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-SODtherefore 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-α, and interleukin-1. To understand the underlying regulatory mechanisms controllingMn-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 Krüppel-like factor. These studies have revealed the complex promoter architecture of a highly regulated cytoprotective gene.