Heat shock gene regulation by nascent polypeptides and denatured proteins: hsp70 as a potential autoregulatory factor.

Heat shock genes encode proteins (hsp's) that play important structural roles under normal circumstances and are essential to the cells' ability to survive environmental insults. Evidence is presented herein that transcriptional regulation of hsp gene expression is linked with the regulation of overall protein synthesis as well as with the accumulation of proteins denatured by stressful events. The factor that connects the three processes appears to be one of the hsp's, presumably a member(s) of the hsp70 family. Biochemical experiments demonstrate that complexes containing hsp70 and heat shock transcription factor, the specific regulator of hsp gene activity, are formed in the cells.

Abnormal proteins serve as eukaryotic stress signals and trigger the activation of heat shock genes.

Heat shock protein (hsp) genes, a group of ubiquitous genes, are activated by various metabolic stresses. The suggestion that denaturation of intracellular proteins may be produced by the metabolic stresses and then signal the activation of the hsp genes was examined by co-injection of purified proteins and hsp genes into frog oocytes. Activation of hsp genes was observed if the proteins were denatured prior to injection but not if they were introduced in their native form. Furthermore, the activation of hsp genes by abnormal proteins and by heat shock appears to occur by a common mechanism. A model for the transcriptional regulation of the genes is based on competition for degradation between abnormal intracellular proteins and a labile regulatory factor.

Expression of functional cell-cell channels from cloned rat liver gap junction complementary DNA.

An oocyte expression system was used to test the relation between a complementary DNA (cDNA) clone encoding the liver gap junction protein and cell-cell channels. Total liver polyadenylated messenger RNA injected into oocytes induced cell-cell channels between paired oocytes. This induction was blocked by simultaneous injection of antisense RNA transcribed from the gap junction cDNA. Messenger RNA selected by hybridization to the cDNA clone and translated in oocyte pairs yielded a higher junctional conductance than unselected liver messenger RNA. Cell-cell channels between oocytes were also formed when the cloned cDNA was expressed under the control of a heat-shock promoter. A concentration-dependent induction of channels was observed in response to injection with in vitro transcribed gap junction messenger RNA. Thus, the liver gap junction cDNA encodes a protein that is essential for the formation of functional cell-cell channels.

Activation of human heat shock genes is accompanied by oligomerization, modification, and rapid translocation of heat shock transcription factor HSF1.

Transcriptional activity of heat shock (hsp) genes is controlled by a heat-activated, group-specific transcription factor(s) recognizing arrays of inverted repeats of the element NGAAN. To date genes for two human factors, HSF1 and HSF2, have been isolated. To define their properties as well as the changes they undergo during heat stress activation, we prepared polyclonal antibodies to these factors. Using these tools, we have shown that human HeLa cells constitutively synthesize HSF1, but we were unable to detect HSF2. In unstressed cells HSF1 is present mainly in complexes with an apparent molecular mass of about 200 kDa, unable to bind to DNA. Heat treatment induces a shift in the apparent molecular mass of HSF1 to about 700 kDa, concomitant with the acquisition of DNA-binding ability. Cross-linking experiments suggest that this change in complex size may reflect the trimerization of monomeric HSF1. Human HSF1 expressed in Xenopus oocytes does not bind DNA, but derepression of DNA-binding activity, as well as oligomerization of HSF1, occurs during heat treatment at the same temperature at which hsp gene expression is induced in this organism, suggesting that a conserved Xenopus protein(s) plays a role in this regulation. Inactive HSF1 resides in the cytoplasm of human cells; on activation it rapidly translocates to a soluble nuclear fraction, and shortly thereafter it becomes associated with the nuclear pellet. On heat shock, activatable HSF1, which might already have been posttranslationally modified in the unstressed cell, undergoes further modification. These different process provide multiple points of regulation of hsp gene expression.

Genes for Drosophila small heat shock proteins are regulated differently by ecdysterone.

