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Three Amino Acid Substitutions Selectively Disrupt the Activation but Not the Repression Function of the Glucocorticoid Receptor N Terminus*

  • Jorge A. Iñiguez-Lluhí
    Footnotes
    Affiliations
    From the Departments of Cellular and Molecular Pharmacology, and Biochemistry and Biophysics, University of California, San Francisco, California 94143-0450
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  • David Y. Lou
    Affiliations
    From the Departments of Cellular and Molecular Pharmacology, and Biochemistry and Biophysics, University of California, San Francisco, California 94143-0450
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  • Keith R. Yamamoto
    Correspondence
    To whom correspondence should be addressed.
    Affiliations
    From the Departments of Cellular and Molecular Pharmacology, and Biochemistry and Biophysics, University of California, San Francisco, California 94143-0450
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  • Author Footnotes
    * This work was supported by the National Institutes of Health. 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.
    Fellow of the Jane Coffin Childs Memorial Fund for Medical Research.
Open AccessPublished:February 14, 1997DOI:https://doi.org/10.1074/jbc.272.7.4149
      A 210-amino acid region, termed enh2, near the N terminus of the rat glucocorticoid receptor, is necessary for both transcriptional activation and repression. The mechanism(s) of transcriptional regulation conferred by this region, however, are poorly understood. We screened in Saccharomyces cerevisiae a library of random mutants in the enh2 region of a constitutive glucocorticoid receptor derivative and isolated a series of multiply substituted receptors that are specifically defective in transcriptional activation. Although many substitutions in this area were tolerated, three amino acid substitutions (E219K, F220L, and W234R) within a 16-amino acid region were sufficient to disrupt the enh2 transcriptional activation function both in yeast and in mammalian cells. Although this region is rich in acidic residues, the conserved tryptophan at position 234 appears to be a more critical feature for enh2 activity; hydrophobic but not charged residues were tolerated at this position. Notably, the mutants uncoupled the activation and repression functions of enh2, as the activation defective isolates remained competent for repression of AP-1 at the composite response element plfG.

