. Also included are a large number of structurally and functionally related transcription regulatory proteins termed orphan receptors, so named because their natural ligands have not been identified (
      • Ladias J.A.A.
      • Karathanasis K.K.
      ,
      • Wang L.H.
      • Tsai S.Y.
      • Cook R.G.
      • Beattie W.G.
      • Tsai M.J.
      • O'Malley B.W.
      ,
      • Wang L.H.
      • Ing N.H.
      • Tsai S.Y.
      • O'Malley B.W.
      • Tsai M.J.
      ). Chicken ovalbumin upstream promoter-transcription factor (COUP-TF)1 is one of the most studied of the orphan receptors. COUP-TFI (also termed EAR3) and COUP-TFII (also termed ARP-1) are closely related transcription factors that are expressed ubiquitously and are involved in the regulation of several important biological processes, such as neurogenesis, organogenesis, cell fate determination, and metabolic homeostasis (
      • Kastner P.M.
      • Mark M.
      • Chambon P.
      ,
      • Mangelsdorf D.J.
      • Thummel C.
      • Beato M.
      • Herrlich P.
      • Schuts G.
      • Umesono K.
      • Blumberg P.
      • Mark M.
      • Chambon P.
      ,
      • Mangelsdorf D.J.
      • Evans R.M.
      ). Targeted disruptions of mouse COUP-TFI or COUP-TFII result in perinatal and embryonic lethality, respectively (
      • Tsai S.Y.
      • Tsai M.-J.
      ,
      • Qiu Y.
      • Pereira F.A.
      • DeMayo F.J.
      • Lydon J.P.
      • Tsai S.Y.
      • Tsai M.-J.
      ). COUP-TFs are also involved in the regulation of the transcription of genes that encode various metabolic enzymes such as phosphoenolpyruvate carboxykinase (PEPCK; EC 4.1.1.32), cholesterol 7α-hydroxylase (CYP7A; EC 1.14.13.17) (
      • Stroup D.
      • Crestani M.
      • Chiang J.Y.L.
      ), and mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase (EC 4.1.3.5) (
      • Rodriguez J.C.
      • Ortiz J.A.
      • Hegardt F.G.
      • Haro D.
      ).
      COUP-TFs homodimerize or heterodimerize with retinoid X receptor and a few other nuclear receptors and bind to a wide variety of response elements that contain imperfect AGGTCA direct repeats separated by a variable number of nucleotides (
      • Tsai S.Y.
      • Tsai M.-J.
      ,
      • Cooney A.J.
      • Tsai S.Y.
      • O'Malley B.W.
      • Tsai M.-J.
      ). Although COUP-TF was initially identified as an activator of the chicken ovalbumin gene, COUP-TFs generally serve as negative regulators in conjunction with other nuclear hormone receptors such as retinoic acid receptor, thyroid hormone receptor, vitamin D receptor, peroxisome proliferator-activated receptor, and hepatocyte nuclear factor 4 (HNF-4) (
      • Cooney A.J.
      • Tsai S.Y.
      • O'Malley B.W.
      • Tsai M.-J.
      ,
      • Ladias J.A.A.
      • Cladaras-Hadzopoulou M.
      • Kardassis D.
      • Cardot P.
      • Cheng J.
      • Zannis V.I.
      • Cladaras C.
      ,
      • Leng X.
      • Cooney S.Y.
      • Tsai S.Y.
      • Tsai M.-J.
      ). Several mechanisms account for the repressive effects of COUP-TFs. COUP-TFs bind to a number of nuclear hormone receptor response elements and, thus, compete with receptors for these DNA elements. COUP-TFs may also repress transcription by forming nonproductive complexes with retinoid X receptor, the essential heterodimer partner of a number of nuclear hormone receptors. In addition, at least two direct mechanisms of repression have been described. In one example, termed active repression, COUP-TFs bind to specific DNA elements and repress by forming a direct interaction with corepressors such as nuclear receptor corepressor and silencing mediator for retinoic acid and thyroid hormone receptor (SMRT) (
      • Shibata H.
