Heat stress transcription factors (Hsfs) are the central regulators of defense response to heat stress. We identified a total of 25 rice Hsf genes by genome-wide analysis of rice (Oryza sativa L.) genome, including the subspecies of O. japonica and O. indica. Proteins encoded by OsHsfs were divided into three classes according to their structures. Digital Northern analysis showed that OsHsfs were expressed constitutively. The expressions of these OsHsfs in response to heat stress and oxidative stress differed among the members of the gene family. Promoter analysis identified a number of stress-related cis-elements in the promoter regions of these OsHsfs. No significant correlation, however, was found between the heat-shock responses of genes and their cis-elements. Overall, our results provide a foundation for future research of OsHsfs function.
Heat shock; Transcription factors; Rice; Protein structure; Expression analysis
Two members of the heat shock transcription factor (HSF) family, HSF1 and HSF2, both function as transcriptional activators of heat shock gene expression. However, the inducible DNA-binding activities of these two factors are regulated by distinct pathways. HSF1 is activated by heat shock and other forms of stress, whereas HSF2 is activated during hemin-induced differentiation of human K562 erythroleukemia cells, suggesting a role for HSF2 in regulating heat shock gene expression under nonstress conditions such as differentiation and development. To understand the distinct regulatory pathways controlling HSF2 and HSF1 activities, we have examined the biochemical and physical properties of the control and activated states of HSF2 and compared these with the properties of HSF1. Our results reveal that the inactive, non-DNA-binding forms of HSF2 and HSF1 exist primarily in the cytoplasm of untreated K562 cells as a dimer and monomer, respectively. This difference in the control oligomeric states suggests that the mechanisms used to control the DNA-binding activities of HSF2 and HSF1 are distinct. Upon activation, both factors acquire DNA-binding activity, oligomerize to a trimeric state, and translocate into the nucleus. Interestingly, we find that simultaneous activation of both HSF2 and HSF1 in K562 cells subjected to hemin treatment followed by heat shock results in the synergistic induction of hsp70 gene transcription, suggesting a novel level of complex regulation of heat shock gene expression.
Organisms respond to circumstances threatening the cellular protein homeostasis by activation of heat-shock transcription factors (HSFs), which play important roles in stress resistance, development, and longevity. Of the four HSFs in vertebrates (HSF1-4), HSF1 is activated by stress, whereas HSF2 lacks intrinsic stress responsiveness. The mechanism by which HSF2 is recruited to stress-inducible promoters and how HSF2 is activated is not known. However, changes in the HSF2 expression occur, coinciding with the functions of HSF2 in development. Here, we demonstrate that HSF1 and HSF2 form heterotrimers when bound to satellite III DNA in nuclear stress bodies, subnuclear structures in which HSF1 induces transcription. By depleting HSF2, we show that HSF1-HSF2 heterotrimerization is a mechanism regulating transcription. Upon stress, HSF2 DNA binding is HSF1 dependent. Intriguingly, when the elevated expression of HSF2 during development is mimicked, HSF2 binds to DNA and becomes transcriptionally competent. HSF2 activation leads to activation of also HSF1, revealing a functional interdependency that is mediated through the conserved trimerization domains of these factors. We propose that heterotrimerization of HSF1 and HSF2 integrates transcriptional activation in response to distinct stress and developmental stimuli.
The heat shock response of Arabidopsis thaliana is dependent upon a complex regulatory network involving twenty-one known transcription factors and four heat shock protein families. It is known that heat shock proteins (Hsps) and transcription factors (Hsfs) are involved in cellular response to various forms of stress besides heat. However, the role of Hsps and Hsfs under cold and non-thermal stress conditions is not well understood, and it is unclear which types of stress interact least and most strongly with Hsp and Hsf response pathways. To address this issue, we have analyzed transcriptional response profiles of Arabidopsis Hsfs and Hsps to a range of abiotic and biotic stress treatments (heat, cold, osmotic stress, salt, drought, genotoxic stress, ultraviolet light, oxidative stress, wounding, and pathogen infection) in both above and below-ground plant tissues.
