Human Heat Shock Transcription Factor 1 Research Articles

Structure of human heat-shock transcription factor 1 in complex with DNA.

Heat-shock transcription factor 1 (HSF1) has a central role in mediating the protective response to protein conformational stresses in eukaryotes. HSF1 consists of an N-terminal DNA-binding domain (DBD), a coiled-coil oligomerization domain, a regulatory domain and a transactivation domain. Upon stress, HSF1 trimerizes via its coiled-coil domain and binds to the promoters of heat shock protein-encoding genes. Here, we present cocrystal structures of the human HSF1 DBD in complex with cognate DNA. A comparative analysis of the HSF1 paralog Skn7 from Chaetomium thermophilum showed that single amino acid changes in the DBD can switch DNA binding specificity, thus revealing the structural basis for the interaction of HSF1 with cognate DNA. We used a crystal structure of the coiled-coil domain of C. thermophilum Skn7 to develop a model of the active human HSF1 trimer in which HSF1 embraces the heat-shock-element DNA.

A Direct Regulatory Interaction between Chaperonin TRiC and Stress-Responsive Transcription Factor HSF1

Heat shock transcription factor 1 (HSF1) is an evolutionarily conserved transcription factor that protects cells from protein-misfolding-induced stress and apoptosis. The mechanisms by which cytosolic protein misfolding leads to HSF1 activation have not been elucidated. Here, we demonstrate that HSF1 is directly regulated by TRiC/CCT, a central ATP-dependent chaperonin complex that folds cytosolic proteins. A small-molecule activator of HSF1, HSF1A, protects cells from stress-induced apoptosis, binds TRiC subunits in vivo and in vitro, and inhibits TRiC activity without perturbation of ATP hydrolysis. Genetic inactivation or depletion of the TRiC complex results in human HSF1 activation, and HSF1A inhibits the direct interaction between purified TRiC and HSF1 in vitro. These results demonstrate a direct regulatory interaction between the cytosolic chaperone machine and a critical transcription factor that protects cells from proteotoxicity, providing a mechanistic basis for signaling perturbations in protein folding to a stress-protective transcription factor.

Overexpression of heat shock transcription factor 1 enhances the resistance of melanoma cells to doxorubicin and paclitaxel

BackgroundHeat Shock Transcription Factor 1 (HSF1) is activated under stress conditions. In turn, it induces expression of Heat Shock Proteins (HSPs), which are well-known regulators of protein homeostasis. Elevated levels of HSF1 and HSPs were observed in many types of tumors. The aim of the present study was to determine whether HSF1 could have an effect on the survival of cancer cells treated with chemotherapeutic cytotoxic agents.MethodsWe constructed mouse (B16F10) and human (1205Lu, WM793B) melanoma cells overexpressing full or mutant form of human HSF1: a constitutively active one with a deletion in regulatory domain or a dominant negative one with a deletion in the activation domain. The impact of different forms of HSF1 on the expression of HSP and ABC genes was studied by RT-PCR and Western blotting. Cell cultures were treated with increasing amounts of doxorubicin, paclitaxel, cisplatin, vinblastine or bortezomib. Cell viability was determined by MTT, and IC50 was calculated. Cellular accumulation of fluorescent dyes and side population cells were studied using flow cytometry.ResultsCells overexpressing HSF1 and characterized by increased HSPs accumulation were more resistant to doxorubicin or paclitaxel, but not to cisplatin, vinblastine or bortezomib. This resistance correlated with the enhanced efflux of fluorescent dyes and the increased number of side population cells. The expression of constitutively active mutant HSF1, also resulting in HSPs overproduction, did not reduce the sensitivity of melanoma cells to drugs, unlike in the case of dominant negative form expression. Cells overexpressing a full or dominant negative form of HSF1, but not a constitutively active one, had higher transcription levels of ABC genes when compared to control cells.ConclusionsHSF1 overexpression facilitates the survival of melanoma cells treated with doxorubicin or paclitaxel. However, HSF1-mediated chemoresistance is not dependent on HSPs accumulation but on an increased potential for drug efflux by ABC transporters. Direct transcriptional activity of HSF1 is not necessary for increased expression of ABC genes, which is probably mediated by HSF1 regulatory domain.

