Heat Shock Factor 1 Contains Two Functional Domains That Mediate Transcriptional Repression of the c-fos and c-fms Genes*

Yue Xie, Rong Zhong, Changmin ChenDagger , and Stuart K. Calderwood§

From the Dana Farber Cancer Institute, Harvard Medical School, and the Dagger  Department of Medicine, Molecular and Cellular Biology Laboratory, Beth Israel and Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02115

Received for publication, October 4, 2002, and in revised form, November 26, 2002

    ABSTRACT
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ABSTRACT
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EXPERIMENTAL PROCEDURES
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Heat shock factor 1 (HSF1), in addition to its pivotal role as a regulator of the heat shock response, functions as a versatile gene repressor. We have investigated the structural domains involved in gene repression using mutational analysis of the hsf1 gene. Our studies indicate that HSF1 contains two adjacent sequences located within the N-terminal half of the protein that mediate the repression of c-fos and c-fms. One region (NF) appears to be involved in quenching transcriptional activation factors on target promoters and binds to the basic zipper transcription factor NF-IL6 required for activation of c-fms and IL-1beta . The NF domain encompasses the leucine zipper 1 and 2 sequences as well as the linker domain between the DNA binding and leucine zipper regions. The function of this domain in gene repression is highly specific for HSF1, and the homologous region from conserved family member HSF2 does not restore repressive function in HSF2/HSF1 chimeras. In addition, HSF2 is not capable of binding to NF-IL6. The NF domain, although necessary for repression, is not sufficient, and a second region (REP) occupying a portion of the regulatory domain is required for repression. Neither domain functions independently, and both are required for repression. Furthermore, we constructed dominant inhibitors of c-fos repression by HSF1, which also blocked the repression of c-fms and IL-1beta , suggesting a shared mechanism for repression of these genes by HSF1. Our studies suggest a complex mechanism for gene repression by HSF1 involving the binding to and quenching of activating factors on target promoters. Mapping the structural domains involved in this process should permit further characterization of molecular mechanisms that mediate repression.

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Heat shock factor 1 (HSF1)1 is the regulator of heat shock protein (hsp) gene transcription and controls the response to protein stress conserved in eukaryotic cells (1-7). HSF1 senses exposure to stresses such as heat shock at least partially by monitoring the presence of denatured and aggregated proteins in cells (8, 9). Upon activation, HSF1 trimerizes and binds to the promoters of hsp genes in a hyper-phosphorylated form competent to activate transcription (2, 5, 10-14).

We have found that, in addition to activating the transcription of hsp genes, HSF1 acts as a repressor of non-heat shock genes (15-21). Heat shock inhibits the transcription of many inducible genes involved in macrophage activation and the acute phase response, including interleukin 1beta (IL-1beta ), tumor necrosis factor alpha  (TNFalpha ), and c-fms through the mediation of HSF1 (15, 17, 18, 20-22). In addition, other inducible genes not involved in the specialized function of macrophages, including the immediate early genes c-fos and urokinase plasminogen activator (uPA) are repressed by heat shock and HSF1 (16). Indeed repression by HSF1 may be a conserved property in eukaryotes as evidenced by the finding that developmental loci in Drosophila, which become repressed during heat shock, are associated with the Drosophila HSF homologue (23). This capacity for gene repression is specific for HSF1 within the hsf family in mammalian cells and is not a property of HSF2 (16, 20). We have examined in most detail the mechanism of gene repression by HSF1 in monocytes responding to bacterial endotoxin exposure. We find that the pro-inflammatory IL-1beta gene is repressed by HSF1 and that this response is mediated by HSF1 binding to and quenching the activating effect of an essential factor on the IL-1beta promoter, nuclear factor of interleukin 6 (NF-IL6/C/EBPbeta ), which regulates the transcription of many genes in myeloid cells (15, 20, 21, 24). Our previous studies (24) show that HSF1 binds to NF-IL6 both in vitro and in vivo through the basic zipper (bZIP) region. The bZIP region contains the leucine zipper dimerization domain and DNA binding region common to many bZIP families of transcription factors (25). The bZIP region mediates cooperative interactions with a number of other essential transcription factors including, in the case of the IL-1beta promoter, Spi1/PU.1 (20, 24, 26-28). HSF1 appears to repress the IL-1beta promoter by a mechanism that involves HSF1 binding to a functional heat shock element (HSE) and interaction with NF-IL6; HSF1 then blocks essential cooperative interactions between NF-IL6 and PU.1 required for IL-1beta promoter activation (15, 20). A similar mechanism appears to be involved in HSF1 repression of the c-fms gene (20). The HSF1-NF-IL6 interaction may constitute a molecular mechanism involved in the multiple levels of cross-talk between the heat shock response and the innate immune/acute phase response in myeloid cells (20, 29, 30). Indeed, NF-IL6 activation caused either by overexpression of the protein from transfected expression plasmid or by bacterial endotoxin stimulation of endogenous NF-IL6 leads to the repression of the hsp70b promoter through a mechanism that appears to involve directly HSF1-NF-IL6 binding (21). It is not known whether other members of the bZIP family including C/EBP, AP-1 binding, or activating transcription factor/cAMP-response element-binding protein family proteins can interact with HSF1 (31). Such an interaction could potentially be involved in the repression of genes such as c-fos or TNFalpha in which NF-IL6 does not play a major role in transcriptional activation.

In the work presented here we have examined the mechanisms of gene repression by HSF1 using mutational analysis to map repression domains. We have found that gene repression is conferred by sequences in the N-terminal half of the protein and that the C terminus, which contains the major trans-activation domains, does not play a major role in repression. We have identified two regions in the N terminus of HSF1 essential in the repression of c-fos and c-fms, including a region (amino acids 229-279) needed for repression but not required for NF-IL6 binding and a sequence (amino acids 106-205) essential for NF-IL6 binding. We next developed a dominant interfering negative construct (HSF1-(1-205)) that competitively inhibits c-fos repression by HSF1. This construct was also found to inhibit repression of the IL-1beta promoter and the c-fms promoter by HSF1 suggesting the presence of a common repression domain within HSF1 that acts on a range of target promoters.

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Cells and Constructs-- Chinese hamster ovaricytes, CHO K1, were obtained from the American Type Tissue Culture Collection and maintained in Ham's F-12 medium supplemented with 10% fetal bovine serum and 2.0 mM L-glutamine.

The c-fms promoter reporter gene, pGLfms, contains the 500-bp promoter sequence of the murine c-fms gene (21). The IL-1beta core promoter reporter gene, pGL3il-1beta , contains the sequence of -59 to +12 of the human IL-1beta gene (20). Both reporter genes were constructed using the commercially available pGL3.Basic plasmid (Promega, Madison, WI). The c-fos reporter gene, pGLfos, and the hsp70B promoter reporter gene, pGLhsp70B, were constructed as described previously (16). The transfection efficiency control vector, pCMV-beta Gal, contains the beta -galactosidase-coding sequence controlled by the cytomegalovirus (CMV) promoter.

