Abstract
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.
Highlights
Reactive oxygen species produced during both normal cellular function and, most importantly, as a consequence of the inflammatory response, have been implicated in the initiation and propagation of a variety of pathological states (1, 2)
Since de novo transcription is necessary for the induction of Mn-superoxide dismutases (SODs) by LPS, TNF-␣, or IL-1, we evaluated alterations in the chromatin structure of manganese SOD (Mn-SOD) in response to these mediators
In order to exclude the possibility that induction of human growth hormone (hGH) is somehow due to the vector itself, we demonstrated that LPS, TNF-␣, and IL-1 had no effect on a vector containing the minimal thymidine kinase promoter (TKGH) as compared with the results with a 4.5-kb fragment of the Mn-SOD promoter (Eco/E, Fig. 8)
Summary
(Received for publication, October 1, 1998, and in revised form, November 5, 1998). Shiuhyang Kuo‡, Sarah E. Mn-SOD expression is dramatically regulated in a variety of cells by numerous proinflammatory mediators, including lipopolysaccharide, tumor necrosis factor-␣, and interleukin-1. In vitro footprinting data implied that five of the binding sites may be occupied by a combination of Sp1 and gut-enriched Kruppel-like factor These studies have revealed the complex promoter architecture of a highly regulated cytoprotective gene. Mn-SOD contains a GC-rich promoter lacking a TATA- and a CAAT-box This promoter architecture was originally associated with housekeeping genes that are constitutively expressed (22). In order to explore the mechanism of the transcriptional regulation of this unique TATA- and CAAT-less gene promoter, we first investigated alterations in the chromatin structure of rat Mn-SOD by mapping regions hypersensitive (HS) to cleav-. Our findings have provided evidence for the molecular architecture within the promoter of this TATA and CAAT-less gene
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