Masako Tanabe

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.

Arrest of spermatogenesis in mice expressing an active heat shock transcription factor 1.

In mammals, testicular temperature is lower than core body temperature, and the vulnerable nature of spermatogenesis to thermal insult has been known for a century. However, the primary target affected by increases in temperature is not yet clear. We report here that male mice expressing an active form of heat shock transcription factor 1 (HSF1) in the testis are infertile due to a block in spermatogenesis. The germ cells entered meiotic prophase and were arrested at pachytene stage, and there was a significant increase in the number of apoptotic germ cells in these mice. In wild‐type mice, a single heat exposure caused the activation of HSF1 and similar histological changes such as a stage‐specific apoptosis of pachytene spermatocytes. These results suggest that male infertility caused by thermal insult is at least partly due to the activation of HSF1, which induces the primary spermatocytes to undergo apoptosis.

The Mammalian HSF4 Gene Generates Both an Activator and a Repressor of Heat Shock Genes by Alternative Splicing

The expression of heat shock genes is controlled at the level of transcription by members of the heat shock transcription factor family in vertebrates. HSF4 is a mammalian factor characterized by its lack of a suppression domain that modulates formation of DNA-binding homotrimer. Here, we have determined the exon structure of the human HSF4 gene and identified a major new isoform, HSF4b, derived by alternative RNA splicing events, in addition to a previously reported HSF4a isoform. In mouse tissues HSF4b mRNA was more abundant than HSF4a as examined by reverse transcription-polymerase chain reaction, and its protein was detected in the brain and lung. Although both mouse HSF4a and HSF4b form trimers in the absence of stress, these two isoforms exhibit different transcriptional activity; HSF4a acts as an inhibitor of the constitutive expression of heat shock genes, and hHSF4b acts as a transcriptional activator. Furthermore HSF4b but not HSF4a complements the viability defect of yeast cells lacking HSF. Moreover, heat shock and other stresses stimulate transcription of target genes by HSF4b in both yeast and mammalian cells. These results suggest that differential splicing of HSF4 mRNA gives rise to both an inhibitor and activator of tissue-specific heat shock gene expression.

Disruption of the HSF3 gene results in the severe reduction of heat shock gene expression and loss of thermotolerance

The vertebrate genome encodes a family of heat shock factors (HSFs 1–4) of which the DNA‐binding and transcriptional activities of HSF1 and HSF3 are activated upon heat shock. HSF1 has the properties of a classical HSF and exhibits rapid activation of DNA‐binding and transcriptional activity upon exposure to conditions of heat shock and other stresses, whereas HSF3 typically is activated at higher temperatures and with distinct delayed kinetics. To address the role of HSF3 in the heat shock response, null cells lacking the HSF3 gene were constructed by disruption of the resident gene by somatic recombination in an avian lymphoid cell line. Null cells lacking HSF3, yet expressing normal levels of HSF1, exhibited a severe reduction in the heat shock response, as measured by inducible expression of heat shock genes, and did not exhibit thermotolerance. At intermediate heat shock temperatures, where HSF1 oligomerizes to an active trimer in wild‐type cells, HSF1 remained as an inert monomer in the HSF3 null cell line. HSF3 null cells were restored to a nearly normal heat shock‐responsive state by reintroduction of an exogenous HSF3 gene. These results reveal that HSF3 has a dominant role in the regulation of the heat shock response and directly influences HSF1 activity.

The DNA-binding properties of two heat shock factors, HSF1 and HSF3, are induced in the avian erythroblast cell line HD6.

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.

