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• W2092985720 abstract "Tumor necrosis factor-α (TNFα) is a pivotal early mediator of host defenses that is essential for survival in infections. We previously reported that exposing macrophages to febrile range temperatures (FRT) (38.5–40 °C) markedly attenuates TNFα expression by causing abrupt and premature cessation of transcription. We showed that this inhibitory effect of FRT is mediated by an alternatively activated repressor form of heat shock factor 1 (HSF-1) and that a fragment of the TNFα gene comprising a minimal 85-nucleotide (nt) proximal promoter and the 138-nt 5′-untranslated region (UTR) was sufficient for mediating this effect. In the present study we have used an electrophoretic mobility shift assay (EMSA) to identify a high affinity binding site for HSF-1 in the 5′-UTR of the TNFα gene and have used a chromosome immunoprecipitation assay to show that HSF-1 binds to this region of the endogenous TNFα gene. Mutational inactivation of this site blocks the inhibitory effect of overexpressed HSF-1 on activity of the minimal TNFα promoter (−85/+138) in Raw 264.7 murine macrophages, identifying this site as an HSF-1-dependent repressor. However, the same mutation fails to block repression of a full-length (−1080/+138) TNFα promoter construct by HSF-1 overexpression, and HSF-1 binds to upstream sequences in the regions −1080/−845, −533/−196, and −326/−39 nt in EMSA, suggesting that additional HSF-1-dependent repressor elements are present upstream of the minimal −85-nt promoter. Furthermore, although mutation of the HSF-1 binding site in the minimalTNFα promoter construct abrogates HSF-1-mediated repression, the same mutation fails to abrogate repression of this construct by high levels of HSF-1 overexpression or exposure to 39.5 °C. This suggests that HSF-1 might repress TNFα transcription through redundant mechanisms, some of which might not require high affinity binding of HSF-1. Tumor necrosis factor-α (TNFα) is a pivotal early mediator of host defenses that is essential for survival in infections. We previously reported that exposing macrophages to febrile range temperatures (FRT) (38.5–40 °C) markedly attenuates TNFα expression by causing abrupt and premature cessation of transcription. We showed that this inhibitory effect of FRT is mediated by an alternatively activated repressor form of heat shock factor 1 (HSF-1) and that a fragment of the TNFα gene comprising a minimal 85-nucleotide (nt) proximal promoter and the 138-nt 5′-untranslated region (UTR) was sufficient for mediating this effect. In the present study we have used an electrophoretic mobility shift assay (EMSA) to identify a high affinity binding site for HSF-1 in the 5′-UTR of the TNFα gene and have used a chromosome immunoprecipitation assay to show that HSF-1 binds to this region of the endogenous TNFα gene. Mutational inactivation of this site blocks the inhibitory effect of overexpressed HSF-1 on activity of the minimal TNFα promoter (−85/+138) in Raw 264.7 murine macrophages, identifying this site as an HSF-1-dependent repressor. However, the same mutation fails to block repression of a full-length (−1080/+138) TNFα promoter construct by HSF-1 overexpression, and HSF-1 binds to upstream sequences in the regions −1080/−845, −533/−196, and −326/−39 nt in EMSA, suggesting that additional HSF-1-dependent repressor elements are present upstream of the minimal −85-nt promoter. Furthermore, although mutation of the HSF-1 binding site in the minimalTNFα promoter construct abrogates HSF-1-mediated repression, the same mutation fails to abrogate repression of this construct by high levels of HSF-1 overexpression or exposure to 39.5 °C. This suggests that HSF-1 might repress TNFα transcription through redundant mechanisms, some of which might not require high affinity binding of HSF-1. Tumor necrosis factor-α (TNFα) 1The abbreviations used are:TNFαtumor necrosis factor αLPSbacterial endotoxin lipopolysaccharideHSPheat shock proteinHSFheat shock factorrHSFrecombinant HSFHREheat shock response elementUTRuntranslated regionILinterleukinEMSAelectrophoretic mobility shift assayFRTfebrile range temperaturentnucleotide(s)NFnuclear factorChIPchromosomal immunoprecipitationSTATsignal transducers and activators of transcription 1The abbreviations used are:TNFαtumor necrosis factor αLPSbacterial endotoxin lipopolysaccharideHSPheat shock proteinHSFheat shock factorrHSFrecombinant HSFHREheat shock response elementUTRuntranslated regionILinterleukinEMSAelectrophoretic mobility shift assayFRTfebrile range temperaturentnucleotide(s)NFnuclear factorChIPchromosomal immunoprecipitationSTATsignal transducers and activators of transcription is an early pivotal mediator expressed in response to infection and injury (1Beutler B. J. Invest. Med. 1995; 43: 227-235PubMed Google Scholar). Although TNFα is essential for optimal host defense, persistent or inappropriately high TNFα expression has grave consequences, including multiorgan failure and death (2Chollet-Martin S. Montravers P. Gilbert C. Elbim C. Desmonts J.M. Fagon J.Y. Gougerot-Pocidalo M.A. Am. Rev. Respir. Dis. 1993; 164: 990-996Google Scholar, 3Beutler B. Milsark I. Cerami A. Science. 