Transcriptional activation of the tumor necrosis factor alpha-inducible zinc finger protein, A20, is mediated by kappa B elements

• Published in: The Journal of biological chemistry 1992-09, Vol.267 (25), p.17971-17976
• Main Authors: KRIKOS, A, LAHERTY, C. D, DIXIT, V. M
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Base Sequence; Biological and medical sciences; Cell Line; DNA-Binding Proteins; Endothelium, Vascular - physiology; Female; Fundamental and applied biological sciences. Psychology; Gene Expression Regulation - drug effects; Genomic Library; Humans; Intracellular Signaling Peptides and Proteins; Molecular and cellular biology; Molecular genetics; Molecular Sequence Data; NF-kappa B - metabolism; Nuclear Proteins; Oligodeoxyribonucleotides; Placenta - physiology; Plasmids; Poly A - genetics; Poly A - isolation & purification; Pregnancy; Promoter Regions, Genetic; Protein Biosynthesis; Proteins - genetics; Restriction Mapping; RNA - genetics; RNA - isolation & purification; RNA, Messenger; Transcription, Genetic; Transcription. Transcription factor. Splicing. Rna processing; Transfection; Tumor Necrosis Factor alpha-Induced Protein 3; Tumor Necrosis Factor-alpha - pharmacology; Zinc Fingers - genetics
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/s0021-9258(19)37138-8

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.