HSF1 is required for cellular adaptation to daily temperature fluctuations.

The heat shock response (HSR) is a universal mechanism of cellular adaptation to elevated temperatures and is regulated by heat shock transcription factor 1 (HSF1) or HSF3 in vertebrate endotherms, such as humans, mice, and chickens. We here showed that HSF1 and HSF3 from egg-laying mammals (monotremes), with a low homeothermic capacity, equally possess a potential to maximally induce the HSR, whereas either HSF1 or HSF3 from birds have this potential. Therefore, we focused on cellular adaptation to daily temperature fluctuations and found that HSF1 was required for the proliferation and survival of human cells under daily temperature fluctuations. The ectopic expression of vertebrate HSF1 proteins, but not HSF3 proteins, restored the resistance in HSF1-null cells, regardless of the induction of heat shock proteins. This function was associated with the up-regulation of specific HSF1-target genes. These results indicate the distinct role of HSF1 in adaptation to thermally fluctuating environments and suggest association of homeothermic capacity with functional diversification of vertebrate HSF genes.

Regulation of HSF1 transcriptional complexes under proteotoxic stress: Mechanisms of heat shock gene transcription involve the stress-induced HSF1 complex formation, changes in chromatin states, and formation of phase-separated condensates: Mechanisms of heat shock gene transcription involve the stress-induced HSF1 complex formation, changes in chromatin states, and formation of phase-separated condensates.

Environmental, physiological, and pathological stimuli induce the misfolding of proteins, which results in the formation of aggregates and amyloid fibrils. To cope with proteotoxic stress, cells are equipped with adaptive mechanisms that are accompanied by changes in gene expression. The evolutionarily conserved mechanism called the heat shock response is characterized by the induction of a set of heat shock proteins (HSPs), and is mainly regulated by heat shock transcription factor 1 (HSF1) in mammals. We herein introduce the mechanisms by which HSF1 tightly controls the transcription of HSP genes via the regulation of pre-initiation complex recruitment in their promoters under proteotoxic stress. These mechanisms involve the stress-induced regulation of HSF1-transcription complex formation with a number of coactivators, changes in chromatin states, and the formation of phase-separated condensates through post-translational modifications.

The Mediator subunit MED12 promotes formation of HSF1 condensates on heat shock response element arrays in heat-shocked cells.

Upon heat shock, activated heat shock transcription factor 1 (HSF1) binds to the heat shock response elements (HSEs) in the promoters of mammalian heat shock protein (HSP)-encoding genes and recruits the preinitiation complex and coactivators, including Mediator. These transcriptional regulators may be concentrated in phase-separated condensates around the promoters, but they are too minute to be characterized in detail. We herein established HSF1-/- mouse embryonic fibroblasts harbouring HSP72-derived multiple HSE arrays and visualized the condensates of fluorescent protein-tagged HSF1 with liquid-like properties upon heat shock. Using this experimental system, we demonstrate that endogenous MED12, a subunit of Mediator, is concentrated in artificial HSF1 condensates upon heat shock. Furthermore, the knockdown of MED12 markedly reduces the size of condensates, suggesting an important role for MED12 in HSF1 condensate formation.

HSF1 phosphorylation establishes an active chromatin state via the TRRAP-TIP60 complex and promotes tumorigenesis.

Transcriptional regulation by RNA polymerase II is associated with changes in chromatin structure. Activated and promoter-bound heat shock transcription factor 1 (HSF1) recruits transcriptional co-activators, including histone-modifying enzymes; however, the mechanisms underlying chromatin opening remain unclear. Here, we demonstrate that HSF1 recruits the TRRAP-TIP60 acetyltransferase complex in HSP72 promoter during heat shock in a manner dependent on phosphorylation of HSF1-S419. TRIM33, a bromodomain-containing ubiquitin ligase, is then recruited to the promoter by interactions with HSF1 and a TIP60-mediated acetylation mark, and cooperates with the related factor TRIM24 for mono-ubiquitination of histone H2B on K120. These changes in histone modifications are triggered by phosphorylation of HSF1-S419 via PLK1, and stabilize the HSF1-transcription complex in HSP72 promoter. Furthermore, HSF1-S419 phosphorylation is constitutively enhanced in and promotes proliferation of melanoma cells. Our results provide mechanisms for HSF1 phosphorylation-dependent establishment of an active chromatin status, which is important for tumorigenesis.

MED12 interacts with the heat-shock transcription factor HSF1 and recruits CDK8 to promote the heat-shock response in mammalian cells.