1985; 229: 869-871Crossref PubMed Scopus (1878) Google Scholar, 4Cross A.S. Sadoff J.C. Kelly N. Bernton E. Gemski P. J. Exp. Med. 1989; 169: 2021-2027Crossref PubMed Scopus (159) Google Scholar, 5Cross A. Asher L. Seguin M. Yuan L. Kelly N. Hammack C. Sadoff J. J. Clin. Invest. 1995; 96: 676-686Crossref PubMed Scopus (109) Google Scholar). The pleiotropic nature of TNFα has lead to the evolution of stringent and redundant regulatory mechanisms imposed at transcriptional, translational, and posttranslational levels (6Sariban E. Imamura K. Luebbers R. Kufe D. J. Clin. Invest. 1988; 81: 1506-1510Crossref PubMed Scopus (182) Google Scholar, 7Jongeneel C.V. Shakhov A.N. Nedospasov S.A. Cerottini J.-C. Eur. J. Immunol. 1989; 19: 549-552Crossref PubMed Scopus (24) Google Scholar, 8Biragyn A. Nedospasov S.A. J. Immunol. 1995; 155: 674-683PubMed Google Scholar, 9Lieberman A.P. Pitha P.M. Shin M.L. J. Exp. Med. 1990; 172: 989-992Crossref PubMed Scopus (55) Google Scholar, 10Ensor J.E. Crawford E.K. Hasday J.D. Am. J. Physiol. 1995; 269: C1140-C1146Crossref PubMed Google Scholar, 11Beutler B. Krochin N. Milsark I.W. Luedke C. Cerami A. Science. 1986; 232: 977-979Crossref PubMed Scopus (1013) Google Scholar). tumor necrosis factor α bacterial endotoxin lipopolysaccharide heat shock protein heat shock factor recombinant HSF heat shock response element untranslated region interleukin electrophoretic mobility shift assay febrile range temperature nucleotide(s) nuclear factor chromosomal immunoprecipitation signal transducers and activators of transcription tumor necrosis factor α bacterial endotoxin lipopolysaccharide heat shock protein heat shock factor recombinant HSF heat shock response element untranslated region interleukin electrophoretic mobility shift assay febrile range temperature nucleotide(s) nuclear factor chromosomal immunoprecipitation signal transducers and activators of transcription We reported that exposure to febrile range hyperthermia suppresses TNFα expression in murine peritoneal macrophages, Kupffer cells, precision-cut liver slices, the murine Raw 264.7 macrophage cell line, human monocyte-derived macrophages, and the THP1 monocyte cell line (10Ensor J.E. Crawford E.K. Hasday J.D. Am. J. Physiol. 1995; 269: C1140-C1146Crossref PubMed Google Scholar, 12Ensor J.E. Wiener S.M. McCrea K.A. Viscardi R.M. Crawford E.K. Hasday J.D. Am. J. Physol. 1994; 266: C967-C974Crossref PubMed Google Scholar, 13Jiang Q. DeTolla L. Kalvakolanu I. Van Roojien N. Singh I.S. Fitzgerald B. Cross A.S. Hasday J.D. Infect. Immun. 1999; 67: 1539-1546Crossref PubMed Google Scholar, 14Jiang Q. DeTolla L. Kalvakolanu I. Fitzgerald B. Hasday J.D. Am. J. Physiol. 1999; 276: R1653-R1660Crossref PubMed Google Scholar, 15Singh I.S. Calderwood S. Kalvakolanu I. Viscardi R. Hasday J.D. J. Biol. Chem. 2000; 275: 9841-9848Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 16Fairchild K.D. Viscardi R.M. Hester L. Singh I.S. Hasday J.D. J. Interferon Cytokine Res. 2000; 20: 1049-1055Crossref PubMed Scopus (89) Google Scholar). We showed that the predominant mechanism of suppression of TNFα expression is by an abrupt and early cessation of TNFα transcription, and that the TNFα gene sequence between −85 and +138 is sufficient to confer temperature responsiveness in murine macrophages (15Singh I.S. Calderwood S. Kalvakolanu I. Viscardi R. Hasday J.D. J. Biol. Chem. 2000; 275: 9841-9848Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). We also showed that the heat stress-activated transcription factor, heat shock transcription factor 1 (HSF-1) is activated at febrile range temperatures (FRT) to an alternate DNA binding form that acts as a repressor of gene expression (15Singh I.S. Calderwood S. Kalvakolanu I. Viscardi R. Hasday J.D. J. Biol. Chem. 2000; 275: 9841-9848Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar) and binds to the minimal temperature-responsive TNFα gene sequence (nt −85/+138). Furthermore, overexpression of HSF-1 represses the activity of a luciferase reporter construct-driven minimal TNFα promoter sequence (15Singh I.S. Calderwood S. Kalvakolanu I. Viscardi R. Hasday J.D. J. Biol. Chem. 2000; 275: 9841-9848Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). HSF-1 is activated in response to various chemical and thermal stresses through a cascade of posttranslational modifications, including trimerization, nuclear translocation, DNA binding, and phosphorylation of its transactivation domain, the result being the activation of heat shock protein gene transcription (17Sorger P.K. Cell. 1991; 65: 363-365Abstract Full Text PDF PubMed Scopus (513) Google Scholar, 18Sarge K.D. Murphy S.P. Morimoto R.I. Mol. Cell. Biol. 1993; 13: 1392-1407Crossref PubMed Scopus (744) Google Scholar, 19Cotto J.J. Kline M. Morimoto R.I. J. Biol. Chem. 1996; 271: 3355-3358Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar). HSF-1 binds to conserved regulatory sequences known as heat shock response elements (HRE), a pentanucleotide nGAAn element that forms stable binding sites for HSF-1 when oriented in inverted dyad repeats (20Perisic O. Xiao H. Lis J.T. Cell. 1989; 59: 797-806Abstract Full Text PDF PubMed Scopus (324) Google Scholar). Recently, several