The HSF1 expression plasmid, pcDNA3.1(-)/HSF1, contains the human hsf1 coding sequence driven by the CMV promoter in mammalian expression vector pcDNA3.1(-) (Invitrogen). A series of C-terminal truncation mutants derived from HSF1, which result in deletions of amino acid residues from the C terminus (amino acid 529) to amino acids 479, 429, 379, 329, 279, 264, 249, 229, 205, and 179 were generated by PCR-based mutagenesis using pcDNA3.1(-)/HSF1 as the template. N- and C-terminal deletion mutants, HSF1-(106-279) and HSF1-(146-279), and the internal deletion mutant, HSF1-(Delta 215-278), were generated similarly. The N- and C-terminal deletion mutants were cloned into a plasmid encoding the His6 tag. The HSF1/HSF2 chimeras, HSF1-(1-327)/HSF2-(344-536), HSF2-(1-222)/HSF1-(225-281), HSF2-(1-222)/HSF1-(225-529), and HSF2-(1-222)/HSF1-(279-529), were constructed by fusing PCR-amplified fragments from HSF1 and HSF2 cDNA templates using standard techniques. The expression plasmid for the full-length NF-IL6, pcDNA3.1(-)/NF-IL6, was derived by cloning the entire NF-IL6 cDNA into pcDNA3.1(-). A truncated form of NF-IL6, pcDNA3.1(-)/NF-IL6bZIP, was prepared from an internal SplI deletion (amino acids 41-205) of the trans-activation domain from the full-length NF-IL6 cDNA that retained the intact basic zipper (b-ZIP) region (32). The pcDNA3.1 (+)/HSF-2A, which contains the coding sequence of HSF-2A, was used in in vitro protein interaction assays as control. The expression plasmid for the GST/HSF1 fusion protein contains the full-length HSF1 coding sequence inserted in-frame downstream of the coding sequence for glutathione S-transferase in the pGEX vector (Amersham Biosciences). The expression plasmid for the GST-NF-IL6b-ZIP fusion protein contains the truncated NF-IL6 cDNA inserted in pGEX vector and is designated as GST/NF-IL6b-ZIP. The control plasmid, pGEX-2T, was used to produce GST control protein.

Transfection Methods and Assays for Luciferase and beta -Galactosidase-- For promoter activity analysis, transient transfection was carried out using the liposomal transfection reagent N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium salts (Roche Molecular Biochemicals). Unless specified in the figure legends, cells were plated in 24-well tissue culture plates at 4 × 104/well and cultured for 18 h before being transfected with 0.4 µg/well of promoter reporter construct. As the control for transfection efficiency, 0.2 µg/well of pCMV-beta Gal expression vector was simultaneously transfected. For co-expression assays, a total of 0.4 µg/well expression vector for transcription factors was used. Cells were harvested 18-24 h after transfection, and the luciferase activity and beta -galactosidase expression levels were assayed according to the manufacturer's protocols (Promega). The promoter activities were normalized in relative light units per milliunits of beta -galactosidase activity.

Western Analysis-- Nuclear or whole cell extracts were prepared and subjected to SDS-PAGE by using standard methodology. Proteins were then transferred electrophoretically onto polyvinylidene difluoride (Immobilon) membranes (Millipore) as described (33). The membranes were then blocked by incubation in 1× phosphate-buffered saline containing 5% nonfat dried milk and incubated at 4 °C with a specific antibody against the N terminus of HSF1 (for C-terminal deletion mutants), against the C terminus of HSF1 (for chimeras and mutants containing complete C terminus), or against His6 tag (for mutants with both N- and C-terminal deletions). The membranes were then washed and incubated with a second antibody coupled to horseradish peroxidase (Vector Laboratories). Antigen-antibody complexes were detected by chemiluminescence (ECL, Amersham Biosciences).

In Vitro Transcription and Translation of HSF1 and NF-IL6-- HSF1 and NF-IL6 were produced in vitro from pcDNA3.1(-)/HSF1, pcDNA3.1 (-)/NF-IL6, and pcDNA3.1(-)/NF-IL6bZIP using a TNT Quick T7 Transcription/Translation kit according to manufacturer's protocol (Promega). HSF1 constructs generated by PCR as described above and cloned into the eukaryotic expression vectors (which contain promoters for in vitro transcription) were used as the templates for in vitro production of truncated proteins for "pull-down" analysis. The in vitro translated proteins were checked for size and integrity by SDS-PAGE analysis and for function by assaying the binding properties to oligonucleotides containing specific binding motifs for HSF1 and NF-IL6 using electrophoretic mobility shift assay (EMSA).

EMSA-- Nuclear extracts were prepared from cells using nuclear and cytoplasmic extraction reagents (Pierce). Briefly, cells were incubated for 10 min on ice in 200 µl of CERI solution containing 0.75 mM phenylmethylsulfonyl fluoride, 2.0 µg/ml aprotinin and leupeptin, 20 mM NaF, and 2.0 mM Na3VO4. 11 µl of CERII solution was than added, and cytoplasmic extracts were collected by centrifugation at 12,000 × g for 5 min. Nuclear pellets were lysed in 100 µl of nuclear extraction reagent solution containing 2 mM phenylmethylsulfonyl fluoride, 2.0 µg/ml aprotinin and leupeptin. Extracts were then aliquoted and stored at -80 °C.

The oligonucleotide probes were synthesized and labeled by end filling with 32P. Consensus HSE from human hsp70A gene, 5'-CACCTCGGCTGGAATATTCCCGACCTGGCAGCCGA-3', was used in EMSA.

Each binding mixture (12 µl) for EMSA contained 2.0 µl of nuclear extract or 10 µl of in vitro translated protein, 2.0 µg of bovine serum albumin, 2.0 µg of poly(dI-dC), 0.5-1.0 ng of labeled double-stranded oligonucleotide probe, 12 mM HEPES, 12% glycerol, 0.12 mM EDTA, 0.9 mM MgCl2, 0.6 mM dithiothreitol, 0.6 mM phenylmethylsulfonyl fluoride, and 2.0 µg/ml aprotinin and leupeptin (pH 7.9). Final concentrations of KCl in the binding mixture were defined for optimal binding of each oligonucleotide. Samples were incubated at room temperature for 15 min, and complexes were then analyzed by electrophoresis on 4.5% non-denaturing polyacrylamide gels. The protein-DNA complexes were visualized by autoradiography.

In Vitro Protein Interaction Assay-- To produce GST fusion proteins and control GST protein, 250-ml cultures of Escherichia coli DH5alpha cells expressing GST/NF-IL6b-ZIP fusion protein, GST/HSF1 fusion protein, or GST control protein were incubated by shaking at 37 °C until the A600 reached 0.4-0.6. Isopropyl-beta -D-thiogalactopyranoside was then added to the bacterial culture to a final concentration of 0.5 mM in order to induce GST fusion protein expression. GST proteins were prepared as described previously (34). For each in vitro protein binding reaction, 50 pmol of GST fusion protein or GST control protein was immobilized on glutathione-Sepharose beads and then incubated with 20 µl of in vitro translated, 35S-labeled proteins in 500 µl of binding buffer containing 20 mM Tris-HCl (pH 8.0), 100 mM NaCl, 10 mM EDTA, 2.5% Nonidet P-40, 1.0 mM dithiothreitol, 2.0 mM phenylmethylsulfonyl fluoride, 2.0 µg/ml aprotinin, and 5.0 µg/ml leupeptin. The binding reaction was carried out at 4 °C for 30 min with gentle rocking. The protein-GST beads were then washed five times with binding buffer and analyzed on 10% SDS-PAGE gel. As input controls, 1 µl each of in vitro translation samples was run in parallel with relevant binding reactions.

