Human Hsp70 Promoter Research Articles

Basal level transcription of the human hsp86 gene is directed by intron-based elements.

The heat shock response includes the syntheses of three major size-classes of heat shock proteins (hsps) designated as class 20, 70 and 90 hsps, respectively. In contrast to the class 20 and 70 hsp genes, those coding for the class 90 hsps are already actively transcribed in the non-induced state. We have shown that the increased basal level transcription of the human hsp86 gene depends on the presence of the first intron. Furthermore, intron 1 of the hsp86 gene was also capable of conferring increased basal level transcription on the human hsp70 promoter. Finally, the intron I-based sequence elements of the hsp86 gene mediated an effective rescue of 5'-truncated hsp70 and hsp86 promoters. In contrast, such a rescue of inactivated promoters was not obtained with the viral SV40 gene enhancer which is often also capable of stimulating basal level expression. Element(s) which direct the basal transcription of the human hsp86 gene at normal physiological temperatures are located within the first intron of the gene. It is conceivable that this intron-dependent activation of basal hsp86 transcription reflects a fundamental requirement for this protein in the context of cellular growth and metabolism under non-stress conditions.

Human cytomegalovirus immediate-early 2 (IE2) protein can transactivate the human hsp70 promoter by alleviation of Dr1-mediated repression

The immediate-early 1 and 2 (IE1 and IE2, respectively) proteins of human cytomegalovirus are known transcription factors, which regulate the expression of viral and cellular genes. Transcriptional activation by IE2 is dependent on the presence of a TATA motif in target promoters, and IE2 can interact directly with the TATA-binding protein (TBP) component of TFIID. TBP is known to be the target for transcriptional repression by the cellular Dr1 protein, and this factor has been shown to repress expression from the hsp70 promoter in vivo. Since this promoter is up-regulated by IE2, we asked whether the effects of Dr1 can be overcome by IE2. We report here that IE2 can overcome Dr1-mediated repression of the hsp70 promoter in vivo and that IE2 can interact with Dr1 in vivo and in vitro. We also demonstrate a previously unreported activity of Dr1, inhibition of DNA binding by TBP, and show that IE2 is able to overcome this inhibition in vitro, suggesting a mechanism for the TATA dependency of IE2-mediated trans activation.

Displacement of sequence-specific transcription factors from mitotic chromatin

The general inhibition in transcriptional activity during mitosis abolishes the stress-inducible expression of the human hsp70 gene. Among the four transcription factors that bind to the human hsp70 promoter, the DNA-binding activities of three (C/EBP, GBF, and HSF1) were normal, while Sp1 showed reduced binding activity in mitotic cell extracts. In vivo footprinting and immunocytochemical analyses revealed that all of the sequence-specific transcription factors were displaced from promoter sequences as well as from bulk chromatin during mitosis. The correlation of transcription factor displacement with chromatin condensation suggests an involvement of chromatin structure in mitotic repression. However, retention of DNase I hypersensitivity suggests that the hsp70 promoter was not organized in a canonical nucleosome structure in mitotic chromatin. Displacement of transcription factors from mitotic chromosomes could present another window in the cell cycle for resetting transcriptional programs.

Stable overexpression of human HSF-1 in murine cells suggests activation rather than expression of HSF-1 to be the key regulatory step in the heat shock gene expression.

