Heat Shock Transcription Factor Genes Research Articles

Characterization of alternatively spliced transcripts encoding heat shock transcription factor in cultured cells of the cabbage armyworm, Mamestra brassicae

A gene encoding heat shock transcription factor (HSF) was cloned and sequenced from cultured cells of the cabbage armyworm, Mamestra brassicae. The cDNA potentially encoded a 699-aa protein, with a calculated molecular weight of 77.8 kDa. Deduced amino acid identities to HSFs from Aedes aegypti and Drosophila melanogaster were 36 and 34%, respectively. Analysis of the genomic DNA revealed eight exons and three optional exons: a, b, and c. Exon a contained a premature in-frame stop codon that would generate a truncated protein. When the cells were exposed to high temperature or cadmium, no particular alternative transcripts showed significant up- or down-regulated expression relative to the total amount of the transcripts. These results suggest that alternative splicing may not be a principal mechanism for regulation of M. brassicae HSF gene expression in response to heat shock and cadmium.

A novel HSF4gene mutation (p.R405X) causing autosomal recessive congenital cataracts in a large consanguineous family from Pakistan

BackgroundHereditary cataracts are most frequently inherited as autosomal dominant traits, but can also be inherited in an autosomal recessive or X-linked fashion. To date, 12 loci for autosomal recessive cataracts have been mapped including a locus on chromosome 16q22 containing the disease-causing gene HSF4 (Genbank accession number NM_001040667). Here, we describe a family from Pakistan with the first nonsense mutation in HSF4 thus expanding the mutational spectrum of this heat shock transcription factor gene.MethodsA large consanguineous Pakistani family with autosomal recessive cataracts was collected from Quetta. Genetic linkage analysis was performed for the common known autosomal recessive cataracts loci and linkage to a locus containing HSF4 (OMIM 602438) was found. All exons and adjacent splice sites of the heat shock transcription factor 4 gene (HSF4) were sequenced. A mutation-specific restriction enzyme digest (HphI) was performed for all family members and unrelated controls.ResultsThe disease phenotype perfectly co-segregated with markers flanking the known cataract gene HSF4, whereas other autosomal recessive loci were excluded. A maximum two-point LOD score with a Zmax = 5.6 at θ = 0 was obtained for D16S421. Direct sequencing of HSF4 revealed the nucleotide exchange c.1213C > T in this family predicting an arginine to stop codon exchange (p.R405X).ConclusionWe identified the first nonsense mutation (p.R405X) in exon 11 of HSF4 in a large consanguineous Pakistani family with autosomal recessive cataract.

Maturation of the nodule-specific transcript MsHSF1c in Medicago sativa may involve interallelic trans-splicing

In nonplant species, many heat-shock transcription factors (HSFs) undergo spatiotemporal-specific alternative splicing. However, little is known about the spatiotemporal-specific splicing of HSFs in plants. Previously, we reported that the alfalfa HSF gene MsHSF1 undergoes multiple alternative splicing events in various tissues. Here, we identified another spliced transcript isoform, MsHSF1c, containing a 177-base tandem repeat, and showed that the low-abundance MsHSF1c is a nodule-specific transcript of MsHSF1. We also found that MsHSF1 presents multiple alleles with single-base variations and the expression of MsHSF1 alleles has allele-specific differences in alfalfa nodules. Because single-base variations at position 1006 change the AT of MsHSF1b to GT in MsHSF1b-3, creating a pair of donor/acceptor sites with the AG of MsHSF1b/1b-1 at position 827–828 for pre-mRNA splicing, we suggest that MsHSF1c may be generated by trans-splicing between alleles MsHSF1b-3 and MsHSF1b or MsHSF1b-1. These results provide new insight into the role of tissue-specific contribution in the transcription of plant HSF genes.

