Heat Shock Factor Protein Research Articles

Silencing heat shock factor 1 by small interfering RNA abrogates heat shock-induced cardioprotection against ischemia–reperfusion injury in mice

Induction of heat shock factor 1 (HSF1) is known to associate with cellular response to divergent pathophysiological stresses including whole body hyperthermia (WBH) and ischemia-reperfusion. However, a direct cause-effect relationship between HSF1 activation and cytoprotection induced by myocardial preconditioning has not been conclusively established, mainly due to the limitations of available experiment tools. In the present studies, we used a novel approach to block HSF1 with small interfering RNA (siRNA) technique in vivo. Male adult ICR mice were treated intraperitoneally with amine (vehicle) or siRNA specific to HSF1 (siRNA-HSF1). Three days later, WBH preconditioning protocol (rectal temperature 42 °C for 15 min) was applied to these mice under light anesthesia. WBH preconditioning resulted in 2.7-fold and 3.4-fold increase in cardiac HSF1 mRNA and protein expression respectively 2 hours after WBH, which was inhibited in the siRNA-treated mice. The silencing effect of siRNA on HSF1 was associated with complete loss of the infarct- limiting protection by WBH preconditioning after 48 hours. Pretreatment with siRNA-HSF1 had no effect on infarct size in the sham control animals as compared with the amine-treated group. DNA micro-array analysis revealed that siRNA-HSF1 caused a general inhibition on multiple members of HSP family, except Hsp32, Hsp47 and Hsp60. In addition, the silencing effect of siRNA on HSF1 and HSPs gene expression was transient and its inhibitory effect disappeared by 10 days after treatment. siRNA-HSF1 also impaired the thermotolerance of the heat shocked mice as indicated by higher mortality following WBH. For the first time, we have applied siRNA technique in the field of myocardial preconditioning to demonstrate HSF1 activation as an essential step in WBH preconditioning against cardiac ischemia-reperfusion injury.

α-Lipoic Acid Inhibits Endotoxin-stimulated Expression of iNOS and Nitric Oxide Independent of the Heat Shock Response in RAW 264.7 Cells

The heat shock response protects against sepsis-induced mortality, organ injury, cardiovascular dysfunction, and apoptosis. Several inducers of the heat shock response, such as hyperthermia, sodium arsenite, and pyrollidine dithiocarbonate, inhibit NF-κB activation and nitric oxide formation. The antioxidant lipoic acid (LA) has recently been found to inhibit NF-κB activation and nitric oxide formation. We therefore tested the hypothesis that LA induces a heat shock response. To test this hypothesis, we determined whether exposure to LA affects expression of both heat shock protein 70 (HSP-70) and nuclear heat shock factor-1 (HSF-1) in lipopolysaccharide (LPS) stimulated macrophages. LA and hyperthermia attenuated LPS-induced increases in nuclear NF-κB, iNOS protein, and media nitrite concentrations. LPS and hyperthermia increased HSP-70 concentrations 8-fold and 20-fold, respectively. No effect of LA treatment alone on HSP-70 protein expression was detected. Likewise, no effect of LA on HSF-1 protein expression was detected. These data suggest that LA inhibits LPS-induced activation of iNOS in macrophages independent of the heat shock response.

Molecular determinants of responses to myocardial ischemia/reperfusion injury: focus on hypoxia-inducible and heat shock factors.

During the past several years much new evidence has accumulated regarding the molecular and biochemical mechanisms underlying cardiac responses to hypoxia and to ischemia/reperfusion injury. Studies have involved cell culture, and ex vivo and in vivo preparations. This review focuses on regulation of two transcription factors that are thought to be important in these processes, hypoxia-inducible factor1alpha (HIF-1alpha) and heat shock factor (HSF). Both of these molecules are expressed acutely and chronically in response to hypoxia and ischemia/reperfusion, and both have numerous targets that comprise part of integrated response to ischemic injury aimed at promoting cell survival. Emphasis is placed on new mechanisms of action that regulate HIF-1alpha, HSF, and heat shock proteins as key responses to hypoxia and ischemia, and possible approaches to therapy based on these data are discussed.

HSF1 modulation of Hsp70 mRNA polyadenylation via interaction with symplekin.

