Transcription Of Heat Shock Genes Research Articles

Activation of heat shock factor 1 in rat brain during cerebral ischemia or after heat shock.

Recently, many studies have demonstrated the induction of stress proteins in the mammalian nervous system under various pathological conditions. These altered genetic programs may function to protect individual cells against stressful conditions. However, little is known about the molecular mechanisms regulating these stress responses in animals. We report here the activation of a heat shock factor (HSF) in the rat brain during cerebral ischemia or after heat shock. Gel mobility shift assays revealed an increase in DNA binding activity to the heat shock element (HSE) during the early phases of ischemia. Supershift experiments using specific antisera against HSF1 and HSF2 showed that the ischemia-induced HSE-binding activity was mainly due to HSF1. In the heat-shocked brain, HSF1 was also activated, and the HSE-binding activity was higher in the cerebellum than in the cerebral cortex or hippocampus; Western blot analysis also showed that HSF1 was more abundant in the cerebellum than in the other two brain regions. Our results indicate that heat shock gene transcription is regulated by the activation of HSF1 in both cerebral ischemia and heat shock, and that different brain regions display differential sensitivities in their stress response. The cellular signals for heat shock gene transcription under in vivo pathological conditions will also be discussed.

Isolation and characterization of six heat shock transcription factor cDNA clones from soybean

Thermal stress in soybean seedlings causes the activation of pre-existing heat shock transcription factor proteins (HSFs). Activation results in the induction of DNA binding activity which leads to the transcription of heat shock genes. From a soybean cDNA library we have isolated cDNA clones corresponding to six HSF genes. Two HSF genes are expressed constitutively at the transcriptional level, and the remaining four are heat-inducible. Two of the heat inducible genes are also responsive to cadmium stress. Comparative analysis of HSF sequences indicated higher conservation of the DNA binding domain among plant HSFs than those from yeast or other higher eukaryotes. The putative plant HSF oligomerization domain contains hydrophobic heptapeptide repeats characteristic of coiled coils and seems to exist in two structural variants. The carboxy-terminal domains are reduced in size and the C-terminal heptad repeat is degenerate.

Hepatic Heat Shock and Acute-phase Gene Expression Are Induced Simultaneously after Celiotomy in the Anesthetized Pig

The liver plays a central role in the whole organism's response to injury. Expression of hepatic acute-phase and heat-shock genes likely contributes to the restoration of homeostasis after stressful events. However, after prolonged ischemia, hepatic transcription of heat-shock genes can exclude the simultaneous transcription of acute-phase genes. The issue of whether hepatic 72-kd heat-shock protein (hsp72) gene expression is induced under perioperative conditions that do not result in prolonged liver ischemia and whether this might further affect the expression of the acute-phase reactant inter-alpha-trypsin inhibitor (alpha-Ti) was examined. Pigs were anesthetized with sodium pentobarbital and ketamine hydrochloride, tracheally intubated, and their lungs ventilated. After celiotomy, a hepatic biopsy sample was obtained. Arterial blood pressure, cardiac output, and total hepatic blood flow were measured. Subsequent biopsies were obtained at 1, 2, 3, 4, and 6 h after the initial biopsy. Arterial norepinephrine concentrations were measured using high-pressure liquid chromatography. Nuclear runoff (run on) analysis and Northern blotting were applied to estimate changes in hsp72 and alpha-Ti gene transcription rates and RNA levels. Western blotting was used to estimate changes in hsp72 levels. Hemodynamic parameters did not change significantly over time. Arterial norepinephrine concentrations were increased at all time points. Hepatic hsp72 RNA levels increased up to sixfold while nuclear runoff assays did not detect significant changes in hsp72 gene transcription rates. The increases in hsp72 RNA levels correlated with accumulation of hsp72 (up to sevenfold). Increases in alpha-Ti transcription rates up to 42-fold were associated with respective increases in alpha-Ti RNA levels (up to 17-fold). These data demonstrate that hepatic expression of hsp72 is not confined to conditions that lead to prolonged liver ischemia but is also part of the response of the liver to surgery under general anesthesia. Furthermore, these conditions are permissive for the simultaneous RNA expression of the acute-phase reactant alpha-Ti.