Genetic Selection for Constitutively Trimerized Human HSF1 Mutants Identifies a Role for Coiled-Coil Motifs in DNA Binding

Human heat shock transcription factor 1 (HSF1) promotes the expression of stress-responsive genes and is a critical factor for the cellular protective response to proteotoxic and other stresses. In response to stress, HSF1 undergoes a transition from a repressed cytoplasmic monomer to a homotrimer, accumulates in the nucleus, binds DNA, and activates target gene transcription. Although these steps occur as sequential and highly regulated events, our understanding of the full details of the HSF1 activation pathway remains incomplete. Here we describe a genetic screen in humanized yeast that identifies constitutively trimerized HSF1 mutants. Surprisingly, constitutively trimerized HSF1 mutants do not bind to DNA in vivo in the absence of stress and only become DNA binding competent upon stress exposure, suggesting that an additional level of regulation beyond trimerization and nuclear localization may be required for HSF1 DNA binding. Furthermore, we identified a constitutively trimerized and nuclear-localized HSF1 mutant, HSF1 L189P, located in LZ3 of the HSF1 trimerization domain, which in response to proteotoxic stress is strongly compromised for DNA binding at the Hsp70 and Hsp25 promoters but readily binds to the interleukin-6 promoter, suggesting that HSF1 DNA binding is in part regulated in a locus-dependent manner, perhaps via promoter-specific differences in chromatin architecture. Furthermore, these results implicate the LZ3 region of the HSF1 trimerization domain in a function beyond its canonical role in HSF1 trimerization.

Deciphering Human Heat Shock Transcription Factor 1 Regulation via Post-Translational Modification in Yeast

Heat shock transcription factor 1 (HSF1) plays an important role in the cellular response to proteotoxic stresses. Under normal growth conditions HSF1 is repressed as an inactive monomer in part through post-translation modifications that include protein acetylation, sumoylation and phosphorylation. Upon exposure to stress HSF1 homotrimerizes, accumulates in nucleus, binds DNA, becomes hyper-phosphorylated and activates the expression of stress response genes. While HSF1 and the mechanisms that regulate its activity have been studied for over two decades, our understanding of HSF1 regulation remains incomplete. As previous studies have shown that HSF1 and the heat shock response promoter element (HSE) are generally structurally conserved from yeast to metazoans, we have made use of the genetically tractable budding yeast as a facile assay system to further understand the mechanisms that regulate human HSF1 through phosphorylation of serine 303. We show that when human HSF1 is expressed in yeast its phosphorylation at S303 is promoted by the MAP-kinase Slt2 independent of a priming event at S307 previously believed to be a prerequisite. Furthermore, we show that phosphorylation at S303 in yeast and mammalian cells occurs independent of GSK3, the kinase primarily thought to be responsible for S303 phosphorylation. Lastly, while previous studies have suggested that S303 phosphorylation represses HSF1-dependent transactivation, we now show that S303 phosphorylation also represses HSF1 multimerization in both yeast and mammalian cells. Taken together, these studies suggest that yeast cells will be a powerful experimental tool for deciphering aspects of human HSF1 regulation by post-translational modifications.

Targeting Misfolded Proteins to Fight Neurodegenerative Diseases

Targeting Misfolded Proteins to Fight Neurodegenerative Diseases

Celastrols as Inducers of the Heat Shock Response and Cytoprotection

Alterations in protein folding and the regulation of conformational states have become increasingly important to the functionality of key molecules in signaling, cell growth, and cell death. Molecular chaperones, because of their properties in protein quality control, afford conformational flexibility to proteins and serve to integrate stress-signaling events that influence aging and a range of diseases including cancer, cystic fibrosis, amyloidoses, and neurodegenerative diseases. We describe here characteristics of celastrol, a quinone methide triterpene and an active component from Chinese herbal medicine identified in a screen of bioactive small molecules that activates the human heat shock response. From a structure/function examination, the celastrol structure is remarkably specific and activates heat shock transcription factor 1 (HSF1) with kinetics similar to those of heat stress, as determined by the induction of HSF1 DNA binding, hyperphosphorylation of HSF1, and expression of chaperone genes. Celastrol can activate heat shock gene transcription synergistically with other stresses and exhibits cytoprotection against subsequent exposures to other forms of lethal cell stress. These results suggest that celastrols exhibit promise as a new class of pharmacologically active regulators of the heat shock response.

Redox-dependent regulation of the conformation and function of human heat shock factor 1.