Induction of heat shock protein (HSP) gene expression by stress is initiated by binding of HSF1 to HSP gene promoters to increase their transcription. The cytoprotective functions of these HSPs are essential for cell survival, and thus it is critical that inducible HSP gene expression be executed rapidly and efficiently. Here we report an interaction between heat shock factor 1 (HSF1) and symplekin, a protein known to form a complex with the polyadenylation factors CstF and CPSF. HSF1-symplekin complexes are detected only after stress treatment, and these two proteins co-localize in punctate nuclear structures in stressed cells. HSF1 also complexes in a stress-induced manner with the 3' processing factor CstF-64. Interfering with HSF1-symplekin interaction by overexpressing a non-DNA-binding mutant HSF1 protein significantly decreases Hsp70 mRNA polyadenylation in stressed cells, supporting the functional role for HSF1 in promoting 3' processing of this transcript. Importantly, this was also found to result in a significant loss of Hsp70 protein induction and increased cell death in response to stress exposure. These results indicate that the HSF1-symplekin interaction functions as a mechanism for recruiting polyadenylation factors to HSP genes to enhance the efficiency/kinetics of production of mature Hsp mRNA transcripts to achieve the critical cellular need for rapid HSP expression after stress. Thus, HSF1 regulates HSP gene expression at not one but two different steps of the expression pathway, functioning both as a transcription factor and a polyadenylation stimulatory factor.

Identification of Xenopus heat shock transcription factor-2: conserved role of sumoylation in regulating deoxyribonucleic acid-binding activity of heat shock transcription factor-2 proteins.

Heat shock transcription factor (Hsf)-1 and Hsf2 are members of the heat shock factor (HSF) protein family involved in heat shock protein (hsp) gene regulation, a regulation that is critical for the ability of cells to survive exposure to stress conditions. Although the role of Hsf1 in binding and activating transcription of hsp gene promoters in response to cell stress is well established, how Hsf2 enhances stress-induced hsp expression is not understood. To gain an insight into the critical conserved features of the regulation and function of Hsf2, we have identified and characterized the Hsf2 protein from Xenopus laevis. We found that, similar to its human counterpart, Xenopus Hsf2 is sumoylated at lysine 82 and that, as it does in human Hsf2, the modification event of the small ubiquitin-related modifier 1 functions to increase the deoxyribonucleic acid-binding activity of this transcription factor in Xenopus. These results indicate that sumoylation is an evolutionarily conserved modification of Hsf2 proteins, supporting the position of this modification as a critical regulator of Hsf2 function.

JNK phosphorylates the HSF1 transcriptional activation domain: role of JNK in the regulation of the heat shock response.

The role of c-Jun NH2-terminal kinase (JNK) signaling cascade in the stress-inducible phosphorylation of heat shock factor 1 (HSF1) was investigated using known agonists and antagonists of JNK. We showed that treatment of HeLa cells with MG132, a proteasome inhibitor and known INK activator, caused the transcriptional activation domain of HSF1 to be targeted and phosphorylated by JNK2 in vivo. Dose-response and time course studies of the effects of heat shock and anisomycin treatment showed a close correlation of the activation of JNK and hyperphosphorylation of HSF1. SB203580 inhibited INK at the 100 microM concentration and significantly reduced the amount of hyperphosphorylated HSF1 upon heat shock or anisomycin treatment. SB203580 and dominant-negative JNK suppress hsp70 promoter-driven reporter gene expression selectively at 45 degrees C but not at 42 degrees C heat stress, suggesting that JNK would be preferentially associated with the protective heat shock response against severe heat stress. The possibility that JNK-mediated phosphorylation of HSF1 may selectively stabilize the HSF1 protein and confers protection to cells under conditions of severe stress is discussed.

Pervanadate induces the hyperphosphorylation but not the activation of human heat shock factor 1.

