In vivo architecture of the manganese superoxide dismutase promoter

Mitochondrial manganese superoxide dismutase (Mn-SOD) is the primary cellul ar defense against damaging superoxide radicals generated by aerobic metabo lism and as a consequence of inflammatory disease. Elevated expression of M n-SOD therefore provides a potent cytoprotective advantage during acute inf lammation. Mn SOD contains a GC-rich and TATA/CAAT-less promoter characteri stic of a housekeeping gene. In contrast, however, Mn-SOD expression is dra matically regulated in a variety of cells by numerous proinflammatory media tors, including lipopolysaccharide, tumor necrosis factor-alpha, and interl eukin-1. To understand the underlying regulatory mechanisms controlling Mn- SOD expression, me utilized DNase I-hypersensitive (HS) site analysis, whic h revealed seven hypersensitive sites throughout the gene. Following high r esolution DNase I HS site analysis, the promoter was found to contain five HS subsites, including a subsite that only appears following stimulus treat ment. Dimethyl sulfate in vivo footprinting identified 10 putative constitu tive protein-DNA binding sites in the proximal Mn-SOD promoter as well as t wo 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 Kru ppel-like factor. These studies have revealed the complex promoter architec ture of a highly regulated cytoprotective gene.