    RESULTS AND DISCUSSION
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Functional Domains within HSF1 That Mediate Repression-- As our previous studies showed that heat shock represses the transcription of multiple genes, including c-fos, uPA, IL-1beta , TNFalpha , and c-fms through a mechanism involving interactions between HSF1 and the target promoters of these genes, we have investigated potential structural domains within HSF1 that mediate repression. We first examined a series of deletion mutants generated from the human hsf1 gene as well as chimeras between hsf1 and the structurally related hsf2 gene. We generated C-terminal truncation mutants by depleting successive 50 codon segments from the 3' terminus of the hsf1 coding region and expressed the fragments in CHO-K1 cells in vivo using a eukaryotic expression vector (Fig. 1A). After expression in vivo, the mutants were then tested for their ability to repress the activity of co-transfected target promoter-reporter constructs. For this purpose, cells were first transfected with a luciferase reporter construct driven by the human c-fos core promoter and an expression vector for c-fos activator, H-Ras (16). The c-fos core promoter has fairly low basal activity, and the activity is induced by exposure of cells to growth factors such as epidermal growth factor (35, 36). H-ras transfection substitutes for growth factor activation by mimicking the signaling effects of cellular Ras activated by ligand-bound growth factor receptors, a system described by us previously (16). We showed previously (16) that heat shock inhibits serum-induced c-fos expression. We have observed that wild-type HSF1 is a very effective repressor of the c-fos promoter previously activated either by H-Ras expression or serum stimulation and reduces activity of the reporter luciferase by over 90% (16) (Fig. 1). We used the Ras-activated c-fos promoter as a well defined model system of choice in these studies due to this susceptibility of repression by HSF1. In addition, as c-fos repression by HSF1 does not involve binding to HSE, we were able to concentrate on domains required for functional repression without the complication of taking into account the already characterized DNA binding domain (16). Our control studies indicated that neither heat shock nor HSF1 overexpression affect Ras synthesis and that the effects of heat and HSF1 are on factors proximal to the c-fos promoter (16). These effects appear to be downstream of extracellular signal-regulated protein kinase as indicated by our studies showing that extracellular signal-regulated kinase activity is actually stimulated rather than inhibited by heat shock in CHO cells.2 When the C-terminal deletion mutants of HSF1 were co-expressed with the c-fos promoter, we found that sequences from HSF1 could be deleted from the C terminus spanning codon 529 to codon 279 without significant loss of ability to repress Ras-induced activity (Fig. 1B). Wild-type HSF1 repressed c-fos by over 90% (Fig. 1B, compare lane 2 with lane 1), and this repression was similar in each mutant up to HSF1-(1-279) (lanes 3-7). Although the 1-279 fragment efficiently represses the c-fos promoter (Fig. 1B, lane 7), this mutation has lost all ability to activate the hsp70B promoter as indicated in our control studies (Fig. 1D, compare lanes 2 and 7), largely due to removal of the C-terminal trans-activation domains (amino acids 379-529). By contrast, overexpression of wild-type HSF1 activates the hsp70B promoter (Fig. 1D). Thus, the transcriptional activation domains of HSF1 do not play a major role in trans-repression of target genes. Further deletion from the C terminus led to the loss of ability to repress the c-fos promoter (Fig. 1B). Much of the potency of HSF1 to repress c-fos was lost by deletion from codon 279 to 264, and repression was abolished by deletion to codon 205 (Fig. 1B). We also carried out similar experiments with the c-fms promoter activated by co-transfection with the factor NF-IL6 (Fig. 1C).


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Fig. 1.   The HSF1-(1-279) domain is sufficient for transcriptional repression. A, serial C-terminal deletion mutants of HSF1 were constructed as described under "Experimental Procedures" and used in transient transfection assays to determine domain(s) that are responsible for gene repression. B, we first examined repression of Ras-activated c-fos activity by HSF1 co-transfection. Cells were co-transfected with Ras expression plasmid, pGLfos and pCMV-bGal transfection efficiency control either alone (lane 1) or with HSF1 constructs as described under "Experimental Procedures." Incubations were carried out in triplicate, and luciferase activities were corrected for transfection efficiency. The whole experiment was repeated (reproducibly) 3 times, and the mean values of relative luciferase activity are plotted ± 1 S.D. The columns showing c-fos activity in HSF1 co-transfection experiments (lanes 2-12 in B) are plotted beneath the figures in A representing the structure of the HSF1 constructs transfected in each incubation. C, we have examined the effect of the HSF1 deletion constructs on NF-IL6-activated c-fms transcription. Cells were transfected with pGLfms reporter gene and pCMV-bGal plus NF-IL6 expression vector together alone (lane 1) or with the various HSF1 C-terminal deletion mutants (lanes 2-12) in the same order as in B. The complete experiment was repeated (reproducibly) 3 times, and mean values of relative luciferase activity are plotted ± 1 S.D. D, to control for the effects of the various HSF1 constructs on transcriptional activation of hsp promoters, the same mutant constructs were also tested by co-transfection with the stress-inducible hsp70B reporter construct pGLhsp70B, in the same order as in lanes 1-12 in B. As before, luciferase values were normalized to transfection efficiency, and the experiment was repeated (reproducibly) 3 times, and mean values of relative luciferase activity are plotted ± 1 S.D. In all experiments presented here, plasmid DNA from empty expression vector was added to achieve equal amounts of total DNA in each transfection. E, the expression levels of the HSF1 mutants in transfectants in an experiment carried out parallel to B were determined by Western analysis of the whole cell lysates with antibody against the N terminus of HSF1 (52). Control cultures and transfectants were washed 3 times in ice-cold phosphate-buffered saline and lysed in SDS-PAGE sample buffer, and equal amounts of protein were fractionated by 10% SDS-PAGE prior to electrophoretic transfer and Western analysis as described under "Experimental Procedures." Similar results were obtained in duplicate Western analyses carried out in parallel with the incubations in C. F, in addition the ability of selected mutants to bind HSE elements from the hsp70B promoter was determined. Wild-type HSF1 and the various deletion mutants were in vitro translated, and the proteins synthesized in this way were incubated with 32P-labeled HSE and DNA-protein interaction determined by EMSA as described under "Experimental Procedures." A representative autoradiograph showing the relative positions of complexes between HSF1 and HSE is included. For comparison, the 1st lane shows a HSF1-HSE complex (marked with HSF-1) formed in an incubation of 32P-labeled HSE with nuclear extract from heat-shocked CHO cells. G, we have compared the relative quantities of the HSE-HSF complexes by densitometric analysis of the x-ray film autoradiographs. As many of the bands on the EMSA were not singlets, we quantitated the most intense band on the autoradiograph.

