Binding of the transcription factor interferon regulatory factor-1 to the inducible nitric-oxide synthase promoter

Nitric oxide production in a variety of inflammatory conditions is dependent on the synthesis of the enzyme, inducible nitric-oxide synthase (iNOS). The gene for this enzyme is regulated by a number of inflammatory cytokines, including interferon-gamma. Transcriptional activation of the gene is dependent on the interferon-gamma-induced transcription factor, interferon regulatory factor-1 (IRF-1). Using a 99-base pair segment of the iNOS gene promoter encompassing nucleotides -979 to -881, a region essential for gene activation by cytokines, we show that with increasing concentrations of added IRF-1, a monomeric then a dimeric complex form. Molecular footprinting analysis shows that the factor binds initially to a canonical IRF-1 site as a monomer. The region of binding is then extended both in a 5' and 3' direction on formation of the dimeric complex, with additional contacts in the minor groove of DNA. Binding of the second molecule of IRF-1 is dependent on the presence of the initial bound protein. Sequential binding of IRF-1 to form a dimeric complex has not been described previously, and we show that formation of this dimeric complex is essential for full activation of the iNOS gene by cytokines in vascular smooth muscle cells.