studies have shown that HSF-1 can also act as a negative regulator of certain non-heat shock genes, including interleukin(IL)-1β, c-fos,urokinase, and TNFα (15Singh I.S. Calderwood S. Kalvakolanu I. Viscardi R. Hasday J.D. J. Biol. Chem. 2000; 275: 9841-9848Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 21Cahill C.M. Warerman W.R. Xie Y. Auron P.E. Calderwood S.K. J. Biol. Chem. 1996; 271: 24874-24879Abstract Full Text Full Text PDF PubMed Google Scholar, 22Chen C. Xie Y. Stevenson M.A. Auron P.E. Calderwood S.K. J. Biol. Chem. 1997; 272: 26803-26806Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). In the case of IL-1β, this effect requires stable binding of HSF-1 to the IL-1β promoter adjacent to an essential NF-IL-6 binding element (21Cahill C.M. Warerman W.R. Xie Y. Auron P.E. Calderwood S.K. J. Biol. Chem. 1996; 271: 24874-24879Abstract Full Text Full Text PDF PubMed Google Scholar), whereas, in the case of urokinase and c-fos the effect apparently does not require stable DNA binding (22Chen C. Xie Y. Stevenson M.A. Auron P.E. Calderwood S.K. J. Biol. Chem. 1997; 272: 26803-26806Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). The minimal temperature-responsive TNFα gene sequence (−85/+138) was also inhibited by HSF-1 overexpression, but this sequence did not contain a complete cognate HSF-1 binding sequence. It does, however, contain multiple nGAAn elements positioned in critical locations, including adjacent to an essential Sp-1 binding site, at the transcription start site, and 35 nucleotides downstream of the transcription start site. In the present study we have identified the high affinity binding site for HSF-1 in the minimal temperature-responsive TNFα gene sequence and showed that inactivating the site by mutation reverses the repression of this promoter fragment by HSF-1 overexpression. All oligonucleotides were synthesized by Invitrogen, Gaithersburg, MD. Fig. 1 (see below) shows the sequences of each oligonucleotide used for electrophoretic mobility shift assays (EMSA). Complementary oligonucleotides were synthesized, annealed, and used as probe for EMSA. Primers for PCR-directed mutagenesis were Luc_5, 5′-ctttatgtttttggcgtcttca-3′ (5′ pGL3 backbone); Luc_3, 5′-ctagcaaaataggctgtccc-3′ (luciferase open reading frame); 49_Mut forward primer, 5′-ggggagaacagaaactccaccccatcttggaaatagctc-3′ and 49_Mut reverse primer, 5′-gagctatttccaagatggggtggagtttctgttctcccc3′, respectively. For EMSA spanning of the −1080 to −85 region of the TNFα gene sequence five partially overlapping PCR amplified fragments used as probes: I, −1080 to −845; II, −889 to −652; III, −686 to −494; IV, −533 to −196; and V, −326 to −39. The respective forward and reverse primers used for amplification were −1080/−845 (5′-ttggtccatgggatccg-3′ and 5′-ccccggtcttccaaggattcccctcccccaccctcc-3′), −889/−652 (5′-ttaggagtgggagggtggg-3′ and 5′-tcagccctgggaattcacggacctcac-3′), −686/−494 (5′-gaaggcttgtgaggtccgt-3′ and 5′-ggagacatgatattgaggag-3′), −533/−196 (5′-actcaaacagggggctttccctcctca-3′ and 5′-ggggacacccaggcatcaaggaatctctccccc-3′), and −326/−39 (5′-gtcctatacaacacacacac-3′ and 5′-tagcccttggggaagagggc-3′). The amplified PCR fragments were gel-purified and 32P-labeled for use as probe in EMSA. TNFα promoter-luciferase reporter constructs used in the study have been described earlier (15Singh I.S. Calderwood S. Kalvakolanu I. Viscardi R. Hasday J.D. J. Biol. Chem. 2000; 275: 9841-9848Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). Three constructs in the pGL3 vector (Promega, Madison, WI), namelypTNF −1080/+138 (−1080 to +138 nt),pTNF −244+138 (−244/+138 nt), andpTNF −85/+138 (−85/+138 nt), respectively, were used in this study. For mutated constructs, mutations were introduced into pTNF −1080/+138 plasmid (Mut-pTNF −1080/+138) using a modification of the overlap extension PCR method. In the first step, the 5′ and 3′ partially overlapping fragments containing the desired mutant were amplified using Pfu DNA polymerase. Outside primers Luc_5 and Luc_3 that correspond to flanking pGL3 backbone sequences and complementary internal primers (49_Mut forward and reverse primers), which generated a 36-nt overlap in the gene fragment and introduced a 3-base (GAA to CCC at +50 to +52 nt) substitution (see Fig. 4), were used for the amplification. Following first step PCR, the products were gel-purified and the second amplification was performed with equal concentrations of the two first step products as template and the outside primers Luc5 and Luc3 using Taq polymerase. The final product was digested with KpnI and HindIII, gel-purified, and cloned into the KpnI/HindIII site of pGL3. Mutated constructs corresponding topTNF −244/+138 and pTNF −85/+138(Mut-pTNF −244/+138 andMut-pTNF −85/+138, respectively) were prepared by amplifying the respective fragments fromMut-pTNF −1080/+138 using Taq polymerase and cloning the product into pGL3 vector as described earlier (15Singh I.S. Calderwood S. Kalvakolanu I. Viscardi R. Hasday J.D. J. Biol. Chem. 2000; 275: 9841-9848Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). The sequences of all constructs were confirmed by automated dideoxy sequencing (University of Maryland Biopolymer Core Laboratories). A