      INTRODUCTION

      In animal cells, the effects of steroid hormones on the genome are mediated by members of the intracellular receptor superfamily, a vast collection of proteins endowed with the capacity to regulate the transcription of an equally diverse array of target genes during development and in response to specific physiological and pathological cues (
      • Mangelsdorf D.J.
      • Thummel C.
      • Beato M.
      • Herrlich P.
      • SchÜtz G.
      • Umesono K.
      • Blumberg B.
      • Kastner P.
      • Mark M.
      • Chambon P.
      • Evans R.M.
      ,
      • Tsai M.-J.
      • O'Malley B.W.
      ). The glucocorticoid receptor (GR) is responsible for the effects of glucocorticoids and constitutes a prototype for this family of transcriptional regulators (
      • Yamamoto K.R.
      ,
      • Beato M.
      • Herrlich P.
      • SchÜtz G.
      ). Upon ligand binding to its C-terminal region, GR is recruited to gene enhancers and promoters via a central zinc-binding region that is capable of recognizing specific DNA sequences termed glucocorticoid response elements (GREs). Once in the vicinity of a promoter, the receptor can mediate either stimulatory or inhibitory influences on transcription (
      • Yamamoto K.R.
      • Pearce D.
      • Thomas J.
      • Miner J.N.
      • McKnight S.L.
      • Yamamoto K.R.
      Transcriptional Regulation.
      ,
      • Diamond M.I.
      • Miner J.N.
      • Yoshinaga S.K.
      • Yamamoto K.R.
      ,
      • Jonat C.
      • Rahmsdorf H.J.
      • Herrlich P.
      • Park K.-K.
      • Cato A.C.B.
      • Gebel S.
      • Ponta H.
      • Herrlich P.
      ). The direction of the effect appears to be dictated by the nature of the response element recognized by the receptor and by interactions with other sequence-specific transcription factors (
      • Miner J.N.
      • Yamamoto K.R.
      ,
      • Starr D.B.
      • Matsui W.
      • Thomas J.R.
      • Yamamoto K.R.
      ,
      • Lefstin J.A.
      • Thomas J.R.
      • Yamamoto K.R.
      ).
      In contrast to the relatively high amino acid sequence conservation in the zinc-binding and ligand-binding domains, the N-terminal regions of intracellular receptors are divergent both in size and sequence (
      • Segraves W.A.
      ,
      • Detera-Wadleigh S.D.
      • Fanning T.G.
      ). This implies that the N-terminal regions may contribute strongly to the class specificity of otherwise closely related receptors. Deletion of the C-terminal ligand-binding regions of steroid receptors yields constitutive (hormone-independent) transcriptional activators, implying that the N-terminal regions harbor autonomous transcriptional activation functions (
      • Godowski P.J.
      • Rusconi S.
      • Miesfeld R.
      • Yamamoto K.R.
      ). Insertion and deletion analysis (
      • Hollenberg S.M.
      • Evans R.M.
      ,
      • Giguère V.
      • Hollenberg S.M.
      • Rosenfeld M.G.
      • Evans R.M.
      ) as well as fusions to heterologous DNA-binding domains (
      • Godowski P.J.
      • Picard D.
      • Yamamoto K.R.
      ) circumscribed the activation function (termed enh2 or τ-1) to a region flanked by amino acids 108 and 317 in the rat GR. A prevalence of negatively charged residues, together with cross synergy and interference assays, led to the classification of enh2 as a so-called “acidic activation domain” (
      • Tasset D.
      • Tora L.
      • Fromental C.
      • Scheer E.
      • Chambon P.
      ). As with other activators, however, the features of this region essential for transcriptional activation have been difficult to define (
      • Godowski P.J.
      • Picard D.
      • Yamamoto K.R.
      ,
      • Miesfeld R.
      • Sakai D.
      • Inoue A.
      • Schena M.
      • Godowski P.J.
      • Yamamoto K.R.
      • Ringold G.
      Steroid Hormone Action, UCLA Symposium on Molecular and Cellular Biology.
      ,
      • Miesfeld R.
      • Godowski P.J.
      • Maler B.A.
      • Yamamoto K.R.
      ).
      In addition to its transcriptional stimulation activity, GR has the potential to inhibit transcription driven by other activators, such as AP-1 (
      • Diamond M.I.
      • Miner J.N.
      • Yoshinaga S.K.
      • Yamamoto K.R.
      ,
      • Jonat C.
      • Rahmsdorf H.J.
      • Herrlich P.
      • Park K.-K.
      • Cato A.C.B.
      • Gebel S.
      • Ponta H.
      • Herrlich P.
      ,
      • KÖnig H.
      • Ponta H.
      • Rahmsdorf H.J.
      • Herrlich P.
      ,
      • Heck S.
      • Kullmann M.
      • Gast A.
      • Ponta H.
      • Rahmsdorf H.J.
      • Herrlich P.
      • Cato A.C.
      ). For example, at plfG, a composite GRE from the proliferin gene, transcriptional activation by the cJun-cFos heterodimeric form of AP-1 is repressed by the hormone-bound GR (
      • Yamamoto K.R.
      • Pearce D.
      • Thomas J.
      • Miner J.N.
      • McKnight S.L.
      • Yamamoto K.R.
      Transcriptional Regulation.
      ,
      • Diamond M.I.
      • Miner J.N.
      • Yoshinaga S.K.
      • Yamamoto K.R.