Genes for small heat shock proteins (hsp27 to hsp22) are activated in late third-instar larvae of Drosophila melanogaster in the absence of heat stress. This regulation has been simulated in cultured Drosophila cells in which the genes are activated by the addition of ecdysterone. Sequence elements (HERE) involved in ecdysterone regulation of the hsp27 and hsp23 genes have been defined by transfection studies and have recently been identified as binding sites for ecdysterone receptor. We report here that the hsp27 and hsp23 genes are regulated differently by ecdysterone. The hsp27 gene is activated rapidly by ecdysterone, even in the absence of protein synthesis. In contrast, high-level expression of the hsp23 gene begins only after a lag of about 6 h, is dependent on the continuous presence of ecdysterone, and is sensitive to low concentrations of protein synthesis inhibitors. Transfection experiments with reporter constructs show that this difference in regulation is at the transcriptional level. Synthetic hsp27 or hsp23 HERE sequences confer hsp27- or hsp23-type ecdysterone regulation on a basal promoter. These findings indicate that the hsp27 gene is a primary, and the hsp23 gene is mainly a secondary, hormone-responsive gene. Ecdysterone receptor is implied to play a role in the regulation of both genes.

Ecdysterone receptor is a sequence-specific transcription factor involved in the developmental regulation of heat shock genes.

Purification of ecdysterone receptor from Drosophila melanogaster to apparent homogeneity is reported. Purified receptor binds specifically to several sequences in the promoters of the developmentally active hsp27 and hsp23 heat shock genes that were previously implied in ecdysterone regulation of the genes and that share limited homology among themselves and with mammalian steroid receptor binding sites. Some of these elements confer ecdysterone regulation on a basal promoter in transfected cells, acting in a synergistic fashion. Transcription in vitro of promoters containing such elements is stimulated up to 100-fold by added purified ecdysterone receptor, depending on receptor dosage and the number of elements present. Transcriptional enhancement requires sequence-specific binding of receptor to template promoters which facilitates the formation of a preinitiation complex. Ecdysterone stimulates DNA binding of the receptor in vitro.

Multiple layers of regulation of human heat shock transcription factor 1.

Upon heat stress, monomeric human heat shock transcription factor 1 (hHSF1) is converted to a trimer, acquires DNA-binding ability, is transported to the nucleus, and becomes transcriptionally competent. It was not known previously whether these regulatory changes are caused by a single activation event or whether they occur independently from one another, providing a multilayered control that may prevent inadvertant activation of hHSF1. Comparison of wild-type and mutant hHSF1 expressed in Xenopus oocytes and human HeLa cells suggested that retention of hHSF1 in the monomeric form depends on hydrophobic repeats (LZ1 to LZ3) and a carboxy-terminal sequence element in hHSF1 as well as on the presence of a titratable factor in the cell. Oligomerization of hHSF1 appears to induce DNA-binding activity as well as to uncover an amino-terminally located nuclear localization signal. A mechanism distinct from that controlling oligomerization regulates the transcriptional competence of hHSF1. Components of this mechanism were mapped to a region, including LZ2 and nearby sequences downstream from LZ2, that is clearly separated from the carboxy-terminally located transcription activation domain(s). We propose the existence of a fold-back structure that masks the transcription activation domain in the unstressed cell but is opened up by modification of hHSF1 and/or binding of a factor facilitating hHSF1 unfolding in the stressed cell. Activation of hHSF1 appears to involve at least two independently regulated structural transitions.

Key features of heat shock regulatory elements.

The promoters of heat shock protein genes are among the best-studied inducible eucaryotic promoters. Regions responsible for heat regulation have been identified previously by deletion experiments with several different heat shock genes. In this paper the critical importance of two novel features of heat shock regulatory elements was investigated. First, the elements were modular and, as a consequence, displayed a characteristic 5-nucleotide periodicity produced by multiple GAA blocks that were arranged in alternating orientations and at 2-nucleotide intervals. Functional heat shock regulatory elements appeared to include three or more of these blocks. Second, the nucleotides at the two positions immediately upstream from GAA segments played an important role in defining the competence of regulatory elements.

Massive heat-shock polypeptide synthesis in late chicken embryos: convenient system for study of protein synthesis in highly differentiated organisms.