In this study, we evaluated the effects of pervanadate, a tyrosine phosphatase inhibitor, on the regulation and function of heat-shock factor 1 (HSF1) in HeLa cells. We showed that 50-100 microM pervanadate induced the hyperphosphorylation of the latent HSF1, as demonstrated by a retarded mobility of the HSF1 protein in SDS-polyacrylamide gel electrophoresis and as supported by the reversal of this mobility shift upon treatment of the cell extract with acid phosphatase. Pervanadate by itself had no effect on the monomeric stoichiometry and DNA-binding activity of HSF1. Upon heat shock, the pervanadate-induced hyperphosphorylated HSF1 formed DNA-binding trimers and translocated into the nuclear compartment. At high concentration (approximately 500 microM), pervanadate also induced the tyrosine phosphorylation of many cellular proteins and blunted the heat-induced transcription of hsp 70. N-acetyl cysteine inhibited these effects of pervanadate, suggesting a redox-based mechanism for its activity. Analysis of the activation of mitogen-activated protein kinases (MAPKs) using antibodies specific for the phospho-form (activated) of the kinases in Western blot showed that pervanadate activated extracellular signal-regulated kinase (ERK1/2), c-Jun-N-terminal kinase 1/2 (JNK1/2), and p-38 kinase. Pharmacological inhibitors of the ERK1/2 kinase pathway or the p38 kinase had little or no effect on the pervanadate-induced hyperphosphorylation of HSF1. Our results show that hyperphosphorylation of hHSF1 can occur prior to and independent of other events involved in the activation of hHSF1. The possibility that activation of the MAPK signaling cascade, notably JNK, may contribute to the hyperphosphorylation of human HSF1 (hHSF1) is discussed.

SIR repression of a yeast heat shock gene: UAS and TATA footprints persist within heterochromatin.

Previous work has suggested that products of the Saccharomyces cerevisiae Silent Information Regulator (SIR) genes form a complex with histones, nucleated by cis-acting silencers or telomeres, which represses transcription in a position-dependent but sequence-independent fashion. While it is generally thought that this Sir complex works through the establishment of heterochromatin, it is unclear how this structure blocks transcription while remaining fully permissive to other genetic processes such as recombination or integration. Here we examine the molecular determinants underlying the silencing of HSP82, a transcriptionally potent, stress-inducible gene. We find that HSP82 is efficiently silenced in a SIR-dependent fashion, but only when HMRE mating-type silencers are configured both 5' and 3' of the gene. Accompanying dominant repression are novel wrapped chromatin structures within both core and upstream promoter regions. Strikingly, DNase I footprints mapping to the binding sites for heat shock factor (HSF) and TATA-binding protein (TBP) are strengthened and broadened, while groove-specific interactions, as detected by dimethyl sulfate, are diminished. Our data are consistent with a model for SIR repression whereby transcriptional activators gain access to their cognate sites but are rendered unproductive by a co-existing heterochromatic complex.

Rapid activation of heat shock factor-1 DNA binding by H 2O 2 and modulation by glutathione in human neuroblastoma and Alzheimer's disease cybrid cells

Because cellular signaling systems are critical mediators of responses to oxidative stress, a condition associated with neurodegenerative disorders, the redox-dependent regulation of heat shock factor-1 (HSF-1) was investigated in human neuroblastoma SH-SY5Y cells. Exposure of cells to 200 μM H 2O 2 caused a rapid increase in HSF-1 DNA binding that was evident within 10 min, and caused a robust increase that reached levels 8-fold the basal activity. In comparison, the transcription factors, activator protein-1 (AP-1) and early growth response-1 (EGR-1), were activated more slowly and to a lesser extent. Activation of HSF-1 DNA binding activity was associated with a cytosolic to nuclear translocation of HSF-1 protein, and was detected with concentrations of H 2O 2 of 100 μM and greater. Intracellular glutathione modulated H 2O 2-induced HSF-1 DNA binding activity, as depletion of glutathione caused HSF-1 to be activated with lower concentrations of H 2O 2 (25 μM) and supplementation of glutathione blocked HSF-1 activation by 100 to 400 μM H 2O 2. Alzheimer's disease (AD) and control cybrid cells (SH-SY5Y cells in which the mitochondria were replaced with platelet mitochondria from AD or matched control subjects) were used to test the effects of the chronic oxidative stress caused by the excessive production of reactive oxygen intermediates (ROIs) in AD cybrids on HSF-1 activity. Basal and maximal (induced by H 2O 2 in glutathione-depleted cells) HSF-1 DNA binding activity were lower in AD than control cybrids, suggesting that the cells had compensated for excessive ROIs. These results indicate that the activation of HSF-1 is highly sensitive to oxidative stress and is regulated by endogenous antioxidant mechanisms.

