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Abstract

The heat shock transcription factor 1 is the main regulator of the mammalian heat shock response. The synthesis of molecular chaperones, required for proper folding, refolding and disaggregation of proteins is dependent on the activation of HSF1 from an inactive monomer into a DNA-bound trimer. The mechanism of trimerization of HSF1 was extensively studied, and recent progress led to speculate over a model in which the trimerization is dependent on temperature and concentration. Also, the existence of an equilibrium between active and inactive HSF1 under non-stress conditions was proposed, explaining the expression of target genes of HSF1 in the absence of cellular stress. The attenuation phase of the heat shock response was proposed to be regulated through a negative feed-back mechanism in which the molecular chaperone complex Hsp70/Hsp40 represses the transcription activity of trimeric HSF1. The mechanism of this attenuation as well as the nature of the cochaperones interacting with HSF1 during that phase of the heat shock response are still unknown. The aim of this work was to identify new interaction partners of HSF1 during the attenuation phase, to characterize these interactors, and to establish their role in the heat shock cycle. The identification part was performed by generating a stable human cell line constitutively in a state close to the attenuation phase. This was achieved by overexpressing a tagged version of HSF1 in these cells, significantly increasing its concentration in the cytoplasm, leading to a concentration-dependent trimerization in the absence of stress. Moreover, this cell line was shown to be resistant to mild heat shock, indicating that these cells have an increased pool of molecular chaperones. Immunoprecipitation and shotgun proteomics identified Hsp70, Hsc70, Bag2 and Bag4 as main interaction partners of HSF1. The two first proteins of the list have been known for decades to interact with the transcription factor during the attenuation phase, but the Hsp70 nucleotide exchange factor Bag2 and Bag4 were interesting new potential partners, as these interactions were confirmed to be direct, and not only through the Hsp70 chaperone. Full-length Bag2 and the Bag domain of Bag4 were purified, and their activity as nucleotide exchange factor was investigated. Surprisingly, the Bag domain of Bag4 proved to be much more efficient than the full length Bag2, and both proteins were able to stimulate the ADP-release of Hsc70 better than of Hsp70. Both purified proteins were also able to support refolding of heat-denaturated luciferase in the presence of Hsc70 and the J-domain protein Hdj1. In this experiment, differences between Bag2 and the Bag domain of Bag4 were small. 9 Finally, the roles of these two nucleotide exchange factors were investigated in vivo. The overexpression of both full-length Bag2 and Bag 4 proteins in U2-OS cells led to a strong activation of heat shock gene transcription, even in the absence of cellular stress. The repression of HSF1 by the Hsp70/Hsp40 complex is most probably short-lived, as the overexpression of the two Bag proteins stimulates the release of trimeric HSF1, hence allowing the transcription of heat shock genes and the subsequent production of molecular chaperones to continue. Fragments of Bag2 and Bag4, when overexpressed, could not stimulate the heat shock response, hinting at the fact that both Bag domain and N-terminal fragment are necessary for this effect. Taken together, these results are an additional indication of the existence of an equilibrium between active and inactive HSF1, equilibrium which is displaced towards active trimeric HSF1 in the case of cellular stress.