In cultured eucaryotic cells, heat treatments specifically induced the rapid synthesis of the so-called heat-shock polypeptides. To ascertain the physiological importance of this phenomenon for highly differentiated organisms, we attempted to determine whether the heat-shock response occurs in a living endothermic organism at extreme temperatures, and if so, whether the response is organ specific. We developed a procedure to label proteins efficiently in 5- to 18-day-old chicken embryos. Heat-shock polypeptides of identical sizes of 85,000, 70,000, and 25,000 daltons were synthesized predominantly in chicken embryo fibroblasts and in many different organs of 18-day-old embryos at 42.5 to 44 degrees C.

Activation of the DNA-binding ability of human heat shock transcription factor 1 may involve the transition from an intramolecular to an intermolecular triple-stranded coiled-coil structure.

Heat stress regulation of human heat shock genes is mediated by human heat shock transcription factor hHSF1, which contains three 4-3 hydrophobic repeats (LZ1 to LZ3). In unstressed human cells (37 degrees C), hHSF1 appears to be in an inactive, monomeric state that may be maintained through intramolecular interactions stabilized by transient interaction with hsp70. Heat stress (39 to 42 degrees C) disrupts these interactions, and hHSF1 homotrimerizes and acquires heat shock element DNA-binding ability. hHSF1 expressed in Xenopus oocytes also assumes a monomeric, non-DNA-binding state and is converted to a trimeric, DNA-binding form upon exposure of the oocytes to heat shock (35 to 37 degrees C in this organism). Because endogenous HSF DNA-binding activity is low and anti-hHSF1 antibody does not recognize Xenopus HSF, we employed this system for mapping regions in hHSF1 that are required for the maintenance of the monomeric state. The results of mutagenesis analyses strongly suggest that the inactive hHSF1 monomer is stabilized by hydrophobic interactions involving all three leucine zippers which may form a triple-stranded coiled coil. Trimerization may enable the DNA-binding function of hHSF1 by facilitating cooperative binding of monomeric DNA-binding domains to the heat shock element motif. This view is supported by observations that several different LexA DNA-binding domain-hHSF1 chimeras bind to a LexA-binding site in a heat-regulated fashion, that single amino acid replacements disrupting the integrity of hydrophobic repeats render these chimeras constitutively trimeric and DNA binding, and that LexA itself binds stably to DNA only as a dimer but not as a monomer in our assays.

Organization of the Drosophila melanogaster hsp70 heat shock regulation unit.

Expression from the Drosophila melanogaster hsp70 promoter was controlled by a regulatory unit that was composed of two sequence elements that resembled the heat shock consensus sequence. The unit functioned in both orientations and at different distances from downstream promoter sequences. Each element of the unit alone was essentially inactive. Association of two elements resulted in a dramatic increase of transcription from the hsp70 promoter. This synergistic effect was independent of the relative orientation of the elements and, to a large extent, of the distance between them. Duplication of a region containing only one element also yielded a highly active, heat-regulated promoter. Genes with three to five elements were three to four times more active than those with a single regulatory unit.

The heat shock consensus sequence is not sufficient for hsp70 gene expression in Drosophila melanogaster.

A hybrid gene in which the expression of an Escherichia coli beta-galactosidase gene was placed under the control of a Drosophila melanogaster 70,000-dalton heat shock protein (hsp70) gene promoter was constructed. Mutant derivatives of this hybrid gene which contained promoter sequences of different lengths were prepared, and their heat-induced expression was examined in D. melanogaster and COS-1 (African green monkey kidney) cells. Mutants with 5' nontranscribed sequences of at least 90 and up to 1,140 base pairs were expressed strongly in both cell types. Mutants with shorter 5' extensions (of at least 63 base pairs) were transcribed and translated efficiently in COS-1 but not at all in D. melanogaster cells. Thus, in contrast to the situation in COS-1 cells, the previously defined heat shock consensus sequence which is located between nucleotides 62 and 48 of the hsp70 gene 5' nontranscribed DNA segment is not sufficient for the expression of the D. melanogaster gene in homologous cells. A second consensus-like element 69 to 85 nucleotides upstream from the cap site is postulated to be also involved in the heat-induced expression of the hsp70 gene in D. melanogaster cells.

