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

Departments of 1 Biochemistry and Molecular Biology, 2 Neuroscience, and 4 Pediatrics, University of Florida, Gainesville, Florida 32610; and 3 Department of Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania 15261 Transcription of the human inducible nitric oxide synthase ( iNOS ) gene is regulated by inflammatory cytokines in a tissue-specific manner. To determine whether differences in cytokine-induced mRNA levels between pulmonary epithelial cells (A549) and hepatic biliary epithelial cells (AKN-1) result from different protein or DNA regulatory mechanisms, we identified cytokine-induced 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 an overlapping yet cell type-specific pattern. Using in vivo footprinting and ligation-mediated PCR to detect potential DNA or protein interactions, we examined one promoter region near 5 kb containing both constitutive and cytokine-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, performed on chromatin in situ, identify a portion of the molecular mechanisms regulating transcription of the human iNOS gene in both lung- and liver-derived epithelial cells. nitric oxide synthase; deoxyribonuclease I-hypersensitive sites; inflammatory cytokines; lung epithelial cells; liver epithelial cells