Cytokine-induced changes in chromatin structure and in vivo footprints in the inducible NOS promoter

Transcription of the human inducible nitric oxide synthase (iNOS) gene is r egulated by inflammatory cytokines in a tissue-specific manner. To determin e whether differences in cytokine-induced mRNA levels between pulmonary epi thelial cells (A549) and hepatic biliary epithelial cells (AKN-1) result fr om different protein or DNA regulatory mechanisms, we identified cytokine-i nduced changes in DNase I-hypersensitive (HS) sites in 13 kb of the iNOS 5' -flanking region. Data showed both constitutive and inducible HS sites in a n overlapping yet cell type-specific pattern. Using in vivo footprinting an d ligation-mediated PCR to detect potential DNA or protein interactions, we examined one promoter region near -5 kb containing both constitutive and c ytokine-induced HS sites. In both cell types, three in vivo footprints were present in both control and cytokine-treated cells, and each mapped within a constitutive HS site. The remaining footprint appeared only in response to cytokine treatment and mapped to an inducible HS site. These studies, pe rformed on chromatin in situ, identify a portion of the molecular mechanism s regulating transcription of the human iNOS gene in both lung- and liver-d erived epithelial cells.