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 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.
Mellott
J K
JK
Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, Florida 32610, USA.
Nick
H S
HS
Waters
M F
MF
Billiar
T R
TR
Geller
D A
DA
Chesrown
S E
SE
D001483:Base Sequence / Q000235:genetics; D001653:Bile Ducts, Intrahepatic / Q000166:cytology / Q000378:metabolism; D002460:Cell Line; D002843:Chromatin / Q000235:genetics*; D002874:Chromosome Mapping; D016207:Cytokines / Q000494:pharmacology / Q000502:physiology*; D018983:DNA Footprinting; D004847:Epithelial Cells / Q000378:metabolism; D005796:Genes / Q000187:drug effects; D006801:Humans; D008168:Lung / Q000166:cytology / Q000378:metabolism; D008969:Molecular Sequence Data; D019001:Nitric Oxide Synthase / Q000235:genetics*; D052247:Nitric Oxide Synthase Type II; D011401:Promoter Regions, Genetic / Q000187:drug effects / Q000235:genetics*; D012016:Reference Values
D002843:Chromatin; D016207:Cytokines; C496319:NOS2 protein, human; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II