Activated and promoter-bound heat-shock transcription factor 1 (HSF1) induces RNA polymerase II recruitment upon heat shock, and this is facilitated by the core Mediator in Drosophila and yeast. Another Mediator module, CDK8 kinase module (CKM), consisting of four subunits including MED12 and CDK8, plays a negative or positive role in the regulation of transcription; however, its involvement in HSF1-mediated transcription remains unclear. We herein demonstrated that HSF1 interacted with MED12 and recruited MED12 and CDK8 to the HSP70 promoter during heat shock in mammalian cells. The kinase activity of CDK8 (and its paralog CDK19) promoted HSP70 expression partly by phosphorylating HSF1-S326 and maintained proteostasis capacity. These results indicate an important role for CKM in the protection of cells against proteotoxic stress.

Testicular localization of activating transcription factor 1 and its potential function during spermatogenesis†.

Activating transcription factor 1 (ATF1), belonging to the CREB/ATF family of transcription factors, is highly expressed in the testes. However, its role in spermatogenesis has not yet been established. Here, we aimed to elucidate the impact of ATF1 in spermatogenesis by examining the expression pattern of ATF1 in mice and the effect of ATF1 knockdown in the mouse testes. We found that ATF1 is expressed in various organs, with very high levels in the testes. Immunohistochemical staining showed that ATF1 was localized in the nuclei of spermatogonia and co-localized with proliferating cell nuclear antigen. In ATF1-deficient mice, the seminiferous tubules of the testis contained cells at all developmental stages; however, the number of spermatocytes was decreased. Proliferating cell nuclear antigen expression was decreased and apoptotic cells were rare in the seminiferous tubules. These results indicate that ATF1 plays a role in male germ cell proliferation and sperm production.

HSF1 is required for induction of mitochondrial chaperones during the mitochondrial unfolded protein response.

The mitochondrial unfolded protein response (UPRmt ) is characterized by the transcriptional induction of mitochondrial chaperone and protease genes in response to impaired mitochondrial proteostasis and is regulated by ATF5 and CHOP in mammalian cells. However, the detailed mechanisms underlying the UPRmt are currently unclear. Here, we show that HSF1 is required for activation of mitochondrial chaperone genes, including HSP60, HSP10, and mtHSP70, in mouse embryonic fibroblasts during inhibition of matrix chaperone TRAP1, protease Lon, or electron transfer complex 1 activity. HSF1 bound constitutively to mitochondrial chaperone gene promoters, and we observed that its occupancy was remarkably enhanced at different levels during the UPRmt . Furthermore, HSF1 supported the maintenance of mitochondrial function under the same conditions. These results demonstrate that HSF1 is required for induction of mitochondrial chaperones during the UPRmt , and thus, it may be one of the guardians of mitochondrial function under conditions of impaired mitochondrial proteostasis.

The pericentromeric protein shugoshin 2 cooperates with HSF1 in heat shock response and RNA Pol II recruitment.

The recruitment of RNA polymerase II (Pol II) to core promoters is highly regulated during rapid induction of genes. In response to heat shock, heat shock transcription factor 1 (HSF1) is activated and occupies heat shock gene promoters. Promoter-bound HSF1 recruits general transcription factors and Mediator, which interact with Pol II, but stress-specific mechanisms of Pol II recruitment are unclear. Here, we show in comparative analyses of HSF1 paralogs and their mutants that HSF1 interacts with the pericentromeric adaptor protein shugoshin 2 (SGO2) during heat shock in mouse cells, in a manner dependent on inducible phosphorylation of HSF1 at serine 326, and recruits SGO2 to the HSP70 promoter. SGO2-mediated binding and recruitment of Pol II with a hypophosphorylated C-terminal domain promote expression of HSP70, implicating SGO2 as one of the coactivators that facilitate Pol II recruitment by HSF1. Furthermore, the HSF1-SGO2 complex supports cell survival and maintenance of proteostasis in heat shock conditions. These results exemplify a proteotoxic stress-specific mechanism of Pol II recruitment, which is triggered by phosphorylation of HSF1 during the heat shock response.

Acute HSF1 depletion induces cellular senescence through the MDM2-p53-p21 pathway in human diploid fibroblasts.