      • Nawaz Z.
      • Tsai S.Y.
      • O'Malley B.W.
      • Tsai M.-J.
      ). Finally, COUP-TFs can repress transcription by directly binding to the ligand binding domain of nuclear hormone receptors, a process called transrepression (
      • Leng X.
      • Cooney S.Y.
      • Tsai S.Y.
      • Tsai M.-J.
      ,
      • Achatz G.
      • Speckmayer R.
      • Hauser C.
      • Sandhofer F.
      • Paulweber B.
      ).
      COUP-TFs also activate transcription in various promoter contexts (
      • Stroup D.
      • Crestani M.
      • Chiang J.Y.L.
      ,
      • Rodriguez J.C.
      • Ortiz J.A.
      • Hegardt F.G.
      • Haro D.
      ,
      • Kadowaki Y.
      • Toyoshima K.
      • Yamamoto T.
      ,
      • Ktistaki E.
      • Talianidis I.
      ,
      • Kimura A.
      • Nishiyori A.
      • Murakami T.
      • Tsukamoto T.
      • Hata S.
      • Osumi T.
      • Okamura R.
      • Mori M.
      • Takiguchi M.
      ,
      • Lazennec G.
      • Kern L.
      • Valotaire Y.
      • Salbert G.
      ,
      • Power S.C.
      • Cereghini S.
      ,
      • Sawaya B.
      • Rohr O.
      • Aunis D.
      • Schaeffer E.
      ). COUP-TFII stimulates the transcriptional activity of the rat CYP7A promoter by binding to the nucleotide sequence located between −74 and −54 (relative to the transcription start site), which contains a direct repeat of two hormone response element half-sites separated by 4 nucleotides (a DR4) (
      • Stroup D.
      • Crestani M.
      • Chiang J.Y.L.
      ). Furthermore, COUP-TFs function as accessory factors for the hormonal response of some genes, such as the glucocorticoid response of the PEPCK and the estrogen response of the trout estrogen receptor gene (
      • Lazennec G.
      • Kern L.
      • Valotaire Y.
      • Salbert G.
      ). Finally, COUP-TFs also can function as coactivators. For instance, COUP-TFs potentiate HNF-4-mediated transactivation in the hepatocyte nuclear factor 1α (HNF-1α) gene promoter through direct interaction with HNF-4 and without a requirement for DNA binding (
      • Ktistaki E.
      • Talianidis I.
      ). A similar coactivator function has been described in the vHNF (also termed HNF-1-β) gene promoter, where COUP-TFs do not bind DNA directly but potentiate the transcriptional activity of the vHNF gene promoter through a DNA-independent interaction with Oct-1 (
      • Power S.C.
      • Cereghini S.
      ).
      Our interest is directed to the role COUP-TF plays in the hormonal regulation of the PEPCK gene. PEPCK catalyzes the conversion of oxaloacetate to phosphoenolpyruvate, a rate-controlling step in hepatic gluconeogenesis. PEPCK gene transcription is positively regulated by glucocorticoids, glucagon (cAMP) and retinoic acid, whereas insulin inhibits the transcription of the PEPCK gene (
      • Granner D.K.
      • Pilkis S.
      ,
      • O'Brien R.M.
      • Bonovich M.T.
      • Forest C.D.
      • Granner D.K.
      ,
      • Hanson R.W.
      • Reshef L.
      ). Induction of transcription of the PEPCK gene by glucocorticoids is achieved through a complex glucocorticoid response unit (GRU) (see Fig. 1). The GRU includes, as a linear array from 5′ to 3′, two glucocorticoid accessory factor binding sites, gAF1 and gAF2,
      We originally designated the accessory factor elements AF1, AF2, and AF3 in order of their discovery. The subsequent definition of transactivation domains of the nuclear receptor subfamily as AF1 and AF2 (
      • Nagpal S.
      • Friant S.
      • Nakshatri H.
      • Chambon P.
