Abstract
Chaperone synthesis in response to proteotoxic stress is dependent on a family of transcription factors named heat shock factors (HSFs). The two main factors in this family, HSF1 and HSF2, are co-expressed in numerous tissues where they can interact and form heterotrimers in response to proteasome inhibition. HSF1 and HSF2 exhibit two alternative splicing isoforms, called α and β, which contribute to additional complexity in HSF transcriptional regulation, but remain poorly examined in the literature. In this work, we studied the transcriptional activity of HSF1 and HSF2 splicing isoforms transfected into immortalized Mouse Embryonic Fibroblasts (iMEFs) deleted for both Hsf1 and Hsf2, under normal conditions and after proteasome inhibition. We found that HSF1α is significantly more active than the β isoform after exposure to the proteasome inhibitor MG132. Furthermore, we clearly established that, while HSF2 had no transcriptional activity by itself, short β isoform of HSF2 exerts a negative role on HSF1β-dependent transactivation. To further assess the impact of HSF2β inhibition on HSF1 activity, we developed a mathematical modelling approach which revealed that the balance between each HSF isoform in the cell regulated the strength of the transcriptional response. Moreover, we found that cellular stress such as proteasome inhibition could regulate the splicing of Hsf2 mRNA. All together, our results suggest that relative amounts of each HSF1 and HSF2 isoforms quantitatively determine the cellular level of the proteotoxic stress response.
Highlights
Proteasome is a major protein complex responsible for regulated degradation of intracellular proteins, and its activity is modified in many disorders
To verify that the difference observed in transactivation abilities was not due to a defect in the level of expression of the transcription factors, immunoblots were performed with extracts from Hsf1.22/2 immortalized Mouse Embryonic Fibroblasts (iMEFs) that had been co-transfected with an increased quantity of pCR3.1-HSF1a or pCR3.1-HSF1b, in addition to pEGFP used as a transfection efficiency control (Figure 1B)
HSF1a protein became detectable when 3.125 ng of vector were transfected per 1000 cells, whereas HSF1b was detected after transfection of a lower amount of vector (0.78 ng/1000 cells), suggesting that the b isoform was more stable than the a isoform, but less effective from a transcriptional point of view
Summary
Proteasome is a major protein complex responsible for regulated degradation of intracellular proteins, and its activity is modified in many disorders. We have shown that proteasome subunit expression was regulated by heat shock factors [2]. Heat shock factor 1 (HSF1) and heat shock factor 2 (HSF2) belong to the family of transcription factors, which are essential for the expression of heat shock proteins (Hsps) in response to protein insults. Proteasome inhibition, but not heat shock, activates HSF2 [4]. It was shown that after treatment with MG132, a classical proteasome inhibitor, HSF1 and HSF2 can form heterotrimers and bind to DNA [5]. The exact role of such heterotrimers is not yet fully understood, but it was proposed that HSF2 could act as a modulator of HSF1 activity [6,7]
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