      ,
      • Miner J.N.
      • Yamamoto K.R.
      ). Exploiting the inability of the closely related mineralocorticoid receptor to repress in this context, Pearce and Yamamoto (
      • Pearce D.
      • Yamamoto K.R.
      ) generated receptor chimeras that demonstrated a requirement for the N-terminal region of GR for repression from plfG. Thus, a single region of GR harbors determinants for transcriptional activation and repression.
      The mechanisms by which GR achieves activation or repression are unknown. It has been suggested, however, that both chromatin-dependent and -independent mechanisms of activation are at work. That is, transcriptional activation by GR expressed in Saccharomyces cerevisiae requires the Swi1, Swi2, and Swi3 proteins (
      • Yoshinaga S.K.
      • Peterson C.L.
      • Herskowitz I.
      • Yamamoto K.R.
      ), part of a multiprotein Swi/Snf complex that may be involved in chromatin remodeling (
      • Kingston R.E.
      • Bunker C.A.
      • Imbalzano A.N.
      ). Similarly, in human cells, GR activity is potentiated by a mammalian Swi2 homolog (
      • Muchardt C.
      • Yaniv M.
      ). Moreover, a screen for genomic mutations causing loss of GR function in yeast yielded swp73, an additional member the Swi/Snf complex (
      • Cairns B.R.
      • Levinson R.S.
      • Yamamoto K.R.
      • Kornberg R.D.
      ). On the other hand, in vitro studies with “naked” DNA templates suggest that the enh2 region of GR may stimulate transcription by a mechanism independent of chromatin (
      • Freedman L.P.
      • Yoshinaga S.K.
      • Vanderbilt J.N.
      • Yamamoto K.R.
      ). Studies with various activators suggest that activation may involve interactions with components of the basal transcription machinery either directly or via accessory factors or coactivators (
      • Tjian R.
      • Maniatis T.
      ,
      • Zawel L.
      • Reinberg D.
      ,
      • Guarente L.
      ). Biochemical- and interaction-based assays have yielded several molecules that physically interact with particular steroid receptors, including GR (
      • Hong H.
      • Kohli K.
      • Trivedi A.
      • Johnson D.L.
      • Stallcup M.R.
      ,
      • Cavailles V.
      • Dauvois S.
      • L'Horset F.
      • Lopez G.
      • Hoare S.
      • Kushner P.J.
      • Parker M.G.
      ,
      • Baniahmad C.
      • Nawaz Z.
      • Baniahmad A.
      • Gleeson M.A.
      • Tsai M.J.
      • O'Malley B.W.
      ,
      • Onate S.A.
      • Tsai S.Y.
      • Tsai M.J.
      • O'Malley B.W.
      ,
      • Eggert M.
      • Mows C.C.
      • Tripier D.
      • Arnold R.
      • Michel J.
      • Nickel J.
      • Schmidt S.
      • Beato M.
      • Renkawitz R.
      ,
      • Folkers G.E.
      • van der Saag P.T.
      ,
      • Lee J.W.
      • Ryan F.
      • Swaffield J.C.
      • Johnston S.A.
      • Moore D.D.
      ,
      • Lee J.W.
      • Choi H.S.
      • Gyuris J.
      • Brent R.
      • Moore D.D.
      ,
      • vom Baur E.
      • Zechel C.
      • Heery D.
      • Heine M.J.S.
      • Garnier J.M.
      • Vivat V.
      • Le Douarin B.
      • Gronemeyer H.
      • Chambon P.
      • Losson R.
      ,
      • Zeiner M.
      • Gehring U.
      ,
      • LeDouarin B.
      • Zechel C.
      • Garnier J.M.
      • Lutz Y.
      • Tora L.
      • Pierrat P.
      • Heery D.
      • Gronemeyer H.
      • Chambon P.
      • Losson R.
      ). Most of those factors interact with the receptor C-terminal region, which appear also to carry transcriptional regulatory activities. The functional significance and role in transcriptional activation for most of these interactions, however, remain largely unknown.
      Efforts to elucidate the transcriptional regulatory mechanisms of the N-terminal region of GR would be aided by point mutants that distinguish activation from repression “surfaces,” and that could be used to test the functional significance of physical interactions with potential targets. One approach to the characterization of activation domains has been to mutate frequently represented amino acid residues, such as glutamine, proline, or those with acidic side chains. This strategy, however, may fail to identify residues important for function (
      • Courey A.J.
      • Tjian R.
      ,
      • Gill G.
      • Pascal E.
      • Tseng Z.H.
      • Tjian R.
      ,
      • Triezenberg S.J.
      ). In this report, we describe a genetic approach in S. cerevisiae in which we screened a large set of rat GR derivatives carrying multiple substitutions in enh2 for mutants that are specifically defective in transcriptional activation.

      Acknowledgments

      We thank D. B. Starr and J. Lefstin for providing plasmids, M. D. Krstic and C. Jamieson for the communication of results, and the members of the Yamamoto laboratory for discussion and assistance. We also appreciate helpful comments on the manuscript by B. Darimont, R. Grosschedl, I. Herskowitz, E. O'Shea, D. B. Starr, R. Tjian, and M. d. M. Vivanco-Ruiz.

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