A mathematical model of the hsp70 regulation in the cell

A mathematical model of the regulation process of the heat shock protein hsp70 in the cell is presented. The model describes the damaging effect of elevated temperature on proteins; the interaction of free hsp70 with injured proteins and its chaperone role in nascent protein translation; the relation between the amount of free hsp70 and the formation of the activated trimer form of the heat shock factor protein (HSF) the binding of activated HSF with the heat shock elements on the DNA; the transcription of mRNA of hsp70 and the synthesis of hsp70. The reaction of the model to a temporal rise in temperature shows an initial decline and a subsequent sharp rise to an ultimately increased level of free hsp70 in the cell. The response of the model to both a single and two consecutive heat shocks appears to closely resemble experimental data on hsp70 synthesis. This general agreement demonstrates the structure of the model to be sound and suitable as a basis for further modelling the complex tolerance mechanism of the cell.

Heat shock factor-1 protein in heat shock factor-1 gene-transfected human epidermoid A431 cells requires phosphorylation before inducing heat shock protein-70 production.

Heat shock factor-1 (HSF1) is a transcriptional factor that binds to heat shock elements located on the promoter region of heat shock protein genes. The purpose of this study was to further investigate the regulation of the expression of the heat shock protein-70 (HSP-70) gene. The HSF1 gene was inserted into pCDNA3 plasmid and then transfected into human epidermoid A431 cells using the CaOP3 method. Control cells were transfected with vector alone. Expression of HSP-70, HSF1, and HSF2 genes and protein were determined. We found a significant increase in the expression of the HSF1 gene, but not HSP-70 and HSF2 genes, in the HSF1 gene-transfected cells. The amount of HSF1-heat shock element complex was significantly increased in both the nucleus and cytosol in HSF1 gene-transfected cells, indicating increased synthesis of HSF1. The amount of HSP-72 in these cells did not change. Therefore, overexpression of HSF1 protein failed to initiate transcription of the HSP-70 gene. Subsequently, we treated the cells with 1 microM PMA (a protein kinase C stimulator), and HSP-70 mRNA and protein were measured at 1 or 4 h of the treatment, respectively. The levels of both HSP-70 mRNA and HSP-72 protein were significantly increased in nontransfected and transfected cells; the levels of HSP-72 in HSF1 gene-transfected cells were greater than that found in the vector-transfected cells. The PMA-induced increase in HSP-72 protein peaked 8 h after treatment with PMA and returned to baseline levels at 72 h. This increase was blocked by a PKC inhibitor, staurosporine. After treatment with PMA, HSF1 translocated quickly from cytosol to nucleus. The results suggest that phosphorylation of newly synthesized HSF1 and possibly of other factors are necessary for the induction of HSP-72. Activation of PKC can cause phosphorylation of HSF1, which leads to an enhanced but transient increase in HSP-70 production.

The heat shock factor and mitochondrial Hsp70 are necessary for survival of heat shock in Saccharomyces cerevisiae

A heat shock recovery assay on solid medium (Nwaka et al. (1995) J. Biol. Chem. 270, 10193–10198) as well as the classical cell counting method were used to investigate the function of some heat shock proteins in thermotolerance. We show that expression of intact heat shock factor protein (HSF), which regulates the stress induced expression of heat shock proteins (HSPs), is necessary for recovery from heat shock. A HSF1 mutant ( hsf1-m3) which does not induce the expression of some heat shock proteins at heat stress (37–40°C) is defective in recovery after heat shock at 50–52 C compared to a corresponding wild-type strain in both stationary and exponentially growing cells. Using two temperature sensitive mutants of the mitochondrial Hsp70 ( ssc1–2 and ssc1–3) encoded by the SSC1 gene, we show that the ssc1–3 mutant, which has a mutation in the ATPase domain, is defective in recovery after heat shock in contrast to the ssc1–2 mutant, which has a mutation in the peptide binding domain. Different binding capacities for unfolded proteins are shown to be the molecular reason for the observed phenotypes. The thermotolerance defect of the hsf1-m3 and ssc1–3 mutants is demonstrated for both glucose and glycerol media.

