Tumor necrosis factor alpha and interleukin-1beta regulate the murine manganese superoxide dismutase gene through a complex intronic enhancer involving C/EBP-beta and NF-kappaB.
Manganese superoxide dismutase (MnSOD), a tumor necrosis factor (TNF)-inducible reactive oxygen-scavenging enzyme, protects cells from TNF-mediated apoptosis. To understand how MnSOD is regulated, transient transfections of promoter-reporter gene constructions, in vitro DNA binding assays, and in vivo genomic footprint (IVGF) analysis were carried out on the murine MnSOD gene. The results of this analysis identified a 238-bp region of intron 2 that was responsive to TNF and interleukin-1beta (IL-1). This TNF response element (TNFRE) had the properties of a traditional enhancer element that functioned in an orientation- and position-independent manner. IVGF of the TNFRE revealed TNF- and IL-1-induced factor occupancy of sites that could bind NF-kappaB and C/EBP. The 5' portion of the TNFRE bound C/EBP-beta in vitro and was both necessary and sufficient for TNF responsiveness with the MnSOD promoter or with a heterologous promoter when in an upstream position. The 3' end of the TNFRE bound both NF-kappaB and C/EBP but was not necessary for TNF responsiveness with the MnSOD promoter. However, this 3' portion of the TNFRE was required for the TNFRE to function as a downstream enhancer with a heterologous promoter. These data functionally separate the MnSOD TNFRE into a region responsible for TNF activation and one that mediates induction when it is downstream of a promoter.
Jones
P L
PL
Department of Microbiology and Immunology, Emory University School of Medicine, Emory University, Atlanta, Georgia 30322, USA.
Ping
D
D
Boss
J M
JM
D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D022762:CCAAT-Enhancer-Binding Proteins; D002874:Chromosome Mapping; D017931:DNA Primers / Q000235:genetics; D004268:DNA-Binding Proteins / Q000378:metabolism; D004742:Enhancer Elements, Genetic; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D006801:Humans; D007375:Interleukin-1 / Q000494:pharmacology*; D007438:Introns; D051379:Mice; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000378:metabolism; D009687:Nuclear Proteins / Q000378:metabolism; D016133:Polymerase Chain Reaction; D012689:Sequence Homology, Nucleic Acid; D013482:Superoxide Dismutase / Q000235:genetics*; D014158:Transcription, Genetic / Q000187:drug effects; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D022762:CCAAT-Enhancer-Binding Proteins; D017931:DNA Primers; D004268:DNA-Binding Proteins; D007375:Interleukin-1; D016328:NF-kappa B; D009687:Nuclear Proteins; D014409:Tumor Necrosis Factor-alpha; D013482:Superoxide Dismutase