Synergistic activation of transcription is mediated by the N-terminal domain of Drosophila fushi tarazu homeoprotein and can occur without DNA binding by the protein.

Synergistic activation of transcription by Drosophila segmentation genes in tissue culture cells provides a model with which to study combinatorial regulation. We examined the synergistic activation of an engrailed-derived promoter by the pair-rule proteins paired (PRD) and fushi tarazu (FTZ). Synergistic activation by PRD requires regions of the homeodomain or adjacent sequences, and that by FTZ requires the first 171 residues. Surprisingly, deletion of the FTZ homeodomain does not reduce the capacity of the protein for synergistic activation, although this mutation abolishes any detectable DNA-binding activity. This finding suggests that FTZ can function through protein-protein interactions with PRD or other components of the homeoprotein transcription complex, adding a new layer of mechanisms that could underlie the functional specificities and combinatorial regulation of homeoproteins.

Selective induction of human heat shock gene transcription by the adenovirus E1A gene products, including the 12S E1A product.

We have previously shown that the human 70-kilodalton heat shock protein gene (hsp70) is induced by the adenovirus E1A gene product and during the S-G2 phase of the cell cycle. In this study, we investigated the effect of E1A on the expression of other human hsp genes. A gene encoding one form of the hsp89 protein (hsp89 alpha) was activated during an adenovirus infection with kinetics similar to those of activation of hsp70. The induction required a functional E1A gene. However, the hsp89 transcript was not cell cycle regulated. Genes encoding another form of hsp89 and the hsp27 protein were not induced by E1A or during the cell cycle. Further examination of hsp70 expression revealed a greater complexity than previously seen. S1 nuclease analysis using an hsp70 cDNA as well as a distinct hsp70 genomic clone demonstrated three related hsp70 transcripts; two were induced by E1A, and one was not. Both of the E1A-inducible genes were regulated during the cell cycle. All three were induced by heat shock. These results suggest common aspects of control among certain members of this family of cellular genes distinct from heat shock control. Finally, using viruses that express the individual E1A proteins, we found that the hsp70 gene is induced by the 12S and the 13S E1A products. The efficiency of induction by the 12S product was somewhat less than that by the 13S product but only by a factor of less than 2. This is in contrast to the induction of early viral genes, for which the 13S product is considerably more efficient than the 12S product.

Feedback regulation of the heat shock response.

The heat shock response is triggered primarily by nonnative proteins accumulating in a stressed cell and results in increased expression of heat shock proteins (Hsps), i.e., of chaperones capable of participating in the refolding or elimination of nonnative proteins. Best known is the transcriptional part of this response that is mediated predominantly by heat shock factor 1 (HSF1). HSF1 activity is regulated at different levels by Hsps and co-chaperones and is modulated further by a number of mechanisms involving other stress-regulated aspects of cell metabolism.

Hybrid plasmids carrying part of the Rous sarcoma virus-specific leader sequence.

'Strong-stop' DNA, complementary to the 5'-terminal 101 nucleotides of Rous sarcoma virus genomic RNA and subgenomic mRNAs can be used to isolate RSV-specific mRNA from infected cells. To analyse leader sequences of these mRNAs and to study the mechanism of their translation, relatively large quantities of strong-stop DNA are required. The construction of four plasmids carrying useful strong-stop DNA sequences for mRNA isolation is described. One such hybrid plasmid carries a DNA insertion containing sequences derived from a region starting 42 nucleotides from the 5'-end of RSV RNA, and ending at the tRNATry primer attachment site. Since this plasmid lacks the 20-nucleotide terminal repeat sequence it can be used for the specific isolation of 5'-end fragments of RSV-specific mRNAs and of complementary DNA transcripts of these fragments.

Nucleotide sequence analysis of the Drosophila small heat shock gene cluster at locus 67B.

