title: A high affinity HSF-1 binding site in the 5'-untranslated region of the murine tumor necrosis factor-alpha gene is a transcriptional repressor.
abstract: Tumor necrosis factor-alpha (TNFalpha) 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 degrees C) markedly attenuates TNFalpha 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 TNFalpha 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 TNFalpha gene and have used a chromosome immunoprecipitation assay to show that HSF-1 binds to this region of the endogenous TNFalpha gene. Mutational inactivation of this site blocks the inhibitory effect of overexpressed HSF-1 on activity of the minimal TNFalpha 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) TNFalpha 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 minimal TNFalpha 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 degrees C. This suggests that HSF-1 might repress TNFalpha transcription through redundant mechanisms, some of which might not require high affinity binding of HSF-1.
authors.affiliation: Department of Medicine, Division of Pulmonary and Critical Care Medicine, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA. isingh@umaryland.edu
authors.forename: Ishwar S
authors.initials: IS
authors.lastname: Singh
authors.forename: Ju-Ren
authors.initials: JR
authors.lastname: He
authors.forename: Stuart
authors.initials: S
authors.lastname: Calderwood
authors.forename: Jeffrey D
authors.initials: JD
authors.lastname: Hasday
mesh_terms: D020121:5' Untranslated Regions; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D004252:DNA Mutational Analysis; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D005982:Glutathione Transferase / Q000378:metabolism; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics; D000076249:Heat Shock Transcription Factors; D006801:Humans; D008264:Macrophages / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D013696:Temperature; D014157:Transcription Factors; D014158:Transcription, Genetic; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics* / Q000378:metabolism
chemical_list: D020121:5' Untranslated Regions; D004268:DNA-Binding Proteins; D018840:HSP70 Heat-Shock Proteins; D000076249:Heat Shock Transcription Factors; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D014157:Transcription Factors; D014409:Tumor Necrosis Factor-alpha; D005982:Glutathione Transferase