Role of bacterial infection in the epigenetic regulation of Wnt antagonist WIF1 by PRC2 protein EZH2.
Role of bacterial infection in the epigenetic regulation of Wnt antagonist WIF1 by PRC2 protein EZH2.
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Zeste同源物增强子2（EZH 2）通过组蛋白H3赖氨酸-27-三甲基化（H3 K27 me 3）抑制基因转录。啮齿类柠檬酸杆菌（Citrobacter rodentium，CR）通过异常调节Wnt/β-catenin信号通路促进隐窝增生和肿瘤发生。我们旨在研究EZH 2在细菌感染后表观遗传学调节Wnt/β-catenin信号传导中的作用。NIH：用CR（108 cfu）感染Swiss远系繁殖和ApcMin/+小鼠; BLT 1 −/−ApcMin/+小鼠、AOM/DSS处理的小鼠和去识别的人腺癌样品是结肠癌模型。在用野生型而不是突变型CR感染后，在第6天和第12天（高峰增生）隐窝中升高的EZH 2水平分别与H3 K27 me 3和β-连环蛋白水平的增加一致。染色质免疫沉淀显示EZH 2和H3 K27 me 3占据WIF 1（Wnt抑制因子-1）启动子，导致WIF 1 mRNA和蛋白表达降低。在通过siRNA或EZH 2抑制剂DZNep单独或与HDAC抑制剂SAHA组合敲低EZH 2后，WIF 1启动子活性显著增加，而EZH 2的过表达减弱了WIF 1报告子活性。SET结构域突变体（F681 Y）的异位过表达几乎完全挽救了WIF 1报告基因活性并部分挽救了WIF 1蛋白水平，而H3 K27 me 3水平显著减弱，表明完整的甲基转移酶活性是EZH 2依赖性效应所需的。有趣的是，虽然EZH 2敲低细胞中β-连环蛋白水平较低，但F681 Y突变体仅表现出β-连环蛋白水平的部分降低。除EZH 2外，感染后第6天和第12天隐窝中miR-203表达的增加与其靶WIF 1水平的降低相关;原代结肠细胞中miR-203的过表达降低了WIF 1 mRNA和蛋白水平。在CR感染的ApcMin/+小鼠的息肉中，EZH 2和β-连环蛋白水平升高，同时WIF 1表达降低，这与BLT 1 −/−ApcMin/+、AOM/DSS和人类腺癌中记录的变化一致。因此，EZH 2诱导的WIF 1表达下调可能部分调节了Wnt/β-连环蛋白依赖性隐窝增生对CR感染的应答。
The Enhancer of Zeste Homolog-2 (EZH2) represses gene transcription through histone H3 lysine-27-trimethylation (H3K27me3). Citrobacter rodentium (CR) promotes crypt hyperplasia and tumorigenesis by aberrantly regulating Wnt/β-catenin signaling. We aimed at investigating EZH2’s role in epigenetically regulating Wnt/β-catenin signaling following bacterial infection. NIH:Swiss outbred and ApcMin/+ mice were infected with CR (108cfu); BLT1−/−ApcMin/+ mice, AOM/DSS-treated mice and de-identified human adenocarcinoma samples were models of colon cancer. Following infection with wild type but not mutant CR, elevated EZH2 levels in the crypt at days-6 and 12 (peak hyperplasia) coincided with increases in H3K27me3 and β-catenin levels, respectively. Chromatin immunoprecipitation revealed EZH2 and H3K27me3’s occupancy on WIF1 (Wnt Inhibitory Factor-1) promoter resulting in reduced WIF1 mRNA and protein expression. Following EZH2 knockdown via siRNA or EZH2-inhibitor DZNep either alone or in combination with HDAC inhibitor SAHA, WIF1 promoter activity increased significantly while overexpression of EZH2 attenuated WIF1-reporter activity. Ectopic overexpression of SET domain mutant (F681Y) almost completely rescued WIF1 reporter activity and partially rescued WIF1 protein levels while H3K27me3 levels were significantly attenuated suggesting that an intact methyltransferases activity is required for EZH2-dependent effects. Interestingly, while β-catenin levels were lower in EZH2-knocked-down cells, F681Y mutants exhibited only partial reduction in β-catenin levels. Besides EZH2, increases in miR-203 expression in the crypts at days-6 and 12 post-infection correlated with reduced levels of its target WIF1; overexpression of miR-203 in primary colonocytes decreased WIF1 mRNA and protein levels. Elevated levels of EZH2 and β-catenin with concomitant decrease in WIF1 expression in the polyps of CR-infected ApcMin/+ mice paralleled changes recorded in BLT1−/−ApcMin/+, AOM/DSS and human adenocarcinomas. Thus, EZH2-induced downregulation of WIF1 expression may partially regulate Wnt/β-catenin-dependent crypt hyperplasia in response to CR infection.
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