All stress treatments interact with Hsf and Hsp response pathways to varying extents, suggesting considerable cross-talk between heat and non-heat stress regulatory networks. In general, Hsf and Hsp expression was strongly induced by heat, cold, salt, and osmotic stress, while other types of stress exhibited family or tissue-specific response patterns. With respect to the Hsp20 protein family, for instance, large expression responses occurred under all types of stress, with striking similarity among expression response profiles. Several genes belonging to the Hsp20, Hsp70 and Hsp100 families were specifically upregulated twelve hours after wounding in root tissue, and exhibited a parallel expression response pattern during recovery from heat stress. Among all Hsf and Hsp families, large expression responses occurred under ultraviolet-B light stress in aerial tissue (shoots) but not subterranean tissue (roots).
Our findings show that Hsf and Hsp family member genes represent an interaction point between multiple stress response pathways, and therefore warrant functional analysis under conditions apart from heat shock treatment. In addition, our analysis revealed several family and tissue-specific heat shock gene expression patterns that have not been previously described. These results have implications regarding the molecular basis of cross-tolerance in plant species, and raise new questions to be pursued in future experimental studies of the Arabidopsis heat shock response network.
HSF2 regulates proteostasis capacity against febrile-range thermal stress, which provides temperature-dependent mechanisms of cellular adaptation to thermal stress. Furthermore, HSF2 has a strong impact on disease progression of Huntington's disease R6/2 mice, suggesting that it could be a promising therapeutic target for protein misfolding diseases.
Heat shock response is characterized by the induction of heat shock proteins (HSPs), which facilitate protein folding, and non-HSP proteins with diverse functions, including protein degradation, and is regulated by heat shock factors (HSFs). HSF1 is a master regulator of HSP expression during heat shock in mammals, as is HSF3 in avians. HSF2 plays roles in development of the brain and reproductive organs. However, the fundamental roles of HSF2 in vertebrate cells have not been identified. Here we find that vertebrate HSF2 is activated during heat shock in the physiological range. HSF2 deficiency reduces threshold for chicken HSF3 or mouse HSF1 activation, resulting in increased HSP expression during mild heat shock. HSF2-null cells are more sensitive to sustained mild heat shock than wild-type cells, associated with the accumulation of ubiquitylated misfolded proteins. Furthermore, loss of HSF2 function increases the accumulation of aggregated polyglutamine protein and shortens the lifespan of R6/2 Huntington's disease mice, partly through αB-crystallin expression. These results identify HSF2 as a major regulator of proteostasis capacity against febrile-range thermal stress and suggest that HSF2 could be a promising therapeutic target for protein-misfolding diseases.
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.
Appropriate responses of organisms to heat stress are essential for their survival. In eukaryotes, adaptation to high temperatures is mediated by heat shock transcription factors (HSFs). HSFs regulate the expression of heat shock proteins, which function as molecular chaperones assisting in protein folding and stability. In many model organisms a great deal is known about the products of hsf genes. An important exception is the filamentous fungus and model eukaryote Neurospora crassa. Here we show that two Neurospora crassa genes whose protein products share similarity to known HSFs play different biological roles. We report that heat shock factor 1 (hsf1) is an essential gene and that hsf2 is required for asexual development. Conidiation may be blocked in the hsf2 knockout (hsf2KO) strain because HSF2 is an integral element of the conidiation pathway or because it affects the availability of protein chaperones. We report that genes expressed during conidiation, for example fluffy, conidiation-10, and repressor of conidiation-1 show wild-type levels of expression in a hsf2KO strain. However, consistent with the lack of macroconidium development, levels of eas are much reduced. Cultures of the hsf2KO strain along with two other aconidial strains, the fluffy and aconidial-2 strains, took longer than the wild type to recover from heat shock. Altered expression profiles of hsp90 and a putative hsp90-associated protein in the hsf2KO strain after exposure to heat shock may in part account for its reduced ability to cope with heat stress.