Transcription of the heat shock genes is regulated by the activation of the heat shock transcription factor (HSF-1). After heat shock, HSF-1 forms oligomers and binds to the heat shock element (HSE), which consists of several repeats of NGAAN located in the promoter region of the heat shock genes. HSF-1 is then phosphorylated, leading to the enhanced transcription of the heat shock genes likely by transactivation. We have stably overexpressed the human heat shock transcription factor-1 (HSF-1) in murine cells to investigate whether the regulation of the expression of the heat shock genes may partly reside at the level of HSF-1 expression. Human HSF-1 cDNA was cloned into a retroviral vector (pvhhsf-1) and was overexpressed in a murine fibroblast cell line. The overexpressed human HSF-1 is found in both the cytoplasm and nucleus of control cells but is translocated into the nucleus upon heat shock. Electrophoretic mobility shift analysis suggests that the human HSF-1 has constitutive DNA binding ability and its DNA binding ability is increased upon heat shock. Cross-linking experiments indicate that the overexpressed human HSF-1 is mainly a monomer under control conditions and forms oligomers upon heat shock. Immunoblotting shows that the human HSF-1 is phosphorylated upon heat shock and its apparent molecular weight is shifted up by at least 10 kDa. In spite of both the DNA binding ability and phosphorylation, the overexpression of human HSF-1 does not increase the transcription of murine HSP-70 mRNA or increase the synthesis of other HSPs after heat shock beyond that observed in control untransfected cells. An exception is the enhanced synthesis of a 47-50 kDa protein after heat shock and an apparent lack of induction of one HSP-70 kDa species when the protein pattern is analyzed by isoelectric focusing. Interestingly, cells overexpressing human HSF-1 show a 4-fold increase in the basal expression of luciferase when the plasmids containing the human HSP-70 promoter ligated to the luciferase reporter gene are transiently expressed in these cells. Murine cells overexpressing human HSF-1 are more resistant to the cytotoxic effects of heat when compared to the control untransfected cells, but the kinetics of thermotolerance development and decay is similar between HSF-1 transfected and untransfected cells. In conclusion, human HSF-1 protein in murine fibroblasts is modified in a similar fashion as the endogenous mouse HSF-1 after heat shock. However, the overexpression of HSF-1 does not result in overproduction of heat shock proteins after heat shock, perhaps because these cells contain abundant amounts of endogenous HSF-1.

C-Myb-induced trans-activation mediated by heat shock elements without sequence-specific DNA binding of c-Myb.

The c-myb proto-oncogene product (c-Myb) can transactivate the human hsp70 promoter in a transient cotransfection assay. The present studies have demonstrated that the heat shock element (HSE) in the hsp70 promoter mediates trans-activation by c-Myb. Mutagenesis of the DNA sequence in HSE indicated that the NGAAN motif is necessary for not only the heat shock response but also the c-Myb-induced trans-activation. The HSE in the hsp70 promoter does not contain a c-Myb-binding site, implying that the sequence-specific DNA binding of c-Myb is not required for the HSE-dependent trans-activation by c-Myb. We had demonstrated that a disruption of the leucine zipper motif in the central portion of the c-Myb molecule increased the degree of c-Myb-induced trans-activation of the promoter containing c-Myb-binding sites, suggesting that a putative inhibitor binds to c-Myb through this leucine zipper (Kanie-Ishii, C., MacMillan, E. M., Nomura, T., Sarai, A., Ramsay, R. G., Aimoto, S., Ishii, S., and Gonda, T. J. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 3088-3092). However, disruption of the leucine zipper in c-Myb abolished the HSE-dependent trans-activation by c-Myb, whereas deletion of the transcriptional activation domain containing acidic amino acids in c-Myb did not abolish the HSE-dependent trans-activation by c-Myb. These results suggest that c-HSEs by interacting with unidentified trans-acting factor(s) but not by a direct binding to the promoter through its DNA-binding domain.

Regulation of the Human hsp70 Promoter by p53

The tumor suppressor p53 is a nuclear phosphoprotein with characteristics of a transcription factor. It displays sequence-specific DNA binding, contains a potent transactivation domain, and has been implicated as both a transcriptional activator and a repressor. Transcription of the human hsp70 gene is stimulated by adenovirus E1a protein. This E1a transactivation of the hsp70 promoter is mediated by CCAAT binding factor (CBF). It is demonstrated here that p53 both represses transcription from the human hsp70 promoter and also interacts with CBF. Thus, the repression of the hsp70 promoter by p53 may be mediated by direct protein-protein interaction with CBF. These results suggest that protein-protein interaction between p53 and specific transcription factors may be an additional mechanism by which p53 regulates gene expression.

Induction of the human growth hormone gene placed under human hsp70 promoter control in mouse cells: a quantitative indicator of metal toxicity.

