Abstract
The heat shock response is a universal transcriptional response to proteotoxic stress orchestrated by heat shock transcription factor Hsf1 in all eukaryotic cells. Despite over 40 years of intense research, the mechanism of Hsf1 activity regulation remains poorly understood at the molecular level. In metazoa, Hsf1 trimerizes upon heat shock through a leucine‐zipper domain and binds to DNA. How Hsf1 is dislodged from DNA and monomerized remained enigmatic. Here, using purified proteins, we demonstrate that unmodified trimeric Hsf1 is dissociated from DNA in vitro by Hsc70 and DnaJB1. Hsc70 binds to multiple sites in Hsf1 with different affinities. Hsf1 trimers are monomerized by successive cycles of entropic pulling, unzipping the triple leucine‐zipper. Starting this unzipping at several protomers of the Hsf1 trimer results in faster monomerization. This process directly monitors the concentration of Hsc70 and DnaJB1. During heat shock adaptation, Hsc70 first binds to a high‐affinity site in the transactivation domain, leading to partial attenuation of the response, and subsequently, at higher concentrations, Hsc70 removes Hsf1 from DNA to restore the resting state.
Highlights
The heat shock response (HSR) is an ancient transcriptional program, evolved in all organisms to cope with a wide variety of environmental, physiological and developmental stressful conditions that induce an imbalance of protein homeostasis
We identify several binding sites for Hsc[70] within Hsf[1], one of which in the transactivation domain involved in initial attenuation, a second close to the trimerization domain essential for Hsc70mediated monomerization
The interaction of Hsf[1] with heat shock elements (HSE)-containing DNA was very stable and little decrease in fluorescence polarization was observed over 90 min (Fig. 1C) when only buffer, or excess unlabeled control DNA without HSEs was added
Summary
The heat shock response (HSR) is an ancient transcriptional program, evolved in all organisms to cope with a wide variety of environmental, physiological and developmental stressful conditions that induce an imbalance of protein homeostasis. Genetic evidence suggested an involvement of Hsp[90] in HSR regulation in yeast 25, more recent ex vivo data suggest that Hsp[70] is associated with Hsf[1] under non-stress conditions and this interaction is disrupted upon heat shock 26,27. Whether such a model can be adopted for mammalian cells is not clear since Hsf[1] is constitutively trimeric in yeast and does not rely on a monomer-trimer transition for activation 28. The overall degree of sequence identity between yeast and human Hsf[1] is just 17% and the proposed binding sites of Hsp[70] in yeast Hsf[1] are not conserved in human Hsf[1]
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