The cellular heat stress response is well studied in Drosophila in respect to the role of heat shock proteins (Hsp). Hsps are molecular chaperones, highly expressed during and after exposure to numerous stress types. Hsps are all regulated by a common transcription factor, the heat shock factor (HSF), and it is known that HSF is controlling other, so far uncharacterised, heat-responsive genes. In this study, we investigate whether novel candidate genes for heat resistance, identified by microarray experiments, are regulated by HSF. The microarray experiments recently identified several strongly upregulated genes in response to a short, non-lethal heat treatment in Drosophila melanogaster. To test whether or not a subset of these genes are HSF-induced, we studied 11 currently unannotated genes using quantitative polymerase chain reaction on HSF mutant flies with a non-functional HSF at elevated temperatures. We found indication of HSF regulation in most of the studied genes, suggesting a role of these unknown genes in heat tolerance. Surprisingly, some of the genes seemed to be upregulated independent of HSF function. The high induction in response to heat, which mimics the expression profile of Hsps, implies a role in the cellular heat response of these genes as well.
Heat and other environmental insults (stress) cause unfolding of proteins, triggering the activation of heat shock transcription factor HSF (HSF 1 in vertebrates) that, in higher eukaryotes, involves trimerization of the factor and acquisition of heat shock element (HSE) DNA-binding ability. Interaction of activated HSF1 with HSEs in promoters of genes encoding heat shock proteins (Hsps) enhances their expression. It was suggested that Hsp70 may function as the negative regulator of HSF1. In the simplest model, stress-unfolded proteins would complete with monomeric HSF1 for Hsp70 binding. This competetion would result in dissociation of an HSF1-Hsp70 complex, allowing trimerization of released HSF 1 monomers. In support of this model, we present evidence herein that 1) non-activated HSF1 forms a 1:1 complex with Hsp70, 2) both rates of heat-induced appearance of HSF1 oligomers and rates of disappearance of HSF1 heterodimers and monomers decrease when concentrations of unengaged Hsps are increased, and 3) transient overexpression of Hsp70 inhibits heat activation of HSF1.
Heat shock transcriptional factors (Hsfs) play a crucial role in plant responses to biotic and abiotic stress conditions and in plant growth and development. Apple (Malus domestica Borkh) is an economically important fruit tree whose genome has been fully sequenced. So far, no detailed characterization of the Hsf gene family is available for this crop plant.
A genome-wide analysis was carried out in Malus domestica to identify heat shock transcriptional factor (Hsf) genes, named MdHsfs. Twenty five MdHsfs were identified and classified in three main groups (class A, B and C) according to the structural characteristics and to the phylogenetic comparison with Arabidopsis thaliana and Populus trichocarpa. Chromosomal duplications were analyzed and segmental duplications were shown to have occurred more frequently in the expansion of Hsf genes in the apple genome. Furthermore, MdHsfs transcripts were detected in several apple organs, and expression changes were observed by quantitative real-time PCR (qRT-PCR) analysis in developing flowers and fruits as well as in leaves, harvested from trees grown in the field and exposed to the naturally increased temperatures.
The apple genome comprises 25 full length Hsf genes. The data obtained from this investigation contribute to a better understanding of the complexity of the Hsf gene family in apple, and provide the basis for further studies to dissect Hsf function during development as well as in response to environmental stimuli.
Hsf; Malus domestica; Gene expression; High temperature; Apple fruit/ flower
Sequencing of the Arabidopsis genome revealed a unique complexity of the plant heat stress transcription factor (Hsf) family. By structural characteristics and phylogenetic comparison, the 21 representatives are assigned to 3 classes and 14 groups. Particularly striking is the finding of a new class of Hsfs (AtHsfC1) closely related to Hsf1 from rice and to Hsfs identified from frequently found expressed sequence tags of tomato, potato, barley, and soybean. Evidently, this new type of Hsf is well expressed in different plant tissues. Besides the DNA binding and oligomerization domains (HR-A/B region), we identified other functional modules of Arabidopsis Hsfs by sequence comparison with the well-characterized tomato Hsfs. These are putative motifs for nuclear import and export and transcriptional activation (AHA motifs). There is intriguing flexibility of size and sequence in certain parts of the otherwise strongly conserved N-terminal half of these Hsfs. We have speculated about possible exon-intron borders in this region in the ancient precursor gene of plant Hsfs, similar to the exon-intron structure of the present mammalian Hsf-encoding genes.