Genome-wide analysis of heat shock transcription factor families in rice and Arabidopsis

The heat shock transcription factors (HSFs) are the major heat shock factors regulating the heat stress response. They participate in regulating the expression of heat shock proteins (HSPs), which are critical in the protection against stress damage and many other important biological processes. Study of the HSF gene family is important for understanding the mechanism by which plants respond to stress. The completed genome sequences of rice ( Oryza sativa) and Arabidopsis ( Arabidopsis thaliana) constitute a valuable resource for comparative genomic analysis, as they are representatives of the two major evolutionary lineages within the angiosperms: the monocotyledons and the dicotyledons. The identification of phylogenetic relationships among HSF proteins in these species is a fundamental step to unravel the functionality of new and yet uncharacterized genes belonging to this family. In this study, the full complement of HSF genes in rice and Arabidopsis has probably been identified through the genome-wide scan. Phylogenetic analyses resulted in the identification of three major clusters of orthologous genes that contain members belonging to both species, which must have been represented in their common ancestor before the taxonomic splitting of the angiosperms. Further analysis of the phylogenetic tree reveals a possible dicot specific gene group. We also identified nine pairs of paralogs, as evidence for studies on the evolution history of rice HSF family and rice genome evolution. Expression data analysis indicates that HSF proteins are widely expressed in plants. These results provide a solid base for future functional genomic studies of the HSF gene family in rice and Arabidopsis.

Cytosolic HSP90 Regulates the Heat Shock Response That Is Responsible for Heat Acclimation in Arabidopsis thaliana

Plant survival requires the ability to acclimate to heat. When plants are subjected to heat shock, the expression of various genes is induced, and the plants become tolerant of higher temperatures. We found that transient treatment with geldanamycin and radicicol, two heat shock protein 90 (HSP90) inhibitors, induced heat-inducible genes and heat acclimation in Arabidopsis thaliana seedlings. Heat shock reduced the activity of exogenously expressed glucocorticoid receptor (GR). Since GR activity depends on HSP90, this suggests that heat shock reduces cytosolic HSP90 activity in vivo. Microarray analysis revealed that many of the genes that are up-regulated by both heat shock and HSP90 inhibitors are involved in protein folding and degradation, suggesting that the activation of a protein maintenance system is a crucial part of this response. Most of these genes have heat shock response element-like motifs in their promoters, which suggests that heat shock transcription factor (HSF) is involved in the response to HSP90 inhibition. Several HSF genes are expressed constitutively in A. thaliana, including AtHsfA1d. Recombinant AtHsfA1d protein recognizes the heat shock response element motif and interacts with A. thaliana cytosolic HSP90, HSP90.2. Overexpression of a dominant negative form of HSP90.2 induced the heat-inducible gene. Thus, it appears that in the absence of heat shock, cytosolic HSP90 negatively regulates heat-inducible genes by actively suppressing HSF function. Upon heat shock, cytosolic HSP90 is transiently inactivated, which may lead to HSF activation.

Structure and alternative splicing of a heat shock transcription factor gene, MsHSF1, in Medicago sativa

Plant heat shock transcription factors (HSF) are highly complex. In this study, we identified an alfalfa HSF gene MsHSF1 that is composed of four exons and three introns in the encoding region. The intron1–exon2–intron2–exon3–intron3 as an intervening sequence was inserted at the conserved position that separates the coding region for the DNA-binding domain by single intron in other known plant HSF genes. Alternative splicing of MsHSF1 has generated five transcript isoforms. Spliced transcript MsHSF1b consisted of exon1 and exon4, encodes a class A1 HSF protein that can specifically bind to the heat shock elements in vitro. Other four spliced transcripts ( MsHSF1a-1 to 4) consist of exon1, part of the intervening sequence and exon4. These transcripts carry the premature termination codon and are low-abundant. Apparently these transcripts are the targets of nonsense-mediated mRNA decay (NMD). These results provide new insight into roles in the expression regulation of plant HSF genes.

STRESS RESPONSE SUPPRESSOR1 and STRESS RESPONSE SUPPRESSOR2, Two DEAD-Box RNA Helicases That Attenuate Arabidopsis Responses to Multiple Abiotic Stresses

Two genes encoding Arabidopsis (Arabidopsis thaliana) DEAD-box RNA helicases were identified in a functional genomics screen as being down-regulated by multiple abiotic stresses. Mutations in either gene caused increased tolerance to salt, osmotic, and heat stresses, suggesting that the helicases suppress responses to abiotic stress. The genes were therefore designated STRESS RESPONSE SUPPRESSOR1 (STRS1; At1g31970) and STRS2 (At5g08620). In the strs mutants, salt, osmotic, and cold stresses induced enhanced expression of genes encoding the transcriptional activators DREB1A/CBF3 and DREB2A and a downstream DREB target gene, RD29A. Under heat stress, the strs mutants exhibited enhanced expression of the heat shock transcription factor genes, HSF4 and HSF7, and the downstream gene HEAT SHOCK PROTEIN101. Germination of mutant seed was hyposensitive to the phytohormone abscisic acid (ABA), but mutants showed up-regulated expression of genes encoding ABA-dependent stress-responsive transcriptional activators and their downstream targets. In wild-type plants, STRS1 and STRS2 expression was rapidly down-regulated by salt, osmotic, and heat stress, but not cold stress. STRS expression was also reduced by ABA, but salt stress led to reduced STRS expression in both wild-type and ABA-deficient mutant plants. Taken together, our results suggest that STRS1 and STRS2 attenuate the expression of stress-responsive transcriptional activators and function in ABA-dependent and ABA-independent abiotic stress signaling networks.