      ) has created a potentially confusing situation. To avoid having to discuss the interaction of the AF1 (or AF2) transcription domain of the glucocorticoid receptor with the AF1 element (or with the AF2 and AF3 elements) in the GRU, we now designate these elements as gAF1 to denote the glucocorticoid accessory factor 1 element, etc.
      2We originally designated the accessory factor elements AF1, AF2, and AF3 in order of their discovery. The subsequent definition of transactivation domains of the nuclear receptor subfamily as AF1 and AF2 (
      • Nagpal S.
      • Friant S.
      • Nakshatri H.
      • Chambon P.
      ) has created a potentially confusing situation. To avoid having to discuss the interaction of the AF1 (or AF2) transcription domain of the glucocorticoid receptor with the AF1 element (or with the AF2 and AF3 elements) in the GRU, we now designate these elements as gAF1 to denote the glucocorticoid accessory factor 1 element, etc.
      two glucocorticoid receptor (GR) binding sites, GR1 and GR2, and a third accessory factor binding site, gAF3 (
      • Imai E.
      • Stromstedt P.
      • Quinn P.G.
      • Carlstedt-Duke J.
      • Gustafsson J.
      • Granner D.K.
      ,
      • Scott D.K.
      • Mitchell J.A.
      • Granner D.K.
      ). An intact cAMP response element (located between −93 and −86) is also required for a full glucocorticoid response and, thus, is part of the GRU (see Fig. 1). The proteins that mediate the accessory activities through gAF1, gAF2, and gAF3 have been identified. Both HNF-4 and COUP-TF bind the gAF1 element and act as accessory factors for the glucocorticoid response (
      • Hall R.K.
      • Sladek F.M.
      • Granner D.K.
      ). A number of factors bind to gAF2, although only binding of hepatic nuclear factor 3 (HNF-3) specifically correlates with the ability of the gAF2 element to induce the glucocorticoid response (
      • Wang J.-C.
      • Stromstedt P.-E.
      • O'Brien R.M.
      • Granner D.K.
      ). COUP-TF also binds to gAF3 and acts as the accessory factor through this element (
      • Scott D.K.
      • Mitchell J.A.
      • Granner D.K.
      ). Thus, COUP-TF serves as an accessory factor required for the induction of PEPCK gene transcription by glucocorticoids through both the gAF1 element and the gAF3 element (see Fig. 1).
      Figure thumbnail gr1
      Figure 1Schematic diagram of the PEPCK gene promoter GRU. The cis-acting elements and associatedtrans-acting factors required for a complete glucocorticoid response are shown. The location of each cis-element with respect to the transcription start site is shown above the schematic of the PEPCK promoter. Also shown are the sequences of the gAF1 and gAF3 elements and their relative locations. C/EBP, CAAT/enhancer-binding protein.
      The goal of this study is to begin to elucidate the mechanism by which COUP-TF acts as a transactivator of transcription. We first identify the minimal region of the molecule (amino acids 184–423) that confers the transactivation function of COUP-TFI. This is distinct from the previously described repression domain of COUP-TFI (
      • Leng X.
      • Cooney S.Y.
      • Tsai S.Y.
      • Tsai M.-J.
      ). The activation domain mediates gAF1 and gAF3 accessory activity of the glucocorticoid response of the PEPCK gene, is responsible for transactivation through the CYP7A COUP-TFII binding site, and enables COUP-TFI to function as a coactivator for HNF-4. GRIP1 and SRC-1 potentiate the transactivation function of COUP-TFI. However, in a system wherein COUP-TF is repressive, these coactivators cannot convert COUP-TFI into an activator. GRIP1 and SRC-1 interact with COUP-TFI in vivo, and these interactions require the intact activation domain defined in the function experiments.

      DISCUSSION

      COUP-TF, extensively studied as a repressor of transcription, also activates transcription by 1) binding to a nuclear receptor DNA response element and directly activating gene expression, as in the CYP7A gene promoter (
      • Stroup D.
      • Crestani M.
      • Chiang J.Y.L.
