Transcriptional regulation of human hsp70 genes: relationship between cell growth, differentiation, virus infection, and the stress response.
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Bacterial Proteins↗
Biomedical subjects
R I Morimoto
Publications and source records attributed to R I Morimoto.
Cloning and characterization of two mouse heat shock factors with distinct inducible and constitutive DNA-binding ability.
We have cloned two distinct mouse heat shock transcription factor genes, mHSF1 and mHSF2. The mHSF1 and mHSF2 open reading frames are similar in size, containing 503 and 517 amino acids, respectively. Although mHSF1 and mHSF2 are quite divergent overall (only 38% identity), they display extensive homology in the DNA-binding and oligomerization domains that are conserved in the heat shock factors of Saccharomyces cerevisiae, Kluyveromyces lactis, Drosophila, tomato, and human. The ability of these two mouse heat shock factors to bind to the heat shock element (HSE) is regulated by heat. mHSF1 is expressed in an in vitro translation system in an inactive form that is activated to DNA binding by incubation at temperatures greater than 41 degrees C, the same temperatures that activate heat shock factor DNA binding and the stress response in mouse cells in vivo. mHSF2, on the other hand, is expressed in a form that binds DNA constitutively but loses DNA binding by incubation at greater than 41 degrees C. Both mHSF1 and mHSF2 are encoded by single-copy genes, and neither is transcriptionally regulated by heat shock. However, there is a striking difference in the levels of mHSF1 mRNA in different tissues of the mouse.
Amino Acid Sequence↗
Attenuation of the heat shock response in HeLa cells is mediated by the release of bound heat shock transcription factor and is modulated by changes in growth and in heat shock temperatures.
When HeLa S3 cells are subjected to a continuous 42 degrees C heat shock, activation of heat shock transcription factor (HSF) and transcriptional activation of the heat shock genes hsp70, hsp89 alpha, and hsp60 is transient, peaking at 40-60 min of heat shock, and then attenuating. We have used in vivo genomic footprinting to demonstrate that attenuation of hsp70 transcription is mediated by release of bound HSF from the heat shock element (HSE) of the hsp70 gene promoter. Release of bound HSF in vivo occurs at a higher rate than would be predicted from in vitro measurements of dissociation. Attenuation of HSF activation and heat shock gene transcription occurs only when mild heat shock temperatures are employed (42 degrees C); increasing the heat shock temperature by 1 degree C elicits a much higher level of activation, which does not attenuate during a 4-hr heat shock. Surprisingly, altering the temperature at which cells are grown prior to heat shock modulates the magnitude and temporal pattern of the response to a given heat shock temperature. This finding suggests that HSF does not sense temperature directly but, instead, may be responsive to the magnitude of the difference between growth and heat shock temperatures.
Base Sequence↗
Analysis of the specificity and mechanism of transcriptional activation of the human hsp70 gene during infection by DNA viruses.
We have examined the transcriptional regulation of the 70-kDa (70K) heat shock gene family following infection of human and monkey cells with four different DNA viruses: adenovirus type 5 (Ad5), herpes simplex virus type 1 (HSV-1), simian virus 40, and vaccinia virus. Our results indicate that induction of these genes is not a general response to the stress of viral infection but is instead a highly specific response, both with regard to the inducing virus and with regard to the target gene. Of three 70K heat shock genes examined, only hsp70 was induced during viral infection, and induction occurred only after infection by Ad5 and HSV-1. As revealed by genomic footprinting analysis, the mechanism of transcriptional activation of hsp70 during Ad5 or HSV-1 infection does not involve changes in the avidity of binding of basal transcription factors to the hsp70 promoter. In HSV-1-infected HeLa cells, transcriptional activation of hsp70 was quite transient, following which transcription was rapidly repressed; this was accompanied by the release of bound factors from the hsp70 promoter. In addition to the selectivity which characterizes the viral activation of hsp70 transcription, our results indicate that the consequences of this activation, as measured by changes in hsp70 mRNA levels and protein synthesis, are also virus specific.
Adenovirus Early Proteins↗
Flux of the paramyxovirus hemagglutinin-neuraminidase glycoprotein through the endoplasmic reticulum activates transcription of the GRP78-BiP gene.
