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 a lpha (TNF-alpha). A reporter construct containing the 5'-flanking region of the mouse TNF-alpha gene displayed weak activity when transfected into RAW 264.7 macrophage-like cells. The addition of the third intron of TNF-alpha to this construct resulted in an enhancement of CAT protein. This enhanceme nt 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 r eporter gene. Mutations of this site that destroyed potential ets transcrip tion factor binding sites had reduced transcriptional activity. The major t ranscription factor that bound to the oligonucleotide was confirmed to be G ABP by supershift and competition analysis. In RAW264.7 cells, the binding was constitutive, however, in bone marrow-derived macrophages binding activ ity was shown to be interferon-gamma inducible, This may imply a role for e ts transcription factors in the production of TNF-alpha.