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, 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.
Imanishi
D
D
Department of Hematology, Atomic Bomb Disease Institute, Nagasaki University School of Medicine, Japan.
Yamamoto
K
K
Tsushima
H
H
Miyazaki
Y
Y
Kuriyama
K
K
Tomonaga
M
M
Matsuyama
T
T
D000276:Adjuvants, Immunologic / Q000502:physiology; D001665:Binding Sites / Q000235:genetics / Q000276:immunology; D002478:Cells, Cultured; D016207:Cytokines / Q000502:physiology*; D004268:DNA-Binding Proteins / Q000032:analysis / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D005786:Gene Expression Regulation / Q000276:immunology; D006801:Humans; D050856:Interferon Regulatory Factor-1; D007371:Interferon-gamma / Q000378:metabolism / Q000502:physiology; D020014:K562 Cells; D016328:NF-kappa B / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D052002:NF-kappa B p50 Subunit; D010750:Phosphoproteins / Q000032:analysis / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000276:immunology*; D020218:Response Elements / Q000276:immunology*; D051996:Transcription Factor RelA; D014157:Transcription Factors / Q000096:biosynthesis / Q000378:metabolism; D014158:Transcription, Genetic / Q000276:immunology; D014409:Tumor Necrosis Factor-alpha / Q000502:physiology; D020298:U937 Cells
D000276:Adjuvants, Immunologic; D016207:Cytokines; D004268:DNA-Binding Proteins; C494257:IRF1 protein, human; D050856:Interferon Regulatory Factor-1; D016328:NF-kappa B; D052002:NF-kappa B p50 Subunit; D010750:Phosphoproteins; D051996:Transcription Factor RelA; D014157:Transcription Factors; D014409:Tumor Necrosis Factor-alpha; D007371:Interferon-gamma