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Specific Interaction between Tomato HsfA1 and HsfA2 Creates Hetero-oligomeric Superactivator Complexes for Synergistic Activation of Heat Stress Gene Expression*

  • Kwan Yu Chan-Schaminet
    Footnotes
    Affiliations
    From the Department of Molecular Cell Biology, Goethe University, Max-von-Laue-Strasse 9, D-60438 Frankfurt am Main, Germany
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  • Sanjeev K. Baniwal
    Footnotes
    Affiliations
    From the Department of Molecular Cell Biology, Goethe University, Max-von-Laue-Strasse 9, D-60438 Frankfurt am Main, Germany
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  • Daniela Bublak
    Affiliations
    From the Department of Molecular Cell Biology, Goethe University, Max-von-Laue-Strasse 9, D-60438 Frankfurt am Main, Germany
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  • Lutz Nover
    Affiliations
    From the Department of Molecular Cell Biology, Goethe University, Max-von-Laue-Strasse 9, D-60438 Frankfurt am Main, Germany
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  • Klaus-Dieter Scharf
    Correspondence
    To whom correspondence should be addressed: Goethe University, Molecular Cell Biology, Max-von-Laue-Str. 9/N200/R3.05, D-60438 Frankfurt/M., Germany. Tel.: 0049-69-798-29283; Fax: 0049-69-798-29286;
    Affiliations
    From the Department of Molecular Cell Biology, Goethe University, Max-von-Laue-Strasse 9, D-60438 Frankfurt am Main, Germany
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  • Author Footnotes
    * This work was supported by the Deutsche Forschungsgemeinschaft (to L. N. (No 249/4) and K. D. S. (Scha 577/6)).
    The on-line version of this article (available at http://www.jbc.org) contains supplemental Tables S1 and S2 and Figs. S1–S4.
    1 Present address: Charité-Universitätsmedizin Berlin, Institut für Biochemie, Monbijoustr. 2, 10117 Berlin, Germany.
    2 Present address: University of Southern California, Inst. for Genetic Medicine, 2250 Alcazar St., Los Angeles, CA 90033.
Open AccessPublished:June 01, 2009DOI:https://doi.org/10.1074/jbc.M109.007336
      In plants, a family of more than 20 heat stress transcription factors (Hsf) controls the expression of heat stress (hs) genes. There is increasing evidence for the functional diversification between individual members of the Hsf family fulfilling distinct roles in response to various environmental stress conditions and developmental signals. In response to hs, accumulation of both heat stress proteins (Hsp) and Hsfs is induced. In tomato, the physical interaction between the constitutively expressed HsfA1 and the hs-inducible HsfA2 results in synergistic transcriptional activation (superactivation) of hs gene expression. Here, we show that the interaction is strikingly specific and not observed with other class A Hsfs. Hetero-oligomerization of the two-component Hsfs is preferred to homo-oligomerization, and each Hsf in the HsfA1/HsfA2 hetero-oligomeric complex has its characteristic contribution to its function as superactivator. Distinct regions of the oligomerization domain are responsible for specific homo- and hetero-oligomeric interactions leading to the formation of hexameric complexes. The results are summarized in a model of assembly and function of HsfA1/A2 superactivator complexes in hs gene regulation.
      Heat stress transcription factors (Hsfs)
      The abbreviations used are: Hsf
      heat stress transcription factor
      Hsps
      heat stress proteins
      hs
      heat stress
      AHA
      activator motifs: peptide motifs consisting of aromatic, large hydrophobic and acidic amino acid residues
      GFP
      green fluorescent protein
      OD
      oligomerization domain
      DBD
      DNA binding domain
      CTAD
      C-terminal activator domain
      SEC
      size exclusion chromatography
      NLS
      nuclear localization signal
      NES
      nuclear export signal
      HR
      heptad repeat
      wt
      wild type.