Heat shock transcription factor 1 (HSF1) regulates the expression of a wide array of genes, controls the expression of heat shock proteins (HSPs) as well as cell growth. Although acute depletion of HSF1 induces cellular senescence, the underlying mechanisms are poorly understood. Here, we report that HSF1 depletion-induced senescence (HDIS) of human diploid fibroblasts (HDFs) was independent of HSP-mediated proteostasis but dependent on activation of the p53-p21 pathway, partly because of the increased expression of dehydrogenase/reductase 2 (DHRS2), a putative MDM2 inhibitor. We observed that HDIS occurred without decreased levels of major HSPs or increased proteotoxic stress in HDFs. Additionally, VER155008, an inhibitor of HSP70 family proteins, increased proteotoxicity and suppressed cell growth but failed to induce senescence. Importantly, we found that activation of the p53-p21 pathway resulting from reduced MDM2-dependent p53 degradation was required for HDIS. Furthermore, we provide evidence that increased DHRS2 expression contributes to p53 stabilization and HDIS. Collectively, our observations uncovered a molecular pathway in which HSF1 depletion-induced DHRS2 expression leads to activation of the MDM2-p53-p21 pathway required for HDIS.

Poly(ADP-Ribose) Polymerase 1 Promotes the Human Heat Shock Response by Facilitating Heat Shock Transcription Factor 1 Binding to DNA.

The heat shock response (HSR) is characterized by the rapid and robust induction of heat shock proteins (HSPs), including HSP70, in response to heat shock and is regulated by heat shock transcription factor 1 (HSF1) in mammalian cells. Poly(ADP-ribose) polymerase 1 (PARP1), which can form a complex with HSF1 through the scaffold protein PARP13, has been suggested to be involved in the HSR. However, its effects on and the regulatory mechanisms of the HSR are not well understood. Here we show that prior to heat shock, the HSF1-PARP13-PARP1 complex binds to the HSP70 promoter. In response to heat shock, activated and auto-PARylated PARP1 dissociates from HSF1-PARP13 and is redistributed throughout the HSP70 locus. Remarkably, chromatin in the HSP70 promoter is initially PARylated at high levels and decondensed, whereas chromatin in the gene body is moderately PARylated afterwards. Activated HSF1 then binds to the promoter efficiently and promotes the HSR. Chromatin PARylation and HSF1 binding to the promoter are also facilitated by the phosphorylation-dependent dissociation of PARP13. Furthermore, the HSR and proteostasis capacity are reduced by pretreatment with genotoxic stresses, which disrupt the ternary complex. These results illuminate one of the priming mechanisms of the HSR that facilitates the binding of HSF1 to DNA during heat shock.

The HSF1-PARP13-PARP1 complex facilitates DNA repair and promotes mammary tumorigenesis.

Poly(ADP-ribose) polymerase 1 (PARP1) is involved in DNA repair, chromatin structure, and transcription. However, the mechanisms that regulate PARP1 distribution on DNA are poorly understood. Here, we show that heat shock transcription factor 1 (HSF1) recruits PARP1 through the scaffold protein PARP13. In response to DNA damage, activated and auto-poly-ADP-ribosylated PARP1 dissociates from HSF1-PARP13, and redistributes to DNA lesions and DNA damage-inducible gene loci. Histone deacetylase 1 maintains PARP1 in the ternary complex by inactivating PARP1 through deacetylation. Blocking ternary complex formation impairs redistribution of PARP1 during DNA damage, which reduces gene expression and DNA repair. Furthermore, ternary complex formation and PARP1 redistribution protect cells from DNA damage by promoting DNA repair, and support growth of BRCA1-null mammary tumors, which are sensitive to PARP inhibitors. Our findings identify HSF1 as a regulator of genome integrity and define this function as a guarding mechanism for a specific type of mammary tumorigenesis.

HSF1 and HSF3 cooperatively regulate the heat shock response in lizards.

Cells cope with temperature elevations, which cause protein misfolding, by expressing heat shock proteins (HSPs). This adaptive response is called the heat shock response (HSR), and it is regulated mainly by heat shock transcription factor (HSF). Among the four HSF family members in vertebrates, HSF1 is a master regulator of HSP expression during proteotoxic stress including heat shock in mammals, whereas HSF3 is required for the HSR in birds. To examine whether only one of the HSF family members possesses the potential to induce the HSR in vertebrate animals, we isolated cDNA clones encoding lizard and frog HSF genes. The reconstructed phylogenetic tree of vertebrate HSFs demonstrated that HSF3 in one species is unrelated with that in other species. We found that the DNA-binding activity of both HSF1 and HSF3 in lizard and frog cells was induced in response to heat shock. Unexpectedly, overexpression of lizard and frog HSF3 as well as HSF1 induced HSP70 expression in mouse cells during heat shock, indicating that the two factors have the potential to induce the HSR. Furthermore, knockdown of either HSF3 or HSF1 markedly reduced HSP70 induction in lizard cells and resistance to heat shock. These results demonstrated that HSF1 and HSF3 cooperatively regulate the HSR at least in lizards, and suggest complex mechanisms of the HSR in lizards as well as frogs.