The cellular glucose-regulated protein GRP78-BiP is a member of the HSP70 stress family of gene products, and the protein is a resident component of the endoplasmic reticulum, where it is thought to play a role in the folding and oligomerization of secretory and membrane-bound proteins. GRP78-BiP also binds to malfolded proteins, and this may be one mechanism for preventing their intracellular transport. An induction in synthesis of the GRP78-BiP protein occurs in cells infected with paramyxoviruses (R. W. Peluso, R. A. Lamb, and P. W. Choppin, Proc. Natl. Acad. Sci. USA 75:6120-6124, 1978). We have studied the expression and activity of the GRP78-BiP gene and synthesis of the GRP78-BiP protein during infection with the paramyxovirus simian virus 5 (SV5). We wished to identify the viral component capable of causing activation of GRP78-BiP since GRP78-BiP interacts specifically and transiently with the SV5 hemagglutinin-neuraminidase (HN) glycoprotein during HN folding (D. T. W. Ng, R. E. Randall, and R. A. Lamb, J. Cell Biol. 109:3273-3289, 1989). Expression of cDNAs of the SV5 wild-type HN glycoprotein and a mutant form of HN that is malfolded but not the SV5 F glycoprotein or SV5 cytoplasmic proteins P, V, and M caused increased amounts of GRP78-BiP mRNA to accumulate, as detected by nuclease S1 protection assays. As unfolded or malfolded forms of HN cannot be detected to accumulate during SV5 infection, the data suggest that the flux of HN through the ER in SV5-infected cells can cause activation of GRP78-BiP transcription.
Carrier Proteins↗
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.
Base Sequence↗
Surprising features of transcriptional regulation of heat shock genes.
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Animals↗
Heat shock: the role of transient inducible responses in cell damage, transformation, and differentiation.
The stress response is ubiquitous among all organisms of the bacterial, plant, and animal kingdoms. A prominent feature of this response is the synthesis of a discrete set of proteins, known as heat shock proteins, that have been shown to be essential in a plethora of protein biosynthetic and processing reactions, including protein folding and oligomerization, translocation, and secretion. During heat shock and other forms of physiological stress, heat shock proteins act as intracellular sentinels to recognize malfolded proteins. The heat shock response is tightly regulated and encompasses selective transcriptional and translational mechanisms that control the preferential synthesis of stress proteins during physiological stress. Deregulation of stress gene expression is associated with various human diseases. It is likely that the balance of interactions between damaged protein molecules and stress proteins has profound effects that impinge on normal cell growth and differentiation.
Animals↗
cis-acting sequences and trans-acting factors required for constitutive expression of a microinjected HSP70 gene after the midblastula transition of Xenopus laevis embryogenesis.
Microinjected human HSP70 promoter-chloramphenical acetyl transferase (CAT) chimeric genes are constitutively expressed immediately after the midblastula transition of Xenopus embryogenesis. Analysis of a series of 5'-deletion mutants in the HSP70 promoter revealed that sequences within 74 bases of the transcriptional start site were sufficient for strong basal activity. We investigated the role of specific sequences in the basal promoter by injecting HSP70-CAT vectors containing linker-scanner mutations in the basal elements (CCAAT, purine-rich element, GC-element, ATF/AP1, and TATA). Our data reveal that deletion of any of these cis-acting elements in the basal promoter prevents expression after the midblastula stage of development. Furthermore, we have identified specific binding activities in embryonic nuclear extracts that complex with basal promoter elements (CCAAT, ATF, and GC) of the heterologous HSP70 promoter. These trans-acting factors are detectable in nuclear extracts of early blastula embryos, and their respective binding activity increases dramatically after the midblastula transition. The expression of the human HSP70 gene after the midblastula transition of Xenopus embryogenesis requires an array of cis-acting elements, which interact with specific Xenopus transcription factors.
Animals↗
In vitro activation of heat shock transcription factor DNA-binding by calcium and biochemical conditions that affect protein conformation.
The transcription of heat shock genes in response to physiological stress requires activation of heat shock transcription factor (HSF). Although the transcriptional response is most commonly induced by temperature elevation, the biochemical events involved in HSF activation in vivo can also be triggered at normal physiological temperatures by chemicals that inhibit metabolic processes. We have used a HeLa cell-free system in which HSF DNA-binding is activated by conditions that affect protein conformation, including increasing concentrations of hydrogen ions, urea, or nonionic detergents. Treatment with calcium ions also results in a concentration- and time-dependent activation of HSF in vitro. Pretreatment with each of these biochemical conditions reduces the temperature dependence for HSF activation in vitro. These results suggest that HSF is activated either directly by undergoing a conformational change or indirectly through interactions with unfolded proteins.
