INTERACTION OF NF-KAPPA-B AND NFAT WITH THE INTERFERON-GAMMA PROMOTER

Interferon-gamma (IFN-gamma) is a pleiotropic lymphokine whose product ion is restricted to activated T cells and NK cells. Along with other cytokines, IFN-gamma gene expression is inhibited by the immunosuppres sant cyclosporin A. We have previously identified an intronic enhancer region (C3) of the IFN-gamma gene that binds the NF-kappa B 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-kappa B conse nsus site. Sequence analysis of the IFN-gamma promoter revealed the pr esence of two additional C3-related elements (C3-1P and C3-3P). In add ition, an NF-kappa B site (IFN-gamma kappa B) was identified within th e promoter region. Based on this observation, we have analyzed the pot ential role of NF-kappa B and NFAT family members in regulating IFN-ga mma transcription. Electrophoretic mobility shift assay analysis demon strated that after T cell activation, the p50 and p65 NF-kappa B subun its bind specifically to the newly identified IFN-gamma kappa B and C3 -related sites. In addition, we identified the NFAT proteins as a comp onent of the inducible complexes that bind to the C3-3P site. Site-dir ected mutagenesis and transfection studies demonstrate that calcineuri n-inducible transcriptional factors enhance the transcriptional activi ty of the IFN-gamma promoter through the cyclosporin-sensitive C3-3P s ite, whereas NF-kappa B proteins functionally interact with the C3-rel ated sites. In addition, when located downstream to the beta-galactosi dase gene driven by the IFN-gamma promoter, the intronic C3 site worke d in concert with both the IFN-gamma kappa B and the C3-3P site to enh ance gene transcription. These results demonstrate that the coordinate activities of NFAT and NF-kappa B proteins are involved in the molecu lar mechanisms controlling IFN-gamma gene transcription.