Role of Heat Shock Factor 1 in Conserving Cholesterol Transportation in Leydig Cell Steroidogenesis via Steroidogenic Acute Regulatory Protein.

Testicular testosterone synthesis begins with cholesterol transport into mitochondria via steroidogenic acute regulatory (StAR) protein in Leydig cells. Acute heat stress is known to obstruct testicular steroidogenesis by transcriptional repression of StAR. In contrast, chronic heat stress such as cryptorchidism or varicocele generally does not affect testicular steroidogenesis, suggesting that Leydig cells adapt to heat stress and retain their steroid synthesis ability. However, the mechanisms of the stress response in steroid-producing cells are unclear. We examined the relationship between the heat stress response and heat shock factor 1 (HSF1), which protects cells from proteotoxic stress by inducing heat shock protein as a molecular chaperone. The influences of HSF1 deficiency on cholesterol transport by StAR and the expression of steroidogenic enzymes under chronic heat stress were studied in testes of HSF1-knockout (HSF1KO) mice with experimental cryptorchidism. StAR protein in wild-type-cryptorchid mice was transiently decreased after induction of cryptorchidism and then gradually returned to basal levels. In contrast, StAR protein in HSF1KO mice continued to decrease and failed to recover, resulting in impaired serum testosterone. StAR messenger RNA was not decreased with cryptorchidism, indicating that posttranslational modification of StAR, not its transcription, was obstructed in cryptorchidism. Other steroidogenic enzymes, including CYP11A1, 3ß-HSD, and CYP17A1, were not decreased. Lipid droplets were increased in the cytosol of HSF1KO-cryptorchid mice, suggesting dysfunctional cholesterol transportation. These findings provide insight into the role of HSF1 in Leydig cell steroidogenesis, suggesting that it maintains cholesterol transport by recovering StAR under chronic heat stress.

Variations in brain defects result from cellular mosaicism in the activation of heat shock signalling.

Repetitive prenatal exposure to identical or similar doses of harmful agents results in highly variable and unpredictable negative effects on fetal brain development ranging in severity from high to little or none. However, the molecular and cellular basis of this variability is not well understood. This study reports that exposure of mouse and human embryonic brain tissues to equal doses of harmful chemicals, such as ethanol, activates the primary stress response transcription factor heat shock factor 1 (Hsf1) in a highly variable and stochastic manner. While Hsf1 is essential for protecting the embryonic brain from environmental stress, excessive activation impairs critical developmental events such as neuronal migration. Our results suggest that mosaic activation of Hsf1 within the embryonic brain in response to prenatal environmental stress exposure may contribute to the resulting generation of phenotypic variations observed in complex congenital brain disorders.

A study of hearing function and histopathologic changes in the cochlea of the type 2 diabetes model Tsumura Suzuki obese diabetes mouse.

OBJECTIVES: This study used Tsumura Suzuki Obese Diabetes (TSOD) mice as a spontaneous type 2 diabetes model and Tsumura Suzuki Non-obesity (TSNO) mice as controls to investigate factors involved in the onset of hearing impairment. METHOD: Body weight, blood glucose levels, and auditory brainstem responses (ABRs) were measured. The cochleae were excised and evaluated histopathologically. RESULTS: The TSOD mice showed significant hyperglycemia at 2-7 months and severe obesity at 5-10 months; significantly elevated ABR thresholds at 8-10 months; and the capillary lumens in the cochlea stria vascularis were narrower in the TSOD mice than in the TSNO mice. At 17 months, India ink vascular staining of the TSOD mice's cochleae revealed decreased capillary density in the stria vascularis. The vascular area of capillaries in the stria vascularis and the vascular area were significantly smaller in TSOD mice. Histopathological analysis showed vessel wall thickening in the modiolus and narrowed capillaries in the stria vascularis, suggesting reduced blood flow to the inner ear. CONCLUSION: The diabetes mice model used in our study showed early age-associated hearing loss, and histopathology showed findings of vessel wall thickening in the modiolus, narrowing of capillaries in the stria vascularis, and chronically reduced blood flow in the cochlea.

Anti-TNF-α Agent Infliximab and Splenectomy Are Protective Against Renal Ischemia-Reperfusion Injury.