Base Sequence↗
Identification of cytosolic peroxisome proliferator binding protein as a member of the heat shock protein HSP70 family.
Clofibrate and many of its structural analogues induce proliferation of peroxisomes in the hepatic parenchymal cells of rodents and certain nonrodent species including primates. This induction is tissue specific, occurring mainly in the liver parenchymal cells and to a lesser extent in the kidney cortical epithelium. The induction of peroxisomes is associated with a predictable pleiotropic response, characterized by hepatomegaly, and increased activities and mRNA levels of certain peroxisomal enzymes. Using affinity chromatography, we had previously isolated a protein that binds to clofibric acid. We now show that this protein is homologous with the heat shock protein HSP70 family by analysis of amino acid sequences of isolated peptides from trypsin-treated clofibric acid binding protein and by cross-reactivity with a monoclonal antibody raised against the conserved region of the 70-kDa heat shock proteins. The clofibric acid-Sepharose column could bind HSP70 proteins isolated from various species, which could then be eluted with either clofibric acid or ATP. Conversely, when a rat liver cytosol containing multiple members of the HSP70 family was passed through an ATP-agarose column, and eluted with clofibric acid, only P72 (HSC70) was eluted. These results suggest that clofibric acid, a peroxisome proliferator, preferentially interacts with P72 at or near the ATP binding site.
Amino Acid Sequence↗
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.
Base Sequence↗
Cell cycle-dependent association of HSP70 with specific cellular proteins.
In asynchronous populations of HeLa cells maintained at control or heat shock temperatures, HSP70 levels and its subcellular distribution exhibit substantial heterogeneity as demonstrated by indirect immunofluorescence with HSP70-specific monoclonal antibodies. Of particular interest is a subpopulation of cells in which the characteristic nuclear accumulation and nucleolar association of HSP70 is not detected after heat shock treatment. This apparent variation in the heat shock response is not observed when synchronized cells are examined. In this study, we demonstrate that three monoclonal antibodies to HSP70, in particular, do not detect nucleolar-localized HSP70 in heat-shocked G2 cells. This is not due to an inability of G2 cells to respond to heat shock as measured by increased HSP70 mRNA and protein synthesis, or due to a lack of accumulation of HSP70 after heat shock in G2. Rather the epitopes recognized by the various antibodies appear to be inaccessible, perhaps due to the association of HSP70 with other proteins. Non-denaturing immunoprecipitations with these HSP70-specific antibodies suggest that HSP70 may interact with other cellular proteins in a cell cycle-dependent manner.
Antibodies, Monoclonal↗
Mutational analysis of the human HSP70 protein: distinct domains for nucleolar localization and adenosine triphosphate binding.
The human HSP70 gene was modified in vitro using oligonucleotide-directed mutagenesis to add sequences encoding a peptide from the testis-specific form of human lactate dehydrogenase (LDH) to the carboxy terminus of HSP70. The peptide-tagged HSP70 can be distinguished from the endogenous HSP70 protein using an LDH peptide-specific antiserum in indirect immunofluorescence assays of cells transiently transfected with an expression vector containing the tagged HSP70 gene regulated by the human HSP70 promoter. A series of deletion mutants within the HSP70 protein coding region were generated. Using double-label indirect immunofluorescence with the LDH peptide-specific antiserum and HSP70-specific mAbs, we compared the intracellular distribution of the deletion mutants to that of endogenous HSP70. We have determined that sequences in the carboxy terminus of HSP70 are necessary for proper nucleolar localization after heat shock. In contrast, sequences in the amino terminus of HSP70 are responsible for the ATP-binding ability of the protein. Mutants that were unable to bind ATP, however, still displayed nucleolar association, indicating that ATP binding is apparently not required for interaction with substrate. Additional support that HSP70 appears to be composed of at least two domains follows from the results of trypsin digestions of wild type and mutant HSP70. Protease digestion of the mutant HSP70 proteins identified a region of HSP70 that, when deleted, affected HSP70 conformation.
Adenosine Triphosphate↗
E1a transactivation of the human HSP70 promoter is mediated through the basal transcriptional complex.