      4The abbreviations used are: Hsf
      heat stress transcription factor
      Hsps
      heat stress proteins
      hs
      heat stress
      AHA
      activator motifs: peptide motifs consisting of aromatic, large hydrophobic and acidic amino acid residues
      GFP
      green fluorescent protein
      OD
      oligomerization domain
      DBD
      DNA binding domain
      CTAD
      C-terminal activator domain
      SEC
      size exclusion chromatography
      NLS
      nuclear localization signal
      NES
      nuclear export signal
      HR
      heptad repeat
      wt
      wild type.
      are the terminal components of signal transduction chains mediating the activation of genes responsive to heat stress (hs) and a large number of other environmental or chemical stressors (
      • Morimoto R.I.
      ,
      • Schöffl F.
      • Prändl R.
      • Reindl A.
      ,
      • Baniwal S.K.
      • Bharti K.
      • Chan K.Y.
      • Fauth M.
      • Ganguli A.
      • Kotak S.
      • Mishra S.K.
      • Nover L.
      • Port M.
      • Scharf K.D.
      • Tripp J.
      • Weber C.
      • Zielinski D.
      • von Koskull-Döring P.
      ,
      • von Koskull-Döring P.
      • Scharf K.D.
      • Nover L.
      ). Besides their central role in response to stress-related stimuli, Hsfs are also involved in the regulation of cell growth and survival under normal physiological and developmental conditions (for review see Refs.
      • von Koskull-Döring P.
      • Scharf K.D.
      • Nover L.
      ,
      • Akerfelt M.
      • Trouillet D.
      • Mezger V.
      • Sistonen L.
      ,
      • Morimoto R.I.
      ). Remarkably, the Hsf-controlled stress response system is conserved throughout the eukaryotic kingdom. Hsfs are maintained in an inactive state under normal growth conditions controlled by interaction with molecular chaperones, but they are rapidly activated under stress conditions (
      • Morimoto R.I.
      ,
      • Schöffl F.
      • Prändl R.
      • Reindl A.
      ,
      • Wu C.
      ,
      • Nakai A.
      ,
      • Nover L.
      • Bharti K.
      • Döring P.
      • Mishra S.K.
      • Ganguli A.
      • Scharf K.D.
      ,
      • Voellmy R.
      • Boellmann F.
      ). As a result, heat stress proteins (Hsps) are synthesized, many of which act as molecular chaperones protecting proteins against stress damage or assisting in their folding, intracellular distribution, and degradation (
      • Young J.C.
      • Agashe V.R.
      • Siegers K.
      • Hartl F.U.
      ,
      • Bukau B.
      • Weissman J.
      • Horwich A.
      ,
      • Millar A.H.
      • Whelan J.
      • Small I.
      ,
      • Han J.H.
      • Batey S.
      • Nickson A.A.
      • Teichmann S.A.
      • Clarke J.
      ,
      • Horwich A.L.
      • Fenton W.A.
      • Chapman E.
      • Farr G.W.
      ,
      • Oreb M.
      • Tews I.
      • Schleiff E.
      ).
      Heat stress-inducible genes in eukaryotes share conserved promoter elements (heat shock elements, HSE) with the consensus motif (AGAAn)(nTTCT) (
      • Pelham H.R.
      • Bienz M.
      ,
      ). Similar to other transcriptional activators, Hsfs have a modular structure (
      • von Koskull-Döring P.
      • Scharf K.D.
      • Nover L.
      ,
      • Pirkkala L.
      • Nykänen P.
      • Sistonen L.
      ) with an N-terminal conserved DNA binding domain with a central helix-turn-helix motif and a domain with extended heptad repeats of hydrophobic amino acid residues (HR-A/B region) required for oligomerization. The C-terminal domains of Hsfs are more divergent, and many of them include localization signals for nuclear import (NLS) (
      • Lyck R.
      • Harmening U.
      • Höhfeld I.
      • Scharf K.D.
      • Nover L.
      ) and export (NES) (
      • Heerklotz D.
      • Döring P.
      • Bonzelius F.
      • Winkelhaus S.
      • Nover L.
      ) as well as short peptide motifs with aromatic, large hydrophobic, and acidic amino acid residues (AHA motifs) for the activator function (
      • Treuter E.
      • Nover L.
      • Ohme K.
      • Scharf K.D.
      ,
      • Kotak S.
      • Port M.
      • Ganguli A.
      • Bicker F.
      • von Koskull-Döring P.
