Transcriptional activation of the tumor necrosis factor alpha-inducible zinc finger protein, A20, is mediated by kappa B elements.
A20 was first identified as a tumor necrosis factor (TNF) primary response transcript encoding a 790-amino acid protein with a unique zinc finger motif. Recently, A20 was shown to protect cells from TNF-induced cytotoxicity in a variety of cell lines. Nuclear run-on studies previously established that TNF activates A20 at the transcriptional level. To further characterize the mechanism by which TNF activates the A20 gene, we have cloned the A20 5'-flanking sequences and identified TNF-responsive elements within the promoter. The transcription initiation site was mapped by both primer extension and S1 nuclease protection experiments to a position 4.2 kilobases (kb) upstream of the initiator methionine; the first and second exon were separated by a 3.9-kb intron. Sequences upstream of the transcription start site were 76% GC-rich and contained six Sp1 binding sites and a TATA-like sequence at -29 but lacked a consensus CCAAT site. Transfection of Jurkat T-cells with an array of A20 promoter CAT constructs showed that two kappa B elements residing at -54 and -66 were required for induction by TNF. Supporting this notion, DNA electrophoretic mobility shift assays using nuclear extracts from unstimulated and TNF-stimulated Jurkat cells demonstrated kappa B-specific binding of a TNF-activated factor to an end-labeled probe containing the two A20 kappa B sequences. Finally, evidence obtained from cotransfection experiments showed that A20 negatively regulated its own expression.
Krikos
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A
Department of Pathology, University of Michigan Medical School, Ann Arbor 48109-0602.
Laherty
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CD
Dixit
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D000595:Amino Acid Sequence; D001483:Base Sequence; D002460:Cell Line; D004268:DNA-Binding Proteins; D004730:Endothelium, Vascular / Q000502:physiology; D005260:Female; D005786:Gene Expression Regulation / Q000187:drug effects; D015698:Genomic Library; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000378:metabolism*; D009687:Nuclear Proteins; D009838:Oligodeoxyribonucleotides; D010920:Placenta / Q000502:physiology; D010957:Plasmids; D011061:Poly A / Q000235:genetics / Q000302:isolation & purification; D011247:Pregnancy; D011401:Promoter Regions, Genetic; D014176:Protein Biosynthesis; D011506:Proteins / Q000235:genetics*; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D012333:RNA, Messenger; D015183:Restriction Mapping; D014158:Transcription, Genetic; D014162:Transfection; D000072598:Tumor Necrosis Factor alpha-Induced Protein 3; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*; D016335:Zinc Fingers / Q000235:genetics*
D004268:DNA-Binding Proteins; D047908:Intracellular Signaling Peptides and Proteins; D016328:NF-kappa B; D009687:Nuclear Proteins; D009838:Oligodeoxyribonucleotides; D011506:Proteins; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D011061:Poly A; D012313:RNA; C076529:TNFAIP3 protein, human; D000072598:Tumor Necrosis Factor alpha-Induced Protein 3