Our previous studies showed that heat shock also blocks the transcription of the endogenous c-fms gene, and both heat shock and HSF1 overexpression inhibit the activity of transfected c-fms promoter reporter constructs (20, 21). We have therefore carried out similar experiments on the c-fms promoter to those on the c-fos promoter described above, using experimental conditions defined in our earlier publication (21). The results of the deletion experiments were essentially similar, in that HSF1 strongly represses c-fms activity (Fig. 1C, lanes 1 and 2), and sequences from HSF1 could be deleted from the C terminus spanning codon 529 to codon 279 without significant loss of ability to repress c-fms Fig. 1C (lanes 2-7). Our previous experiments indicate that HSF1 does not affect expression of NF-IL6 and that repression is due to interaction of HSF1 and NF-IL6 on the c-fms promoter (14). c-fms repression was, however, partially reversed in some of the C-terminal deletion mutants that contain a fragment of the C-terminal trans-activation domain (Fig. 1C, lanes 3-6). We have shown that HSF1 physically binds to NF-IL6 and antagonizes the trans-activation of IL-1beta and c-fms genes by NF-IL6 (20, 21). The partial loss of c-fms repression in HSF1 mutants with removal of a portion of the trans-activation domain may be due to the interference of HSF1/NF-IL6 physical and functional interaction by the remaining tail of the trans-activation domain (this is discussed in more detail later). Indeed, when this region was completely removed from the mutants, as in HSF1-(1-279), the repression of NF-IL6-activated c-fms transcription by HSF1-(1-279) was equally effective as wild-type HSF1 (Fig. 1C). Further deletion of HSF1 to amino acid 229 effectively destroyed the capacity to repress c-fms (Fig. 1C). Examination of the relative expression of the deletion mutants by Western analysis with an antibody to the HSF1 N terminus shows that the HSF1 mutants were expressed efficiently and at equivalent levels (Fig. 1E). Endogenous HSF1 can be seen in each lane at an approximate mass of 69 kDa (Fig. 1E). The HSF1 deletion mutants contain an N-terminal His tag and therefore the transfected, untruncated HSF1 (lane marked 1-529) migrates more slowly than the endogenous HSF1 (Fig. 1E). In addition the HSF1 deletion mutants were functionally active and capable of binding to heat shock elements (HSE) in the EMSA assay, indicating that loss of repression in certain of the mutants was not due to the production of unstable or dysfunctional peptide fragments (Fig. 1F). Deletion mutant 1-229, which we show above has lost all ability to repress either c-fos or c-fms, binds avidly to HSE; deletion mutant HSF1-(1-179) loses all DNA binding capacity, while still being expressed to high level in cells (Fig. 1, E and F). Overall, these experiments suggest that the amino acid sequence between amino acids 229 and 279, which we have designated the REP domain and which includes a portion of the transcriptional regulatory domain (amino acid 221-310), is essential for gene repression by HSF1. (In the case of the c-fos promoter, the N-terminal boundary of the REP sequence may be slightly different involving amino acids 205-279; Fig. 1B.) HSE binding activity was much greater in HSF1 truncation mutants with a deletion of 150 or more amino acids (Fig. 1, F and G). This is due to the deletion in these constructs of leucine zipper 4, a motif located between amino acids 379 and 429, that functions as an intramolecular inhibitor of trimerization and DNA binding (37). Wild-type HSF1, and the 1-479 and 1-429 mutants, demonstrate decreased HSF-HSE binding activity (Fig. 1G) due to the inhibitory presence of leucine zipper 4 in these constructs.

To test the role of the HSF1 REP domain as an essential repression domain, we next investigated the effect of an HSF1 mutant with an internal deletion of the majority of this domain (amino acids 215-278) (Fig. 2). Our experiments show that excision of this region results in a partial reversal of repression of c-fos (by 70%) and c-fms (by 60%) promoter activity, whereas the activation of hsp70B promoter was not affected in this deletion mutant, which activates hsp70B efficiently (lane 5, Fig. 2, B-D). (Compare lane 1, which shows promoter activity in the absence of HSF1, with lane 2, in the presence of wild-type HSF1, and lane 5, in the presence of internally deleted HSF1-(1-214)/-(279-529).) These experiments indicate that the amino acid 215-278 region of HSF1 is important for trans-repression but is dispensable in hsp70B trans-activation by HSF1 overexpression. We then tested the potency of the REP region as a portable repression domain that could function independently from the remainder of the HSF1 molecule. This was tested by examining the ability of HSF2/HSF1 chimeras to repress c-fos and c-fms (Fig. 2). HSF2 was deemed a suitable protein for these studies because it is a member of the hsf family that lacks the ability to repress c-fos or c-fms (compare lanes 2 and 3 in Fig. 2, B and C) (16, 21). We have prepared chimeric constructs between the region of the hsf2 gene encoding the N-terminal core (DNA binding domain/trimerization domain, amino acids 1-222) and the test domains derived from the hsf1 gene. HSF1 and HSF2 are most similar in sequence in the N-terminal core domain, which contains the highly conserved DNA binding and trimerization domains (6). As HSF1 trimerization is required for gene repression, we also chose the HSF2 N terminus for its potential to supply an essential trimerization/DNA binding domain that might be needed for gene repression. However, chimeras containing HSF2-(1-222) fused to either the REP domain (HSF2-(1-222)/HSF1-(225-281)) or the entire C terminus of HSF1 (HSF2-(1-222)/HSF1-(225-529)) were equally ineffective in repressing the c-fos and c-fms promoters. (Compare the effects of wild-type HSF1 (lane 2) with HSF2-(1-222)/HSF1-(225-281) (lane 6) and HSF2-(1-222)/HSF1-(225-529) (lane 7).) Similarly, HSF2-(1-222)/HSF1-(279-529) was also ineffective in repressing both promoters (lane 8 in Fig. 2, B and C). However, the mutants containing the complete trans-activation domain of HSF1 (225-529 or 279-529) fused to the N terminus of HSF2 activate the hsp70B promoter as efficiently as the wild-type HSF1, indicating that the mutants were appropriated folded in vivo and functioned effectively in the context of the hsp70B promoter (Fig. 2D). Conversely, replacing the entire HSF1 trans-activation domain with the C-terminal 192 amino acid residues of HSF2, HSF1-(1-327)/HSF2-(344-536), does not decrease the repression of c-fos and c-fms but completely abolishes the ability to activate hsp70B (Fig. 2). This result is consistent with the C-terminal deletion experiments shown in Fig. 1, when deletion of the trans-activation domain abolished activation of hsp70B but retained repression of c-fos and c-fms. These findings therefore suggest an essential role for residues within the N-terminal core domain of HSF1 that are not conserved in HSF2 as well as the essential REP domain (between amino acids 205 and 279) in c-fos and c-fms repression but not in hsp70B activation (Fig. 2, B-D). Analysis of protein levels in the transfectants indicates that HSF1 wild type, HSF1-(1-214)/-(279-529), HSF2-(1-222)/HSF1-(225-529), and HSF2-(1-222)/HSF1-(279-529) are expressed efficiently in the cells as determined by Western analysis (Fig. 2E). However, we were unable to examine expression levels of HSF2-(1-222)/HSF1-(225-281) as it contains neither the HSF1 N nor C terminus, and our efforts to make antibodies to domains within the N terminus of HSF2 have proven unsuccessful. In addition, HSF2/HSF1 chimeric constructs prepared as His tag or HA fusions were likewise not detected by Western analysis (data not shown). We were, however, able to detect efficient expression (and HSE binding) of HSF2-(1-222)/HSF1-(225-281) using EMSA analysis of nuclear extracts from cells transfected with this construct as well as wild-type HSF1, HSF2, and the other mutants (Fig. 2F). These proteins containing the N terminus of HSF2 bind to HSE, and the intensity of the HSF-HSE bands was similar to the intensity of the HSF-HSE band seen in heat-shocked control cells (Fig. 2, F and G). The HSE binding activity in cells expressing the chimera containing the HSF1 N terminus and the HSF2 C terminus (HSF1-(1-327)/HSF2-(344-536)) was relatively strong and far exceeded HSF-HSE binding both in heat-shocked cells and in the other transfected constructs (Fig. 1F). The mechanism behind this is unclear but does not involve enhanced protein expression of this construct as may be observed in Fig. 2E.


