Identification of a novel cytokine response element in the human IFN regulatory factor-1 gene promoter

The present study investigates the regulatory mechanisms involved in the cooperation between IFN-gamma and TNF-alpha to promote transcription from IFN regulatory factor-1 (IRF-1), A transient transfection analysis revealed that the region between -218 and -144, where +1 is the transcription start site, as well as previously reported downstream elements, pp kappa B and IFN-gamma activation site/kappa B, were required for the optimal response to the two cytokines, A subsequent DNase I footprint analysis showed that the region between -171 and -144 was inducibly protected with stimulation by TNF-alpha, and this protection was significantly enhanced with the combination of IFN-gamma and TNF-alpha, In an EMSA with the protected region as a probe, a TNF-alpha-inducible complex (C1) and an IFN-gamma-inducible complex (C2), but no synergy-specific DNA-protein complexes, were recognized. The C1 complex consisted of a pre-existing factor (p65/p50), whereas time C2 complex consisted of a newly synthesized IRF-1-related factor. A methylation interference assay revealed the critical G residues (from -167 to -151) for the DNA-protein complex formation specific to the cytokine response, and within this region the novel kappa B sequence, the promoter distal kappa B (pd kappa B) element (5'-GGGGAAG TAC3'), was identified. Because the base substitutions over the pd kappa B region (from -171 to -144) affected not only the TNF-alpha-response but also that of IFN-gamma this region might contribute to the cooperative action of the NF-kappa B subunits with the IRF-1-related factor. Finally, we demonstrated that none of the cf-acting elements, pp kappa B, pd kappa B, or IFN-gamma activation site/kappa B, is dispensable for the optimal synergism in response to IFN-gamma and TNF-alpha.