Sodium Salicylate and Yeast Heat Shock Gene Transcription

The anti-inflammatory drug sodium salicylate modulates the activity of specific transcription factors in humans. Here we show that salicylate and sorbate, another organic acid, stimulate DNA binding by yeast heat shock transcription factor (HSF) in vivo. Surprisingly, salicylate inhibits heat shock gene transcription even in cells induced by a prior heat shock. This inhibition of transcription occurs at a step after HSF and transcription factor IID binding but before promoter melting by RNA polymerase. Salicylate appears to generate a tight binding but activation-impotent HSF by cytoplasmic acidification, since inhibiting proton efflux from cells triggers this same DNA binding and inhibition of heat shock gene expression.

Transcription of heat shock gene loci versus non-heat shock loci in Chironomus polytene chromosomes: evidence for heat-induced formation of novel putative ribonucleoprotein particles (hsRNPs) in the major heat shock puffs

The heat shock response of Chironomus polytene chromosomes was reexamined. The in vivo effects of heat shock on chromosomal [3H]uridine labeling, RNA polymerase II distribution and ribonucleoprotein (RNP) formation were investigated. One primary result is a clarification of the number and location of chromosomal sites strongly induced by treatment at 37 degrees C for 60 min. In total, seven major heat shock loci were identified by transcription autoradiography in Chironomus tentans: I-20A, II-16B, II-10C, II-4B, II-1C, III-12B, and IV-5C. Secondly, combining immunofluorescence with transcription autoradiography, I find RNA polymerase II occurring after heat shock at multiple chromosomal sites that were also active under normal conditions (20 degrees C). Furthermore, the results demonstrate conclusively that the presence of RNA polymerase II at heat shock and non-heat shock loci is generally correlated with [3H]uridine labeling during heat shock. These latter results extend and corroborate previous findings. Thirdly, the most striking result of this study was revealed in ultrathin sections of puffs by electron microscopy: I discerned a site-specific ultrastructural difference in putative RNP particles between heat shock versus non-heat shock loci. At least three of the seven induced major heat shock puffs (I-20A, III-12B, IV-5C) were observed to contain globular particles that were different, i.e. significantly larger, 250-1,000 A in diameter with a prominent 500-750 A class, than RNP particles of other loci under non-heat shock conditions. These large heat shock puff particles presumably represent nascent or newly synthesized heat shock RNA associated with protein(s) to form heat shock RNPs (hsRNPs). This finding suggests the possible involvement of novel RNPs (hsRNPs) in transcriptional regulation or heat shock RNA turnover and may stimulate further molecular investigations on this subject in both cell physiological and structural terms. I conclude that the locus-specific putative hsRNPs are an intrinsic property of greatly increased heat shock gene transcription.

A distinct segment of the sigma 32 polypeptide is involved in DnaK-mediated negative control of the heat shock response in Escherichia coli.

Induction of heat shock proteins in Escherichia coli is caused by a transient increase in the cellular level of sigma 32 (the rpoH gene product), a protein required for transcription of heat shock genes. Both increased synthesis and stabilization of sigma 32 contribute to the increase in sigma 32. We previously showed that heat-induced translation of sigma 32-beta-galactosidase fusion protein encoded by an rpoH-lacZ gene fusion was mediated by an mRNA secondary structure formed between two 5'-proximal segments (A and B) of rpoH coding sequence spanning some 200 nt. We now report that a portion of the sigma 32 polypeptide that corresponds to further downstream (designated region C) is involved in the DnaK-mediated negative control resulting in the shutoff of heat-induced synthesis and degradation of fusion protein. Gene fusions carrying the 5' half (433 nt) or more of the rpoH coding sequence exhibited normal shutoff of synthesis, and the fusion proteins produced were very unstable, like authentic sigma 32; both the shutoff of synthesis and the instability of protein were markedly affected by the dnaK and dnaJ mutations. In contrast, gene fusions carrying < or = 364 nt (lacking region C) and a fusion carrying most of the rpoH sequence but with a frameshift mutation specifically affecting region C exhibited little or no shutoff and produced stable proteins. These results indicate that a distinct segment of sigma 32 plays a critical role in the negative feedback control of sigma 32. The control may be exerted during or after completion of sigma 32 synthesis mediated by interaction between nascent or mature sigma 32 and DnaK/DnaJ proteins.