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Fig. 2.   HSF1 residues in the N-terminal core domain and the 215-279 region are essential for repression. A, HSF1 with an internal deletion and HSF1/HSF2 chimeras were constructed as described under "Experimental Procedures" and used in transient transfection assays to determine domain(s) that are responsible for gene repression. A, solid bars represent sequences from HSF1 and gray bars are from HSF2. We next examined the effects of transfection of these constructs on Ras activated c-fos (B) and NF-IL6 activated c-fms transcription (C) essentially as described in Fig. 1 and "Experimental Procedures." Incubations were carried out in triplicate, and luciferase activities were corrected for transfection efficiency. Similarly, each experiment was repeated (reproducibly) 3 times, and mean values of relative luciferase activity are plotted ± 1 S.D. The columns showing c-fos and c-fms activity in HSF co-transfection experiments (lanes 2-8 in B and C) are plotted beneath the figures in A representing the structure of the HSF1/HSF2 constructs transfected in each incubation. D, we tested for the effects of the HSF constructs on the hsp70B reporter, with experimental replication and data analysis as in B and C. E, the expression levels of the HSF1-containing constructs were next examined by Western analysis using an antibody directed against the C terminus of HSF1 (52). F, expression of the HSF2-containing constructs could be observed using EMSA. Wild-type HSF1 and the various mutants were transiently transfected into cells, and nuclear extracts from the cells were incubated with 32P-labeled HSE and DNA-protein interaction determined by EMSA as described under "Experimental Procedures." A representative autoradiograph showing the relative positions of complexes between the HSF and HSE is included. For comparison, the 2nd lane shows an HSF-HSE complex (lane HS) formed in an incubation of 32P-labeled HSE with nuclear extract from heat-shocked CHO cells, and the 3rd lane shows a similar incubation that has also been incubated with anti-HSF1 antibody. A number of nonspecific complexes of lower electrophoretic mobility were also observed in the gels. These seemed to be more abundant in the transfected cells for unknown reasons (4th to 8th lanes). All experiments were performed three times in triplicate with highly reproducible findings. G, we have compared the relative quantities of the HSE-HSF complexes by densitometric analysis of the x-ray film autoradiographs. We have shown the relative density values for untreated controls, heat-shocked cells, and cells transfected with the HSF2-HSF1 chimeric constructs HSF2-(1-222)/HSF1-(225-281), HSF2-(1-222)/HSF1-(225-529), and HSF2-(1-222)/HSF1-(279-529) (designated H2/225-281, H2/225-529, and H2/279-529 in the figure). The anti-HSF1 antibody supershifted band and the HSF1-(1-327)/HSF2-(344-536) band were not included in the analysis.

N-terminal Sequences in HSF1 Are Required for Repression-- We next attempted to use simple N-terminal truncation from codon 1 in the open reading frame of hsf1, in a similar approach to the C-terminal deletion experiments, to characterize N-terminal residues essential for repression. However, these experiments were complicated by the fact that all the N-terminal truncation mutants we prepared non-specifically repressed the c-fos promoter as well as control promoters, probably by a squelching mechanism (38). Because squelching is likely to be mediated by the trans-activation domains of HSF1 competing for downstream transcriptional co-activators, we tried the alternative approach of using an HSF1 construct lacking the activation domains as a starting template for making N-terminal deletion mutants of HSF1. As shown previously (Fig. 1, B and C), the HSF1-(1-279) construct lacks the trans-activation domains but is entirely proficient in c-fos and c-fms repression, and this construct was therefore used as a starting template to prepare the N-terminal deletion mutants. Indeed, HSF1-(1-279) repressed the c-fos promoter, but the repression was diminished by deletion of amino acids 1-105 from this sequence (HSF1-(106-279)) and abolished by further deletion of amino acids 1-145 (HSF1-(146-279)) (Fig. 3). Control experiments indicated that these constructs based on HSF1-(1-279) do not non-specifically inhibit the activity of control promoters such as the CMV immediate early and beta -actin promoters.3 Amino acid residues 1-145 of HSF1 encompass the complete sequence of the DNA binding domain (amino acids 10-81), the linker region, and part of the trimerization domain (amino acids 137-203), which contains two leucine zipper structures (6, 39-42). We have shown previously that HSF1 binding to the c-fos promoter and amino acids 1-50 are not required for c-fos repression by HSF1 (16). Our results therefore indicate an essential role in gene repression for amino acids 106-146, which compose a proportion of linker domain and read into the first leucine zipper of the trimerization domain (Fig. 3). In a recent study, we have also demonstrated that serine residue 195 plays an important role in maintaining transcriptional repression by HSF1.4 Serine 195 locates adjacent to the hydrophobic residues of the second leucine zipper. These data suggest that the leucine zippers in the trimerization domain are required for the repression.


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Fig. 3.   N-terminal sequences are required for transcriptional repression by HSF1. The sequences of HSF1 mutant constructs based on the HSF1-(1-279) template, containing further N-terminal deletions, are shown in diagrammatic form in A. We examined the effects of transiently transfecting wild-type HSF1 (lane 1), HSF1-(1-279), and mutants 106-279 and 146-279 to determine domain(s) essential for repression of Ras-activated c-fos (B) and NF-IL6-activated c-fms transcription (C). The columns showing c-fos and c-fms activity in HSF1 co-transfection experiments (lanes 2-6 in B and C) are plotted below the figures in A representing the structure of the HSF1 constructs transfected in each incubation. Relative effectiveness of the transfectants can be observed by comparison with lane 1, which shows promoter activity in the absence of co-transfected HSF1. The same mutant constructs were also tested for effects on the trans-activation of hsp genes using the hsp70B reporter construct in D. Transfection conditions and transfection efficiency controls were performed as under "Experimental Procedures" and Fig. 1. All experiments were performed three times in triplicate with reproducible results.

