ETS transcription factors regulate an enhancer activity in the third intron of TNF-α

We describe an enhancer site in the third intron of tumor necrosis factor α (TNF-α). A reporter construct containing the 5'-flanking region of the mouse TNF-α gene displayed weak activity when transfected into RAW264.7 macrophage-like cells. The addition of the third intron of TNF-α 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-γ inducible. This may imply a role for ets transcription factors in the production of TNF-α.