      ), 2) binding to a DNA element and indirectly influencing expression in the context of several other transcription factors, as in the PEPCK gene GRU (
      • Scott D.K.
      • Mitchell J.A.
      • Granner D.K.
      ,
      • Hall R.K.
      • Sladek F.M.
      • Granner D.K.
      ), and 3) by forming a protein-protein interaction with a DNA-bound factor, such as with HNF-4 in the HNF-1α gene promoter (
      • Ktistaki E.
      • Talianidis I.
      ). We show here by analyzing a number of deletion mutations that span the COUP-TFI molecule that a broad region located between amino acids 184–423 is required for these three activation functions. Furthermore, this same broad region is required for the interaction of COUP-TFI with the coactivators SRC-1 and GRIP1. It seems likely that amino acids scattered throughout this segment form the surface(s) required for direct transactivation and for the interaction with other factors. Although all three functions are equally disrupted by these mutations, we cannot conclude that precisely the same domains are involved in each.
      It is perhaps not surprising that the entire E/F domain, also referred to as the ligand binding domain, is required for activation since, in other members of the nuclear receptor superfamily, this domain must be intact for transactivation (
      • Darimont B.D.
      • Wagner R.L.
      • Apriletti J.W.
      • Stallcup M.R.
      • Kushner P.J.
      • Baxter J.D.
      • Fletterick R.J.
      • Yamamoto K.R.
      ). In addition to transactivation, this domain provides other functions, including nuclear localization and interactions with various coactivators (
      • Moras D.
      • Gronemeyer H.
      ). Although COUP is an orphan receptor that does not bind a known ligand, the other functions of the ligand binding domain may have been compromised by the different mutations that were made in the E/F domain. The coactivators GRIP1 and SRC-1 interact with the activation function 2 (AF2) domain near the C terminus of many nuclear receptors (
      • Darimont B.D.
      • Wagner R.L.
      • Apriletti J.W.
      • Stallcup M.R.
      • Kushner P.J.
      • Baxter J.D.
      • Fletterick R.J.
      • Yamamoto K.R.
      ). It is noteworthy that the C-terminal 15 amino acids of COUP-TFI that are required for transactivation are not within the domain of COUP-TFI that is homologous with the AF2 domain.
      There is significant overlap in the amino acid segments of COUP·TF1 that are involved in the opposing actions of transcription repression and activation. The C-terminal 15 amino acid segment of the protein, which is dispensable for repression (see Fig. 8 and Ref.
      • Leng X.
      • Cooney S.Y.
      • Tsai S.Y.
      • Tsai M.-J.
      ), is essential for the activation functions. On the other hand, a deletion of the C-terminal 35 amino acids, which are apparently required for transactivation, also renders COUP-TFI incapable of serving as a repressor. The corepressors, nuclear receptor corepressor and SMRT, bind to this 35-amino acid segment and confer the repression activity of COUP-TFI (
      • Shibata H.
      • Nawaz Z.
      • Tsai S.Y.
      • O'Malley B.W.
      • Tsai M.-J.
      ). It is not clear whether other segments of the molecule are also involved in repression. By contrast, the E/F domains of COUP-TFII, including the C-terminal 15 amino acids, are required for active repression (
      • Achatz G.
      • Speckmayer R.
      • Hauser C.
      • Sandhofer F.
      • Paulweber B.
      ). We have not studied COUP-TFII as a transactivator, but the accumulated evidence suggests that some very subtle differences between the two COUP molecules account for quite different biologic responses.
      Repression and transactivation are presumably facilitated through protein-protein interactions that occur on the E/F region of COUP-TFI. Given that both activation and repression require overlapping sequences, there must be a mechanism in place that allows different sets of proteins to interact with COUP-TFI. By this view, either a coactivator complex or a corepressor complex is bound to the activation or repression domains within the ligand binding domain of COUP-TFI. It is possible that an equilibrium exists between these two complexes so that an excess of one complex or the other determines the direction of the response. This explanation seems unlikely for several reasons. First, if an equilibrium between coactivator and the corepressor complexes determines the direction of the COUP-TFI response, then at a particular time in a given cell all the COUP-TFI binding sites should mediate either a positive or a negative response. This is obviously not the case (e.g. compare Figs. 5 and 8). Furthermore, we show here that overexpression of SRC-1 and GRIP1 fails to switch COUP-TFI from a repressor to an activator in at least one specific instance.
