Transcriptional activation of the tumor necrosis factor alpha-inducible zinc finger protein, A20, is mediated by kappa B elements.
Transcriptional activation of the tumor necrosis factor alpha-inducible zinc finger protein, A20, is mediated by kappa B elements.
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DOI：
10.1016/s0021-9258(19)37138-8
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发表时间：
1992-09
期刊：
影响因子：
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通讯作者：
A. Krikos;C. D. Laherty;V. Dixit
中科院分区：
文献类型：
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作者：
A. Krikos;C. D. Laherty;V. Dixit
A20 最初被鉴定为肿瘤坏死因子 (TNF) 初级反应转录物，编码具有独特锌指基序的 790 个氨基酸蛋白质。最近，A20 在多种细胞系中被证明可以保护细胞免受 TNF 诱导的细胞毒性。核连续研究先前证实 TNF 在转录水平激活 A20。为了进一步表征 TNF 激活 A20 基因的机制，我们克隆了 A20 5'侧翼序列并鉴定了启动子内的 TNF 响应元件。通过引物延伸和 S1 核酸酶保护实验将转录起始位点定位到起始子蛋氨酸上游 4.2 千碱基 (kb) 的位置；第一个和第二个外显子被 3.9 kb 的内含子分开。转录起始位点上游的序列富含 76% GC，并包含 6 个 Sp1 结合位点和 -29 处的 TATA 样序列，但缺乏共有的 CCAAT 位点。用一系列 A20 启动子 CAT 构建体转染 Jurkat T 细胞表明，TNF 诱导需要位于 -54 和 -66 的两个 kappa B 元件。使用未刺激和 TNF 刺激的 Jurkat 细胞核提取物进行的 DNA 电泳迁移率变动分析证明了 TNF 激活因子与包含两个 A20 kappa B 序列的末端标记探针的 kappa B 特异性结合，支持了这一观点。最后，从共转染实验中获得的证据表明，A20 负向调节其自身的表达。
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.