We present here evidence that redox-dependent thiol-disulfide exchange can provide a mechanism regulating the conformation and activity of the human heat shock transcription factor 1 (HSF1). Diamide and dithiothreitol were reagents used respectively to promote and reverse disulfide cross-link, and the resolution and detection of redox conformers of HSF1 were done according to recently published methods [Manalo, D. J., and Liu, A. Y.-C. (2001) J. Biol. Chem. 276, 23554-23561]. We showed that preincubation of the latent HSF1 monomer with diamide inhibited the in vitro heat-induced activation and trimerization of HSF1 and caused the formation of ox-hHSF1, a compact, disulfide cross-linked HSF1 conformer detectable in immuno-Western blot assay. These effects of diamide were dose-dependent and were rapidly and quantitatively reversed by dithiothreitol. Cysteine site-specific mutants of HSF1 were constructed and used to determine which of the five cysteine residues may be engaged in disulfide cross-link. Analysis of the in vitro transcribed and translated HSF1 proteins showed that while mutation of C1 (amino acid 36) and C2 (amino acid 103) had no effect on the redox sensitivity of HSF1, the mutation of C3 (amino acid 153) or double mutation of C4 and C5 (amino acids 373 and 378, respectively) to serine rendered HSF1 insensitive to diamide and prevented its conversion to ox-HSF1. HSF1 with a single cysteine to serine mutation at either the C4 or C5 position gave different ox-HSF1 conformers in the presence of diamide, suggesting that C3 could be disulfide cross-linked to either C4 or C5. The possibility that HSF1 may have integrated redox chemistry of cysteine sulfhydryl into its functional response in higher mammalian cells is discussed.

A Novel Association between the Human Heat Shock Transcription Factor 1 (HSF1) and Prostate Adenocarcinoma

A search for differentially expressed genes in a pair of nonmetastatic (PC-3) versus metastatic variant (PC-3M) human prostate carcinoma cell lines led to identification of the human heat shock factor (HSF1) as an overexpressed gene product in PC-3M cells. Analysis of primary prostate cancer specimens indicated that HSF1 is generally up-regulated in most of the malignant prostate epithelial cells relative to the normal prostate cells. Among the known effectors of HSF1 action, constitutive levels of HSP70 and HSP90 are not significantly altered by the naturally elevated expression of HSF1 as in PC-3M cells or by transduced overexpression of HSF1 in PC-3 cells. The basal levels of HSP27 in both cases are, however, consistently increased by two- to threefold. With respect to response to heat shock, high basal concentration of HSP90 is not further enhanced in these cells, and HSP70 is up-regulated irrespective of HSF1 level. Heat shock, however, causes an increase in HSP27 when HSF1 is up-regulated, except when the expression of HSF1 is already too high. These results document for the first time that HSF1 is overexpressed in human prostate cancer cells, at least one consequence of which in the prostate cancer cell lines tested is stimulation of both basal and stress-induced expression of HSP27, an important factor in cell growth, differentiation, or apoptosis.

Enhanced protein denaturation in indomethacin-treated cells

Indomethacin, a potent anti-inflammatory drug, activates the DNA-binding activity of human heat shock transcription factor 1 (HSF1), but this is insufficient to elevate heat shock gene expression. However, indomethacin pretreatment leads to a complete heat shock response at temperatures that are by themselves insufficient. Here, we showed that the heat-induced loss of enzymatic activity of a nuclear or a cytoplasmic luciferase expressed in murine cells was enhanced when cells had been pretreated with indomethacin. Additionally, in these cells the 70-kDa constitutive heat shock protein exhibited an enhanced aggregation in the presence of indomethacin. Similarly an increase in the aggregation of beta-galactosidase was observed. These data suggest that indomethacin at moderate temperatures accelerates the presence of denatured proteins in the cell, thus lowering the temperature threshold for a heat shock response.

Dual regulation of a heat shock promoter during embryogenesis: stage-dependent role of heat shock elements.