Targeting the Heat Shock Factor 1 by RNA Interference: A Potent Tool to Enhance Hyperthermochemotherapy Efficacy in Cervical Cancer

Carcinoma of the uterine cervix is one of the highest causes of mortality in female cancer patients worldwide, and improved treatment options for this type of malignancy are highly needed. Local hyperthermia has been successfully used in combination with systemic administration of cisplatin-based chemotherapy in phase I/II clinical studies. Heat-induced expression of cytoprotective and antiapoptotic heat shock proteins (HSP) is a known complication of hyperthermia, resulting in thermotolerance and chemoresistance and hindering the efficacy of the combination therapy. Heat shock transcription factor 1 (HSF1) is the master regulator of heat-induced HSP expression. In the present report, we used small interfering RNA (siRNA) to silence HSF1 and to examine the effect of HSF1 loss of function on the response to hyperthermia and cisplatin-based chemotherapy in HeLa cervical carcinoma. We have identified the 322-nucleotide to 340-nucleotide HSF1 sequence as an ideal target for siRNA-mediated HSF1 silencing, have created a pSUPER-HSF1 vector able to potently suppress the HSF1 gene, and have generated for the first time human cancer cell lines with stable loss of HSF1 function. We report that, although it surprisingly does not affect cancer cell sensitivity to cisplatin or elevated temperatures up to 43 degrees C when administered separately, loss of HSF1 function causes a dramatic increase in sensitivity to hyperthermochemotherapy, leading to massive (>95%) apoptosis of cancer cells. These findings indicate that disruption of HSF1-induced cytoprotection during hyperthermochemotherapy may represent a powerful strategy to selectively amplify the damage in cancer cells and identify HSF1 as a promising therapeutic target in cervical carcinoma.

Identification and characterization of a novel heat shock transcription factor gene, GmHsfA1, in soybeans (Glycine max)

Plants have a large family of HSFs with different roles in the heat shock response that mediate the expression of HSP regulated genes. The HSF encoding genes are easily identified by their highly conserved modular structure and motifs. In the present study, a putative GmHsfA1 was identified and characterized from the soybean expressed sequence tag (EST) database by sequence comparison with the functionally well-characterized LpHsfA1 and rapid amplification of cDNA ends (RACE). Multiple alignment showed that the amino acid sequence of GmHSFA1, matching best with LpHSFA1 (52.2% similarity), was obviously different from that of each of several HSFA1s from other plant species. The GmHsfA1 has a constitutive expression profile in the different tissues examined. The overexpression of GmHsfA1 in transgenic soybean plants led to the activation of GmHsp70 under normal temperature and the overexpression of GmHsp70 under high temperature. Furthermore, transgenic soybean plants with GmHsfA1 overexpression showed obvious enhancement of thermotolerance under heat stress in comparison with non-transgenic plants. The experimental results suggested that GmHSFA1 is a novel and functional heat-shock transcription factor.

The role of heat shock transcription factor 1 in the genome-wide regulation of the mammalian heat shock response.

Previous work has implicated heat shock transcription factor 1 (HSF1) as the primary transcription factor responsible for the transcriptional response to heat stress in mammalian cells. We characterized the heat shock response of mammalian cells by measuring changes in transcript levels and assaying binding of HSF1 to promoter regions for candidate heat shock genes chosen by a combination of genome-wide computational and experimental methods. We found that many heat-inducible genes have HSF1 binding sites (heat shock elements, HSEs) in their promoters that are bound by HSF1. Surprisingly, for 24 heat-inducible genes, we detected no HSEs and no HSF1 binding. Furthermore, of 182 promoters with likely HSE sequences, we detected HSF1 binding at only 94 of these promoters. Also unexpectedly, we found 48 genes with HSEs in their promoters that are bound by HSF1 but that nevertheless did not show induction after heat shock in the cell types we examined. We also studied the transcriptional response to heat shock in fibroblasts from mice lacking the HSF1 gene. We found 36 genes in these cells that are induced by heat as well as they are in wild-type cells. These results provide evidence that HSF1 does not regulate the induction of every transcript that accumulates after heat shock, and our results suggest that an independent posttranscriptional mechanism regulates the accumulation of a significant number of transcripts.