Role of HSF1 Binding to NF-IL6 in Gene Repression-- Our previous studies (20, 21) on repression of the IL-1beta and c-fms genes by HSF1 suggest a mechanism of HSF1 repression involving direct binding of HSF1 to the bZIP region of NF-IL6, indicating a specific molecular target for repression. We have further investigated the potential role of HSF1/NF-IL6 binding in repression (Fig. 4). We examined the in vitro binding of HSF1 deletion mutants to a glutathione S-transferase fusion protein containing the bZIP region of NF-IL6, which we have shown previously to bind HSF1 (20, 21). We initially used HSF1-(1-279) as the starting template because the C-terminal amino acid of this fragment appears to be close to the C-terminal boundary of the repression domain (Fig. 1). As can be seen, both wild-type HSF1 and HSF1-(1-279), produced by in vitro transcription/translation in rabbit reticulocyte lysate, bind avidly to the NF-IL6 construct GST/NF-IL6bZIP in the pull-down experiments (Fig. 4, A and B). In addition, we observed avid binding to GST/NF-IL6bZIP of the in vitro translated deletion mutants HSF1-(1-264), -(1-249), -(1-229), and -(1-205). However, NF-IL6 binding activity was not detected after further deletion to amino acid 179 (Fig. 4B, lane 18), even though this polypeptide is synthesized efficiently in vitro and can be expressed stably in cells in vivo (Fig. 1E). This experiment suggests that amino acids between 179 and 205, comprising most of leucine zipper 2, are essential for HSF1 interaction with NF-IL6. HSF1 deletion to amino acid 179 also inhibited the capacity to bind to HSE in the EMSA assay, although the protein was still expressed abundantly in cells (Fig. 1, E and F). This region in HSF1 may thus be required for both formation of DNA-binding trimers and for interaction with NF-IL6. Association with NF-IL6 was not observed in HSF1 mutants 272-529, 205-529, and 146-529 (data not shown). However, weak binding with NF-IL6 was observed with the HSF1-(106-279) mutant (Fig. 4C). The relative avidities of the HSF1 constructs that bind to GST/NF-IL6bZIP are shown in Fig. 4D. These experiments suggest that HSF1 binds to the bZIP region of NF-IL6 through a region roughly delineated to amino acid residues 106-205 that we have designated as the NF domain. Although these studies were carried out in vitro, our previous studies show that HSF1 and NF-IL6 interact in vivo and can be co-immunoprecipitated with anti-HSF1 and anti-NF-IL6 antibodies (20). This interaction may partially explain the requirement for sequences in the amino acids 106-279 region of HSF1 for gene repression. Clearly, however, NF-IL6 binding by HSF1 is not sufficient in itself for repression as the residues in the REP domain (between 205 and 279), which are essential for repression (Fig. 1), are not required for NF-IL6 binding (Fig. 4). The REP domain appears to play a role in gene repression that is independent of NF-IL6 binding.


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Fig. 4.   HSF1 binds to NF-IL6 through a region containing amino acid residues 179-205. A, the NF-IL6 fragment NF-IL6bZIP was fused to GST and GST/NF-IL6bZIP used as bait to detect the binding of wild-type, full-length 35S-labeled HSF1. In vitro translated HSF1 was incubated with either GST/NF-IL6bZIP or, as a control, with wild-type GST. Samples eluting from the GST or GST fusion protein were then separated by 10% SDS-PAGE, and the relative migration of the 35S-labeled proteins was detected by x-ray film autoradiography. Lane 1 shows the labeled proteins in the in vitro translation input; lane 2 is the eluate from wild-type GST control protein, and lane 3 shows the proteins eluted from GST/NF-IL6bZIP. The upper band corresponds to full-length HSF1; the lower bands are alternatively translated HSF1 products. B, we show the binding of the deletion mutants HSF1-(1-279), HSF1-(1-264), HSF1-(1-249), HSF1-(1-229), HSF1-(1-205), or HSF1-(106-279) to GST/NF-IL6bZIP using similar conditions to A. For each deletion mutant there are 3 lanes on the gel, with the 1st lane showing the labeled proteins in the in vitro translation input, the 2nd lane showing the eluate from wild-type GST, and the 3rd lane showing the proteins eluted from GST/NF-IL6bZIP. Thus lanes 1, 4, 7, 10, 13, and 16 show the input controls; lanes 2, 5, 8, 11, 14, and 17 are GST controls; and lanes 3, 6, 9, 12, 15, and 18 are proteins eluted from GST/NF-IL6bZIP. C, a similar experiment is shown using the HSF1 mutant HSF1-(106-279). As in A, lane 1 shows the labeled proteins in the in vitro translation input; lane 2 is the eluate from wild-type GST control protein, and lane 3 shows the proteins eluted from GST/NF-IL6bZIP. D, we have compared the relative binding of HSF1 and deletion mutants to GST/NF-IL6bZIP by densitometric analysis of the x-ray film autoradiographs. As many of the HSF1 bands were doublets, for each HSF1 isoform, we quantitated the upper band on the autoradiograph, corresponding to the full-length in vitro translated HS1 construct. In these experiments, the GST/NF-IL6bZIP bound, respectively, 5.1, 5.7, 5.7, 7.1, 6.5, 4.5, and 2.6% of the 35S-labeled constructs 1-279, HSF1-(1-264), HSF1-(1-249), HSF1-(1-229), HSF1-(1-205), wild-type HSF1, and HSF1-(106-279). All experiments were performed three times with reproducible results.

Development of Dominant Interfering Negative Constructs That Inhibit Repression by HSF1-- To test further the mechanism of gene repression by HSF1, we examined the ability of mutants null for repression to inhibit repression by wild-type HSF1. We have discovered two HSF1 fragments with null activity for c-fos repression when transfected into cells but which competitively inhibit gene repression by wild-type HSF1. These are HSF1-(1-205) and HSF1-(146-279). We chose these two fragments because, although HSF1-(1-205) contains the NF domain but not REP, HSF1-(146-279) encompasses the REP domain but only part of NF. Intracellular expression of either fragment competitively inhibited the c-fos repression mediated by HSF1 (Fig. 5, A and B). These HSF1 fragments may block repression by interacting with HSF1 itself or with proteins on the target promoter to overcome HSF1-mediated repression. These reagents allowed us to test the generality of the mechanisms of gene repression between different target genes, and we next examined the ability of HSF1-(1-205) to prevent HSF1 repression of either the IL-1beta or c-fms promoters (Fig. 5, C and D). Both promoters were repressed by HSF1 as shown previously, and co-transfection with HSF1-(1-205) prevented the repression in a dose-dependent manner (Fig. 5, C and D). In the case of c-fms, increasing amounts of HSF1-(1-205) not only alleviated HSF1-induced repression but also increased c-fms activity to 3-fold higher than in cells that had not been exposed to wild-type HSF1 (Fig. 5C). The mechanism behind this finding is not apparent, although it is possible that endogenous HSF1 may exert a background repressive effect on c-fms that is reversed by the competitive inhibitor HSF1-(1-205). This HSF1 truncation mutant, HSF1-(1-205) which binds avidly to NF-IL6 (Fig. 4), would be expected to compete for NF-IL6 with HSF1 within the cell, and this could be at least part of the mechanism for the c-fms superinduction at high levels of co-transfected HSF1-(1-205) (Fig. 5C). Similar results were obtained with IL-1beta , which is also NF-IL6-dependent (Fig. 5D). HSF1-(1-205) caused an even more pronounced super-induction which may also be due to the reversal of HSF1 repression by endogenous HSF1 (Fig. 5D). It is notable that this effect was not seen in the c-fos experiments (Fig. 5B). Another possibility for some of these effects could be direct binding of HSF1-(1-205) to wild-type, endogenous HSF1. This explanation, however, seems less likely in light of our in vitro findings that association of HSF1 with truncation mutants HSF1 205, 229 and 279 is very inefficient.5 Overall, however, these experiments using HSF1-(1-205) suggest a broadly similar common mechanism for the repression of diverse target promoters by HSF1.


