Molecular mechanism of the regulation of the mammalian heat shock transcription factor Hsf1

• Applicant: Professor Dr. Matthias Peter Mayer
• Subject Area: Biochemistry ; Biophysics ; Structural Biology
• Term: since 2021
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 468811147

The heat shock response is a universal, cell-autonomous transcriptional program. It is induced by an imbalance of protein homeostasis and orchestrated in all eukaryotic cells by heat shock transcription factor Hsf1. Through this function Hsf1 is in metazoa at center stage of many physiological and pathophysiological processes like post-embryonic development and aging, cancer and neurodegeneration. The activity of Hsf1 is controlled by intrinsic stress-induced conformational changes, by a large number of post-translational modifications, and by molecular chaperones and other interacting proteins. Hsf1 exerts its function by releasing RNA polymerase II that is paused at the transcription start site in heat shock genes. Despite over 40 years of intense research, the mechanism of Hsf1 activity regulation at a molecular level remains poorly understood. Metazoan Hsf1 consists of a DNA binding domain, leucine zipper-like heptad-repeat regions A and B (HR-A/B) functioning as trimerization domain, a regulatory domain, a third heptad repeat region (HR-C), repressing trimerization, and a transactivation domain. In a previously DFG funded project we demonstrated that Hsf1 is a thermosensor, the response of which is proportional to the severity and the length of the heat stress. Using purified proteins, we demonstrated that trimeric Hsf1 is dissociated from DNA in vitro by Hsc70 and DnaJB1 by monomerization of Hsf1. Hsc70 monomerizes Hsf1 trimers by successive cycles of entropic pulling, unzipping the three-stranded leucine-zipper.In this project we have four aims. (1) We want to gain insights into the structure of monomeric, dimeric and oligomeric Hsf1 using x-ray crystallography and cryo-electron microscopy. (2) We want to develop Hsf1 activity sensors based on bioluminescence resonance energy transfer (BRET). Such sensors could be used for screening for drugs that inhibit Hsf1 activation or Hsc70-mediated Hsf1 inactivation. (3) We want to analyse the modulation of the core Hsf1 activation-attenuation cycle by posttranslational modifications in the trimerization domain and adjacent to the Hsc70 binding site that is essential for Hsf1 monomerization. (4) We want to study the interaction of the reported Hsf1 activator and inhibitor HSBP1 and SSBP1 with Hsf1 by biochemical assays and hydrogen exchange mass spectrometry to get a molecular understanding of their activity. With aims (3) and (4) we want to elucidate how the core activation-attenuation cycle of Hsf1 activity integrates multiple input signals and is tuned to the needs of the cell.

• DFG Programme: Research Grants