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Opposite Orientations of a Transcription Factor Heterodimer Bind DNA Cooperatively with Interaction Partners but Have Different Effects on Interferon-β Gene Transcription*

  • Veronica Burns
    Affiliations
    Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, Michigan 48109
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  • Tom Klaus Kerppola
    Correspondence
    To whom correspondence should be addressed: Department of Biological Chemistry, University of Michigan Medical School, 1150 W. Medical Center Dr., Ann Arbor, MI, 48109-0650. Tel.: 734-764-3554; Fax: 734-936-9353
    Affiliations
    Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, Michigan 48109
    Search for articles by this author
  • Author Footnotes
    * This work was supported, in whole or in part, by National Institutes of Health Grant R01-DA030339 (to T. K. K.) and by the University of Michigan Molecular Biophysics Training Grant (to V. E. B.).
    This article contains supplemental Figs. S1–S14 and Table S1.
Open AccessPublished:July 27, 2012DOI:https://doi.org/10.1074/jbc.M112.374462
      ATF2-Jun, IRF3, and HMGI recognize a composite regulatory element within the interferon-β enhancer (IFNb). Cooperative ATF2-Jun-IRF3 complex formation at IFNb has been proposed to require a fixed orientation of ATF2-Jun binding. Our results show that ATF2-Jun heterodimers bound IFNb in both orientations alone and in association with IRF3 and HMGI. Two sets of symmetrically located amino acid residues in ATF2 and Jun facilitated the interactions between heterodimers bound in opposite orientations and IRF3 at IFNb. IRF3 and HMGI bound IFNb in association with both orientations of ATF2-Jun heterodimers with the same cooperativity. ATF2-Jun heterodimers that bound IFNb in opposite orientations in vitro had different effects on interferon-β gene transcription when they were co-expressed with IRF3 in cultured cells. These heterodimers had different transcriptional activities at different endogenous genes. Different regions of ATF2 and Jun mediated their orientation-dependent transcriptional activities at different genes. These studies revealed that cooperative DNA binding does not require a unique nucleoprotein complex configuration, and that transcription factor complexes that bind the same enhancer in different configurations can have different transcriptional activities.

      Introduction

      Transcription initiation is controlled by multi-protein transcription factor complexes assembled at regulatory elements and at the site of transcription initiation. Cooperative DNA binding by transcription factors that bind to closely juxtaposed sequences (composite regulatory elements) are generally thought to require a specific arrangement of the transcription factors on DNA. Conversely, many mechanisms of transcription activation, including changes in chromatin structure and covalent protein modifications are not thought to require a specific arrangement of transcription factors on DNA.
      Some heterodimeric transcription factors can bind their recognition sequences in two opposite orientations (
      • Glover J.N.
      • Harrison S.C.
      Crystal structure of the heterodimeric bZIP transcription factor c-Fos-c-Jun bound to DNA.
      ,
      • Leonard D.A.
      • Kerppola T.K.
      DNA bending determines Fos-Jun heterodimer orientation.
      ). Interactions between such heterodimers and transcription factors that bind adjoining sequences are generally thought to require a specific orientation of heterodimer binding. For instance, cooperative DNA binding by Fos-Jun, Fos-Activating Transcription Factor 2 (ATF2),
      The abbreviations used are: ATF2
      Activating Transcription Factor 2
      Jun
      c-Jun
      IRF3
      Interferon Regulatory Factor 3
      bZIP
      basic-region leucine zipper
      gelFRET
      gel-based fluorescence resonance energy transfer.
      or ATF2-Jun heterodimers with Nuclear Factor of Activated T cells 1 (NFAT1) at composite regulatory elements imposes a fixed orientation of heterodimer binding (
      • Chen L.
      • Oakley M.G.
      • Glover J.N.
      • Jain J.
      • Dervan P.B.
      • Hogan P.G.
      • Rao A.
      • Verdine G.L.
      Only one of the two DNA-bound orientations of AP-1 found in solution cooperates with NFATp.
      ,
      • Chen L.
      • Glover J.N.
      • Hogan P.G.
      • Rao A.
      • Harrison S.C.
      Structure of the DNA-binding domains from NFAT, Fos and Jun bound specifically to DNA.
