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.

In vivo architecture of the manganese superoxide dismutase promoter.

Mitochondrial manganese superoxide dismutase (Mn-SOD) is the primary cellular defense against damaging superoxide radicals generated by aerobic metabolism and as a consequence of inflammatory disease. Elevated expression of Mn-SOD therefore provides a potent cytoprotective advantage during acute inflammation. Mn-SOD contains a GC-rich and TATA/CAAT-less promoter characteristic of a housekeeping gene. In contrast, however, Mn-SOD expression is dramatically regulated in a variety of cells by numerous proinflammatory mediators, including lipopolysaccharide, tumor necrosis factor-alpha, and interleukin-1. To understand the underlying regulatory mechanisms controlling Mn-SOD expression, we utilized DNase I-hypersensitive (HS) site analysis, which revealed seven hypersensitive sites throughout the gene. Following high resolution DNase I HS site analysis, the promoter was found to contain five HS subsites, including a subsite that only appears following stimulus treatment. Dimethyl sulfate in vivo footprinting identified 10 putative constitutive protein-DNA binding sites in the proximal Mn-SOD promoter as well as two stimulus-specific enhanced guanine residues possibly due to alterations in chromatin structure. In vitro footprinting data implied that five of the binding sites may be occupied by a combination of Sp1 and gut-enriched Kr uppel-like factor. These studies have revealed the complex promoter architecture of a highly regulated cytoprotective gene.

Isolation and characterization of radiation-reduced hybrids containing portions of the proximal long arm of the human X chromosome: identification of hybrids containing the Menkes' disease locus.

The proximal long arm of the human X chromosome (Xcen----Xq13) encompasses an estimated 23 megabases of DNA and contains numerous identified genetic loci. In order to generate a highly enriched source of DNA from this region, radiation-reduced human-hamster hybrids were constructed and screened to identify those that contained at least part of proximal Xq. Eight such hybrids were identified and characterized by Southern blot and fluorescence in situ hybridization analyses to determine more precisely the human DNA complement in each. One hybrid contains the entire proximal long arm and will be useful for mapping Xcen----Xq13 in its entirety and for localizing genes within this region. Another hybrid contains a smaller portion of the proximal long arm that includes the region reported to contain the gene for Menkes' disease.