glutathione S-transferase-human HSF-1 fusion construct in pGEX-2T (Amersham Biosciences, Inc., Piscataway, NJ) was expressed and purified over a glutathione-Sepharose 4B column (Amersham Biosciences, Inc.) according to the manufacturer's instructions. The fusion protein was cleaved using thrombin, and the purified HSF-1 protein was used in the study. The Raw 264.7 mouse macrophage cell line was purchased from the American Type Cell Collection (ATCC, Rockville, MD) and maintained in complete RPMI 1640 medium supplemented with 50 units/ml penicillin, 50 μg/ml streptomycin, 2 mml-glutamine, 1 mm sodium pyruvate, 10 mm HEPES buffer (Invitrogen, Gaithersburg, MD), pH 7.3, and containing 10% defined fetal bovine serum (fetal bovine serum, HyClone, Logan, UT) at 37 °C in 5% CO2-enriched air. Cells were routinely tested for Mycoplasma infection using a commercial assay system (MycoTest, Invitrogen), and new cultures were established monthly from frozen stocks. All media and reagents contained less than 0.1 ng/ml endotoxin as determined byLimulus amebocyte lysate assay (Associates of Cape Cod, Falmouth, MA). Cell viability was determined by trypan blue dye exclusion. Cells were stimulated with LPS that was prepared by trichloroacetic acid precipitation from Escherichia coli0111:B4 (Difco, Detroit, MI). Nuclear extract from Raw cells were prepared according to the method of Schreiber et al. (23Schreiber E. Matthias P. Muller M.M. Schaffner W. Nucleic Acids Res. 1989; 17: 6419Crossref PubMed Scopus (3912) Google Scholar) as we previously described (15Singh I.S. Calderwood S. Kalvakolanu I. Viscardi R. Hasday J.D. J. Biol. Chem. 2000; 275: 9841-9848Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar), and total protein was measured using a commercial reagent (Bio-Rad, Hercules, CA) with bovine serum albumin as standard. Double-stranded oligonucleotides were radiolabeled using T4 polynucleotide kinase (Promega) and [γ-32P]ATP according to the manufacturer's protocol. EMSA reactions containing 5 μg of nuclear extract or the indicated amount of recombinant HSF-1, 0.035 pmol of radiolabeled oligonucleotide, 1 μg of poly(dI/dC), 10 mm Tris-HCl, pH 7.8, 10% glycerol, 60 mm NaCl, 1 mm EDTA, and 1 mm dithiothreitol in volume of 20 μl were incubated at room temperature for 30 min. Where indicated, excess unlabeled competitor double-stranded oligonucleotide or 1 μl of anti-HSF-1 antibody (Santa Cruz Biotechnology, Santa Cruz, CA) were incubated with the nuclear extracts for 30 min at room temperature before the addition of the radiolabeled probe. The DNA·protein complexes were then electrophoretically resolved on 4% nondenaturing polyacrylamide gels. The dried gels were analyzed by phosphorimaging (PhosphorImager, Molecular Dynamics) and subsequently exposed to x-ray film. ChIP assay was performed using a kit from Upstate Biotechnology Inc. (Lake Placid, NY). Unless otherwise stated, all reagents were provided in the kit. In brief, Raw 264.7 cells were incubated at 39.5 °C for 1 h and fixed by adding formaldehyde (Sigma Chemical Co., St. Louis, MO) to the medium to a final concentration of 1%. After 15 min the cells were washed with phosphate-buffered saline containing 1 μmphenylmethylsulfonyl fluoride and 2 μg/ml aprotinin and were collected by centrifugation. Cell pellets were resuspended in SDS lysis buffer and sonicated for three 10-s bursts using a Branson Sonifier 450 (duty cycle and output settings were 30 and 3, respectively). Sonicated cell lysates were diluted 10-fold using ChIP dilution buffer and precleared for 1 h at 4 °C using 80 μl of a 50% salmon sperm DNA saturated protein A-agarose beads. Immunoprecipitation was carried out at 4 °C overnight, and immune complexes were collected with salmon sperm DNA saturated protein A-agarose beads. Antibodies used included two rabbit anti-HSF-1 antibodies (from Santa Cruz Biotechnologies and Stressgen) or, to control for nonspecific interaction, rabbit anti-IL-13 (R&D) or no antibody. After washing three times with immune complex wash buffer and twice with TE buffer, the complexes were eluted with 0.1 m NaHCO3 and 1% SDS. Protein-DNA cross-links were reverted by incubating at 65 °C for 4 h, and after proteinase K digestion, DNA was extracted with phenol-chloroform and precipitated using ethanol. PCR was performed (30 cycles, denaturing at 94 °C for 45 s, annealing at 63 °C for 30 s, and extension at 72 °C for 45 s) using primers specific for the murine TNFα sequence between −85 and +138: 5′-ggatcctgtgctagcttccgagggttgaatgaga (forward) and 5′-ttcgaagcttggagatgtgcgccttg (reverse). As a positive control, immunoprecipitated DNA was also amplified using PCR primers specific for an HRE-containing 180-nt fragment of the murineHSP70 promoter: 5′-aactccgattactcaagggaggc (forward) and 5′-gattctgagtagctgtcagcg (reverse) using the same PCR conditions as forTNFα except for a 60 °C annealing temperature. Cells were transfected using FuGENE 6 (Roche Molecular Biochemicals). 