Heat shock transcription factor 1 (HSF1) mediates the induction of heat shock protein gene expression in cells exposed to elevated temperature and other stress conditions. In response to stress HSF1 acquires DNA binding ability and localizes to nuclear stress granules, but the molecular mechanisms that mediate these events are not understood. We report that HSF1 undergoes stress-induced modification at lysine 298 by the small ubiquitin-related protein called SUMO-1. Antibodies against SUMO-1 supershift the HSF1 DNA-binding complex, and modification of HSF1 in a reconstituted SUMO-1 reaction system causes conversion of HSF1 to the DNA-binding form. HSF1 colocalizes with SUMO-1 in nuclear stress granules, which is prevented by mutation of lysine 298. Mutation of lysine 298 also results in a significant decrease in stress-induced transcriptional activity of HSF1 in vivo. This work implicates SUMO-1 modification as an important modulator of HSF1 function in response to stress.

In contrast to most genomic DNA in mitotic cells, the promoter regions of some genes, such as the stress-inducible hsp70i gene that codes for a heat shock protein, remain uncompacted, a phenomenon called bookmarking. Here we show that hsp70i bookmarking is mediated by a transcription factor called HSF2, which binds this promoter in mitotic cells, recruits protein phosphatase 2A, and interacts with the CAP-G subunit of the condensin enzyme to promote efficient dephosphorylation and inactivation of condensin complexes in the vicinity, thereby preventing compaction at this site. Blocking HSF2-mediated bookmarking by HSF2 RNA interference decreases hsp70i induction and survival of stressed cells in the G ₁ phase, which demonstrates the biological importance of gene bookmarking.

Heat shock transcription factor 2 (HSF2) is a transcription factor that regulates heat shock protein gene expression, but the mechanisms regulating the function of this factor are unclear. Here we report that HSF2 is a substrate for modification by the ubiquitin-related protein SUMO-1 and that HSF2 colocalizes in cells with SUMO-1 in nuclear granules. Staining with anti-promyelocytic leukemia antibodies indicates that these HSF2-containing nuclear granules are PML bodies. Our results identify lysine 82 as the major site of SUMO-1 modification in HSF2, which is located in a “wing” within the DNA-binding domain of this protein. Interestingly, SUMO-1 modification of HSF2 results in conversion of this factor to the active DNA binding form. This is the first demonstration that SUMO-1 modification can directly alter the DNA binding ability of a transcription factor and reveals a new mechanism by which SUMO-1 modification can regulate protein function.

Heat shock factor 2 (HSF2) functions as a transcriptional regulator of heat shock protein gene expression in mammalian cells undergoing processes of differentiation and development. Our previous studies demonstrated high regulated expression and unusual constitutive DNA-binding activity of the HSF2 protein in mouse testes, suggesting that HSF2 functions to regulate heat shock protein gene expression in spermatogenic cells. The purpose of this study was to test whether HSF2 regulation in testes is associated with alterations in the HSF2 polypeptide expressed in testes relative to other mouse tissues. Our results show that mouse cells express not one but two distinct HSF2 proteins and that the levels of these HSF2 isoforms are regulated in a tissue-dependent manner. The testes express predominantly the 71-kDa HSF2-alpha isoform, while the heart and brain express primarily the 69-kDa HSF2-beta isoform. These isoforms are generated by alternative splicing of HSF2 pre-mRNA, which results in the inclusion of an 18-amino-acid coding sequence in the HSF2-alpha mRNA that is skipped in the HSF2-beta mRNA. HSF2 alternative splicing is also developmentally regulated, as our results reveal a switch in expression from the HSF2-beta mRNA isoform to the HSF2-alpha isoform during testis postnatal developmental. Transfection analysis shows that the HSF2-alpha protein, the predominant isoform expressed in testis cells, is a more potent transcriptional activator than the HSF2-beta isoform. These results reveal a new mechanism for the control of HSF2 function in mammalian cells, in which regulated alternative splicing is used to modulate HSF2 transcriptional activity in a tissue-dependent manner.

Consumption of a low-protein, high-carbohydrate diet induces a striking increase in circulating fibroblast growth factor-21 (FGF21), which is associated with improved cardiometabolic health and increased longevity. Increased lifespan during this dietary protein “dilution” has been explained by resource-mediated trade-offs between reproduction and survival, such that fecundity is optimized at a greater relative intake of proteins/carbohydrates. The magnitude of this trade-off is thought to be sex-dependent. In this study, we tested the hypothesis that metabolic responses to dietary protein dilution are likewise dependent on sex. We maintained age-matched adult male and female C57BL/6J mice on isocaloric diets containing 22% fat and differing in the ratio of protein/carbohydrate. The normal protein (NP) control diet contained 18% protein and 60% carbohydrate by kcal. The protein diluted (PD) diet contained 4% protein and 74% carbohydrate. Consistent with previous reports, PD males gained less weight and less fat than did normal protein controls and exhibited both improved glucose tolerance and decreased plasma lipids. In contrast, these metabolic benefits were absent among age-matched females maintained on the same diets. Likewise, whereas circulating FGF21 was increased up to 66-fold among PD male mice, this was substantially blunted among female counterparts. Sex differences in energy balance, glucose control, and plasma FGF21 were reversed upon ovariectomy. Collectively, our findings support that female mice are relatively less sensitive to the metabolic improvements observed following dietary protein dilution. This is accompanied by blunted circulating levels of FGF21 and requires an intact female reproductive system.