Potential targets for HSF1 within the preinitiation complex.
Protein-protein interactions between human heat shock transcription factor 1 (hHSF1) and general transcription factors TFIIA-gamma, TFIIB, TBP, TAF(II)32, and TAF(II)55 and positive coactivator PC4 were characterized in order to identify potential targets of contact in the transcriptional preinitiation complex. These contacts represent one of the final steps in the signal transfer of heat stress to the transcriptional apparatus. TATA-binding protein (TBP) and transcription factor IIB (TFIIB) were identified as major targets for HSF1 transcriptional activation domains AD1 and AD2 based on in vitro interaction assays. TBP showed affinity for AD2 and a fragment containing AD1, while the core domain of TFIIB interacted primarily with the AD1 fragment. Interactions were also detected between full-length HSF1 and the small subunit (gamma) of TFIIA. PC4 interacted weakly with HSF2 and showed even less affinity for HSF1. Coimmunoprecipitation of transiently expressed TBP in HeLa cells demonstrated that HSF1 AD2 and AD1+AD2 are able to bind TBP in vivo. Assays based on transcriptional interference confirmed predictions that both TBP and TFIIB can interact with HSF1 activation domains in HeLa cells. The negative regulatory region (NR) of HSF1 did not interact with any general factors tested in vitro but did bind TFIID in nuclear extracts through contacts that probably involve TATA associated proteins (TAFs). These results suggest a model for transcriptional regulation by HSF1 that involves a shift between formation of dysfunctional TFIID complexes with the NR and transcriptionally competent complexes with the C-terminal activation domains.
Yuan
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Department of Microbiology and Cell Science, Program in Plant Molecular and Cellular Biology, University of Florida, Gainesville 32611-0700, USA.
Gurley
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WB
D015153:Blotting, Western; D002467:Cell Nucleus / Q000378:metabolism; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D017930:Genes, Reporter; D006367:HeLa Cells; D000076249:Heat Shock Transcription Factors; D006360:Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D018869:Heat-Shock Response; D006801:Humans; D017874:Immediate-Early Proteins; D008565:Membrane Proteins; D008957:Models, Genetic; D011233:Precipitin Tests; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism; D035181:TATA-Box Binding Protein; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism; D035561:Transcription Factor TFIIA; D035581:Transcription Factor TFIIB; D035362:Transcription Factor TFIID; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D019473:Transcription Factors, TFII / Q000378:metabolism; D015533:Transcriptional Activation / Q000235:genetics*; D014162:Transfection
D004268:DNA-Binding Proteins; C000619706:HSF1 protein, human; D000076249:Heat Shock Transcription Factors; D006360:Heat-Shock Proteins; C490043:IFRD2 protein, human; D017874:Immediate-Early Proteins; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D035181:TATA-Box Binding Protein; D015534:Trans-Activators; D035561:Transcription Factor TFIIA; D035581:Transcription Factor TFIIB; D035362:Transcription Factor TFIID; D014157:Transcription Factors; D019473:Transcription Factors, TFII; D004247:DNA