Kuzmin Igor, Geil Laura, Gibson Lauren, Cavinato Tiziana, Loukinov Dmitry, Lobanenkov Victor, Lerman Michael I
Basic Research Program, SAIC-Frederick, Inc., Frederick, MD 21702, USA.
J Mol Biol. 2005 Feb 18;346(2):411-22. doi: 10.1016/j.jmb.2004.11.066. Epub 2004 Dec 22.
Immune responses to invading pathogens are mediated largely through a family of transmembrane Toll-like receptors and modulated by a number of downstream effectors. In particular, a family of four interleukin 1 receptor-associated kinases (IRAK) regulates responsiveness to bacterial endotoxins. Pharmacological targeting of particular IRAK components may be beneficial for treatment of bacterial infections. Here, we studied transcriptional regulation of the human IRAK2 gene. Analysis of the IRAK2 promoter region reveals putative binding sites for several transcriptional factors, including ZIP (EGR1 and SP1), CTCF and AP-2beta. Deletion of the ZIP or AP-2 sites did not significantly affect IRAK2 promoter activity in naive and endotoxin-treated mononuclear cells, in dormant and activated Jurkat T-cells, in lung and kidney cells. In contrast, we found that CTCF plays a major role in IRAK2 transcription. An electrophoretic mobility shift assay of the DNA fragments containing the IRAK2 CpG island, revealed a single high-affinity binding site for the transcriptional regulator and a chromatin insulator protein, CTCF. This assay revealed a CTCF-binding site within the mouse Irak2 promoter. The presence of the CTCF protein in human IRAK2 promoter was confirmed by chromatin immunoprecipitation assay. Specific residues that interacted with the CTCF protein, were identified by methylation interference assay. In all cell lines analyzed, including cells of lung, renal, monocytic and T-cell origin, the IRAK2 luciferase reporter construct, containing an intact CTCF-binding site, showed strong promoter activity. However, IRAK2 promoter activity was decreased dramatically for the constructs with a mutated CTCF-binding site.
对入侵病原体的免疫反应主要通过跨膜Toll样受体家族介导，并由许多下游效应器调节。特别是，四个白细胞介素1受体相关激酶（IRAK）家族调节对细菌内毒素的反应性。对特定IRAK成分进行药物靶向治疗可能对细菌感染的治疗有益。在这里，我们研究了人类IRAK2基因的转录调控。对IRAK2启动子区域的分析揭示了几个转录因子的推定结合位点，包括ZIP（EGR1和SP1）、CTCF和AP-2β。在未处理和经内毒素处理的单核细胞、休眠和活化的Jurkat T细胞、肺和肾细胞中，删除ZIP或AP-2位点对IRAK2启动子活性没有显著影响。相比之下，我们发现CTCF在IRAK2转录中起主要作用。对含有IRAK2 CpG岛的DNA片段进行的电泳迁移率变动分析，揭示了转录调节因子和染色质绝缘子蛋白CTCF的一个单一高亲和力结合位点。该分析揭示了小鼠Irak2启动子内的一个CTCF结合位点。通过染色质免疫沉淀分析证实了人类IRAK2启动子中CTCF蛋白的存在。通过甲基化干扰分析鉴定了与CTCF蛋白相互作用的特定残基。在所有分析的细胞系中，包括肺、肾、单核细胞和T细胞来源的细胞，含有完整CTCF结合位点的IRAK2荧光素酶报告构建体显示出很强的启动子活性。然而，对于具有突变CTCF结合位点的构建体，IRAK2启动子活性显著降低。