Heat shock response, heat shock transcription factor and cell aging.

A characteristic feature of aging is a progressive impairment in the ability to adapt to environmental challenges. The purpose of this article is to review the evidence of an attenuated response to heat and physiological stresses in a number of mammalian aging model systems, including the human diploid fibroblasts in culture, whole animals and animal-derived cells and cell cultures, as well as peripheral blood mononuclear cells obtained from human donors. Analyses of the regulation and function of heat shock factor 1 (HSF1), a transcription factor that mediates the response to heat shock, showed that while the relative abundance of both the hsf1 transcript and the HSF1 protein did not change as a function of age, the responsiveness of HSF1 to heat-induced activation, as measured by its trimerization and ability to bind to the heat shock element consensus sequence, was inversely related to the age of the cells used. Given the fundamentally important role of heat shock proteins (HSPs) in many aspects of protein homeostasis and signal transduction it seems likely that the inability, or compromised ability, of aging cells and organisms to activate HSF1 and produce HSPs in response to stress would contribute to the well-known increase in morbidity and mortality of the aged when challenged.

Expression of heat shock factor and heat shock protein 70 genes during maize pollen development

We have analysed the expression of heat shock protein 70 (HSP70) and heat shock factor (HSF) gene during maize pollen development, HSFs being the transcriptional activators of hsp genes. In order to eliminate the sporophytic tissues of anthers, we have isolated homogeneous cell populations corresponding to five stages of maize pollen development from microspores to mature pollen. We show that in the absence of heat stress, hsp70 genes are highly expressed late-bicellular pollen as compared to other stages. HSP70 transcripts are significantly accumulated in response to a heat shock at the late microspore stage but to a much lower extent than in vegetative tissues. The latest stages of pollen development, i.e. mid-tricellular and mature pollen, do not exhibit heat-induced accumulation of HSP70 transcripts. Therefore, we analysed the expression of hsf genes throughout pollen development. We demonstrate that at least three hsf genes are expressed in maize and that transcripts corresponding to one hsf gene, whose expression is independent of temperature in somatic as well as in microgametophytic tissues, are present at similar levels throughout pollen development. In addition, we show that the expression of the two other hsf genes is heat-inducible in maize vegetative tissues and is not significantly increased after heat shock at any stage of pollen development. These results indicate that the loss of hsp gene expression at late stages of pollen development is not due to a modification of hsf gene expression at the mRNA level and that hsf gene expression is differentially regulated in vegetative and microgametophytic tissues.

Differences in DNA sequence recognition by the heat-shock factors of Drosophila melanogaster and the parasitic helminth Schistosoma mansoni

It was recently shown that schistosome extracts contain heat-shock factor (HSF) activity that correlates with the pattern of hsp70 mRNA levels at different developmental stages of the parasite (Levy-Holtzman and Schechter (1994) Parasitology 108, 35–42). To extend our understanding of the HSF activity revealed in extracts of Schistosoma mansoni (Sm), it was further analyzed by competition experiments and compared with the well characterized HSF of Drosophila melanogaster (Dm). The interactions of HSF in Sm extracts (SmHSF) and HSF of Dm (DmHSF) with 32P-labeled heat shock element (HSE) probes, with and without unlabeled competitor DNA probes (HSE-related oligos), were analyzed by gel retardation assay. The binding and inhibition studies demonstrated that SmHSF and DmHSF differ in HSE sequence recognition: an array of three mGAAn inverted repeats according to the ideal consensus sequence (nGAAnnTTCnnGAAn) is recognized by DmHSF, but not by SmHSF. In the schistosome, binding is attained only when the third pentamer is a variant, composed of nG T An instead of nG A An. The presence of this variant in the promoter of the hsp70 gene of the parasite suggests coevolution of the variant sequence together with the SmHSF which interacts efficiently with the variant, but not with the ideal HSE sequence. Further inhibition studies revealed additional differences between SmHSF and DmHSF in recognition of the first and second nGAAn pentamers of HSE. In analogy to other systems of ligand-protein interactions, we propose that the complementarity between the HSE ligand and the HSF protein is higher in SmHSF, as compared to DmHSF.

