Identification of a Novel Cytokine Response Element in the Human IFN Regulatory Factor-1 Gene Promoter

• Published in: The Journal of immunology (1950) Vol. 165; no. 7; pp. 3907 - 3916
• Main Authors: Imanishi, Daisuke, Yamamoto, Kazuo, Tsushima, Hideki, Miyazaki, Yasushi, Kuriyama, Kazutaka, Tomonaga, Masao, Matsuyama, Toshifumi
• Format: Journal Article
• Language: English
• Published: United States Am Assoc Immnol 01.10.2000
• Subjects: Adjuvants, Immunologic - physiology; Binding Sites - genetics; Binding Sites - immunology; Cells, Cultured; Cytokines - physiology; DNA-Binding Proteins - analysis; DNA-Binding Proteins - biosynthesis; DNA-Binding Proteins - chemistry; DNA-Binding Proteins - genetics; DNA-Binding Proteins - isolation & purification; DNA-Binding Proteins - metabolism; Gene Expression Regulation - immunology; Humans; Interferon Regulatory Factor-1; Interferon-gamma - metabolism; Interferon-gamma - physiology; IRF-1 protein; K562 Cells; NF-kappa B - biosynthesis; NF-kappa B - isolation & purification; NF-kappa B - metabolism; NF-kappa B p50 Subunit; Phosphoproteins - analysis; Phosphoproteins - genetics; Promoter Regions, Genetic - immunology; Response Elements - immunology; Transcription Factor RelA; Transcription Factors - biosynthesis; Transcription Factors - metabolism; Transcription, Genetic - immunology; Tumor Necrosis Factor-alpha - physiology; U937 Cells
• ISSN: 0022-1767; 1550-6606
• DOI: 10.4049/jimmunol.165.7.3907

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, ppkappaB and IFN-gamma activation site/kappaB, 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 the 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 kappaB sequence, the promoter distal kappaB (pdkappaB) element (5'-GGGGAAG TAC-3'), was identified. Because the base substitutions over the pdkappaB 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-kappaB subunits with the IRF-1-related factor. Finally, we demonstrated that none of the cis-acting elements, ppkappaB, pdkappaB, or IFN-gamma activation site/kappaB, is dispensable for the optimal synergism in response to IFN-gamma and TNF-alpha.