Arabidopsis hot mutants define multiple functions required for acclimation to high temperatures.

Plants acquire thermotolerance to lethal high temperatures if first exposed to moderately high temperature or if temperature is increased gradually to an otherwise lethal temperature. We have taken a genetic approach to dissecting acquired thermotolerance by characterizing loss-of-function thermotolerance mutants in Arabidopsis. In previous work, we identified single recessive alleles of four loci required for thermotolerance of hypocotyl elongation, hot1-1, hot2-1, hot3-1, and hot4-1. Completed screening of M2 progeny from approximately 2500 M1 plants has now identified new alleles of three of these original loci, along with three new loci. The low mutant frequency suggests that a relatively small number of genes make a major contribution to this phenotype or that other thermotolerance genes encode essential or redundant functions. Further analysis of the original four loci was performed to define the nature of their thermotolerance defects. Although the HOT1 locus was shown previously to encode a major heat shock protein (Hsp), Hsp101, chromosomal map positions indicate that HOT2, 3, and 4 do not correspond to major Hsp or heat shock transcription factor genes. Measurement of thermotolerance at different growth stages reveals that the mutants have growth stage-specific heat sensitivity. Analysis of Hsp accumulation shows that hot2 and hot4 produce normal levels of Hsps, whereas hot3 shows reduced accumulation. Thermotolerance of luciferase activity and of ion leakage also varies in the mutants. These data provide the first direct genetic evidence, to our knowledge, that distinct functions, independent of Hsp synthesis, are required for thermotolerance, including protection of membrane integrity and recovery of protein activity/synthesis.

During ischemia-reperfusion in rat kidneys, heat shock response is not regulated by expressional changes of heat shock factor 1.

Ischemia-reperfusion injury is known to induce the inducible form of the 70 kDa heat shock protein HSP70i (or HSP72) mainly via rapid activation of heat shock transcription factor 1 (HSF1). However, little is known about the regulation of the HSF1 gene. We therefore studied the time course of HSF1 mRNA transcription and its relation to the expression pattern of the HSP70i mRNA in the renal cortex, this being the most vulnerable and functionally most important part of the kidney, after different periods of unilateral renal ischemia (10-180 min) and reperfusion (up to 60 min) in male Wistar rats (10 weeks old). Immediately after ischemia there was a significant induction of HSP70i genes. While HSP70i expression constantly increased (up to 4-fold) during reperfusion, even to a higher extent with prolongation of ischemia, HSF1 mRNA remained constitutively expressed under all conditions. Thus, we conclude that during ischemia-reperfusion in rat kidneys, the heat shock response is regulated by other means than expressional changes of HSF1.

During ischemia-reperfusion in rat kidneys, heat shock response is not regulated by expressional changes of heat shock factor 1

Abstract Ischemia-reperfusion injury is known to induce the inducible form of the 70 kDa heat shock protein HSP70i (or HSP72) mainly via rapid activation of heat shock transcription factor 1 (HSF1). However, little is known about the regulation of the HSF1 gene. We therefore studied the time course of HSF1 mRNA transcription and its relation to the expression pattern of the HSP70i mRNA in the renal cortex, this being the most vulnerable and functionally most important part of the kidney, after different periods of unilateral renal ischemia (10–180 min) and reperfusion (up to 60 min) in male Wistar rats (10 weeks old). Immediately after ischemia there was a significant induction of HSP70i genes. While HSP70i expression constantly increased (up to 4-fold) during reperfusion, even to a higher extent with prolongation of ischemia, HSF1 mRNA remained constitutively expressed under all conditions. Thus, we conclude that during ischemia-reperfusion in rat kidneys, the heat shock response is regulated by other means than expressional changes of HSF1.

A heat shock transcription factor in pea is differentially controlled by heat and virus replication.