Transgenic tobacco expression was analysed of chimeric genes with point mutations in the heat shock element (HSE) arrays of a small heat shock protein (sHSP) gene from sunflower: Ha hsp17.7 G4. The promoter was developmentally regulated during zygotic embryogenesis and responded to heat stress in vegetative tissues. Mutations in the HSE affected nucleotides crucial for human heat shock transcription factor 1 (HSF1) binding. They abolished the heat shock response of Ha hsp17.7 G4 and produced expression changes that demonstrated dual regulation of this promoter during embryogenesis. Thus, whereas activation of the chimeric genes during early maturation stages did not require intact HSE, expression at later desiccation stages was reduced by mutations in both the proximal (-57 to -89) and distal (-99 to -121) HSE. In contrast, two point mutations in the proximal HSE that did not severely affect gene expression during zygotic embryogenesis, eliminated the heat shock response of the same chimeric gene in vegetative organs. Therefore, by site-directed mutagenesis, it was possible to separate the heat shock response of Ha hsp17.7 G4 from its developmental regulation. The results indicate the co-existence, in a single promoter, of HSF-dependent and -independent regulation mechanisms that would control sHSP gene expression at different stages during plant embryogenesis.

Analysis of the phosphorylation of human heat shock transcription factor-1 by MAP kinase family members.

The activation of heat shock transcription factor-1 (HSF-1) after treatment of mammalian cells with stresses such as heat shock, heavy metals, or ethanol induces the synthesis of heat shock proteins. HSF-1 is phosphorylated at normal growth temperature and is hyperphosphorylated upon stress. We recently presented evidence that HSF-1 can be phosphorylated by the mitogen activated protein kinase, ERK1, and that such phosphorylation appears to negatively regulate the activity of HSF-1. In this report, we have tested the ability of ERK1 to phosphorylate various HSF-1 deletion mutants. Our results show that ERK1 phosphorylation is dependent on a region of HSF-1 extending from amino acids 280 to 308. This region contains three serine residues that are potential ERK1 phosphorylation sites. The region falls within a previously defined regulatory domain of HSF-1. The possibility of protein kinases other than ERK1 phosphorylating HSF-1 was also examined using in-gel kinase assays. The results show that HSF-1 can be phosphorylated in a ras-dependent manner by other members of the MAP kinase family such as JNK and p38 protein kinases and possibly others.

Hyperphosphorylation of Heat Shock Transcription Factor 1 Is Correlated with Transcriptional Competence and Slow Dissociation of Active Factor Trimers

In the course of its activation by heat and other stresses, the inactive monomer of human heat shock transcription factor 1 (HSF1) is converted to a DNA-binding homotrimer and is hyperphosphorylated. At least four Ser/Thr residues in HSF1 appeared to be inducibly phosphorylated during heat shock. Ser/Thr protein kinase inhibitors inhibited, and protein phosphatase inhibitor calyculin A and phorbol ester enhanced, hsp70-CAT reporter gene expression but not heat shock element DNA binding activity in HeLa cells undergoing a moderate heat shock. Calyculin A (5-20 nM) caused hyperphosphorylation of HSF1, the extent of which was comparable to that produced by moderate to severe heat shock. Upon recovery from a 42 degrees C/30 min-heat shock, HSF1 trimers disassembled quantitatively within 2 h. Calyculin A interfered with the dissociation of HSF1 trimers. Thus, hyperphosphorylation increases the effective half-life of the HSF1 trimer, which may prolong factor activity subsequent to heat shock. Hyperphosphorylation also dramatically stimulated the transactivation function of HSF1: exposure to calyculin A of cells induced to form inactive HSF1 trimers resulted in the conversion of the inactive to active trimers. Given that deletion of certain sequences renders HSF1 constitutively active, these results suggested that the activation of HSF1 trimers by calyculin A was a consequence of hyperphosphorylation of HSF1 rather than of a downstream factor.

Repression of human heat shock factor 1 activity at control temperature by phosphorylation.

Human heat shock transcription factor 1 (HSF1) is responsible for stress-induced transcription of heat shock protein genes. The activity of the HSF1 transcriptional activation domains is modulated by a separate regulatory domain, which confers repression at control temperature and heat inducibility. We show here that two specific proline-directed serine motifs are important for function of the regulatory domain: Mutation of these serines to alanine derepresses HSF1 activity at control temperature, and mutation to glutamic acid, mimicking a phosphorylated serine, results in normal repression at control temperature and normal heat shock inducibility. Tryptic mapping shows that these serines are the major phosphorylation sites of HSF1 at control temperature in vivo. Stimulation of the Raf/ERK pathway in vivo results in an increased level of phosphorylation of these major sites and the regulatory domain is an excellent substrate in vitro for the mitogen-activated MAPK/ERK. We conclude that phosphorylation of the regulatory domain of HSF1 decreases the activity of HSF1 at control temperature, and propose a mechanism for modification of HSF1 activity by growth control signals.

