Potential targets for HSF1 within the preinitiation complex

Protein-protein interactions between human heat shock transcription factor 1 (hHSF1) and general transcription factors TF \\ A-gamma, TF \\ B, TBP, TA F(\\)32, and TAF(\\)55 and positive coactivator PC4 were characterized in o rder to identify potential targets of contact in the transcriptional preini tiation complex. These contacts represent one of the final steps in the sig nal transfer of heat stress to the transcriptional apparatus. TATA-binding protein (TBP) and transcription factor \\ B (TF \\ B) were identified as ma jor targets for HSF1 transcriptional activation domains AD1 and AD2 based o n in vitro interaction assays. TBP showed affinity for AD2 and a fragment c ontaining AD1, while the core domain of TF \\ B interacted primarily with t he AD1 fragment. Interactions were also detected between full-length HSF1 a nd the small subunit (gamma) of TF \\ A. PC4 interacted weakly with HSF2 an d showed even less affinity for HSF1. Coimmunoprecipitation of transiently expressed TBP in HeLa cells demonstrated that HSF1 AD2 and AD1 + AD2 are ab le to bind TBP in vivo. Assays based on transcriptional interference confir med predictions that both TBP and TF \\ B 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 TF \\ D in nuclear extracts through contacts that probably involve TATA associated pro teins (TAFs). These results suggest a model for transcriptional regulation by HSF1 that involves a shift between formation of dysfunctional TF \\ D co mplexes with the NR and transcriptionally competent complexes with the C-te rminal activation domains.