Thiol reducing reagents inhibit the heat shock response. Involvement of a redox mechanism in the heat shock signal transduction pathway.

We evaluated the effects of thiol-reducing agents on the heat shock response in human and rodent cells in culture. Using HeLa cells as an example, we demonstrated that dithiothreitol (DTT,2mM) inhibited the heat (42 degrees C) induced increase in the synthesis of heat shock proteins (HSPs), abundance of mRNA of hsp 70, hsp 70 gene promoter activity, and the heat shock factor (HSF) DNA binding activity. This effect of DTT was specific and attributable to its reducing activity; oxidized DTT was ineffective, and other thiol reducing compounds had the same effect as DTT. Time course and dose-response studies showed that DTT significantly inhibited the heat shock induction of heat shock element binding activity with no preincubation and that 0.6 and 1-2 mM DTT gave half-maximal and maximal inhibition, respectively. The effect of DTT was reversible; removal of the DTT-containing medium prior to heat shock rendered the cells fully responsive. Analysis of the effects of DTT on the regulation and function of HSF suggests that DTT blocked an early and important step in the activation process without having a direct effect on the HSF protein. Thus, DTT inhibited the heat-induced trimerization, phosphorylation, and nuclear translocation of HSF and was also effective against a number of other reagents that are known to activate HSF. On the other hand, DTT did not block the response induced by heat shock at 45 degrees C, and in vitro addition of DTT failed to modulate the DNA binding activity of activated HSF present in cell extracts, suggesting that the HSF protein itself is unlikely to be a direct target of action of DTT. These results, together with the observation that activation of HSF DNA binding activity was attenuated under an anoxic condition and that hydrogen peroxide mimicked the effects of heat shock, suggest the involvement of a redox mechanism as an early and important step in the heat shock signal transduction pathway.

Activation of Drosophila heat shock factor: conformational change associated with a monomer-to-trimer transition.

The induction of heat shock genes in eukaryotic cells is regulated by the transcription factor heat shock factor (HSF). Activation of HSF occurs at two independent levels, DNA binding and the acquisition of transcriptional competence. The binding of HSF to DNA is accomplished by a stress-induced oligomeric switch of HSF protein. We have defined the oligomeric state of the latent and induced forms of HSF by measuring the sedimentation coefficient and the Stokes radius of the protein in Drosophila cell extracts. Calculation of the native molecular mass indicates that the two forms of Drosophila HSF are best described as a monomer and trimer, respectively, of the 77-kDa HSF polypeptide. The monomeric and trimeric states of HSF were verified by chemical cross-linking experiments. The finding of a monomeric composition for the latent form of HSF is incompatible with speculative models which suggest that molecular chaperones such as hsp70 feed back to inhibit trimerization of HSF by forming a stable heteromeric complex. We also found that both HSF monomers and HSF trimers exhibit unusually high frictional ratios, indicating that they have asymmetric shapes. The degree of asymmetry is significantly greater for the HSF trimer, suggesting that the monomer undergoes a conformational change to a more extended structure upon trimerization. These findings are consistent with a model for the inert HSF protein that is based on a monomer constrained by intramolecular coiled-coil interactions between amino- and carboxy-terminal domains.

Activation of Drosophila Heat Shock Factor: Conformational Change Associated with a Monomer-to-Trimer Transition

The induction of heat shock genes in eukaryotic cells is regulated by the transcription factor heat shock factor (HSF). Activation of HSF occurs at two independent levels, DNA binding and the acquisition of transcriptional competence. The binding of HSF to DNA is accomplished by a stress-induced oligomeric switch of HSF protein. We have defined the oligomeric state of the latent and induced forms of HSF by measuring the sedimentation coefficient and the Stokes radius of the protein in Drosophila cell extracts. Calculation of the native molecular mass indicates that the two forms of Drosophila HSF are best described as a monomer and trimer, respectively, of the 77-kDa HSF polypeptide. The monomeric and trimeric states of HSF were verified by chemical cross-linking experiments. The finding of a monomeric composition for the latent form of HSF is incompatible with speculative models which suggest that molecular chaperones such as hsp70 feed back to inhibit trimerization of HSF by forming a stable heteromeric complex. We also found that both HSF monomers and HSF trimers exhibit unusually high frictional ratios, indicating that they have asymmetric shapes. The degree of asymmetry is significantly greater for the HSF trimer, suggesting that the monomer undergoes a conformational change to a more extended structure upon trimerization. These findings are consistent with a model for the inert HSF protein that is based on a monomer constrained by intramolecular coiled-coil interactions between amino- and carboxy-terminal domains.