Since some heat-inducible genes [heat shock (hs) genes] can be induced by virus infection in pea [e.g. Hsp70; Aranda et al. 1996, Proc. Natl Acad. Sci. USA 93, 15289-15293], we have investigated the effect that heat and virus replication may have on the expression of a heat-shock transcription factor gene (Hsf). We have characterized what appears to be the only member of the Hsf family in pea, PsHsfA. Similar to Hsp70, PsHsfA is heat-inducible in vegetative and embryonic tissues, which is concordant with the presence of heat shock elements (HSEs) and stress responsive elements (STREs) on its promoter sequence. The expression of PsHsfA during virus replication was studied in pea cotyledons and leaves, and compared to that of Hsp70. In situ hybridization experiments showed that whereas Hsp70 is induced, there is no detectable increased accumulation of PsHsfA RNA associated with the replication of pea seed-borne mosaic potyvirus (PSbMV). These experiments indicate that there is a selective control of virus-induced hs gene expression, and suggest that different regulatory pathways control hs gene expression during heat shock and virus replication.

Activation of heat shock transcription factor 1 to a DNA binding form during the G(1)phase of the cell cycle.

The heat shock transcription factor (HSF) genes encode proteins that bind to the heat shock elements (HSE) of stress-inducible genes. We have observed the induction of HSF1, the ubiquitous member of the HSF family from a latent cytoplasmic state to a form competent to bind HSE during early G(1)in HeLa cells in the absence of stress. The induction of DNA-binding HSF1 coincided with a burst in cellular protein synthesis in early G(1)and inhibition of this translational peak prevented the formation of DNA binding-activated HSF1. A potential role for HSF1 in cell cycle regulation was suggested by the finding that cell lines stably overexpressing HSF1 showed an increased proportion of G(1)cells relative to other cell cycle phases. However, in contrast to the effects of heat shock, entry into G(1)did not lead to HSF1 hyperphosphorylation or increased activity of a heat shock promoter-reporter gene and did not cause the induction of heat shock protein 70 expression. Thus HSF1, previously implicated in the heat shock response is activated to a DNA binding from in G(1)under non-stress conditions and may play a role in G(1)regulation that does not involve the transcription of heat shock genes.

Heat-Induced Degradation of Per and Tim in Drosophila Bearing a Conditional Allele of the Heat Shock Transcription Factor Gene

Heat pulses elicit dramatic and rapid decreases in the levels of the D. melanogaster period (per) and timeless (tim) proteins (i.e., PER and TIM). To investigate the possible role of the heat shock pathway in this response, we used Drosophila bearing a conditional allele of the hsf gene (termed hsf4), which encodes the heat shock transcription factor (HSF). At all times in a daily cycle, heat-induced decreases in the levels of PER and TIM were similar in wild-type and hsf4 mutant flies. The results strongly suggest that the heat shock pathway contributes little, if any, to the response of the Drosophila circadian clock to heat signals.

Structural Organization and Promoter Analysis of Murine Heat Shock Transcription Factor-1 Gene

Heat shock factor-1 (HSF-1) activates transcription of heat shock proteins in eukaryotes. Several overlapping genomic clones containing the murine HSF-1 gene were isolated from a phage genomic library. Results indicate that the HSF-1 gene contains 13 exons that span at least 30 kilobase pairs. Sequence analysis of the 5'-untranslated region of HSF-1 suggests that it contains sequences of a recently described Bop1 gene in reverse orientation within its first 331 base pairs (bp) upstream of the translation initiation site. The minimal promoter sequence required for HSF-1 basal expression was identified by deletion analysis from -4 kilobase pairs to -331 bp of the promoter fused to a luciferase reporter gene using transient transfection assays. Results indicate that 331 bp upstream of the HSF-1 translation start site is required for maximal basal expression in NIH3T3 and F9 cells. This fragment also results in high levels of luciferase activity in the reverse orientation, that is, 5' to the Bop1 gene, suggesting that this segment is bidirectional and could be utilized for basal expression of both HSF-1 and Bop1 genes. This segment of the promoter contains recognition elements for Sp1 and CCAAT-box binding transcription factors, which when mutated in either sense or antisense orientations to the HSF-1 gene results in a reduction of basal expression by 50-75% relative to wild type, suggesting that these sites are critical for basal expression of both HSF-1 and Bop1 genes.

Different forms of the mRNA encoding the heat-shock transcription factor are expressed during the life cycle of the parasitic helminth Schistosoma mansoni.