Heat shock response in eukaryotes is transcriptionally regulated by conserved heat shock transcription factors (Hsfs). Hsf genes are represented by a large multigene family in plants and investigation of the Hsf gene family will serve to elucidate the mechanisms by which plants respond to stress. In recent years, reports of genome-wide structural and evolutionary analysis of the entire Hsf gene family have been generated in two model plant systems, Arabidopsis and rice. Maize, an important cereal crop, has represented a model plant for genetics and evolutionary research. Although some Hsf genes have been characterized in maize, analysis of the entire Hsf gene family were not completed following Maize (B73) Genome Sequencing Project.
A genome-wide analysis was carried out in the present study to identify all Hsfs maize genes. Due to the availability of complete maize genome sequences, 25 nonredundant Hsf genes, named ZmHsfs were identified. Chromosomal location, protein domain and motif organization of ZmHsfs were analyzed in maize genome. The phylogenetic relationships, gene duplications and expression profiles of ZmHsf genes were also presented in this study. Twenty-five ZmHsfs were classified into three major classes (class A, B, and C) according to their structural characteristics and phylogenetic comparisons, and class A was further subdivided into 10 subclasses. Moreover, phylogenetic analysis indicated that the orthologs from the three species (maize, Arabidopsis and rice) were distributed in all three classes, it also revealed diverse Hsf gene family expression patterns in classes and subclasses. Chromosomal/segmental duplications played a key role in Hsf gene family expansion in maize by investigation of gene duplication events. Furthermore, the transcripts of 25 ZmHsf genes were detected in the leaves by heat shock using quantitative real-time PCR. The result demonstrated that ZmHsf genes exhibit different expression levels in heat stress treatment.
Overall, data obtained from our investigation contributes to a better understanding of the complexity of the maize Hsf gene family and provides the first step towards directing future experimentation designed to perform systematic analysis of the functions of the Hsf gene family.
The heat-shock response, a fundamental defense mechanism against proteotoxic stress, is regulated by a family of heat-shock transcription factors (HSF). In humans HSF1 is considered the central regulator of heat-induced transcriptional responses. The main targets for HSF1 are specific promoter elements (HSE) located upstream of heat-shock genes encoding cytoprotective heat-shock proteins (HSP) with chaperone function. In addition to its cytoprotective function, HSF1 was recently hypothesized to play a more complex role, regulating the expression of non-HSP genes; however, the non-canonical role of HSF1 is still poorly understood. Herein we report that heat-stress promotes the expression of cyclooxygenase-2 (COX-2), a key regulator of inflammation controlling prostanoid and thromboxane synthesis, resulting in the production of high levels of prostaglandin-E2 in human cells. We show that heat-induced COX-2 expression is regulated at the transcriptional level via HSF1-mediated signaling and identify, by in-vitro reporter gene activity assay and deletion-mutant constructs analysis, the COX-2 heat-responsive promoter region and a new distal cis-acting HSE located at position −2495 from the transcription start site. As shown by ChIP analysis, HSF1 is recruited to the COX-2 promoter rapidly after heat treatment; by using shRNA-mediated HSF1 suppression and HSE-deletion from the COX-2 promoter, we demonstrate that HSF1 plays a central role in the transcriptional control of COX-2 by heat. Finally, COX-2 transcription is also induced at febrile temperatures in endothelial cells, suggesting that HSF1-dependent COX-2 expression could contribute to increasing blood prostaglandin levels during fever. The results identify COX-2 as a human non-classical heat-responsive gene, unveiling a new aspect of HSF1 function.
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.