An in vitro test method for general metal toxicity screening was designed, based on the cellular response to stress. The expression of a transfected human growth hormone gene sequence driven by the human heat-shock protein 70 promoter in NIH/3T3 cells was used as marker of noxious contact with metal compounds. Out of a series of 31 metals, 17 were competent for inducing this stress response system. According to the effective concentration and to the intensity of the response, three different clusters of positive compounds emerged and were ranked as strong, intermediate strength and weak inducers. These results correlated well with data from other in vivo and in vitro metal toxicity studies, including LD50 in mice. Apparently the positive/negative compounds also fitted well with data from genotoxicity and carcinogenesis studies on metal salts.

Tributyltin is a potent inducer of the heat shock response in human diploid fibroblasts.

Submicromolar concentrations of tributyltin (TBT), a commercially used organotin compound, were found to induce the expression of several stress proteins, most notably HSP89 and HSP70, in IMR-90 human diploid fibroblasts in a time- and dose-dependent manner. This induction can be demonstrated by quantitation of 1) synthesis of the heat shock proteins (HSPs), 2) relative abundance of mRNA of hsp70, and 3) transient expression of a human hsp70 promoter driven reporter gene. TBT also increased the abundance of mRNA of heme oxygenase, whereas heat shock was without effect. Analysis of protein binding to a consensus heat shock element (HSE) by electrophoretic mobility shift assay suggests that the induction of the heat shock response by TBT was attributable to activation of the heat shock transcription factor (HSTF).

Gene expression following transfection of fish cells

Various genes containing different transcriptional regulatory elements (TRE) and the bacterial marker gene coding for chloramphenicol acetyl transferase were transfected into several fish cell lines to evaluate the efficiency of expression in comparison with mammalian cells. The CMV and RSV TRE were the most efficient non-inducible promoters in directing reporter gene expression. RSV and CMV appeared of similar potency in a stable fish cell line. The human HSP-70 promoter showed high potency in a carp and in a trout cell line after thermal induction. This promoter also induced the synthesis of human growth hormone directed by the corresponding cDNA, but not by the gene. RSV TRE was also able to drive the synthesis of bovine growth hormone when attached directly to the cDNA but not to the gene. These data suggest that non-fish gene TRE can be used to express foreign genes in fish cells or transgenic fish; however, in most cases they are relatively inefficient. The data also suggest that the translation and secretion machinery of fish cells can express efficiently foreign genes but that mammalian introns might be not processed properly in some cases.

Activation of heat shock factor 2 during hemin-induced differentiation of human erythroleukemia cells.

Hemin induces nonterminal differentiation of human K562 erythroleukemia cells, which is accompanied by the expression of certain erythroid cell-specific genes, such as the embryonic and fetal globins, and elevated expression of the stress genes hsp70, hsp90, and grp78/BiP. Previous studies revealed that, as during heat shock, transcriptional induction of hsp70 in hemin-treated cells is mediated by activation of heat shock transcription factor (HSF), which binds to the heat shock element (HSE). We report here that hemin activates the DNA-binding activity of HSF2, whereas heat shock induces predominantly the DNA-binding activity of a distinct factor, HSF1. This constitutes the first example of HSF2 activation in vivo. Both hemin and heat shock treatments resulted in equivalent levels of HSF-HSE complexes as analyzed in vitro by gel mobility shift assay, yet transcription of the hsp70 gene was stimulated much less by hemin-induced HSF than by heat shock-induced HSF. Genomic footprinting experiments revealed that hemin-induced HSF and heat shock-induced HSF, HSF2, and HSF1, respectively, occupy the HSE of the human hsp70 promoter in a similar yet not identical manner. We speculate that the difference in occupancy and/or in the transcriptional abilities of HSF1 and HSF2 accounts for the observed differences in the stimulation of hsp70 gene transcription.

Activation of heat shock factor 2 during hemin-induced differentiation of human erythroleukemia cells.