      The local intranuclear environment may determine the direction of COUP-TFI activity. For example, a given DNA sequence can function as an allosteric effector and influence the activity of a bound transcription factor (
      • Lefstin J.A.
      • Yamamoto K.R.
      ). Indeed, Cooney et al. (
      • Cooney A.J.
      • Tsai S.Y.
      • O'Malley B.W.
      • Tsai M.-J.
      ) demonstrate that COUP-TFI exists in different conformations when bound to different DNA binding sites. Hence, a specific DNA sequence may put COUP-TFI into a conformation that exposes surfaces for binding to either coactivator or corepressor complexes. This plasticity would allow the COUP-TFI binding site from the CYP7A gene promoter, which was used in the present study, to function as a positive element, whereas another DR4 element, with a slightly different sequence, could serve as a negative element when it binds COUP-TFI (
      • Cooney A.J.
      • Leng X.
      • Tsai S.Y.
      • O'Malley B.W.
      • Tsai M.-J.
      ).
      Protein-protein interactions in the context of a specific promoter may also be an important determinant of the direction of COUP-TFI activity. Whether COUP-TFI acts as a coactivator for HNF-4 or as a transrepressor for nuclear hormone receptors such as retinoic acid receptor is determined by protein-protein interactions, which allow COUP-TFI to associate with either SRC-1/GRIP1 or nuclear receptor corepressor/SMRT. The interaction of COUP-TFI with HNF-4 is of interest in this regard. The domain required for the HNF-4 coactivator activity of COUP-TFI is also required for its interaction with SRC-1 and GRIP1 in vivo (Figs. 5 and 7). It is possible that COUP-TFI interacts with the E region of HNF-4 (amino acids 227–271) and thus provides a surface, either directly or indirectly through coactivators, that associates with the basal transcription machinery (
      • Ktistaki E.
      • Talianidis I.
      ). Interestingly, this coactivator activity of COUP-TFI is only seen in DNA sequences that are recognized by HNF-4 but not by COUP-TFI (
      • Ktistaki E.
      • Talianidis I.
      ). Thus, COUP-TFI may only serve as a coactivator when HNF-4 exists in a specific conformation induced by DNA binding. Thus, although GAL4· COUP-TFI acts as a repressor in the context of the (GAL4)5E1bCAT reporter, it acts as a positive accessory factor in the context of the pGAL4gAF1 reporter (compare Figs. 2 and 8). Thus, the action of COUP-TFI is probably determined by the combinatorial effects of the DNA sequence to which it binds and by specific interactions with other proteins, as influenced by specific promoter contexts.
      COUP-TF is an important component of the PEPCK gene GRU. In addition to glucocorticoids, PEPCK gene transcription is regulated by a number of other hormones. Each hormone response is mediated by a set ofcis-elements termed hormone response units. Many of the elements within one hormone response unit are also components of another. In addition, a number of these pleiotropic elements bind different sets of protein complexes. For example, the gAF1 and gAF3 components of the GRU bind COUP-TF (gAF1 can also bind HNF-4) but are also retinoic acid response elements 1 and 2. In this case, these two elements bind retinoic acid receptor/retinoid X receptor heterodimers and together comprise the retinoic acid response unit. It is curious that, although very different combinations of proteins bind, the same DNA contact points are used (
      • Lucas P.C.
      • O'Brien R.M.
      • Mitchell J.A.
      • Davis C.M.
      • Imai E.
      • Forman B.M.
      • Samuels H.H.
      • Granner D.K.
      ,
      • Scott D.K.
      • Mitchell J.A.
      • Granner D.K.