Plant heat stress transcription factors (Hsfs) are the critical components involved in mediating responses to various environmental stressors. However, the detailed roles of many plant Hsfs are far from fully understood. In this study, an Hsf (SlHsfA3) was isolated from the cultivated tomato (Solanum lycopersicum, Sl) and functionally characterized at the genetic and developmental levels. The nucleus-localized SlHsfA3 was basally and ubiquitously expressed in different plant organs. The expression of SlHsfA3 was induced dramatically by heat stress, moderately by high salinity, and slightly by drought, but was not induced by abscisic acid (ABA). The ectopic overexpression of SlHsfA3 conferred increased thermotolerance and late flowering phenotype to transgenic Arabidopsis plants. Moreover, SlHsfA3 played a negative role in controlling seed germination under salt stress. RNA-sequencing data demonstrated that a number of heat shock proteins (Hsps) and stress-associated genes were induced in Arabidopsis plants overexpressing SlHsfA3. A gel shift experiment and transient expression assays in Nicotiana benthamiana leaves demonstrated that SlHsfA3 directly activates the expression of SlHsp26.1-P and SlHsp21.5-ER. Taken together, our results suggest that SlHsfA3 behaves as a typical Hsf to contribute to plant thermotolerance. The late flowering and seed germination phenotypes and the RNA-seq data derived from SlHsfA3 overexpression lines lend more credence to the hypothesis that plant Hsfs participate in diverse physiological and biochemical processes related to adverse conditions.
The heat shock transcription factor Hsf1 of the yeast Saccharomyces cerevisiae regulates expression of genes encoding heat shock proteins and a variety of other proteins as well. To better understand the cellular roles of Hsf1, we screened multicopy suppressor genes of a temperature-sensitive hsf1 mutation. The RIM15 gene, encoding a protein kinase that is negatively regulated by the cyclic AMP-dependent protein kinase, was identified as a suppressor, but Rim15-regulated stress-responsive transcription factors, such as Msn2, Msn4, and Gis1, were unable to rescue the temperature-sensitive growth phenotype of the hsf1 mutant. Another class of suppressors encoded cell wall stress sensors, Wsc1, Wsc2, and Mid2, and the GDP/GTP exchange factor Rom2 that interacts with these cell wall sensors. Activation of a protein kinase, Pkc1, which is induced by these cell wall sensor proteins upon heat shock, but not activation of the Pkc1-regulated mitogen-activated protein kinase cascade, was necessary for the hsf1 suppression. Like Wsc-Pkc1 pathway mutants, hsf1 cells exhibited an osmotic remedial cell lysis phenotype at elevated temperatures. Several of the other suppressors were found to encode proteins functioning in cell wall organization. These results suggest that Hsf1 in concert with Pkc1 regulates cell wall remodeling in response to heat shock.
Vertebrate cells express a family of heat shock transcription factors (HSF1 to HSF4) that coordinate the inducible regulation of heat shock genes in response to diverse signals. HSF1 is potent and activated rapidly though transiently by heat shock, whereas HSF2 is a less active transcriptional regulator but can retain its DNA binding properties for extended periods. Consequently, the differential activation of HSF1 and HSF2 by various stresses may be critical for cells to survive repeated and diverse stress challenges and to provide a mechanism for more precise regulation of heat shock gene expression. Here we show, using a novel DNA binding and detection assay, that HSF1 and HSF2 are coactivated to different levels in response to a range of conditions that cause cell stress. Above a low basal activity of both HSFs, heat shock preferentially activates HSF1, whereas the amino acid analogue azetidine or the proteasome inhibitor MG132 coactivates both HSFs to different levels and hemin preferentially induces HSF2. Unexpectedly, we also found that heat shock has dramatic adverse effects on HSF2 that lead to its reversible inactivation coincident with relocalization from the nucleus. The reversible inactivation of HSF2 is specific to heat shock and does not occur with other stressors or in cells expressing high levels of heat shock proteins. These results reveal that HSF2 activity is negatively regulated by heat and suggest a role for heat shock proteins in the positive regulation of HSF2.
