Effect of T350 phosphorylation on gene repression activity of EZH2 in BJ cells. Homo sapiens

The Polycomb group (PcG) protein enhancer of zeste homolog 2 (EZH2) plays an essential role in histone methylation-mediated epigenetic gene silencing. We demonstrate that under physiological conditions, cyclin-dependent kinase 1 (CDK1) and 2 (CDK2) bind to and phosphorylate EZH2 at threonine 350 (T350) in an evolutionally conserved motif. T350 phosphorylation is required for EZH2 function in silencing of genes that promote differentiation and inhibit proliferation and migration. Blockage of T350 phosphorylation by generating a T350A mutation largely diminishes the global effect of EZH2 on gene silencing. Overall design: Four experiments have been designed. BJ cells were transfected with si-Control, si-EZH2, si-EZH2 plus RNAi resistant lentivirus expressed EZH2, or si-EZH2 plus RNAi resistant lentivirus expressed T350A mutant for 60 hours. Each experiment was triplicated.

多梳蛋白家族（Polycomb group, PcG）的zeste同源增强子2（enhancer of zeste homolog 2, EZH2）在组蛋白甲基化介导的表观遗传基因沉默过程中发挥不可或缺的核心作用。本研究证实，在生理条件下，细胞周期蛋白依赖性激酶1（cyclin-dependent kinase 1, CDK1）与细胞周期蛋白依赖性激酶2（cyclin-dependent kinase 2, CDK2）可结合EZH2，并在进化保守基序内的苏氨酸350位点（threonine 350, T350）对其进行磷酸化修饰。T350位点的磷酸化是EZH2发挥沉默促分化、抑制增殖与迁移相关基因功能的必要条件。通过构建T350A突变体阻断该位点的磷酸化，可大幅削弱EZH2在全基因组范围内的基因沉默调控效应。总体实验设计：将BJ细胞分别转染对照siRNA（si-Control）、靶向EZH2的siRNA（si-EZH2）、si-EZH2联合携带RNAi抗性的野生型EZH2的慢病毒，以及si-EZH2联合携带RNAi抗性的T350A突变体EZH2的慢病毒，培养60小时；每组实验均设置三次生物学重复。