Induction of heat shock proteins by abnormal proteins results from stabilization and not increased synthesis of sigma 32 in Escherichia coli

Accumulation of abnormal proteins in cells of bacteria or eukaryotes can induce synthesis of a set of heat shock proteins. We examined such induction following addition of azetidine (a proline analog) or synthesis of a heterologous protein (human prourokinase) in Escherichia coli. Synthesis of heat shock proteins under these conditions increased almost immediately and continued with increasing rates until it reached a maximum after 30 to 60 min at 30 degrees C. The induction was closely accompanied by an increase in the cellular level of sigma 32 specifically required for transcription of heat shock genes. The increase in sigma 32 initially coincided with increased synthesis of heat shock proteins but then exceeded the latter, particularly following accumulation of prourokinase. The sigma 32 level increase upon either treatment was found to result solely from stabilization of sigma 32, which is ordinarily very unstable, and not from increased synthesis of sigma 32. This is in contrast to what had been found when cells were exposed to a higher temperature, at which both increased synthesis and stabilization of sigma 32 contributed to the increased sigma 32 level. On the basis of these and other findings, we propose that abnormal proteins stabilize sigma 32 by a pathway or a mechanism distinct from that used for the induction of sigma 32 synthesis known to occur at the level of translation. Evidence further suggests that the DnaK chaperone plays a crucial regulatory role in induction of the heat shock response by abnormal proteins.

Suppression of heat-shock protein synthesis by short-chain fatty acids and alcohols

We have shown that ethanol, propanol and butanol (at 0.5–2%) and salts of butyric and propionic acids (at 8–40 mM) all cause a major reduction in heat-shock protein (hsp) synthesis when present in the growth medium of Drosophila cultured cells (Kc and SL2) subjected to either increased temperature or chemical stressors. Inhibition of normal protein synthesis in unstressed cells was comparatively slight, and the usual suppression of synthesis of non-heat-shock proteins in stressed cells was unaffected. Maximum suppression of hsp synthesis occurred only if inhibitors were added before initiation of the stress response, an observation that eliminates the possibility that these findings are due to non-specific, toxic effects. Suppression was accompanied by severely reduced levels of both hsp70 mRNA and active heat-shock factor (HSF). We conclude that the inhibitors act by suppressing the initiation of transcription of heat-shock genes.

The yeast and mammalian Ras pathways control transcription of heat shock genes independently of heat shock transcription factor.

Yeast strains in which the Ras-cyclic AMP (cAMP) pathway is constitutively active are sensitive to heat shock, whereas mutants in which the activity of this pathway is low are hyperresistant to heat shock. To determine the molecular basis for these differences, we examined the transcriptional induction of heat shock genes in various yeast strains. Activation of heat shock genes was attenuated in the strains in which the Ras-cAMP pathway is constitutively active. In contrast, in a strain deficient in cAMP production, several heat shock genes were induced by removal of cAMP from the medium. These results indicate that the Ras-cAMP pathway affects the induction of heat shock genes. In all of the mutants, heat shock transcription factor expression and activity were identical to those in wild-type cells. The response to heat shock in Ha-ras-transformed rat fibroblasts was also studied. While no induction of Hsp68 was observed in Ha-ras-transformed cells, proper regulation of heat shock transcription factor was found. Therefore, in mammals, as in Saccharomyces cerevisiae, the Ras pathway controls the transcription of heat shock genes via a mechanism not involving the heat shock transcription factor.

The yeast and mammalian Ras pathways control transcription of heat shock genes independently of heat shock transcription factor.

Yeast strains in which the Ras-cyclic AMP (cAMP) pathway is constitutively active are sensitive to heat shock, whereas mutants in which the activity of this pathway is low are hyperresistant to heat shock. To determine the molecular basis for these differences, we examined the transcriptional induction of heat shock genes in various yeast strains. Activation of heat shock genes was attenuated in the strains in which the Ras-cAMP pathway is constitutively active. In contrast, in a strain deficient in cAMP production, several heat shock genes were induced by removal of cAMP from the medium. These results indicate that the Ras-cAMP pathway affects the induction of heat shock genes. In all of the mutants, heat shock transcription factor expression and activity were identical to those in wild-type cells. The response to heat shock in Ha-ras-transformed rat fibroblasts was also studied. While no induction of Hsp68 was observed in Ha-ras-transformed cells, proper regulation of heat shock transcription factor was found. Therefore, in mammals, as in Saccharomyces cerevisiae, the Ras pathway controls the transcription of heat shock genes via a mechanism not involving the heat shock transcription factor.