In order to assess the functional roles of heat stress-induced class B-heat shock factors in Arabidopsis, we investigated T-DNA knockout mutants of AtHsfB1 and AtHsfB2b. Micorarray analysis of double knockout hsfB1/hsfB2b plants revealed as strong an up-regulation of the basal mRNA-levels of the defensin genes Pdf1.2a/b in mutant plants. The Pdf expression was further enhanced by jasmonic acid treatment or infection with the necrotrophic fungus Alternaria brassicicola. The single mutant hsfB2b and the double mutant hsfB1/B2b were significantly improved in disease resistance after A. brassicicola infection. There was no indication for a direct interaction of Hsf with the promoter of Pdf1.2, which is devoid of perfect HSE consensus Hsf-binding sequences. However, changes in the formation of late HsfA2-dependent HSE binding were detected in hsfB1/B2b plants. This suggests that HsfB1/B2b may interact with class A-Hsf in regulating the shut-off of the heat shock response. The identification of Pdf genes as targets of Hsf-dependent negative regulation is the first evidence for an interconnection of Hsf in the regulation of biotic and abiotic responses.
Abiotic/environmental stress; transcriptional control and transcription factors; transcriptome analysis; defense responses; disease resistance; Arabidopsis
Chaperone synthesis in response to proteotoxic stress is dependent on a family of transcription factors named heat shock factors (HSFs). The two main factors in this family, HSF1 and HSF2, are co-expressed in numerous tissues where they can interact and form heterotrimers in response to proteasome inhibition. HSF1 and HSF2 exhibit two alternative splicing isoforms, called α and β, which contribute to additional complexity in HSF transcriptional regulation, but remain poorly examined in the literature. In this work, we studied the transcriptional activity of HSF1 and HSF2 splicing isoforms transfected into immortalized Mouse Embryonic Fibroblasts (iMEFs) deleted for both Hsf1 and Hsf2, under normal conditions and after proteasome inhibition. We found that HSF1α is significantly more active than the β isoform after exposure to the proteasome inhibitor MG132. Furthermore, we clearly established that, while HSF2 had no transcriptional activity by itself, short β isoform of HSF2 exerts a negative role on HSF1β-dependent transactivation. To further assess the impact of HSF2β inhibition on HSF1 activity, we developed a mathematical modelling approach which revealed that the balance between each HSF isoform in the cell regulated the strength of the transcriptional response. Moreover, we found that cellular stress such as proteasome inhibition could regulate the splicing of Hsf2 mRNA. All together, our results suggest that relative amounts of each HSF1 and HSF2 isoforms quantitatively determine the cellular level of the proteotoxic stress response.
Inhibition of proteasome-mediated protein degradation machinery is a potent stress stimulus that causes accumulation of ubiquitinated proteins and increased expression of heat shock proteins (Hsps). Hsps play pivotal roles in homeostasis and protection in a cell, through their well-recognized properties as molecular chaperones. The inducible Hsp expression is regulated by the heat shock transcription factors (HSFs). Among mammalian HSFs, HSF1 has been shown to be important for regulation of the heat-induced stress gene expression, whereas the function of HSF2 in stress response is unclear. Recent reports have suggested that both HSF1 and HSF2 are affected during down-regulation of ubiquitin-proteasome pathway (Y. Kawazoe et al., Eur. J. Biochem. 255:356–362, 1998; A. Mathew et al., Mol. Cell. Biol. 18:5091–5098, 1998; D. Kim et al., Biochem. Biophys. Res. Commun. 254:264–268, 1999). To date, however, no unambiguous evidence has been presented as to whether a single specific HSF or multiple members of the HSF family are required for transcriptional induction of heat shock genes when proteasome activity is down-regulated. Therefore, by using loss-of-function and gain-of-function strategies, we investigated the specific roles of mammalian HSFs in regulation of the ubiquitin-proteasome-mediated stress response. Here we demonstrate that HSF1, but not HSF2, is essential and sufficient for up-regulation of Hsp70 expression during down-regulation of the ubiquitin proteolytic pathway. We propose that specificity of HSF1 could be an important therapeutic target during disease pathogenesis associated with abnormal ubiquitin-dependent proteasome function.