Hemin induces nonterminal differentiation of human K562 erythroleukemia cells, which is accompanied by the expression of certain erythroid cell-specific genes, such as the embryonic and fetal globins, and elevated expression of the stress genes hsp70, hsp90, and grp78/BiP. Previous studies revealed that, as during heat shock, transcriptional induction of hsp70 in hemin-treated cells is mediated by activation of heat shock transcription factor (HSF), which binds to the heat shock element (HSE). We report here that hemin activates the DNA-binding activity of HSF2, whereas heat shock induces predominantly the DNA-binding activity of a distinct factor, HSF1. This constitutes the first example of HSF2 activation in vivo. Both hemin and heat shock treatments resulted in equivalent levels of HSF-HSE complexes as analyzed in vitro by gel mobility shift assay, yet transcription of the hsp70 gene was stimulated much less by hemin-induced HSF than by heat shock-induced HSF. Genomic footprinting experiments revealed that hemin-induced HSF and heat shock-induced HSF, HSF2, and HSF1, respectively, occupy the HSE of the human hsp70 promoter in a similar yet not identical manner. We speculate that the difference in occupancy and/or in the transcriptional abilities of HSF1 and HSF2 accounts for the observed differences in the stimulation of hsp70 gene transcription.

C- myc protein complex binds to two sites in human hsp70 promoter region

We investigated the binding of partially purified (enriched) c- myc protein to the human hsp70 promoter region by band shift and ultraviolet crosslinking assays. In the hsp70 promoter region, two sites were found to be homologous to the c- myc protein complex binding sequence in the c- myc gene. These sites are located at positions −230 and −160 bases relative to the transcription initiation site, overlapping with the region reported for the regulation of hsp70 gene expression by c- myc, and upstream of other regulatory sequences including the heat shock element and the serum responsive element. The results shown here suggest that the c- myc protein complex from human HL-60 cells binds to the two sites of the region directly and sequence specifically. It is therefore suggested that the c- myc protein or protein complex contribute to the regulation of hsp70 gene expression by binding directly to the promoter region.

Mammalian promoter element function in the fission yeast Schizosaccharomyces pombe.

We have analyzed the function of several mammalian promoter elements in the fission yeast, Schizosaccharomyces pombe. Mutants of the human HSP70 promoter were introduced into S. pombe as single copy integrants at a specific location. Transcription initiation sites utilized in S. pombe with the HSP70 TATA element were similar to those used in mammalian cells. Of three mammalian TATA elements tested, only the HSP70 TATA element functioned in S. pombe. The adenovirus Ella TATA element had little or no activity in S. pombe, indicating that S. pombe is deficient in the factor(s) necessary for recognition of this element. Of upstream promoter elements tested, the CCAAT, Sp1 binding, ATF binding and heat shock elements were functional in S. pombe. Strains containing mutant promoters fused to the ble gene were used to demonstrate that phleomycin can be used as a graded selection in S. pombe. These data demonstrate that S. pombe should provide a useful system in which to characterize and isolate mammalian factors involved in initiation site determination and transcriptional regulation.

End-filling of an oligonucleotide duplex containing an MDBP site in the human hsp70 promoter inhibits protein·DNA complex formation

A site from the promoter region of the human hsp70 gene binds with a high affinity to the ubiquitous mammalian protein called methylated DNA-binding protein (MDBP) when it is present in a CpG-methylated oligonucleotide duplex with only 14 base-pairs. Binding to this site is dependent upon CpG methylation. Surprisingly, when the same methylated sequence is present in a duplex that has 22 or more base-pairs, binding to this protein is greatly inhibited. Such a requirement for a short duplex region is seen only in certain of the cytosine methylation-dependent binding sites for this protein and is proposed to reflect differences in the conformation of the duplex due to small differences in the nucleotide sequence.

Heat shock-induced interactions of heat shock transcription factor and the human hsp70 promoter examined by in vivo footprinting.

Genomic footprinting of the human hsp70 promoter reveals that heat shock induces a rapid binding of a factor, presumably heat shock transcription factor, to a region encompassing five contiguous NGAAN sequences, three perfect and two imperfect matches to the consensus sequence. Arrays of inverted NGAAN sequences have been defined as the heat shock element. No protein is bound to the heat shock element prior to or after recovery from heat shock. Heat shock does not perturb the binding of factors to other regulatory elements in the promoter which contribute to basal expression of the hsp70 gene.