Stable overexpression of human HSF-1 in murine cells suggests activation rather than expression of HSF-1 to be the key regulatory step in the heat shock gene expression.

Transcription of the heat shock genes is regulated by the activation of the heat shock transcription factor (HSF-1). After heat shock, HSF-1 forms oligomers and binds to the heat shock element (HSE), which consists of several repeats of NGAAN located in the promoter region of the heat shock genes. HSF-1 is then phosphorylated, leading to the enhanced transcription of the heat shock genes likely by transactivation. We have stably overexpressed the human heat shock transcription factor-1 (HSF-1) in murine cells to investigate whether the regulation of the expression of the heat shock genes may partly reside at the level of HSF-1 expression. Human HSF-1 cDNA was cloned into a retroviral vector (pvhhsf-1) and was overexpressed in a murine fibroblast cell line. The overexpressed human HSF-1 is found in both the cytoplasm and nucleus of control cells but is translocated into the nucleus upon heat shock. Electrophoretic mobility shift analysis suggests that the human HSF-1 has constitutive DNA binding ability and its DNA binding ability is increased upon heat shock. Cross-linking experiments indicate that the overexpressed human HSF-1 is mainly a monomer under control conditions and forms oligomers upon heat shock. Immunoblotting shows that the human HSF-1 is phosphorylated upon heat shock and its apparent molecular weight is shifted up by at least 10 kDa. In spite of both the DNA binding ability and phosphorylation, the overexpression of human HSF-1 does not increase the transcription of murine HSP-70 mRNA or increase the synthesis of other HSPs after heat shock beyond that observed in control untransfected cells. An exception is the enhanced synthesis of a 47-50 kDa protein after heat shock and an apparent lack of induction of one HSP-70 kDa species when the protein pattern is analyzed by isoelectric focusing. Interestingly, cells overexpressing human HSF-1 show a 4-fold increase in the basal expression of luciferase when the plasmids containing the human HSP-70 promoter ligated to the luciferase reporter gene are transiently expressed in these cells. Murine cells overexpressing human HSF-1 are more resistant to the cytotoxic effects of heat when compared to the control untransfected cells, but the kinetics of thermotolerance development and decay is similar between HSF-1 transfected and untransfected cells. In conclusion, human HSF-1 protein in murine fibroblasts is modified in a similar fashion as the endogenous mouse HSF-1 after heat shock. However, the overexpression of HSF-1 does not result in overproduction of heat shock proteins after heat shock, perhaps because these cells contain abundant amounts of endogenous HSF-1.

Pharmacological modulation of heat shock factor 1 by antiinflammatory drugs results in protection against stress-induced cellular damage.

The activation of heat shock genes by diverse forms of environmental and physiological stress has been implicated in a number of human diseases, including ischemic damage, reperfusion injury, infection, neurodegeneration, and inflammation. The enhanced levels of heat shock proteins and molecular chaperones have broad cytoprotective effects against acute lethal exposures to stress. Here, we show that the potent antiinflammatory drug indomethacin activates the DNA-binding activity of human heat shock transcription factor 1 (HSF1). Perhaps relevant to its pharmacological use, indomethacin pretreatment lowers the temperature threshold of HSF1 activation, such that a complete heat shock response can be attained at temperatures that are by themselves insufficient. The synergistic effect of indomethacin and elevated temperature is biologically relevant and results in the protection of cells against exposure to cytotoxic conditions.

Stimulation of the DNA-dependent Protein Kinase by RNA Polymerase II Transcriptional Activator Proteins

The DNA-dependent protein kinase (DNA-PK) phosphorylates RNA polymerase II and a number of transcription factors. We now show that the activity of DNA-PK is directly stimulated by certain transcriptional activator proteins, including the human heat shock transcription factor 1 (HSF1) and a transcriptionally active N-terminal 147 amino acid GAL4 derivative. Stimulation of DNA-PK activity required specific sequences in the activator proteins outside the minimal DNA binding domains. The stimulation of DNA-PK activity also required DNA and was greater with DNA containing relevant activator binding sites. Comparison of different HSF binding fragments showed that optimal stimulation occurred when two HSF binding sites were present. Stimulation with HSF and GAL4 was synergistic with Ku protein, another regulator of DNA-PK activity. DNA-PK is tightly associated with the transcriptional template, and an increase in its activity could potentially influence transcription through the phosphorylation of proteins associated with the transcription complex.