4 μg of each test plasmid and 0.5 μg of control (pRL-SV40, Promega) plasmid DNA were mixed with 15 μl of FuGENE 6 in 100 μl of medium. The mixture was incubated at room temperature for 15 min and then added to cells in 60-mm dishes. After 24 h, the cells were split into 24-well plates (1:12 per 60-mm plate). After an additional 24 h, the cells were stimulated with LPS at 37° or 39.5 °C for 6 h. Cells were lysed, and reporter gene expression was analyzed using the Dual Luciferase Reporter assay kit (Promega) according to the manufacturer's protocol. Data are presented as mean ± S.E. Differences between two groups of data were analyzed using the unpaired Student t test. Differences among more than two groups were tested by applying the Fisher protected least significant differences test applied to a one-way analysis of variance. We previously reported that the murine TNFα gene sequence spanning −85 to +138 nt bound HSF-1 in EMSA competition assays and, when transfected into Raw 264.7 macrophages, conferred transcriptional repression by febrile range temperature (FRT; 39.5 °C) or HSF-1 overexpression (15Singh I.S. Calderwood S. Kalvakolanu I. Viscardi R. Hasday J.D. J. Biol. Chem. 2000; 275: 9841-9848Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). The high affinity binding sequence for HSF-1 comprises a minimum of two nGAAn elements arranged as an inverted dyad repeat (20Perisic O. Xiao H. Lis J.T. Cell. 1989; 59: 797-806Abstract Full Text PDF PubMed Scopus (324) Google Scholar) (Fig.1 A). Fig. 1 shows the location of nGAAn elements in the −85 to +138 nt region of the murineTNFα promoter (Fig. 1 C), the sequence of the heat shock response element (HRE) from the human HSP70 promoter (24Wu B.J. Kingston R.E. Morimoto R.I. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 629-633Crossref PubMed Scopus (208) Google Scholar) (Fig. 1 B) and the sequences of each of the TNFα oligonucleotides used for the EMSA analysis of HSF-1 binding (Fig.1 C, underlined sequences). This fragment of theTNFα gene contains 10 nGAAn elements, but none arranged with perfect inverted dyad symmetry. Single nGAAn elements are positioned next to the Sp-1 binding site (−65 nt), at the transcription start site (−8 nt), and at nt +72 and +108 in the 5′-UTR. The 5′-UTR sequence spanning +30 to +68 contains an array of four nGAAn elements, three of which would form a perfect HRE if the “CT” sequence at nt +57 and +58 were inverted to “TC.” In our EMSA analysis, we probed with oligonucleotides spanning −83/−43 and containing three nGAAn elements and the Sp-1 binding sequence, −15/+5 containing one nGAAn and the transcription start site, +30/+68 and containing the imperfect HRE, and +65/+85 and +101/+121, each containing one nGAAn element. The capacity of each nGAAn-containing sequence in the minimal TNFα promoter/5′-UTR to bind HSF-1 was analyzed by EMSA using the oligonucleotides listed in Fig. 1 using the following three-step strategy. First, the capacity of a 100-fold excess of each oligonucleotide to compete and block HSF binding to a consensus HSF binding sequence was analyzed using the sequence from the humanHSP70 promoter as radiolabeled probe and nuclear extracts from Raw 264.7 macrophages exposed to 39.5 °C for 1 h as a source of HSF-1 (Fig. 2 A). Of the murine TNFα oligonucleotides analyzed, only +30/+68 blocked HSF-1 binding to the labeled HSP70 HRE sequence probe (Fig.2 A, lane 5). Competition for binding by thisTNFα oligonucleotide was as complete as that of a comparable concentration of the HSP70 sequence itself (lane 2). By comparison, each of the other TNFα oligonucleotides studied (lanes 3, 4,6, 7) failed to compete for HSF-1 binding. To confirm that HSF-1 binds with high affinity to the +30/+68TNFα sequence, we repeated the EMSA analysis using eachTNFα oligonucleotide as a radiolabeled EMSA probe (Fig.2 B). Of the TNFα sequences studied, only +30/+68 bound HSF-1 (lane 4), forming a complex that comigrated with the complex that formed on the HRE sequence from the HSP70 promoter (lane 1). Supershifting with anti-HSF-1 antibody (lane 7) confirmed that the observed complex contained HSF-1. To further confirm that HSF-1 could directly bind toTNFα +30/+68, we repeated the EMSA analysis with purified recombinant human HSF-1 (Fig. 3).TNFα +30/+68 bound rHSF-1 (lanes 7–10), but to a lesser degree than the HSP70 HRE (lanes 2–5). By comparison, the other TNFα sequence oligonucleotides failed to detectably bind rHSF-1 (data not shown).Figure 3EMSA analysis of recombinant HSF-1 binding to the HSF binding sequence from the human HSP70 promoter and TNFα oligonucleotide +30/+68.Radiolabeled oligonucleotide comprising the HSF binding sequence from the human HSP70 gene (HRE; lanes 1–5) or the TNFα +30/+68 sequence (lanes 6–10) was incubated with the indicated concentration of recombinant human HSF-1. The doublet bands representing the HSF·DNA complex are indicted by the arrows.View Large Image Figure ViewerDownload (PPT) The +30/+68 TNFα sequence contains four nGAAn sites that form two possible partially overlapping HREs centered on nt +49 and +59, respectively (Fig. 1 C). To further define the binding site for HSF-1 in this sequence we replaced the GAA sequences at either 50–52 nt (49_Mut) or 60–62 nt (59_Mut) with CCC (Fig.4 A) and analyzed the ability of the mutated +30/+68 oligonucleotides to bind HSF-1 in an EMSA competition assay (Fig. 4 B). Using the wild-type +30/+68 oligonucleotide as the radiolabeled probe