      ,
      • Diebold R.J.
      • Rajaram N.
      • Leonard D.A.
      • Kerppola T.K.
      Molecular basis of cooperative DNA bending and oriented heterodimer binding in the NFAT1-Fos-Jun-ARRE2 complex.
      ,
      • Ramirez-Carrozzi V.R.
      • Kerppola T.K.
      Control of the orientation of Fos-Jun binding and the transcriptional cooperativity of Fos-Jun-NFAT1 complexes.
      ,
      • Ramirez-Carrozzi V.R.
      • Kerppola T.K.
      Dynamics of Fos-Jun-NFAT1 complexes.
      ). Differences in the preferred orientation of Fos-Jun heterodimer binding at different regulatory elements affect cooperative DNA binding and synergistic transcription activation with NFAT1 (
      • Ramirez-Carrozzi V.R.
      • Kerppola T.K.
      Control of the orientation of Fos-Jun binding and the transcriptional cooperativity of Fos-Jun-NFAT1 complexes.
      ,
      • Ramirez-Carrozzi V.R.
      • Kerppola T.K.
      Dynamics of Fos-Jun-NFAT1 complexes.
      ,
      • Ramirez-Carrozzi V.
      • Kerppola T.
      Asymmetric recognition of nonconsensus AP-1 sites by Fos-Jun and Jun-Jun influences transcriptional cooperativity with NFAT1.
      ).
      The interferon-β enhancer contains closely juxtaposed recognition sequences for ATF2-Jun heterodimers, IRF3 and HMGI (supplemental Fig. S1). Studies using different experimental approaches, including photo-crosslinking, x-ray crystallography, and equilibrium binding assays have produced contradictory interpretations concerning the effects of IRF3 on the orientation of ATF2-Jun heterodimer binding, as well as concerning the cooperativity of complex formation (
      • Falvo J.V.
      • Parekh B.S.
      • Lin C.H.
      • Fraenkel E.
      • Maniatis T.
      Assembly of a functional β interferon enhanceosome is dependent on ATF-2-c-Jun heterodimer orientation.
      ,
      • Panne D.
      • Maniatis T.
      • Harrison S.C.
      Crystal structure of ATF-2/c-Jun and IRF-3 bound to the interferon-β enhancer.
      ,
      • Dragan A.I.
      • Carrillo R.
      • Gerasimova T.I.
      • Privalov P.L.
      Assembling the human IFN-β enhanceosome in solution.
      ). Photo-crosslinking and x-ray crystallography were interpreted to show opposite orientations of ATF2-Jun heterodimer binding in the presence of IRF3, whereas no orientation preference was detected using fluorescence polarization (
      • Falvo J.V.
      • Parekh B.S.
      • Lin C.H.
      • Fraenkel E.
      • Maniatis T.
      Assembly of a functional β interferon enhanceosome is dependent on ATF-2-c-Jun heterodimer orientation.
      ,
      • Panne D.
      • Maniatis T.
      • Harrison S.C.
      Crystal structure of ATF-2/c-Jun and IRF-3 bound to the interferon-β enhancer.
      ,
      • Dragan A.I.
      • Carrillo R.
      • Gerasimova T.I.
      • Privalov P.L.
      Assembling the human IFN-β enhanceosome in solution.
      ). The results from x-ray crystallography and mutational analyses were interpreted to demonstrate that the cooperativity of ATF2-Jun-IRF3 binding was mediated by a change in DNA structure with no requirement for direct contacts between ATF2-Jun and IRF3 (
      • Panne D.
      • Maniatis T.
      • Harrison S.C.
      Crystal structure of ATF-2/c-Jun and IRF-3 bound to the interferon-β enhancer.
      ). In contrast, no binding cooperativity was detected by isothermal titration calorimetry (
      • Dragan A.I.
      • Carrillo R.
      • Gerasimova T.I.
      • Privalov P.L.
      Assembling the human IFN-β enhanceosome in solution.
      ). The effects of HMGI on the configuration and cooperativity of ATF2-Jun and IRF3 binding at the interferon-β enhancer have not been established.