Several cDNAs and a gene encoding the heat-shock transcription factor (HSF) of schistosome were cloned, and multiple forms of the mRNA were found at different developmental stages of the parasite. The encoded protein contained a DNA-binding domain with expected sequence identity (39-58%) to other HSF molecules, and two leucine zipper motifs (LZ123 and LZ4) involved in the oligomerization of HSF. Adult worms express three isoforms of HSF mRNA generated by alternative splicing inside the coding region that contains in-frame splice signals. Introns are not involved in the process since the deleted segments (36 bp or 45 bp) are not flanked by any intron in the gene. Structural variations generated by alternative splicing (insertion of 3 amino acids or 15 amino acids) are continual with LZ4 and added hydrophobic residues are in register with the hydrophobic heptad repeats of LZ4. Structural diversity at the C-terminus of LZ4 may affect the strength of LZ4 interaction with the oligomerization domain (LZ123) and thus modulate the DNA-binding activity of HSF. The conservation of this mechanism in mouse and schistosome may reflect evolutionary pressure to generate multiple HSF species exhibiting functional diversity and capable of responding to different stress signals and physiological signals. Adult worms express HSF mRNA of 2.5 kb, in agreement with the size of the cDNA, while cercariae (developmental stage preceding adult worm) show multiple bands in the range 2.5-3 kb. Available data indicate that the HSF mRNAs of cercariae are inactive. We propose that these mRNA species are generated by an alternative splicing that incorporates introns, which inactivate the mRNA by the insertion of termination codons and/or by shifting of the reading frame. Parasite HSF protein produced in bacteria showed DNA sequence recognition similar to that of HSF in parasite extracts, i.e. the recombinant HSF reacted better with a variant heat-shock element (HSE; one base change in the third NGAAN pentamer of the ideal HSE consensus sequence) than with the ideal HSE. The size of the HSF gene is 12 kb and it is composed of ten exons and nine introns. Excluding the introns, the gene and cDNA show 100% sequence identity. A plant HSF gene contains only a single intron, which matches with the position of intron I2 of schistosome. That the position of this intron is conserved in remote species is indicative of an important function during evolution of the HSF gene.

Targeted Disruption of Heat Shock Transcription Factor 1 Abolishes Thermotolerance and Protection against Heat-inducible Apoptosis

Heat shock transcription factor 1 (HSF1) is a member of the vertebrate HSF family that regulates stress-inducible synthesis of heat shock proteins (HSPs). Although the synthesis of the constitutively expressed and inducible members of the heat shock family of stress proteins correlates with increased cellular protection, their relative contributions in acquired cellular resistance or "thermotolerance" in mammalian cells is presently unknown. We report here that constitutive expression of multiple HSPs in cultured embryonic cells was unaffected by disruption of the murine HSF1 gene. In contrast, thermotolerance was not attainable in hsf1(-/-) cells, and this response was required for protection against heat-induced apoptosis. We conclude that 1) constitutive and inducibly expressed HSPs exhibit distinct physiological functions for cellular maintenance and adaptation, respectively, and 2) other mammalian HSFs or distinct evolutionarily conserved stress response pathways do not compensate for HSF1 in the physiological response to heat shock.

HSF4, a new member of the human heat shock factor family which lacks properties of a transcriptional activator.

Heat shock transcription factors (HSFs) mediate the inducible transcriptional response of genes that encode heat shock proteins and molecular chaperones. In vertebrates, three related HSF genes (HSF1 to -3) and the respective gene products (HSFs) have been characterized. We report the cloning and characterization of human HSF4 (hHSF4), a novel member of the hHSF family that shares properties with other members of the HSF family yet appears to be functionally distinct. hHSF4 lacks the carboxyl-terminal hydrophobic repeat which is shared among all vertebrate HSFs and has been suggested to be involved in the negative regulation of DNA binding activity. hHSF4 is preferentially expressed in the human heart, brain, skeletal muscle, and pancreas. Transient transfection of hHSF4 in HeLa cells, which do not express hHSF4, results in a constitutively active DNA binding trimer which, unlike other members of the HSF family, lacks the properties of a transcriptional activator. Constitutive overexpression of hHSF4 in HeLa cells results in reduced expression of the endogenous hsp70, hsp90, and hsp27 genes. hHSF4 represents a novel hHSF that exhibits tissue-specific expression and functions to repress the expression of genes encoding heat shock proteins and molecular chaperones.