Anti-HSF2 monoclonal antibody, clone TQN513

Recently, many studies have demonstrated the induction of stress proteins in the mammalian nervous system under various pathological conditions. These altered genetic programs may function to protect individual cells against stressful conditions. However, little is known about the molecular mechanisms regulating these stress responses in animals. We report here the activation of a heat shock factor (HSF) in the rat brain during cerebral ischemia or after heat shock. Gel mobility shift assays revealed an increase in DNA binding activity to the heat shock element (HSE) during the early phases of ischemia. Supershift experiments using specific antisera against HSF1 and HSF2 showed that the ischemia-induced HSE-binding activity was mainly due to HSF1. In the heat-shocked brain, HSF1 was also activated, and the HSE-binding activity was higher in the cerebellum than in the cerebral cortex or hippocampus; Western blot analysis also showed that HSF1 was more abundant in the cerebellum than in the other two brain regions. Our results indicate that heat shock gene transcription is regulated by the activation of HSF1 in both cerebral ischemia and heat shock, and that different brain regions display differential sensitivities in their stress response. The cellular signals for heat shock gene transcription under in vivo pathological conditions will also be discussed.

Protein-protein interactions between human heat shock transcription factor 1 (hHSF1) and general transcription factors TF \\ A-gamma, TF \\ B, TBP, TAF(\\)32, and TAF(\\)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 \\ B (TF \\ B) 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 TF \\ B interacted primarily with the AD1 fragment. Interactions were also detected between full-length HSF1 and the small subunit (gamma) of TF \\ A. 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 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 proteins (TAFs). These results suggest a model for transcriptional regulation by HSF1 that involves a shift between formation of dysfunctional TF \\ D complexes with the NR and transcriptionally competent complexes with the C-terminal activation domains.