      ).
      In yeast, Drosophila and nematodes Hsfs are encoded by unique genes, whereas multiple Hsfs exist in vertebrates. Hsf1, one of 3–4 Hsfs identified in vertebrates has been shown to function as ubiquitous regulator of the stress response, while the other members are assumed to have tissue- or development-specific functions (
      • Morimoto R.I.
      ,
      • Pirkkala L.
      • Nykänen P.
      • Sistonen L.
      ).
      In contrast to all other organisms investigated so far, plants possess an extraordinarily complex Hsf family both in terms of total number of representatives (usually more than 20) as well as of their structural and functional diversification. Three classes of plant Hsfs (classes A, B, and C) are defined by peculiarities of their HR-A/B regions (
      • Nover L.
      • Bharti K.
      • Döring P.
      • Mishra S.K.
      • Ganguli A.
      • Scharf K.D.
      ). Evidently, the Hsf mixture changes in a tissue-specific manner and in response to stress treatments (see reviews in Refs.
      • Baniwal S.K.
      • Bharti K.
      • Chan K.Y.
      • Fauth M.
      • Ganguli A.
      • Kotak S.
      • Mishra S.K.
      • Nover L.
      • Port M.
      • Scharf K.D.
      • Tripp J.
      • Weber C.
      • Zielinski D.
      • von Koskull-Döring P.
      ,
      • von Koskull-Döring P.
      • Scharf K.D.
      • Nover L.
      ,
      • Miller G.
      • Mittler R.
      ).
      Remarkably, despite many similarities, our current knowledge indicates plant-specific variations of the Hsf network. (i) In tomato, HsfA1 functions as master regulator of the hs response (
      • Mishra S.K.
      • Tripp J.
      • Winkelhaus S.
      • Tschiersch B.
      • Theres K.
      • Nover L.
      • Scharf K.D.
      ), whereas in Arabidopsis such a master regulator function could not be identified so far. Even the simultaneous knock-down of two HsfA1 isogenes (Hsfs A1a and A1b) had no marked effect on the hs response, and only a subset of hs genes was affected (
      • Lohmann C.
      • Eggers-Schumacher G.
      • Wunderlich M.
      • Schöffl F.
      ). (ii) In contrast to the situation in Arabidopsis, tomato HsfA1 operates upstream of hs-induced expression of chaperones and HsfA2. Hence, HsfA2 accumulates during repeated cycles of heat stress and recovery and becomes the dominant Hsf in thermotolerant cells. Its activity is further controlled by a network of proteins involving HsfA1 and chaperones of the sHsp family influencing its solubility, intracellular localization, and activator function (
      • Heerklotz D.
      • Döring P.
      • Bonzelius F.
      • Winkelhaus S.
      • Nover L.
      ,
      • Scharf K.D.
      • Heider H.
      • Höhfeld I.
      • Lyck R.
      • Schmidt E.
      • Nover L.
      ,
      • Port M.
      • Tripp J.
      • Zielinski D.
      • Weber C.
      • Heerklotz D.
      • Winkelhaus S.
      • Bublak D.
      • Scharf K.D.
      ). Arabidopsis HsfA2 is also hs-inducible and highly expressed after heat stress treatment, but it is different in its physicochemical properties when compared with tomato HsfA2. The analysis of HsfA2 overexpression and knock-out plants of Arabidopsis indicate that HsfA2 functions as enhancer of hs gene expression with specific activity on a subset of Hsf-dependent genes (
      • Nishizawa A.
      • Yabuta Y.
      • Yoshida E.
      • Maruta T.
      • Yoshimura K.
      • Shigeoka S.
      ,
      • Schramm F.
      • Ganguli A.
      • Kiehlmann E.
      • Englich G.
      • Walch D.
      • von Koskull-Döring P.
      ,
      • Charng Y.Y.
      • Liu H.C.
      • Liu N.Y.
      • Chi W.T.
      • Wang C.N.
      • Chang S.H.
      • Wang T.T.
      ).
      Plant Hsfs interact with each other physically and/or functionally with the result of mutual enhancement or repression of their activities (
      • Heerklotz D.
      • Döring P.