Different Thresholds in the Responses of Two Heat Shock Transcription Factors, HSF1 and HSF3

Avian cells express three HSF genes encoding a unique factor, HSF3, as well as homologues of mammalian HSF1 and HSF2. HSF1 is the major factor that mediates the heat shock signal in mammalian cells. We reported previously that cHSF3, as well as cHSF1, is activated by heat shock in chicken cells. In this study, we examined the functional differences between cHSF1 and cHSF3. Comparison of the heat-inducible DNA binding activity of cHSF1 with cHSF3 at various temperatures revealed that the latter was activated at higher temperatures than the former. At a mild heat shock, such as 41 °C, only cHSF1 was activated, whereas both cHSF1 and cHSF3 were activated following a severe heat shock at 45 °C. Heat-inducible nuclear translocation and trimerization were accompanied by DNA binding activity. We also observed that cHSF3 was activated by treating cells with higher concentrations of sodium arsenite compared to cHSF1. The DNA binding activity of cHSF3 by severe heat shock lasted for a longer period than that of cHSF1. Interestingly, the total amount of cHSF3 increased only upon severe heat shock, whereas that of HSF1 decreased. Substantial amounts of cHSF3 remained in the soluble fraction under severe heat shock, whereas cHSF1 rapidly moved to the insoluble fractions in that conditions. Comparison of transcriptional activity of the activation domains of cHSF1 and cHSF3 revealed that the activity of cHSF3 was as strong as that of cHSF1. These findings indicate that there are different thresholds for cHSF1 and cHSF3 and that cHSF3 is involved in the persistent and burst activation of stress genes upon severe stress in chicken cells. Pretreatment of cycloheximide elevated the threshold concentrations of arsenite of both factors. This suggests that denaturation of nascent polypeptides could be the first trigger for the activation of both factors, and the pathways for activation of cHSF1 and cHSF3 may be identical, or at least share some common mechanisms.

Reperfusion Causes Significant Activation of Heat Shock Transcription Factor 1 in Ischemic Rat Heart

BACKGROUND The myocardial protective role of heat shock protein (HSP) has been demonstrated, and there has been increasing interest in stress response in the heart. We examined the DNA-binding activity of heat shock transcription factor (HSF), by which the transcription of heat shock genes is mainly regulated, during heat shock or ischemia/reperfusion in isolated rat heart. METHODS AND RESULTS Rat hearts were isolated and perfused with Krebs-Henseleit buffer by the Langendorff method. Whole-cell extracts were prepared for gel mobility shift assay using oligonucleotides containing the heat shock element, which is present upstream of all heat shock genes. Induction of mRNAs for HSP70, HSP90, and GRP78 (glucose-regulated protein) was examined by Northern blot analysis. Although the activation of HSF during global ischemia was weak and rapidly attenuated, postischemic reperfusion induced a significant activation of HSF. In addition, although HSP70 mRNA was hardly induced during ischemia, its burst induction was detected during postischemic reperfusion. Supershift assays using specific antisera for HSF1 and HSF2 revealed that ischemia/reperfusion as well as heat shock induced the activation of HSF1 in hearts. Although the expression of HSP70 mRNA during heat shock was more vigorous than the expression during ischemia/reperfusion, the induction of HSP90 mRNA in postischemic reperfusion was significantly greater than that in heat shock. CONCLUSIONS Our findings demonstrated that reperfusion causes a significant activation of HSF1 in ischemia-reperfused heart. The striking contrast between the induction of HSP70 mRNA and that of HSP90 mRNA suggests the presence of regulatory mechanisms other than HSF.

Ubiquitous and cell-specific members of the avian small heat shock protein family

Small heat shock proteins (sHsps) have been suggested to act as molecular chaperones for many kinds of substrates and have protective roles in cells exposed to external stresses. Unlike other major Hsps such as Hsp70 and Hsp90, expression of many vertebrate sHsps is restricted to the muscle tissues and/or eye lens. Among the sHsps, the heat‐inducible human Hsp27 (hHsp27) homologue is believed to be expressed ubiquitously in various cell types. Here, we distinguished the chicken homologue of hHsp27 (cHsp24) from the chicken major heat‐inducible protein of molecular size 25 kDa (cHsp25). cHsp25 is not expressed in the absence of stress, but is highly expressed after hyperthermia in all tissues of developing embryos. In contrast, expression of cHsp24 is restricted to some specific tissues even in the presence of stress. Thus, cHsp25 is the first member of the sHsps in vertebrates the expression of which is ubiquitous in tissues exposed to external stresses similar to Hsp70.