and nuclear extracts from Raw 264.7 cells exposed to 39.5 °C for 1 h as a source of HSF-1, we found that unlabeled wild-type +30/+68 added at a 10-fold molar excess was sufficient to abrogate binding to radiolabeled +30/+68 (lane 2). Mutating the nGAAn sequence at +59 (59_Mut, lanes 9–12) did not change the capacity of +30/+68 to compete for HSF-1 binding. In striking contrast, 49_Mut failed to compete for HSF-1 binding when added at 10- to 100-fold excess (lanes 5–7) and only partially competed when added at 1000-fold excess (lane 8). Direct binding of HSF-1 to radiolabeled probe containing the wild-type and mutated +30/+68 sequences (Fig. 3 C) confirmed the results of the EMSA competition analysis. Although 59_Mut formed a complex (lane 4) similar to that formed on wild-type +30/+68 (lane 1), 49_Mut failed to bind HSF-1 under these conditions (lane 3). We extended these observations by introducing the GAA to CCC substitution at +50/−52 into the full-lengthTNFα promoter/5′-UTR (−1080/+138) sequence and analyzing HSF-1 binding to a PCR-amplified −85/+138 fragment of the wild-type and mutated gene (Fig. 5). EMSA was performed by using each PCR product as radiolabeled probe, and nuclear extracts from Raw 264.7 cells exposed to 39.5 °C for 1 h as a source of HSF-1. The wild-type TNFα PCR product formed a complex (lane 1) that was competed for by unlabeled wild-type +30/+68 oligonucleotide (lane 3) but not by 49_Mut oligonucleotide (lane 4) and was supershifted by anti-HSF-1 antibody (lane 5). In contrast, the comparableTNFα sequence containing the GAA to CCC mutation at +50/−52 failed to form a detectable complex (lane 2). Thus, it appears that the HRE-like sequence centered on nt +49 in the 5′-UTR is the only site in the minimal promoter/5′-UTR sequence (−85/+138) that forms high affinity complexes with HSF-1 under these cell-free assay conditions. Although we have demonstrated that HSF-1 binds to naked DNA under artificial cell-free conditions, its ability to repress TNFα transcription in the cell requires it to bind to the endogenous TNFα genein vivo. We used the ChIP assay to determine if HSF-1 binds to the TNFα gene in vivo in Raw 264.7 cells incubated at 39.5 °C for 60 min. DNA was sonicated to yield fragments of ∼500-nt length. PCR with primers specific for the HRE-containing murine HSP70 promoter sequence amplified detectable product of the predicted 180-nt length in samples immunoprecipitated with each of the anti-HSF-1 antibodies (Fig.6, lanes 3, 4), whereas no PCR product was detectable in samples immunoprecipitated without antibody (lane 5) or with an irrelevant rabbit anti-IL-13 antibody (lane 2), thereby validating the ChIP assay in this model. PCR amplification using primers specific for theTNFα sequence spanning −85 to +138, which includes the putative HSF-1 binding site, generated a detectable product of the predicted 223-nt length in samples immunoprecipitated with the anti-HSF-1 antibodies (Fig. 6, lanes 8, 9) but not in samples immunoprecipitated without antibody (lane 10) or with anti-IL-13 antibody (lane 7). The functional significance of abrogating high affinity HSF-1 binding to the TNFα 5′-UTR in comparison with transcriptional repression was analyzed by introducing the GAA to CCC mutation at nt 50–52 into each of threeTNFα promoter-driven luciferase reporter constructs: 1) the minimal promoter/5′-UTR (pTNF −85/+138) (Fig. 7 A); 2) a 382-nt promoter/5′-UTR fragment (pTNF −244/+138) containing the most proximal NF-κB response element (Fig.7 B); and 3) a full-length 1.2-kb promoter/5′-UTR fragment (pTNF −1080/+138) (Fig. 7 C). Each of these promoter fragments was cloned into the NheI/HindIII site of pGL3, transiently transfected into Raw 264.7 cells, and the promoter activity was compared in 37° and 39.5 °C cell cultures and in the presence of increasing concentrations of an HSF-1 expression plasmid at 37 °C (15Singh I.S. Calderwood S. Kalvakolanu I. Viscardi R. Hasday J.D. J. Biol. Chem. 2000; 275: 9841-9848Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). The analysis was performed in both the presence and absence of LPS. Although TNFα promoter activity was higher in the presence of LPS, the pattern of FRT- and HSF-1-induced effects on wild-type and mutated TNFα reporter constructs was comparable in LPS-stimulated and unstimulated cells. The data from the LPS-stimulated cells are shown. HSF-1 overexpression reduced the activity of both wild-typepTNF −1080/+138(Fig. 7 C) andpTNF −85/+138(Fig. 7 A), as we have previously reported (15Singh I.S. Calderwood S. Kalvakolanu I. Viscardi R. Hasday J.D. J. Biol. Chem. 2000; 275: 9841-9848Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar), and similarly reduced the activity of pTNF −244/+138(Fig. 7 B). Mutation of the high affinity HSF-1 binding site (at nt +50/−52) in pTNF −85/+138 seemed to interfere with the inhibitory effect of HSF-1 overexpression (Fig.7 A). At 1 and 2 μg of HSF-1 expression plasmid, the activity of the wild-type pTNF −85/+138 construct was decreased by 28 and 40%, respectively; whereas, activity of the mutant construct was unchanged. However, cotransfection with 3 μg of HSF-1 plasmid or incubation at 39.5 °C inhibited the activity of the wild-type