Heat Shock-Induced Interactions of Heat Shock Transcription Factor and the Human hsp70 Promoter Examined by In Vivo Footprinting

Genomic footprinting of the human hsp70 promoter reveals that heat shock induces a rapid binding of a factor, presumably heat shock transcription factor, to a region encompassing five contiguous NGAAN sequences, three perfect and two imperfect matches to the consensus sequence. Arrays of inverted NGAAN sequences have been defined as the heat shock element. No protein is bound to the heat shock element prior to or after recovery from heat shock. Heat shock does not perturb the binding of factors to other regulatory elements in the promoter which contribute to basal expression of the hsp70 gene.

A cloned human CCAAT-box-binding factor stimulates transcription from the human hsp70 promoter.

The basal promoter of the human hsp70 gene is predominantly controlled by a CCAAT element at position -70 relative to the transcriptional initiation site. We report the isolation of a novel cDNA clone encoding a 114-kDa polypeptide that binds to the CCAAT element of the hsp70 promoter. Expression of this CCAAT-binding factor (CBF) cDNA activated transcription from cotransfected hsp70 promoter-reporter gene constructs in a CCAAT-dependent manner. CCAAT-binding factor shows no homology to the previously identified human CCAAT transcription factor or rat CCAAT/enhancer-binding protein.

A Cloned Human CCAAT-Box-Binding Factor Stimulates Transcription from the Human hsp70 Promoter

The basal promoter of the human hsp70 gene is predominantly controlled by a CCAAT element at position -70 relative to the transcriptional initiation site. We report the isolation of a novel cDNA clone encoding a 114-kDa polypeptide that binds to the CCAAT element of the hsp70 promoter. Expression of this CCAAT-binding factor (CBF) cDNA activated transcription from cotransfected hsp70 promoter-reporter gene constructs in a CCAAT-dependent manner. CCAAT-binding factor shows no homology to the previously identified human CCAAT transcription factor or rat CCAAT/enhancer-binding protein.

Activation of the heat shock transcription factor by hypoxia in mammalian cells.

Members of the stress protein family such as HSP70 are induced in ischemic tissues and may contribute to the ability of cells to survive episodes of transient circulatory insufficiency. However, the biochemical events that lead to this induction, and their degree of similarity with pathways triggered by heat stress, have not been defined. In this study, we demonstrate that transient exposure of cultured C2C12 mouse myogenic cells to a hypoxic atmosphere stimulates DNA binding activity of the heat shock transcription factor through mechanisms that are independent of new protein synthesis. Activation of heat shock transcription factor in hypoxic cells is temporally associated with induction of endogenous HSP70 gene transcription and with induction of a heterologous reporter gene controlled by the human HSP70 promoter. Furthermore, induction of the human HSP70 promoter by hypoxia requires an intact heat shock element, indicating that other cis-acting transcriptional control elements contained within this complex promoter are not sufficient to transduce signals generated within hypoxic cells. These findings provide strong evidence that hypoxia and heat shock induce expression of the HSP70 gene by similar, if not identical, mechanisms.

Maximal stress-induced transcription from the human HSP70 promoter requires interactions with the basal promoter elements independent of rotational alignment.

Transcription of the human HSP70 gene is regulated by a complex array of cis-acting promoter elements that respond to conditions that include normal conditions of cell growth and induction following physiological stress. We have examined the requirements of the basal and inducible promoter elements by using promoter mutations and a transient transfection assay. Multiple forms of stress-induced transcription, including heat shock and incubation with heavy metals or amino acid analogs, are mediated by a single heat shock element (HSE) between -105 and -91 consisting of three contiguous 5-base-pair units, NGAAN, that are inverted relative to adjacent units. Maximal inducible expression requires a fully functional basal promoter. Spacing mutations which alter the relative helical orientation of adjacent genetic elements have only minimal effects on basal and stress-inducible expression and show no effects of periodicity. In addition, placement of the HSE adjacent to the basal promoter removes the requirements for a fully functional basal promoter for maximal stress-inducible expression. These results suggest that factors bound at the HSE and the basal promoter can function through multiple interactions.