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Fig. 5.   HSF1-(147-279) and HSF1-(1-205) act as dominant inhibitors of HSF1 repression of c-fos, c-fms, and il1b. HSF1 mutants, HSF1-(146-279) and HSF1-(1-205), were tested in transient transfection assays for their inhibitory effects on repression by HSF1. Cells were co-transfected with c-fos (A and B), c-fms (C), or IL-1beta (D) reporter genes, alone (1st lane), with the addition of transcriptional activators for each promoter (2nd lane), and with increasing amounts (indicated in the figure) of HSF1-(146-279) (A) or HSF1-(1-205) plasmid DNA (B-D). A and B, the luciferase activity of the c-fos promoter-reporter in cells co-transfected with H-ras expression vector, in the absence of HSF1 co-transfection, was set to 100. C and D, the luciferase activities of the c-fms and IL-1beta reporter genes in cells co-transfected with empty expression vector were used as controls and set to 100, and the activating effect of NF-IL6 co-transfection is shown in the 2nd lane. The 3rd lane shows the repressive effect of HSF1 demonstrated by co-transfecting wild-type HSF1 and NF-IL6. The 4th lane shows the effect of transfection of HSF1-(1-205) alone into cells transfected with NF-IL6. The 5th through 8th lanes show the inhibitory effects of increasing doses of 1-205 on HSF1-mediated repression of the c-fms and IL-1beta reporter genes. As before, luciferase values were normalized to transfection efficiency; the experiment was carried out in triplicate, and mean relative luciferase activity is plotted ± 1 S.D. Experiments were performed three times, with reproducible results.

Our experiments indicate that HSF1 contains at least two closely apposed (if not contiguous) domains that together are sufficient to mediate gene repression. There is the fairly well defined REP domain (represented by amino acids 229-279) that is essential for gene repression (Figs. 1 and 2). Removal of this domain by internal deletion or C-terminal truncation leads to loss of capacity to repress c-fos and c-fms (Figs. 1 and 2). However, this region does not function as a portable repression domain independently of the N-terminal trimerization domain of HSF1, and chimeras constructed by fusing the HSF2 N terminus and the REP domain did not repress target promoters (Fig. 2). The REP domain thus functions in cooperation with other regions in the N terminus of HSF1. N-terminal truncation experiments show that amino acids between 106 and 145 are also essential for repression (Fig. 3). As our previous experiments have suggested a mechanism for HSF1 repression of IL-1beta involving binding to NF-IL6, we have further pursued this mechanism here (20, 21). Our current experiments indicate that NF-IL6 binding maps to the N terminus of HSF1 and that residues in the REP domain are not required (Fig. 4). Instead, residues in the linker and leucine zipper domains are required for NF-IL6 binding (Fig. 4). The N-terminal boundary of the NF-IL6 binding region is close to amino acid 106 as HSF1-(106-279) binds, albeit weakly, to NF-IL6, whereas HSF1-(146-279) loses all binding ability (Fig. 4 and data not shown). The C-terminal boundary of NF-IL6 binding is between amino acids 179 and 205 (Fig. 4). A roughly defined NF-IL6 binding region between amino acids 106 and 205 is thus suggested. Our previous experiments (20, 21) indicated a mechanism for IL-1beta and c-fms repression by HSF1 involving HSF1 binding to NF-IL6 and quenching the cooperative interactions between proteins (NF-IL6 and PU.1/Spi-1) that lead to transcriptional activation of these genes. The NF-IL6 binding "NF" domain may thus constitute a functionally independent repression domain. Indeed, the N-terminal boundary of the NF-IL6 binding region (amino acid 106) corresponded well with N-terminal boundary for gene repression (Figs. 3 and 4). The HSF1-(106-279) construct bound to NF-IL6 only weakly, and this fragment, when expressed in cells, also repressed c-fos and c-fms weakly (Figs. 3 and 4). However, the C-terminal boundary of NF-IL6 binding (amino acid 205) did not correspond to the C-terminal cut-off for repression (amino acid 279). We have thus characterized two repression domains in HSF1, including the REP domain (amino acids 229-279) and the NF domain (amino acids 106-206). Both regions are necessary for repression but not sufficient in themselves. The NF domain contains the leucine zipper 1 trimerization domain, an extended region of hydrophobic heptad repeats (amino acids 137-181) required for stress-induced trimer formation (37, 43). The trimerization domain is therefore essential for both gene activation and repression. However, trimerization alone is clearly not indicated because the N-terminal region of HSF2, which contains an effective trimerization domain, does not substitute for the HSF1 N terminus (Fig. 2). The NF domain also contains the second leucine zipper region (amino acids 183-199) that is not required for trimerization but is involved in the trans-activation step of HSF1 induction (44). We have found that serine 195 located within this region is required for effective gene repression by HSF1 (41, 42), and alanine mutation of this site inhibits repression.4 Leucine zipper 2 may thus play a direct role in gene repression or may be part of the generic uncoiling mechanism required for de-repression of HSF1 (44). The amino acid 106-205 region also contains most of the linker region of HSF1 recently defined by Liu and Thiele (42) as important in regulating the monomer-trimer transition. Their studies indicate that the linker region contains sequences not conserved with HSF2 that may interact with residues within the DNA binding domain (41, 42). These residues in the linker region appear to be required in gene repression and NF-IL6 binding by HSF1 (Figs. 3 and 4). Although binding of the NF domain to NF-IL6 plays a role in IL-1beta and c-fms repression, its role in the repression of other promoters such as c-fos, uPA, and TNFalpha is less clear. However, NF-IL6 belongs to a larger family of transcription factors (bZIP family) including AP1-binding proteins, the C/EBP family, activating transcription factor/cAMP-response element-binding protein, and others (25). The role of HSF1 binding to DNA during gene repression is less clear and appears to vary between target genes. Previous studies (15, 17, 18) show that both the IL-1beta and TNFalpha genes contain functional HSE that are involved in repression by HSF1. The c-fos gene, however, does not contain HSE in the proximal promoter, does not bind avidly to HSF1, and is repressed by a point-mutated HSF1 (HSF1 Leu-22) that fails to bind to HSE sequences (16). It is therefore possible that HSF1 can be recruited to target promoters either through DNA binding, protein-protein association, or both processes (15, 16, 18).