and mutated pTNF −85/+138 construct by a similar extent. HSF-1 overexpression inhibited both wild-type and mutatedpTNF −244/+138 (Fig. 7 B), but the extent of inhibition of the mutated construct was significantly less than that of the wild-type construct at 2 μg (22 versus 42%;p < 0.05) and 3 μg of HSF-1 plasmid (36versus 59%; p < 0.05). Both constructs were inhibited to a similar extent by exposing cells to 39.5 °C. In contrast, in the full-length promoter (pTNF −1080/+138, Fig. 7 C), mutation of the high affinity HSF-1 binding site in the 5′-UTR had no detectable effect on the inhibitory effects of HSF-1 overexpression or exposure to 39.5 °C. The transfection results suggested that additional HSF-1-responsive repressor elements might be present in theTNFα sequence upstream of the minimal promoter. To determine if HSF-1 binds to sequences upstream of −85, we prepared five partially overlapping fragments by PCR amplification spanning the region −39 to −1080. The amplified fragments were radiolabeled and used as a probe in EMSA using purified rHSF-1 (Fig.8). The PCR fragments −1080/−845 (235 bp, lane 1), −533/−196 (337 bp, lane 4), and −326/−39 (287 bp, lane 5) formed complexes with rHSF-1, whereas fragments −889/−652 (237 bp, lane 2) and −686/−494 (192 bp, lane 3) failed to form any detectable complex with rHSF-1. In our earlier studies, we showed that TNFα expression is reduced in human and murine macrophages upon exposure to febrile temperature (10Ensor J.E. Crawford E.K. Hasday J.D. Am. J. Physiol. 1995; 269: C1140-C1146Crossref PubMed Google Scholar, 12Ensor J.E. Wiener S.M. McCrea K.A. Viscardi R.M. Crawford E.K. Hasday J.D. Am. J. Physol. 1994; 266: C967-C974Crossref PubMed Google Scholar, 15Singh I.S. Calderwood S. Kalvakolanu I. Viscardi R. Hasday J.D. J. Biol. Chem. 2000; 275: 9841-9848Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 16Fairchild K.D. Viscardi R.M. Hester L. Singh I.S. Hasday J.D. J. Interferon Cytokine Res. 2000; 20: 1049-1055Crossref PubMed Scopus (89) Google Scholar) and that the effect is largely caused by a reduction in TNFα transcription in the warmer cells. We showed that exposing Raw 264.7 cells to febrile range temperature (39.5 °C) activates the heat stress-activated transcription factor HSF-1, but although HSF-1 in the 39.5 °C cell culture binds to its cognate DNA binding site, it fails to activate transcription from the HSP70 promoter. We showed that HSF-1 activation correlates with cessation of TNFα transcription in 39.5 °C Raw 264.7 cell culture and HSF-1 overexpression reduces TNFα promoter activity in 37 °C Raw 264.7 cell culture. Together, these data offered a persuasive argument that HSF-1 might act as a repressor of macrophage TNFα expression during exposure to febrile temperatures. This hypothesis was further supported by the studies of Xiao et al. (25Xiao X. Zuo X. Davis A.A. McMillan D.R. Curry B.B. Richardson J.A. Benjamin I.J. EMBO J. 1999; 18: 5943-5952Crossref PubMed Scopus (446) Google Scholar) who showed that HSF-1 knockout mice demonstrated exaggerated TNFα expression following challenge with LPS compared with HSF-1-sufficient littermates. We previously showed that HSF-1 binds to the murine TNFα proximal promoter/5′-UTR sequence spanning nt −85 to +138 under EMSA conditions. We have extended these findings in our present study by localizing the HSF-1 binding site between nt +30 and 68 in the 5′-UTR of the murine TNFα gene. The sequence between nt +49 and +58, AGAACATCTT, with the exception of a simple inversion of the 3′-terminal CT dinucleotide (underlined), is the canonical HSF-1 binding sequence. We used EMSA analysis to show that this sequence specifically binds HSF-1 and that mutation of the nGAAn pentanucleotide at nt +49/+53 prevents binding of HSF-1, whereas the same substitution in a downstream GAAn sequence (nt +60/−62) had no effect on HSF-1 binding. However, the inability of DNA·protein complexes to form under EMSA conditions does not necessarily exclude an interaction in vivo. To provide further evidence that HSF-1 might bind to and repress theTNFα gene in vivo, we using the ChIP assay to determine if HSF-1 interacts with the TNFα gene in the living cell. We incubated Raw 264.7 cells for 1 h at 39.5 °C, conditions that we have shown activate HSF-1 to a form that binds HSP70 promoter and TNFα gene sequences under cell-free EMSA conditions. We showed that two different anti-HSF-1 antibodies coimmunoprecipitated both HRE-containing HSP70 promoter sequence and TNFα sequence containing the putative HSF-1 binding site. Negative controls, immunoprecipitated without antibody or with an irrelevant antibody (anti-IL-13) from the same species (rabbit), did not contain detectable HSP70 orTNFα gene fragments demonstrating the specificity of the technique. In transient transfection studies, the TNF−85/+138 reporter construct containing a GAA to CCC substitution at nt +50/−52 was significantly less sensitive to the inhibitory effect of overexpressed HSF-1 than was the wild-type construct. In fact, cotransfection of this mutated construct with 1 or 2 μg of HSF-1 expression plasmid had no inhibitory effect on luciferase activity whereas the wild-type construct was inhibited by 30–40% when cotransfected with the same amount of HSF-1 expression plasmid. These data suggest that HSF-1 represses transcription from the minimal TNFα promoter/5′-UTR fragment, in part, by binding to the HRE-like sequence at +49/−58 nt within the 5′-UTR. The capacity of elements within the 5′-UTR region of genes to repress transcription has been reported to occur in other genes, including the collagen α1 gene and the potassium channel Kv3.1 gene (26Hernandez I. de la Torre P. Rey-Campos J. Garcia I. Sanchez J.A. Munoz R. Rippe R.A. Munoz-Yague T. Solis-Herruzo J.A. DNA Cell Biol. 2000; 19: 341-352Crossref PubMed Scopus (23) Google Scholar, 27Gan L. Hahn S.J. Kaczmarek L.K. J. Neurochem. 