Abstract
Heat shock factor 1 (HSF1) initiates a broad transcriptional response to proteotoxic stress while also mediating a cancer-specific transcriptional program. HSF1 is thought to be regulated by molecular chaperones, including Heat Shock Protein 90 (HSP90). HSP90 is proposed to sequester HSF1 in unstressed cells, but visualization of this interaction in vivo requires protein crosslinking. In this report, we show that HSP90 binding to HSF1 depends on HSP90 conformation and is only readily visualized for the ATP-dependent, N-domain dimerized chaperone, a conformation only rarely sampled by mammalian HSP90. We have used this mutationally fixed conformation to map HSP90 binding sites on HSF1. Further, we show that ATP-competitive, N-domain targeted HSP90 inhibitors disrupt this interaction, resulting in the increased duration of HSF1 occupancy of the hsp70 promoter and significant prolongation of both the constitutive and heat-induced HSF1 transcriptional activity. While our data do not support a role for HSP90 in sequestering HSF1 monomers to suppress HSF1 transcriptional activity, our findings do identify a noncanonical role for HSP90 in providing dynamic modulation of HSF1 activity by participating in removal of HSF1 trimers from heat shock elements in DNA, thus terminating the heat shock response.
Highlights
Heat shock factor 1 (HSF1) is an evolutionarily conserved transcription factor that initiates the cytoprotective heat shock response (HSR)
We assessed the importance of HSP90 conformation for binding to HSF1 and we found that only the ATP-dependent “closed” (e.g., N-domain dimerized) conformational mutants E47A (HSP90α) and E42A (HSP90β) readily co-precipitate full-length HSF1, with HSP90α displaying the stronger interaction
To identify chaperone binding sites in HSF1, we used a series of Flag-tagged HSF1 truncation and internal deletion mutants to interrogate binding of HSP90 and HSP70, including the binding of transiently expressed HSP90αE47A or HSP90β E42A
Summary
Heat shock factor 1 (HSF1) is an evolutionarily conserved transcription factor that initiates the cytoprotective heat shock response (HSR). Through an incompletely defined mechanism, mammalian HSF1 monomers in cytosol are activated and form trimers, translocate into the nucleus, and bind sequences of DNA known as heat shock elements (HSE), ideally represented as nGAAnnTTCnnGAAn2,3. Throughout this process HSF1 is heavily post-translationally modified and interacts with numerous cellular components. The RD contains numerous phosphorylation sites[23] and functions, along with a portion of HR-A/B, to repress the transcriptional activity of NF-IL624 Another heptad repeat (HR-C), C-terminal to the RD, is understood to sequester HR-A/B in an intramolecular interaction that suppresses spontaneous HSF1 trimerization[21]. HSP90 has variously been proposed to function as a sensor, repressor and/or attenuator of the HSR
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