      The mechanisms of transcription activation by the complex assembled at the interferon-β enhancer are incompletely understood. Some of these mechanisms have been proposed to involve covalent protein modifications, whereas others invoke interactions between the complex assembled at the enhancer and the transcription initiation complex at the promoter (
      • Agalioti T.
      • Lomvardas S.
      • Parekh B.
      • Yie J.
      • Maniatis T.
      • Thanos D.
      Ordered recruitment of chromatin modifying and general transcription factors to the IFN-β promoter.
      ,
      • Kim T.K.
      • Kim T.H.
      • Maniatis T.
      Efficient recruitment of TFIIB and CBP-RNA polymerase II holoenzyme by an interferon-β enhanceosome in vitro.
      ,
      • Kim T.K.
      • Maniatis T.
      The mechanism of transcriptional synergy of an in vitro assembled interferon-β enhanceosome.
      ,
      • Munshi N.
      • Agalioti T.
      • Lomvardas S.
      • Merika M.
      • Chen G.
      • Thanos D.
      Coordination of a transcriptional switch by HMGI(Y) acetylation.
      ). Many of these are thought to be mediated by co-factors that do not directly bind to the enhancer (
      • Agalioti T.
      • Lomvardas S.
      • Parekh B.
      • Yie J.
      • Maniatis T.
      • Thanos D.
      Ordered recruitment of chromatin modifying and general transcription factors to the IFN-β promoter.
      ,
      • Kim T.K.
      • Kim T.H.
      • Maniatis T.
      Efficient recruitment of TFIIB and CBP-RNA polymerase II holoenzyme by an interferon-β enhanceosome in vitro.
      ,
      • Munshi N.
      • Agalioti T.
      • Lomvardas S.
      • Merika M.
      • Chen G.
      • Thanos D.
      Coordination of a transcriptional switch by HMGI(Y) acetylation.
      ). A majority of previous studies investigating the mechanisms that regulate interferon-β transcription have been performed using in vitro assays or extrachromosomal reporter genes (
      • Agalioti T.
      • Lomvardas S.
      • Parekh B.
      • Yie J.
      • Maniatis T.
      • Thanos D.
      Ordered recruitment of chromatin modifying and general transcription factors to the IFN-β promoter.
      ,
      • Kim T.K.
      • Kim T.H.
      • Maniatis T.
      Efficient recruitment of TFIIB and CBP-RNA polymerase II holoenzyme by an interferon-β enhanceosome in vitro.
      ,
      • Kim T.K.
      • Maniatis T.
      The mechanism of transcriptional synergy of an in vitro assembled interferon-β enhanceosome.
      ). Studies of endogenous gene regulation are necessary to establish the mechanisms that regulate genes in their normal context.
      We investigated the effects of interactions with IRF3 and HMGI on the orientation of ATF2-Jun heterodimer binding at the interferon-β enhancer and the effects of heterodimer orientation on the cooperativity of complex formation in vitro and on endogenous interferon-β gene transcription in cells. Our results demonstrated that the ATF2-Jun-IRF3-HMGI complex assembled at the interferon-β enhancer in two different configurations that had distinct transcriptional activities.

      DISCUSSION

      The gelFRET analysis of the configurations of bATF2-bJun-iIRF3 and bATF2-bJun-iIRF3-HMGI complexes challenges the conventional view that multi-protein complexes bind composite regulatory elements in a unique and fixed configuration. It is generally recognized that the conformations of nucleoprotein complexes undergo thermal fluctuations. However, these conformations are often assumed to be distributed around a single configuration, and this “average” configuration is thought to be representative of the entire population.
      The discovery that the bATF2-bJun-iIRF3 and bATF2-bJun-iIRF3-HMGI complexes bind the interferon-β enhancer in two stable configurations extends previous observations that heterodimeric transcription factors can bind pseudo-symmetrical recognition sequences in two opposite orientations (
      • Glover J.N.
      • Harrison S.C.
      Crystal structure of the heterodimeric bZIP transcription factor c-Fos-c-Jun bound to DNA.
      ,
      • Leonard D.A.
      • Kerppola T.K.
      DNA bending determines Fos-Jun heterodimer orientation.
      ). The alternative configurations of these nucleoprotein complexes involve large-scale rearrangements of the protein-protein and protein-nucleic acid interfaces in the complex. The large differences between the structures of complexes in which heterodimers bind in opposite orientations predict that they have distinct functions.