We have examined the promoter sequence requirements for E1a transactivation of the human HSP70 gene by using a transient cotransfection assay. A 5' deletion study has defined a basal transcription unit extending to -74 relative to the transcription initiation site which was fully E1a responsive. Further deletion, abolishing a CCAAT element at -67, drastically reduced basal and E1a-induced expression. A linker-scanner analysis has identified four functional elements within the basal transcription unit which may interact with CTF, SP1, TFIID, and an ATF/AP1-like factor. Sequences between -100 and -188 can partially compensate for mutations in these elements. No mutation specifically abolished E1a inducibility. Any reduction in absolute E1a-induced levels was accompanied by a corresponding reduction in absolute basal levels, thereby maintaining a constant relative fold induction. We conclude that E1a transactivation of the human HSP70 promoter does not require any single basal transcription element. We also examined an HSP70 promoter fragment, containing the CCAAT element at -67 and the purine-rich element at -54, out of its normal context by fusing it upstream of a transcriptionally inactive herpes simplex virus thymidine kinase deletion construct containing only the TATA box. The resulting chimeric promoter was fully E1a responsive. Mutagenesis of this promoter fusion demonstrated that the CCAAT element was essential for detectable basal and E1a-induced expression. Mutations in the purine-rich element resulted in an approximately 10-fold elevation in basal levels and rendered the promoter nonresponsive to E1a.
Adenovirus Early Proteins↗
Hemin-induced transcriptional activation of the HSP70 gene during erythroid maturation in K562 cells is due to a heat shock factor-mediated stress response.
Hemin-induced differentiation of the human erythroleukemia cell line K562 results in the expression and accumulation of erythroid-specific gene products such as embryonic and fetal hemoglobins and the elevated synthesis of the major heat shock protein HSP70. This activity was suggested to represent activation of a heat shock gene during erythroid maturation independent of stress induction. In this study, we demonstrate that hemin induces the transcription of two members of the human HSP70 gene family, HSP70 and GRP78 (BiP). However, the induction of HSP70 by hemin showed characteristics consistent with the molecular events associated with a heat shock or stress response. The increase in HSP70 gene transcription was accompanied by induction of the stress-induced form of the heat shock transcription factor. Moreover, a heat shock element was required for the hemin responsiveness of chimeric heat shock promoter-chloramphenicol acetyltransferase genes transiently expressed in transfected K562 cells.
Carrier Proteins↗
HSP70 mRNA translation in chicken reticulocytes is regulated at the level of elongation.
During heat shock of chicken reticulocytes the synthesis of a single heat shock protein, HSP70, increases greater than 10-fold, while the level of HSP70 mRNA increases less than 2-fold during the same period. Comparison of the in vivo levels of HSP70 and beta-globin synthesis with their mRNA abundance reveals that the translation of HSP70 mRNA is repressed in normal reticulocytes and is activated upon heat shock. In its translationally repressed state HSP70 mRNA is functionally associated with polysomes based on sedimentation analysis of polysomes from untreated or puromycin-treated cells and by analysis of in vitro "run-off" translation products using isolated polysomes. Treatment of control and heat shocked cells with the initiation inhibitor pactamycin reveals that elongation of the HSP70 nascent peptide is not completely arrested, but is slower in control cells. Furthermore, the inefficient translation of HSP70 mRNA in vivo is not due to the lack of an essential translation factor; HSP70 mRNA is efficiently translated in chicken reticulocyte translation extracts as well as in heterologous rabbit reticulocyte extracts. Our results reveal that a major control point for HSP70 synthesis in reticulocytes is the elongation rate of the HSP70 nascent peptide.
Animals↗
Mitogen and lymphokine stimulation of heat shock proteins in T lymphocytes.
We have examined the effects of phytohemagglutinin (PHA) and the polypeptide growth factor interleukin 2 (IL-2) on the synthesis of the 70- and 90-kDa heat shock proteins (HSP70 and HSP90, respectively) in human T lymphocytes. Resting T cells (G0) stimulated with PHA responded with a generalized increase in protein synthesis that included HSP70. Gel blot analysis indicated that steady-state levels of HSP70 mRNA were not specifically modulated by PHA. Synthesis of HSP90 protein, however, peaked very rapidly following PHA stimulation and decreased sharply after 1 hr. When IL-2-dependent human T cells, synchronized by IL-2 deprivation, were treated with IL-2, synthesis of HSP70 mRNA was increased as much as 15-fold. HSP70 and HSP90 protein synthesis increased significantly upon IL-2 stimulation of human T lymphocytes. Two distinct members of the ancient family of heat shock genes, HSP70 and HSP90, are shown to be stimulated at the activation and progression stages of lymphocyte mitogenesis, which suggests that genetic mechanisms evolved from primitive stress/adaptation responses may be conserved in mammalian cellular activation.
Cells, Cultured↗