Oligonucleotide inhibition of IL2R alpha mRNA transcription by promoter region collinear triplex formation in lymphocytes.
Oligonucleotide inhibition of IL2R alpha mRNA transcription by promoter region collinear triplex formation in lymphocytes.
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DOI：
10.1093/nar/19.12.3435
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发表时间：
1991-06
影响因子：
14.9
通讯作者：
Frank M. Orson;Douglas W. Thomas;W. Michael McShan;D. Kessler;Michael E. Hogan
中科院分区：
文献类型：
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作者：
Frank M. Orson;Douglas W. Thomas;W. Michael McShan;D. Kessler;Michael E. Hogan
IL2R α 基因 5' 侧翼序列的启动子区域包含对于结合核因子至关重要的增强子元件，这些核因子在 T 淋巴细胞激活后上调转录。 3' 核酸外切酶抗性寡核苷酸（3'A-IL28p，在其 3' 末端由游离胺基终止）被设计为与 IL2R α 启动子区域（-273 至 -246）结合，形成跨越 kappa B 增强子（-266 至 -256）以及大部分血清反应元件（CArG 盒，-251 至 -244）的共线三链体。该寡核苷酸的结合位点特异性在电泳迁移率变动测定中得到了证明，并通过抑制预测与寡核苷酸形成三链体的目标 DNA 片段内的限制性内切核酸酶 (HinfI) 切割来证明。与用相似组成但不同序列的对照寡聚物处理的淋巴细胞相比，在 PHA 刺激后长达 12 小时内，用 3'A-IL28p 预孵育 2 小时的完整正常淋巴细胞相对于其他 mRNA（c-myc、β-肌动蛋白、IL2R β、IL-6）累积的 IL2Rα mRNA 较少。核连续研究表明，IL2R α mRNA 相对于 c-myc 和 β-肌动蛋白的合成速率也通过 3'A-IL28p 处理选择性降低。这些实验表明，通过调节增强子序列中序列特异性共线三链体的形成，可以在活细胞中选择性地调节单个基因的转录。
The promoter region of the IL2R alpha gene 5' flanking sequence contains enhancer elements crucial for binding nuclear factors which upregulate transcription following T lymphocyte activation. A 3' exonuclease resistant oligonucleotide (3'A-IL28p, terminated by a free amine group at its 3' end) was designed to bind to the IL2R alpha promoter region from -273 to -246, forming a collinear triplex spanning the kappa B enhancer (-266 to -256) as well as most of the serum response element (CArG box, -251 to -244). The binding site specificity of this oligonucleotide was demonstrated in electrophoretic mobility shift assays and by inhibition of restriction endonuclease (HinfI) cleavage within the segment of the target DNA predicted to form a triplex with the oligonucleotide. Intact normal lymphocytes, preincubated for 2h with 3'A-IL28p, accumulated less IL2Ralpha mRNA relative to other mRNAs (c-myc, beta-actin, IL2R beta, IL-6) for up to 12h after PHA stimulation, than did lymphocytes treated with a control oligomer of similar composition but different sequence. Nuclear run-on studies demonstrated that the rate of IL2R alpha mRNA synthesis relative to c-myc and beta-actin was also selectively diminished by treatment with 3'A-IL28p. These experiments suggest that transcription of individual genes can be selectively modulated in living cells by sequence specific collinear triplex formation in regulatory enhancer sequences.