      Two independent lines of investigation corroborate the interpretation that bATF2-bJun-iIRF3-HMGI complexes assemble in two distinct configurations at the interferon-β enhancer. First, bATF2-bJun heterodimers bound the interferon-β enhancer in two opposite orientations in association with iIRF3 alone or with iIRF3 and HMGI in the gelFRET assay. Second, iIRF3 and HMGI bound the interferon-β enhancer with same cooperativity in association with bATF2-bJun variants that favor opposite orientations of binding. The two configurations of bATF2-bJun-iIRF3-HMGI complexes that can bind cooperatively to the interferon-β enhancer represent a new mode of cooperative DNA binding at a composite regulatory element that does not require a fixed configuration of protein interactions.
      The phenomenon of dual contact interfaces on opposite faces of heterodimers may be widespread since the residues that mediate protein interactions are often conserved among different members of a protein family (supplemental Fig. S14). We propose to designate interfaces that enable alternative configurations of protein interactions “Janus” interfaces as they provide multiple faces for engagement with interaction partners.
      Different configurations of the same transcription factors can have distinct transcriptional activities at different composite regulatory elements (
      • Schräder M.
      • Müller K.M.
      • Nayeri S.
      • Kahlen J.P.
      • Carlberg C.
      Vitamin D3-thyroid hormone receptor heterodimer polarity directs ligand sensitivity of transactivation.
      ,
      • Scully K.M.
      • Jacobson E.M.
      • Jepsen K.
      • Lunyak V.
      • Viadiu H.
      • Carrière C.
      • Rose D.W.
      • Hooshmand F.
      • Aggarwal A.K.
      • Rosenfeld M.G.
      Allosteric effects of Pit-1 DNA sites on long-term repression in cell type specification.
      ,
      • Tomilin A.
      • Reményi A.
      • Lins K.
      • Bak H.
      • Leidel S.
      • Vriend G.
      • Wilmanns M.
      • Schöler H.R.
      Synergism with the coactivator OBF-1 (OCA-B, BOB-1) is mediated by a specific POU dimer configuration.
      ). Our results extend this principle by demonstrating that transcription factor variants that bind in different configurations can have distinct transcriptional activities at the same composite regulatory element. The mechanisms whereby the orientation of ATF2-Jun heterodimer binding affected interferon-β transcription are likely to reflect orientation-dependent interactions with other factors at the interferon-β gene.
      The amino acid substitutions in ATF2 and Jun that affected the orientation of heterodimer binding at IFNb had opposite effects on the transcription of different endogenous genes. These differences could be due to opposite effects of the substitutions on the orientations of heterodimer binding at different genes, or they could indicate that the mechanisms whereby heterodimer orientation affects transcription vary among different genes. The observation that different regions of ATF2 and Jun determined the effect of the orientation of heterodimer binding on transcription at different genes is consistent with the latter interpretation.
      The consequences of the multiple configurations of transcription factor binding at the interferon-β enhancer and their distinct transcriptional activities for cellular and organismal phenotypes remain unknown. Different cellular processes require either unidirectional or bidirectional action. Some functions long thought to be unidirectional, such as promoter-directed gene transcription, have been recently found to be bidirectional (
      • Core L.J.
      • Waterfall J.J.
      • Lis J.T.
      Nascent RNA sequencing reveals widespread pausing and divergent initiation at human promoters.
      ,
      • Seila A.C.
      • Calabrese J.M.
      • Levine S.S.
      • Yeo G.W.
      • Rahl P.B.
      • Flynn R.A.
      • Young R.A.
      • Sharp P.A.
      Divergent transcription from active promoters.
      ). The structural basis for this bidirectional transcription remains to be established, but could be rooted in the bidirectional binding of transcription regulatory proteins as well as the TATA box-binding protein at their recognition sites (
      • Cox J.M.
      • Hayward M.M.
      • Sanchez J.F.
      • Gegnas L.D.
      • van der Zee S.
      • Dennis J.H.
      • Sigler P.B.
      • Schepartz A.
      Bidirectional binding of the TATA box binding protein to the TATA box.
      ).

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