The yeast heat shock transcription factor (HSF) is regulated by posttranslational modification. Heat and superoxide can induce the conformational change associated with the heat shock response. Interaction between HSF and the chaperone hsp70 is also thought to play a role in HSF regulation. Here, we show that the Ssb1/2p member of the hsp70 family can form a stable, ATP-sensitive complex with HSF—a surprising finding because Ssb1/2p is not induced by heat shock. Phosphorylation and the assembly of HSF into larger, ATP-sensitive complexes both occur when HSF activity decreases, whether during adaptation to a raised temperature or during growth at low glucose concentrations. These larger HSF complexes also form during recovery from heat shock. However, if HSF is assembled into ATP-sensitive complexes (during growth at a low glucose concentration), heat shock does not stimulate the dissociation of the complexes. Nor does induction of the conformational change induce their dissociation. Modulation of the in vivo concentrations of the SSA and SSB proteins by deletion or overexpression affects HSF activity in a manner that is consistent with these findings and suggests the model that the SSA and SSB proteins perform distinct roles in the regulation of HSF activity.
Heat shock factor 1 (HSF1) is an important transcription factor in cellular stress responses, cancer, aging, and developmental processes including gametogenesis. Disruption of Hsf1, together with another HSF family member, Hsf2, causes male sterility and complete lack of mature sperm in mice, but the specific role of HSF1 in spermatogenesis has remained unclear. Here, we show that HSF1 is transiently expressed in meiotic spermatocytes and haploid round spermatids in mouse testis. The Hsf1−/− male mice displayed regions of seminiferous tubules containing only spermatogonia and increased morphological abnormalities in sperm heads. In search for HSF1 target genes, we identified 742 putative promoters in mouse testis. Among them, the sex chromosomal multicopy genes that are expressed in postmeiotic cells were occupied by HSF1. Given that the sex chromatin mostly is repressed during and after meiosis, it is remarkable that HSF1 directly regulates the transcription of sex-linked multicopy genes during postmeiotic repression. In addition, our results show that HSF1 localizes to the sex body prior to the meiotic divisions and to the sex chromocenter after completed meiosis. To the best of our knowledge, HSF1 is the first known transcription factor found at the repressed sex chromatin during meiosis.
Chromatin Regulation; Gene Regulation; Sperm; Spermatogenesis; Transcription; ChIP-Chip; Heat Shock Factor; Meiosis; Multicopy Genes; Sex Chromatin
Members of the heat shock factor (HSF) family are evolutionarily conserved regulators that share a highly homologous DNA-binding domain. In mammals, HSF1 is the main factor controlling the stress-inducible expression of Hsp genes while the functions of HSF2 and HSF4 are less clear. Based on its developmental profile of expression, it was hypothesized that HSF2 may play an essential role in brain and heart development, spermatogenesis, and erythroid differentiation. To directly assess this hypothesis and better understand the underlying mechanisms that require HSF2, we generated Hsf2 knockout mice. Here, we report that Hsf2−/− mice are viable and fertile and exhibit normal life span and behavioral functions. We conclude that HSF2, most probably because its physiological roles are integrated into a redundant network of gene regulation and function, is dispensable for normal development, fertility, and postnatal psychomotor function.
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.
Avian cells express three heat shock transcription factor (HSF) genes corresponding to a novel factor, HSF3, and homologs of mouse and human HSF1 and HSF2. Analysis of the biochemical and cell biological properties of these HSFs reveals that HSF3 has properties in common with both HSF1 and HSF2 and yet has features which are distinct from both. HSF3 is constitutively expressed in the erythroblast cell line HD6, the lymphoblast cell line MSB, and embryo fibroblasts, and yet its DNA-binding activity is induced only upon exposure of HD6 cells to heat shock. Acquisition of HSF3 DNA-binding activity in HD6 cells is accompanied by oligomerization from a non-DNA-binding dimer to a DNA-binding trimer, whereas the effect of heat shock on HSF1 is oligomerization of an inert monomer to a DNA-binding trimer. Induction of HSF3 DNA-binding activity is delayed compared with that of HSF1. As occurs for HSF1, heat shock leads to the translocation of HSF3 to the nucleus. HSF exhibits the properties of a transcriptional activator, as judged from the stimulatory activity of transiently overexpressed HSF3 measured by using a heat shock element-containing reporter construct and as independently assayed by the activity of a chimeric GAL4-HSF3 protein on a GAL4 reporter construct. These results reveal that HSF3 is negatively regulated in avian cells and acquires DNA-binding activity in certain cells upon heat shock.