      • Bonzelius F.
      • Winkelhaus S.
      • Nover L.
      ,
      • Scharf K.D.
      • Heider H.
      • Höhfeld I.
      • Lyck R.
      • Schmidt E.
      • Nover L.
      ,
      • Bharti K.
      • von Koskull-Döring P.
      • Bharti S.
      • Kumar P.
      • Tintschl-Körbitzer A.
      • Treuter E.
      • Nover L.
      ,
      • Baniwal S.K.
      • Chan K.Y.
      • Scharf K.D.
      • Nover L.
      ). Although only a few examples of Hsf cooperation have been investigated in greater detail, remarkable differences in the underlying molecular mechanisms are apparent. The coactivator function of tomato HsfB1 depends on a histone-like motif in its C-terminal domain required for recruitment of the histone acetyltransferase-like protein HAC1, a plant ortholog of the p300/CBP (CREB-binding protein) family. Formation of ternary complexes of HsfA1, HsfB1, and HAC1 with enhanced DNA binding activities does not require physical interaction between the two Hsfs (
      • Bharti K.
      • von Koskull-Döring P.
      • Bharti S.
      • Kumar P.
      • Tintschl-Körbitzer A.
      • Treuter E.
      • Nover L.
      ). In contrast, cooperation between class A Hsfs depends on their oligomerization domains and the formation of hetero-oligomeric complexes. However, despite a similar basic structure of the HR-A/B regions (Fig. 1), interactions between class A Hsfs are highly selective. The interaction of HsfA1 and HsfA2 leads to nuclear retention of HsfA2 and formation of superactivator complexes with enhanced transcriptional activities (
      • Heerklotz D.
      • Döring P.
      • Bonzelius F.
      • Winkelhaus S.
      • Nover L.
      ,
      • Scharf K.D.
      • Heider H.
      • Höhfeld I.
      • Lyck R.
      • Schmidt E.
      • Nover L.
      ). Interestingly, the opposite functional consequence was observed for the HsfA4b/HsfA5 pair. By binding to HsfA4b, HsfA5 impairs DNA binding and consequently the activator function of Hsf4b (
      • Baniwal S.K.
      • Chan K.Y.
      • Scharf K.D.
      • Nover L.
      ).
      Figure thumbnail gr1
      FIGURE 1Domain structure of tomato heat stress transcription factors. Numbers indicate amino acid residues. OD, oligomerization domain corresponding to the hydrophobic heptad repeat region (HR-A/B). The term HsfA1 is used synonymously throughout this report and corresponds to the master regulator HsfA1a in Lycopersicon esculentum (
      • Baniwal S.K.
      • Bharti K.
      • Chan K.Y.
      • Fauth M.
      • Ganguli A.
      • Kotak S.
      • Mishra S.K.
      • Nover L.
      • Port M.
      • Scharf K.D.
      • Tripp J.
      • Weber C.
      • Zielinski D.
      • von Koskull-Döring P.
      ,
      • Mishra S.K.
      • Tripp J.
      • Winkelhaus S.
      • Tschiersch B.
      • Theres K.
      • Nover L.
      • Scharf K.D.
      ). The patterns of AHA motifs are Hsf-specific (
      • Baniwal S.K.
      • Chan K.Y.
      • Scharf K.D.
      • Nover L.
      ,
      • Bharti K.
      • Schmidt E.
      • Lyck R.
      • Heerklotz D.
      • Bublak D.
      • Scharf K.D.
      ,
      • Döring P.
      • Treuter E.
      • Kistner C.
      • Lyck R.
      • Chen A.
      • Nover L.
      ).
      Here we challenge the current model of hetero-oligomerization dependence of the synergistic action of tomato HsfA1 and HsfA2. We demonstrate that multimeric complexes can be formed in the absence of DNA and that transcriptional activity strongly depends on hetero-complex formation. Besides nuclear retention of HsfA2, the combination of specific functional properties of both Hsfs in A1/A2 hetero-complexes is essential for the superactivator function.

      Acknowledgments

      We thank Enrico Schleiff for helpful discussions and support during preparation of the manuscript and Markus Bohnsack for critical proofreading during revision.

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