Although our deletion studies suggest the presence of common sequences within HSF1 that are required for repression of a number of genes, some differences were also found. One marked difference between c-fos and c-fms was noted in the study of C-terminal truncations in Fig. 1, B and C. Deletion through the trans-activation domains of HSF1 led to an inhibition of c-fms repression most marked in mutants HSF1-(1-429) and -(1-379) (Fig. 1C). Further truncation to amino acid 279, producing a construct with the "core repression sequence" 1-279, completely restored the repression capacity (Fig. 1C). These experiments suggest that, in some contexts, sequences in the C terminus can inhibit gene repression. The exact nature of these interactions is unclear, although one possible mechanism might involve the binding of molecular chaperones to C-terminal sequences in HSF1 that can promote repression. It has been shown previously (45) that hsp70 can bind to sequences in the C terminus of HSF1, including amino acids 425-439, and block trans-activation of HSP promoters. Our previous studies (46) have shown that hsp70 acts as a transcriptional co-repressor with HSF1 and that intracellular expression of hsp70 antisense oligonucleotides can block gene repression by HSF1, whereas hsp70 overexpression promotes repression. We suggest that hsp70-containing molecular chaperone complexes that bind to the C terminus of HSF1 override the C-terminal inhibitory sequences and promote gene repression; hsp70 binding is disrupted by C-terminal truncation (45), and this could account for the inhibition of repression observed in some of the C-terminal deletion mutants. This effect appears to be very pronounced in c-fms while less obvious in c-fos (Fig. 1, C and D). It is not clear whether these differences are due to the design of the model systems used to map repression domains or reflect intrinsic differences between the way HSF1 interacts with c-fos and c-fms.

Our experiments therefore suggest a model in which HSF1 represses inducible genes in part by a mechanism involving the quenching of active transcription factors on target promoters (20). In an earlier study of IL-1beta , we showed that HSF1 binds to NF-IL6 in vivo only after NF-IL6 is activated by the proinflammatory lipopolysaccharide and phorbol ester treatment and when HSF1 is activated to a nuclear DNA binding form by heat shock (20). We suggest a mechanism therefore involving the activation of transcription factors during inflammation or mitogenesis, which migrate to the promoters of genes such as IL-1beta , c-fms, or c-fos (Fig. 6). In the cells that subsequently encounter stresses such as febrile heat, HSF1 is activated to a DNA-binding form and migrates to the nucleus as a trimer (37). Activated HSF1 is then recruited to the promoters of these induced genes by bZIP factors that bind to the NF domain (20). In support of this, we have found that NF-IL6 binds to HSF1 and competitively inhibits HSF1 binding to heat shock promoters in vitro and represses the activity of heat shock genes in vivo, whereas HSF1 activation leads to inhibition of NF-IL6-dependent genes (20, 21). HSF1 may thus become tethered to the target promoter through binding to DNA-bound b-ZIP factors and/or by contacting the HSE in genes such as IL-1beta and TNFalpha (15, 18). HSF1 subsequently represses IL-1beta at least partially by quenching essential cooperative interactions between NF-IL6 and Spi.1 (Fig. 6) (20, 24). We have no direct evidence that such a mechanism is involved in the repression of the other promoters. However, the fact that the HSF1 construct 1-205, which contains the NF domain but not the REP domain, acts as a dominant negative inhibitor of repression of IL-1beta , c-fms, and c-fos is suggestive of such a general mechanism (Figs. 1 and 5). However, the molecular mechanisms involved in repression through the REP domain are not clear. The REP domain occupies most of the transcriptional regulatory domain, a region that responds to heat shock and influences the trans-activation domains to induce heat shock promoters (47-49). One possible mechanism for repression by this domain is that the REP domain regulates association of HSF1 with co-repressor molecules (Fig. 6). Recruitment of co-repressor complexes, many of which encode histone deacetylases, has been implicated in the repression of target genes by other factors, most notably the nuclear receptor family (38, 50, 51). Further experiments will explore this possibility.


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Fig. 6.   Two interdependent domains in HSF1 are essential for gene repression. A, regions essential for repression include first the NF domain that binds to NF-IL6 and is essential for functional repression of each gene. There is a second region (REP) not involved in NF-IL6 binding but also essential for repression of target genes. The NF domain overlaps with the linker region between the DNA binding domain (DB) and the leucine zipper regions of the trimerization domain (TD). The REP domain occupies sequences within the regulatory domain (RD), a temperature-sensitive domain that regulates the activation domains (AD1 and AD2). However, AD1 and AD2 are not required for gene repression. B, proposed model for gene repression by HSF1. HSF1 undergoes trimerization after activation by stress and is competent to bind HSE in target genes. The activated HSF1 is also enabled to bind essential transcription factors (TF1) such as NF-IL6 (in the case of the IL-1beta and c-fms promoters) on repressed promoters through the NF domain. We propose that this interaction could recruit HSF1 to promoters repressed by HSF1, and we have shown that such reactions participate in repressing the promoter by quenching interactions of transcription factor 1 with other transcription factors on the enhancer region of the promoter (transcription factor 2) or general transcription factors/co-activators. The second domain (REP) is also essential for gene repression. The function of REP is not known, although we speculate that it could be involved in binding co-repressors, common mediators of gene repression. Gene repression requires both NF and REP domains, and neither has been shown to function as independent, portable repression domains in experiments carried out so far.

In summary, we have found two regions in the HSF1 sequence that are essential in repressing transcription of multiple target genes. These sequences appear to carry out unique functions in gene repression over and above their established roles in HSF1 uncoiling, trimerization, and transcriptional activation in response to stress.

    ACKNOWLEDGEMENTS

We thank Carl Wu and Philip Auron for valuable materials and the faculty at the Harvard Joint Center for Radiation Therapy for support and encouragement in the early part of the studies. We are especially grateful to Phil Auron for many valuable discussions over the years in which this project has developed and to Catherine Cahill who helped to initiate the studies.

    FOOTNOTES

* This work was supported by National Institutes of Health Grants CA47407, CA31303, and CA50642.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed: Center for the Molecular Stress Response, Boston University School of Medicine, 88 E. Newton St., Boston, MA 02118. Tel.: 617-414-1700; Fax: 617-414-1699; E-mail: stuart_calderwood@medicine.bu.edu.

Published, JBC Papers in Press, December 4, 2002, DOI 10.1074/jbc.M210189200

2 M. A. Stevenson and S. K. Calderwood, manuscript in preparation.

3 Y. Xie and S. K. Calderwood, unpublished data.

4 Y. Xie and S. K. Calderwood, manuscript in preparation.

5 H. He and S. K. Calderwood, manuscript in preparation.

    ABBREVIATIONS

The abbreviations used are: HSF1, heat shock factor 1; IL-1beta , interleukin 1beta ; TNFalpha , tumor necrosis factor alpha ; uPA, urokinase plasminogen activator; bZIP, basic zipper; HSE, heat shock element; EMSA, electrophoretic mobility shift assay; GST, glutathione S-transferase; CMV, cytomegalovirus; CHO, Chinese hamster ovary.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
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