ETS transcription factors regulate an enhancer activity in the third intron of TNF-alpha.
We describe an enhancer site in the third intron of tumor necrosis factor alpha (TNF-alpha). A reporter construct containing the 5'-flanking region of the mouse TNF-alpha gene displayed weak activity when transfected into RAW264.7 macrophage-like cells. The addition of the third intron of TNF-alpha to this construct resulted in an enhancement of CAT protein. This enhancement was eliminated if a conserved 20-bp sequence was removed from the intron or if a dominant-negative ets-binding factor was co-transfected with the reporter gene. Mutations of this site that destroyed potential ets transcription factor binding sites had reduced transcriptional activity. The major transcription factor that bound to the oligonucleotide was confirmed to be GABP by supershift and competition analysis. In RAW264.7 cells, the binding was constitutive, however, in bone marrow-derived macrophages binding activity was shown to be interferon-gamma inducible. This may imply a role for ets transcription factors in the production of TNF-alpha.
Department of Microbiology and Immunology, State University of New York Health Science Center at Syracuse, 13210, USA.
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Tomaras
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Foster
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Burrer
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Taffet
D000818:Animals; D001665:Binding Sites; D002454:Cell Differentiation; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D017124:Conserved Sequence; D004268:DNA-Binding Proteins / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004742:Enhancer Elements, Genetic; D051268:GA-Binding Protein Transcription Factor; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D006801:Humans; D007371:Interferon-gamma / Q000378:metabolism / Q000494:pharmacology; D007438:Introns; D008264:Macrophages / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D008809:Mice, Inbred C3H; D008934:Mitogens / Q000494:pharmacology; D009000:Monocytes / Q000166:cytology; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D050783:Proto-Oncogene Proteins c-ets; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics*; D020298:U937 Cells
D004268:DNA-Binding Proteins; D051268:GA-Binding Protein Transcription Factor; C495656:Gabpa protein, mouse; D008934:Mitogens; D011518:Proto-Oncogene Proteins; D050783:Proto-Oncogene Proteins c-ets; D014157:Transcription Factors; D014409:Tumor Necrosis Factor-alpha; D007371:Interferon-gamma; D015500:Chloramphenicol O-Acetyltransferase; D013755:Tetradecanoylphorbol Acetate