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Excess protein synthesis, denatured proteins or molecular crowding spontaneously induce HSF1 trimerisation and activation

ABSTRACT: Stress responses are a key feature of normal physiology and are usurped by cancer cells to ensure enhanced protein synthesis for growth, to compensate for genomic instability, and to protect cancer cells from therapy induced stress. Heat shock factor-1 (HSF1) is a major stress-response transcription factor, and its activity is markedly enhanced in cancer. Stress induces HSF1 conformational changes and post-translational modifications, leading to assembly of active HSF1 trimers that bind DNA to control target gene expression. Although the function of HSF1 in transcription is relatively well-known, the mechanisms leading to HSF1 activation and enhanced stress response in tumours are unclear. To investigate whether HSF1 is a primary sensor of proteotoxic stress in vivo, we studied the range of conditions that can cause HSF1 activation in vitro and in cells, and conditions that prevent its activation. We show that purified recombinant HSF1 adopts a stable monomeric conformation in vitro. Heat stress caused a conformational change and the assembly of HSF1 trimers. Conditions leading to protein denaturation, including heat stress, crowding, Hsp90 inhibition, or proteasome inhibition, all directly lead to HSF1 activation. In contrast, HSF1 activation in vivo is prevented by proteosynthesis inhibition, which reduces the amount of denatured proteins in the cell. These results establish that HSF1 is a direct sensor of proteotoxic stress, independent of post-translational modification, where abrupt environmental changes that cause protein denaturation simultaneously induce a conformational change in monomeric HSF1 leading to its activation. This mechanism explains the universal ability of cells to respond to proteotoxic stress and trigger a protective response when increased chaperone activities are required to restore homeostasis.

INSTRUMENT(S): LTQ Orbitrap Elite

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): B Cell

DISEASE(S): Acute Leukemia

SUBMITTER: Lenka Hernychova

LAB HEAD: MUDr. Petr Muller, Ph.D.

PROVIDER: PXD037662 | Pride | 2024-08-14

REPOSITORIES: Pride

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Project description:Heat shock response (HSR) is critical for survival of organisms undergoing proteotoxic stress. Heat shock factor 1 (HSF1) is widely believed to be the master regulator of this response. Here, we examined the kinetics of the transcriptional response and HSF1 binding changes genome-wide after heat shock (HS) with high sensitivity and high spatial and temporal resolution using PRO-seq and ChIP-seq assays respectively in wild type and in hsf1-deletion mouse embryonic fibroblasts. The transcriptional response is rapid, dynamic, and extensive with activation of several hundred genes and repression of several thousands of genes. Although HSF1 is critically required for classical inducible Heat Shock Protein (HSP) genes, it is not required for the majority of gene activation. HSF1 acts mechanistically to promote RNA polymerase II (Pol II) release from the promoter-proximal pause, while recruitment and initiation of Pol II are predominantly HSF1-independent. Surprisingly, a major class of cytoskeleton genes is immediately (within 2.5 minutes) and transiently activated in an HSF1-independent manner. A second wave of regulation is characterized by robust activation or repression of new sets of genes. The broad repression of thousands of genes, a quarter of which are repressed immediately upon HS and the rest are repressed in the second wave of regulation, is mediated at the level of decreasing Pol II pause release and not at prior steps of Pol II recruitment or initiation. Notably, heat shock does not induce transcription of some previously defined HSP genes, and HSF2 â a homolog of HSF1 â does not compensate for the lack of HSF1. Together, these findings indicate that mammalian cells cope with stress by rapidly inducing pervasive and dramatic changes in transcription that are largely modulated at the step of pause release, and only a fraction of this regulation is HSF1-dependent. Examination of transcriptional regulation before and after heat shock in WT, HSF1-/-, and HSF1&2-/- MEFs

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Excess protein synthesis, denatured proteins or molecular crowding spontaneously induce HSF1 trimerisation and activation

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