A 3' cis-acting element is involved in tumor necrosis factor-alpha gene expression in astrocytes.
Tumor necrosis factor-alpha (TNF-alpha) contributes to demyelinating diseases in the central nervous system. Astrocytes, the major glial cells in the CNS, do not constitutively express TNF-alpha, but the TNF-alpha gene is transcriptionally activated in response to a variety of stimuli, including TNF-alpha itself. Because of the importance of TNF-alpha in the CNS, we examined the mechanisms underlying transcriptional regulation of the TNF-alpha gene in astrocytes. In transient transfection assays, a plasmid construct containing 1.3 kilobase pairs (kb) of 5' flanking sequence of the rat TNF-alpha gene showed high basal activity that could not be further enhanced by TNF-alpha stimulation. A "marked" 10-kb TNF-alpha gene construct, which contains the whole TNF-beta gene with 1.2 kb of 5' flanking sequence, 1.1 kb of intergenic sequence, and the whole TNF-alpha gene with 3 kb of 3' flanking sequence, was able to respond to TNF-alpha stimulation. Analysis of a series of 5' and 3' deletion constructs of the marked TNF-alpha genes demonstrated that upstream sequence elements such as NF-kappaB are not required for TNF-alpha induction and that TNF-alpha responsive elements are located in the 3' flanking region of the TNF-alpha gene. We also found that a TNF-alpha-inducible DNase I-hypersensitive (DH) site is present in this 3' region whose deletion abolishes TNF-alpha inducibility of the marked TNF-alpha gene. Electrophoresis mobility shift assays showed that TNF-alpha-inducible nuclear proteins, consisting of p50 and p65 NF-kappaB proteins, specifically bind to two consecutive NF-kappaB binding sites within the 3' DH site. These results indicate that TNF-alpha-induced TNF-alpha gene expression in astrocytes involves p50 and p65 NF-kappaB proteins binding to downstream NF-kappaB sites and concomitant modulation of the chromatin structure.
Kwon
J
J
Department of Cell Biology, University of Alabama at Birmingham, Birmingham, Alabama 35294-0005, USA.
Lee
S J
SJ
Benveniste
E N
EN
D000818:Animals; D001253:Astrocytes / Q000378:metabolism*; D001483:Base Sequence; D001667:Binding, Competitive; D002478:Cells, Cultured; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D003850:Deoxyribonuclease I / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D051381:Rats; D012045:Regulatory Sequences, Nucleic Acid; D017384:Sequence Deletion; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics*
D016328:NF-kappa B; D014409:Tumor Necrosis Factor-alpha; D015500:Chloramphenicol O-Acetyltransferase; D003850:Deoxyribonuclease I