Interaction of NF-kappaB and NFAT with the interferon-gamma promoter.
Interferon-gamma (IFN-gamma) is a pleiotropic lymphokine whose production is restricted to activated T cells and NK cells. Along with other cytokines, IFN-gamma gene expression is inhibited by the immunosuppressant cyclosporin A. We have previously identified an intronic enhancer region (C3) of the IFN-gamma gene that binds the NF-kappaB protein c-Rel and that shows partial DNA sequence homology with the cyclosporin A-sensitive NFAT binding site and the 3'-half of the NF-kappaB consensus site. Sequence analysis of the IFN-gamma promoter revealed the presence of two additional C3-related elements (C3-1P and C3-3P). In addition, an NF-kappaB site (IFN-gamma kappaB) was identified within the promoter region. Based on this observation, we have analyzed the potential role of NF-kappaB and NFAT family members in regulating IFN-gamma transcription. Electrophoretic mobility shift assay analysis demonstrated that after T cell activation, the p50 and p65 NF-kappaB subunits bind specifically to the newly identified IFN-gamma kappaB and C3-related sites. In addition, we identified the NFAT proteins as a component of the inducible complexes that bind to the C3-3P site. Site-directed mutagenesis and transfection studies demonstrate that calcineurin-inducible transcriptional factors enhance the transcriptional activity of the IFN-gamma promoter through the cyclosporin-sensitive C3-3P site, whereas NF-kappaB proteins functionally interact with the C3-related sites. In addition, when located downstream to the beta-galactosidase gene driven by the IFN-gamma promoter, the intronic C3 site worked in concert with both the IFN-gamma kappaB and the C3-3P site to enhance gene transcription. These results demonstrate that the coordinate activities of NFAT and NF-kappaB proteins are involved in the molecular mechanisms controlling IFN-gamma gene transcription.
Sica
A
A
Laboratory of Experimental Immunology, Division of Basic Sciences, National Cancer Institute, Frederick, Maryland 21702-1201, USA.
Dorman
L
L
Viggiano
V
V
Cippitelli
M
M
Ghosh
P
P
Rice
N
N
Young
H A
HA
D001483:Base Sequence; D001665:Binding Sites; D019703:Calcineurin / Q000502:physiology; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004742:Enhancer Elements, Genetic; D006801:Humans; D007371:Interferon-gamma / Q000235:genetics*; D016328:NF-kappa B / Q000378:metabolism*; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D020569:Proto-Oncogene Proteins c-rel; D013329:Structure-Activity Relationship; D013601:T-Lymphocytes / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
D004268:DNA-Binding Proteins; D016328:NF-kappa B; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins; D011518:Proto-Oncogene Proteins; D020569:Proto-Oncogene Proteins c-rel; D014157:Transcription Factors; D007371:Interferon-gamma; D019703:Calcineurin; D013755:Tetradecanoylphorbol Acetate