Effects of reduced levels of GroE chaperones on protein metabolism: enhanced synthesis of heat shock proteins during steady-state growth of Escherichia coli.

The GroE heat shock proteins (GroEL and GroES) of Escherichia coli represent major molecular chaperones that participate in folding (and assembly) of a variety of proteins and are essential for cell growth at all temperatures. We have examined the effects of reducing the cellular content of GroE on the synthesis and stability of proteins during steady-state growth with near-normal rates. The GroE protein level was manipulated by placing groE under the control of lacUV5 promoter on a multicopy plasmid in a strain lacking the chromosomal groE operon. When this strain was grown with a limited concentration (40 microM) of inducer (IPTG [isopropyl-beta-D-thiogalactopyranoside]) at 37 degrees C, the GroE level and growth rate were comparable to those of the wild type. When cells were depleted of IPTG, they continued to grow at or below 37 degrees C albeit at reduced rates, despite the much-reduced GroE level (ca. 25% of that of wild type). Under these conditions, the cellular contents of at least 13 polypeptides were affected. Among the most striking effects was the enhanced synthesis of a set of heat shock proteins which resulted from the increased level of sigma 32 which is required for transcription of heat shock genes. This increase in the sigma 32 level was brought about by both stabilization and increased synthesis of sigma 32. Other proteins affected by the reduced GroE level included two proteins (enzymes of the Entner-Doudoroff pathway) encoded by the edd-eda operon and the ribosomal protein S6, suggesting that the GroE chaperones are involved in regulating expression of genes for carbohydrate metabolism and in modulating biogenesis or function of the ribosome.

Arachidonate is a potent modulator of human heat shock gene transcription.

Cell and tissue injury activate the inflammatory response through the action(s) of arachidonic acid and its metabolites, leading to the expression of acute-phase proteins and inflammatory cytokines. At the molecular level, little is known how arachidonic acid regulates the inflammatory response. As inflammation is also associated with local increase in tissue temperatures, we examined whether arachidonic acid was directly involved in the heat shock response. Extracellular exposure to arachidonic acid induced heat shock gene transcription in a dose-dependent manner via acquisition of DNA-binding activity and phosphorylation of heat shock factor 1 (HSF1). In addition, exposure of cells to low concentrations of arachidonic acid, which by themselves did not induce HSF1 DNA-binding activity, reduced the temperature threshold for HSF1 activation from elevated temperatures which are not physiologically relevant (> 42 degrees C) to temperatures which can be attained during the febrile response (39-40 degrees C). These results indicate that elevated heat shock gene expression is a direct consequence of an arachidonic acid-mediated cellular response.

Inhibitors of tyrosine and ser/thr phosphatases modulate the heat shock response

Following heat shock the expression of heat shock genes is regulated by the heat shock transcription factor, HSF, known to bind to arrays of the heat shock element, NGAAN, upstream of the heat shock genes. Phosphorylation of HSF is necessary for its activation. We report that the treatment of Chinese hamster HA-1 cells with 250 nM of okadaic acid (OA), a ser/thr phosphatase inhibitor, leads to an increase in activated HSF after heat shock. This is followed by the activation of the transcription of heat shock genes as assayed by the increase in the synthesis of beta-galactosidase in an HA-1 cell line containing the heat shock promoter ligated to the beta-galactosidase gene. To investigate the specificity of OA, we used other phosphatase inhibitors. We found that treatment of HA-1 cells with 500 microM of sodium vanadate, an inhibitor of tyr/phosphatases, resulted in a three to fivefold reduction in HSF activation and binding to the heat shock element following heat shock. Such reduction in HSF activation virtually abolished beta-galactosidase induction. Reduced HSP synthesis was further confirmed by SDS-PAGE and Western blot analysis using anti-HSP-70 and 28 antibodies. Sodium vanadate treatment of heat shocked cells greatly reduced levels of thermotolerance. These results show that ser/thr and specifically tyr/phosphatase inhibitors modulate the signal transduction pathway of HSF activation.