      ). Similarly, the insulin response unit and the cAMP response unit share elements with the GRU, and again the proteins that bind to these elements are specific for the hormone response (
      • O'Brien R.M.
      • Lucas P.C.
      • Forest C.D.
      • Magnuson M.A.
      • Granner D.K.
      ,
      • Liu J.
      • Park E.A.
      • Gurney A.L.
      • Roesler W.J.
      • Hanson R.W.
      ). This overlapping structure of the PEPCK promoter is termed a metabolic control domain, and we hypothesize that this structure allows for a complex, integrated response of the PEPCK gene, which encodes a protein essential for gluconeogenesis, to a wide variety of environmental signals (
      • Scott D.K.
      • Mitchell J.A.
      • Granner D.K.
      ,
      • Sugiyama T.
      • Scott D.K.
      • Wang J.-C.
      • Granner D.K.
      ).
      Our current view is that different hormone response signals result in the recruitment of different sets of proteins to multiple elements within the promoter, which presumably tether different combinatorial sets of coactivators to the promoter (
      • Sugiyama T.
      • Scott D.K.
      • Wang J.-C.
      • Granner D.K.
      ). For example, the glucocorticoid response requires the recruitment of the ligand-bound GR and associated accessory factors and coactivators to the promoter. The retinoic acid response requires the recruitment of retinoic acid receptor/retinoid X receptor and associated coactivators, presumably SRC-1-like coactivators. These processes are, however, exclusive. For example, in the case of the retinoic acid response, the GR and its coactivators are not recruited; hence, the set of proteins bound to the PEPCK promoter during a response to retinoic acid is different from the complexes recruited during a glucocorticoid response. Indeed, the structural requirements of these responses is different, since a 5-base pair insertion between gAF2 and GR1, which effectively rotates the helix by one-half turn, decreases the glucocorticoid response but has no effect on the retinoic acid response (
      • Sugiyama T.
      • Scott D.K.
      • Wang J.-C.
      • Granner D.K.
      ).
      The binding (or absence of binding) of AFs to gAF1, gAF2, and gAF3 has no effect on basal activity (
      • Scott D.K.
      • Mitchell J.A.
      • Granner D.K.
      ,
      • Hall R.K.
      • Sladek F.M.
      • Granner D.K.
      ,
      • Wang J.-C.
      • Stromstedt P.-E.
      • O'Brien R.M.
      • Granner D.K.
      ,
      • Sugiyama T.
      • Scott D.K.
      • Wang J.-C.
      • Granner D.K.
      ), so it was of great interest to determine whether other proteins interact with COUP-TF, HNF-4, and HNF-3 to confer accessory activity to the glucocorticoid response, particularly since these transcription factors are specifically required to confer accessory factor activity from their particular location within the promoter. We have recently demonstrated that HNF-4 binds to both SRC-1 and GRIP1 and that these proteins act as coactivators for HNF-4-mediated transactivation in heterologous promoters and as co-accessory factors for the glucocorticoid response in the PEPCK gene promoter (
      • Wang J.-C.
      • Stafford J.
      • Granner D.K.
      ). Here we show that the same coactivators bind to COUP-TFI and potentiate activation by COUP-TF. Hall et al. (
      • Hall R.K.
      • Sladek F.M.
      • Granner D.K.
      ) demonstrate that either HNF-4 or COUP-TF can act as an accessory factor through the gAF1 element. This observation may now be explained by the observations that both COUP-TF and HNF-4 utilize the same coactivators to promote their transactivation activities (this study and Ref.
      • Wang J.-C.
      • Stafford J.
      • Granner D.K.
      ).
      In summary, we have characterized the domain required for COUP-TF-mediated transcriptional activation in three different systems and have determined that SRC-1 and GRIP1 bind to this domain and serve as coactivators. This takes us a step closer to understanding how the complex PEPCK gene GRU is assembled and how it functions.

      Acknowledgments

      We thank Cathy Caldwell for her excellent technical assistance, Deborah Caplenor Brown for preparation of the manuscript, and Kazuya Yamada for his invaluable advice.

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