BACKGROUND: Renal ischemia-reperfusion (I/R) injury is associated with delayed graft function and results in poor long-term graft survival. We previously showed that splenectomy (SPLN) protects the kidney from I/R injury and reduces serum TNF-α levels. Herein, we further investigated the effects of SPLN on inflammatory responses and tissue injury in renal I/R by examining the expression of major inflammatory cytokines and heat shock protein 70 (HSP70). Because it was shown previously that the anti-TNF-α agent infliximab (IFX) attenuated renal I/R injury, we also investigated whether IFX administration mimics the effects of SPLN. METHODS: The left renal pedicles of adult male Wistar rats were clamped for 45 minutes and then reperfused for 24 hours; right nephrectomy and SPLN were performed immediately. A separate cohort was administered IFX 1 hour before surgery in lieu of SPLN. RESULTS: Serum creatinine and blood urea nitrogen levels were markedly elevated by I/R injury; these increases were significantly reversed by IFX. Furthermore, IFX inhibited the induction of inflammatory cytokines and HSP70 during renal I/R injury. Time-dependent profiles revealed that the expression of inflammatory cytokines was elevated immediately after I/R, whereas levels of HSP70, serum creatinine, and blood urea nitrogen began to rise 3 hours postreperfusion. Macrophages/monocytes were significantly increased in I/R-injured kidneys, but not in those administered IFX. The outcomes of SPLN mirrored those of IFX administration. CONCLUSIONS: Splenectomy and TNF-α inhibition both protect the kidney from I/R injury by reducing the accumulation of renal macrophages/monocytes and induction of major inflammatory cytokines.

Analysis of the heat shock factor complex in mammalian HSP70 promoter.

The heat shock response is characterized by the induction of heat shock proteins (HSPs) and is one of prominent mechanisms that regulate proteostasis capacity in the cell. In mammals, heat shock factor 1 (HSF1) regulates the expression of HSPs transcriptionally in both unstressed and stressed cells. Recent reports show that the HSF1-RPA complex constitutively gains access to nucleosomal DNA in part by recruiting a histone chaperone and a chromatin-remodeling component. Here, we describe the strategies to substitute endogenous HSF1 with ectopically expressed HSF1 or its mutant and to detect the occupancy of HSF1 transcription complex including RPA in vivo on two heat shock response elements located close together in the human or mouse HSP70 promoters by chromatin immunoprecipitation assay with high sensitivity and specificity.

Mitochondrial SSBP1 protects cells from proteotoxic stresses by potentiating stress-induced HSF1 transcriptional activity.

Heat-shock response is an adaptive response to proteotoxic stresses including heat shock, and is regulated by heat-shock factor 1 (HSF1) in mammals. Proteotoxic stresses challenge all subcellular compartments including the mitochondria. Therefore, there must be close connections between mitochondrial signals and the activity of HSF1. Here, we show that heat shock triggers nuclear translocation of mitochondrial SSBP1, which is involved in replication of mitochondrial DNA, in a manner dependent on the mitochondrial permeability transition pore ANT-VDAC1 complex and direct interaction with HSF1. HSF1 recruits SSBP1 to the promoters of genes encoding cytoplasmic/nuclear and mitochondrial chaperones. HSF1-SSBP1 complex then enhances their induction by facilitating the recruitment of a chromatin-remodelling factor BRG1, and supports cell survival and the maintenance of mitochondrial membrane potential against proteotoxic stresses. These results suggest that the nuclear translocation of mitochondrial SSBP1 is required for the regulation of cytoplasmic/nuclear and mitochondrial proteostasis against proteotoxic stresses.

ATF1 modulates the heat shock response by regulating the stress-inducible heat shock factor 1 transcription complex.

The heat shock response is an evolutionally conserved adaptive response to high temperatures that controls proteostasis capacity and is regulated mainly by an ancient heat shock factor (HSF). However, the regulation of target genes by the stress-inducible HSF1 transcription complex has not yet been examined in detail in mammalian cells. In the present study, we demonstrated that HSF1 interacted with members of the ATF1/CREB family involved in metabolic homeostasis and recruited them on the HSP70 promoter in response to heat shock. The HSF1 transcription complex, including the chromatin-remodeling factor BRG1 and lysine acetyltransferases p300 and CREB-binding protein (CBP), was formed in a manner that was dependent on the phosphorylation of ATF1. ATF1-BRG1 promoted the establishment of an active chromatin state and HSP70 expression during heat shock, whereas ATF1-p300/CBP accelerated the shutdown of HSF1 DNA-binding activity during recovery from acute stress, possibly through the acetylation of HSF1. Furthermore, ATF1 markedly affected the resistance to heat shock. These results revealed the unanticipated complexity of the primitive heat shock response mechanism, which is connected to metabolic adaptation.