Cloning and sequencing of heat shock factor (HSF1) cDNA from human retina.

Transcription of heat shock genes takes place in response to the exposure of cells to elevated temperatures or other stress conditions. Heat shock transcription factor (HSF) plays an important role in the activation of such transcription. We report here the identification of human heat shock factor from human retina by RT-PCR and cloning studies. Human retinal poly A+ RNA was reverse transcribed and the cDNAs thus obtained were subjected to PCR using a set of primers designed from the reported cDNA sequence for human HSF1. The amplified product showed 100% sequence identity with the appropriate region of HSF1. A human retinal cDNA library was screened to isolate the full length cDNA for heat shock factor, which showed 100% sequence identity to that reported for human HSF1. Northern blot analysis showed the size of human retinal HSF to be approximately 2.4 kb.

Regulation of the heat-shock response in bacteria.

When bacteria cells are exposed to higher temperature, a set of heat-shock proteins (hsps) is induced rapidly and transiently to cope with increased damage in proteins. The mechanism underlying induction of hsps has been a central issue in the heat-shock response and studied intensively in Escherichia coli. Immediately upon temperature upshift, the cellular level of sigma 32 responsible for transcription of heat-shock genes increases rapidly and transiently. The increase in sigma 32 results from both increased synthesis and stabilization of sigma 32, which is ordinarily very unstable. A clue to further understanding of early regulatory events came from recent analysis of translational induction and subsequent shut-off of sigma 32 synthesis. Whereas a 5'-coding region of mRNA for sigma 32 is involved in the induction mediated by the mRNA secondary structure, a distinct segment of sigma 32 polypeptide further downstream is involved in the DnaK/DnaJ-mediated shut-off and destabilization of sigma 32 that may be mutually interconnected.

Accumulation of secretory protein precursors in Escherichia coli induces the heat shock response

The accumulation of secretory protein precursors, caused either by mutations in secB or secA or by the overproduction of export-defective proteins, results in a two- to fivefold increase in the synthesis of heat shock proteins. In such strains, sigma 32, the alternative sigma factor responsible for transcription of the heat shock genes, is stabilized. The resultant increase in the level of sigma 32 leads to increased transcription of heat shock genes and increased synthesis of heat shock proteins. We have also found that although a secB null mutant does not grow on rich medium at a temperature range of 30 to 42 degrees C, it does grow at 44 degrees C. In addition, we found that a secB null mutant exhibits greater thermotolerance than the wild-type parental strain. Elevated levels of heat shock proteins, as well as some other non-heat shock proteins, may account for the partial heat resistance of a SecB-lacking strain.

Activation of Heat Shock Gene Transcription by Heat Shock Factor 1 Involves Oligomerization, Acquisition of DNA-Binding Activity, and Nuclear Localization and Can Occur in the Absence of Stress

Activation of Heat Shock Gene Transcription by Heat Shock Factor 1 Involves Oligomerization, Acquisition of DNA-Binding Activity, and Nuclear Localization and Can Occur in the Absence of Stress

Activation of Heat Shock Gene Transcription by Heat Shock Factor 1 Involves Oligomerization, Acquisition of DNA-Binding Activity, and Nuclear Localization and Can Occur in the Absence of Stress

Activation of Heat Shock Gene Transcription by Heat Shock Factor 1 Involves Oligomerization, Acquisition of DNA-Binding Activity, and Nuclear Localization and Can Occur in the Absence of Stress

Activation of Heat Shock Gene Transcription by Heat Shock Factor 1 Involves Oligomerization, Acquisition of DNA-Binding Activity, and Nuclear Localization and Can Occur in the Absence of Stress

Activation of Heat Shock Gene Transcription by Heat Shock Factor 1 Involves Oligomerization, Acquisition of DNA-Binding Activity, and Nuclear Localization and Can Occur in the Absence of Stress

Activation of Heat Shock Gene Transcription by Heat Shock Factor 1 Involves Oligomerization, Acquisition of DNA-Binding Activity, and Nuclear Localization and Can Occur in the Absence of Stress

Activation of Heat Shock Gene Transcription by Heat Shock Factor 1 Involves Oligomerization, Acquisition of DNA-Binding Activity, and Nuclear Localization and Can Occur in the Absence of Stress