1999; 73: 1350-1362Crossref PubMed Scopus (16) Google Scholar). Based on the location of the putative repressor site in the murine TNFα gene, only 30–60 nt downstream of the transcription start site, HSF-1 might repress TNFα transcription by blocking RNA polymerase processivity, as has been shown in the phage T7 model system in which binding of the lac repressor 13–15 nucleotides downstream of the initiation site blocks T7 RNA polymerase processivity (28Lopez P.J. Guillerez J. Sousa R. Dreyfus M. J. Mol. Biol. 1998; 276: 861-875Crossref PubMed Scopus (26) Google Scholar). Interestingly, cotransfection with 3 μg of HSF-1 or incubation at 39.5 °C caused comparable reductions in activity of the mutated and wild-type TNF−85/+138 reporter constructs. The capacity of higher HSF-1 levels to repress the TNF−85/+138 construct containing the mutation at +50/+52 suggests that there might be additional pathways through which HSF-1 represses expression of the minimal TNFα promoter. These include lower affinity binding of HSF-1 to non-canonical HSF-1 binding sites elsewhere in this gene fragment that is not detected by EMSA yet still might block transcription complex formation. Alternatively, HSF-1 might repressTNFα transcription without directly binding to DNA. Chenet al. (22Chen C. Xie Y. Stevenson M.A. Auron P.E. Calderwood S.K. J. Biol. Chem. 1997; 272: 26803-26806Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar) have suggested that HSF-1 could interact either with an upstream signal transduction component or with a coactivating factor to specifically repress genes such as c-fos and urokinase-type plasminogen activator. In contrast to the minimal TNFα promoter, the full-length 1.2-kb TNFα promoter construct was comparably inhibited by 1–3 μg of HSF-1, and this inhibition was unaffected by the presence or absence of the GAA to CCC mutation at +50/+52. The intermediate length construct, TNF−244/+138, exhibited a third pattern of responsiveness to the GAA to CCC mutation at +50/−52 and to overexpression of HSF-1. The activity of the wild-type and mutatedTNF−244/+138 was comparably inhibited by the lowest HSF-1 expression but, when cotransfected with 2 and 3 μg of HSF-1 expression plasmid, the wild-type construct was inhibited to a greater extent than the mutated construct. On the other hand, exposure to 39.5 °C reduced the activity of all three constructs, and the repression was unaffected by mutational inactivation of the HSF-1 binding site at nt +49/+58. The different patterns of response of each of the three constructs to HSF-1 overexpression and the mutational inactivation of the HSF-1 binding site at nt +49/−58 suggest that additional sequences upstream of nt −85 might mediate transcriptional repression by HSF-1. Using overlapping ∼200-nt PCR-generated fragments of TNFα upstream sequence, the sequence upstream of nt −85 was scanned for HSF-1 binding. Complex formation with rHSF-1 was detectable to the regions −1080/−845, −533/−196, and −326/−39 suggesting that redundant HSF-1 binding sites might be active in the TNFα gene. Alternatively, or in addition to direct binding to DNA, HSF-1 might interfere with transcription by binding to transcriptional activators as has been shown for STAT (29Stephanou A. Isenberg D.A. Nakajima K. Latchman D.S. J. Biol. Chem. 1999; 274: 1723-1728Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 30Stephanou A. Latchman D.S. Gene Expr. 1999; 7: 311-319PubMed Google Scholar), TFIID, and related factors (31Yuan C.X. Gurley W.B. Cell Stress Chaperones. 2000; 5: 229-242Crossref PubMed Scopus (28) Google Scholar). In summary, we identified a unique HRE-like sequence in the murineTNFα 5′-UTR that binds HSF-1 and is required for HSF-1-mediated transcriptional repression in the minimal mouseTNFα promoter. This offers proof of the concept that HSF-1 can repress gene transcription by binding to the 5′-UTR. Although the 5′-UTR HRE-like sequence of the mouse TNFα gene is not present in the human TNFα 5′-UTR, the results of this study suggest a number of additional redundant pathways through which exposure to febrile range temperature might inhibit transcription of both murine and human TNFα. We thank Dr. Steven Georas for his technical help with the ChIP assay." @default.
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• W2092985720 title "A High Affinity HSF-1 Binding Site in the 5′-Untranslated Region of the Murine Tumor Necrosis Factor-α Gene Is a Transcriptional Repressor" @default.
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