Isolation of a cDNA for HSF2: evidence for two heat shock factor genes in humans.

The heat shock response is transcriptionally regulated by an evolutionarily conserved protein termed heat shock factor (HSF). We report the purification to homogeneity and the partial peptide sequence of HSF from HeLa cells. The peptide sequence was used to isolate a human cDNA with a predicted open reading frame that has homology to the DNA binding domains of both Saccharomyces cerevisiae and Drosophila HSFs. The cDNA directs the synthesis of a protein that binds to the heat shock element with specificity identical to HeLa HSF and stimulates transcription from a heat shock promoter. The expressed protein cross-reacts with anti-HSF antibodies. Surprisingly, however, this cDNA does not encode all of the peptides obtained from purified HeLa HSF. These peptides are encoded by a distinct human cDNA, HSF1, described by Rabindran et al. [Rabindran, S. K., Giorgi, G., Clos, J. & Wu, C. (1991) Proc. Natl. Acad. Sci. USA 88, 6906-6910.] It therefore appears that there is a human heat shock factor gene family and that at least two separate but related HSF proteins regulate the stress response in humans.

Heat shock factor and the heat shock response

Peter K. Sorger Department of Microbiology and Immunology University of California San Francisco, California 94143-0502 The induction of eukaryotic heat shock genes in response to a temperature upshift is mediated by the binding of a transcriptional activator, heat shock factor, to a short highly conserved DNA sequence known as the heat shock element. This review will discuss progress in several or- ganisms in characterizing heat shock factor and will sug- gest preliminary answers to the following questions. How is the DNA-binding activity and transcriptional potency of heat shock factor regulated? How is autoregulation of heat shock protein (hsp) synthesis achieved? What is the mech- anism by which heat shock is sensed? Heat Shock Elements and Their Interaction with Heat Shock Factor Heat shock elements are best described as contiguous arrays of variable numbers of the 5 bp sequence nGAAn arranged in alternating orientation (n denotes less strongly conserved nucleotides that nevertheless may be involved in important DNA-protein interactions; Figure 1; Xiao and Lis, 1988; Amin et al., 1988). At least two nGAAn units are needed for high affinity binding of heat shock factor in vitro, and these may be arranged either head-to-head (nGAAnnTTCn) or tail-to-tail (nTTCnnGAAn; Perisic et al., 1989). How can heat shock factor bind to these structurally distinct sites as well as to heat shock elements with larger numbers of 5 bp units? The answer may lie in the oligo- merit nature of the heat shock factor protein (Figure 2). Heat shock factor from both Saccharomyces cerevisiae (SC-HSF) and Drosophila (D-HSF) associates to form pro- tein trimers in solution and when bound to DNA (Perisic et al., 1989; Sorger and Nelson, 1989). It is not clear, how- ever, whether heat shock factor exists in vivo primarily as a trimeric, hexameric, or possibly even larger complex. Each subunit of a D-HSF multimer is thought to bind to a single nGAAn unit, and the binding to successive units is highly cooperative (Xiao et al., 1991). The coiled-coil structure that has been proposed to form the interface between heat shock factor monomers is inherently three- fold symmetric (see below). Thus, the binding of heat shock factor trimers to DNA (which is inherently two-fold symmetric) would require a flexible hinge between the tri- merization and DNA-binding surfaces of individual mono- mers. Flexibility at this hinge may also be exploited in the binding of subunits to differently oriented nGAAn units. The binding of trimers to adjacent sites is also highly cooperative (Shuey and Parker, 1986): in vitro, the dissoci- ation rate from DNA of a complex of two DNA-bound tri- mers is more than three orders of magnitude lower than that of a single trimer (Xiao et al., 1991). Thus, even when the molarity of sites exceeds that of the heat shock factor protein, heat shock factor preferentially forms large com-