The four small heat shock protein genes of Drosophila melanogaster clustered at cytological locus 67B have been characterized by DNA sequencing. Over 6250 nucleotides, covering the 5', protein-coding and 3' regions of these genes have been determined together with their predicted amino acid sequences. Each gene possesses characteristic eukaryotic 5' and 3' sequence elements and a single uninterrupted protein-coding region. The four encoded polypeptides of 19,700, 20,600, 23,000 and 23,600 Mr share a homologous stretch of 108 amino acid residues, representing 51 to 62% of their lengths. This region is flanked by sequences of dissimilar length and amino acid composition, located mainly at the amino-terminal end, but also at the extreme carboxyl termini of these proteins. The first 14 amino acids exhibit a small degree of homology, both amongst themselves and with some signal peptides and a transmembrane protein. Investigation of the hydrophilic/hydrophobic characteristics of the four polypeptides revealed, within the conserved 108 amino acid stretch, the presence of an alpha-helical region of very prominent local hydrophilicity, which probably represents a surface structural domain common to each protein. Sequence analysis with respect to transcription initiation and termination and possible regulatory signals is discussed together with some structural predictions for the four proteins.

Determinants for the DNase I-hypersensitive chromatin structure 5' to a human HSP70 gene.

A DNase I hypersensitive site was detected in chromatin formed over a human hsp70 gene segment after amplification in COS7 cells. Deletion mutant analysis was used to evaluate the sequence requirements for this chromatin structure. Determinants sufficient to form the hypersensitive site are contained in a 280 base-pair sequence corresponding approximately to the region that is hypersensitive. Deletion of sequences from either end of this region resulted in reduced hypersensitivity, suggesting that multiple genetic elements contribute to the formation of this chromatin structure. As has been reported for other heat shock genes, the hypersensitive chromatin structure is present prior to heat treatment and does not change in intensity or position after heat shock, in spite of the fact that hsp70 gene expression is completely dependent on heat induction. Sequence requirements for hypersensitivity were generally similar to those for heat-induced gene expression when mutant plasmids were tested at low copy number (e.g. in HeLa cells or in COS cells without amplification); however, deletion of sequences between -223 and -162 with respect to the start of transcription abolished the hypersensitive site but had no effect on gene expression. A barrier to exonuclease III digestion was detected within this region (near an imperfect inverted repeat sequence centered at position -202), suggesting that proteins are tightly bound to the DNA at this location.

Cis-acting elements involved in the regulated expression of a human HSP70 gene.

Expression of reporter genes under the control of upstream sequences of a human hsp70 gene was examined in microinjected Xenopus oocytes, transfected monkey COS and human HeLa cells. The genes were strictly heat-regulated in all three cell systems. A 69 nucleotide long segment of hsp70 5' non-transcribed sequence that included at least one functional heat shock regulation sequence was sufficient for heat-controlled expression in Xenopus and monkey cells but not in human HeLa cells. An additional segment of about 200 nucleotides in length was required for optimal activity. This segment contains two heat shock regulation elements, each of which appears to contribute to the overall activity in heat-treated human cells. Upstream non-transcribed sequences of the human hsp70 gene are capable of conferring heat regulation on a heterologous promoter. The potential roles in transcription regulation of a bending center in the TATA box region, a CCAAT-like sequence and some of many potential Sp1 binding sites in the hsp70 5' non-transcribed region were investigated.

Transduction of the stress signal and mechanisms of transcriptional regulation of heat shock/stress protein gene expression in higher eukaryotes.

This review deals with the transcriptional regulation of heat shock or stress genes that are present in every organism studied to date. While some of these genes are expressed constitutively and appear to be involved in basic cellular processes such as protein synthesis and maturation, assembly of protein complexes, and intracellular trafficking, others are normally silent or are expressed at low levels. Expression of the latter genes is enhanced when cells are subjected to stressful conditions such as elevated temperature and other physical and chemical insults or at specific stages of organismal development. The upregulation of these genes correlates with the development of tolerance to subsequent, similar insults. Upregulation following environmental insults appears to be mediated by heat shock transcription factor. This article summarizes what is known about the promoters of regulated stress genes in higher eukaryotes and the mechanisms by which heat shock transcription factor or developmental regulators control their activation. Recent data pointing to a possible connection between the activation of stress genes and general signal transduction pathways are also discussed, as they suggest an integration of the stress response and other cellular control mechanisms.