Transcriptional regulation of the 5 ' proximal promoter of the human manganese superoxide dismutase gene

Manganese superoxide dismutase (MnSOD) is a primary antioxidant enzyme crit ical for maintaining normal cell function and for survival. Previously, we cloned the entire MnSOD gene, including a 0.782-kb 5' DNA sequence, from a human embryonic lung fibroblast cell line, Sequence analysis indicates that the promoter of the human MnSOD gene is TATA-less and CAAT-less, and the D NA sequence immediately upstream from the transcription start site is GC ri ch. To study the function and regulation of the human MnSOD promoter, we cl oned a 257-bp sequence (P7) containing the transcription start site and the 5' CC-rich region. Consensus analysis and DNase I footprinting assay indic ated that P7 contains multiple Sp1- and AP-2-binding sites. Deletions of th e P7 sequence diminished the promoter activity and decreased the response t o Spl protein. The first three Spl consensus sites were required for high p romoter activity in mammalian cells and enhanced promoter activity in Droso phila Schneider Line 2 (SL2) cells. In the SL2 cells, Spl activated the P7 activity in a dose-dependent manner. In contrast, cotransfections with AP-2 expression vector marginally increased P7 activities in human hepatocarcin oma HepG2 cells. The results suggest that Spl is an important regulator for the transcriptional activities of P7, whereas AP-2 is a minor activator fo r P7 and competes with Spl for binding sites which may downregulate P7 func tion.