Role of the intronic enhancer in tumor necrosis factor-mediated induction of manganous superoxide dismutase.

Manganous superoxide dismutase (Mn-SOD), a tumor necrosis factor (TNF)-inducible gene product, plays an important role in removing superoxide anions produced inside mitochondria. Two regulatory regions, the proximal promoter region (PPR), which is upstream from the transcription initiation site, and the TNF-responsive element (TNFRE), which is inside intron 2, are responsible for Mn-SOD expression. To understand how each of these regions contributes to the transcription of Mn-SOD, quantitative reverse transcription-PCR, chromatin immunoprecipitations, and in vivo nuclease sensitivity assays were performed on the murine Mn-SOD gene. These assays demonstrate that Sp1 and nuclear factor (NF)-kappaB p65 are required for Mn-SOD induction by TNF. Sp1 bound the PPR constitutively, whereas NF-kappaB p65 and C/EBP-beta bound the TNFRE only after TNF treatment. Binding of C/EBP-beta to the TNFRE was dependent on the presence of NF-kappaB p65. The chromatin structure within the TNFRE became more accessible to nuclease digestion after TNF treatment. This accessibility required Sp1 and NF-kappaB p65. Treatment of cells with an inhibitor of histone deacetylation, or transient transfection with coactivator-expressing plasmids, enhanced the expression of Mn-SOD. NF-kappaB p65 binding was required for acetylation of histones H3 and H4 at the PPR and the TNFRE. Together, these data suggest communication between the PPR and the TNFRE which involves chromatin remodeling and histone acetylation during the induction process of Mn-SOD in response to TNF.