Architecture of Epigenetic Reprogramming Following Twist1 Mediated Epithelial-Mesenchymal Transition [RNA-seq]. Homo sapiens

Purpose: to characterize epigenetic changes following Twist1 mediated Epithelial-Mesenchymal Transition in human Methods: we characterized the epigenetic and transcriptome landscapes using whole genome transcriptome analysis by RNA-seq, DNA methylation by digital restriction enzyme analysis of methylation (DREAM) and histone modifications by CHIP-seq of H3K4me3 and H3K27me3 in immortalized human mammary epithelial cells relative to cells induced to undergo EMT by Twist1. Results: EMT is accompanied by focal hypermethylation and widespread global DNA hypomethylation, predominantly within transcriptionally repressed gene bodies. At the chromatin level, the number of gene promoters marked by H3K4me3 increases by more than one fifth; H3K27me3 undergoes dynamic genomic redistribution characterized by loss at half of gene promoters and overall reduction of peak size by almost one-half. This is paralleled by increased phosphorylation of EZH2 at serine 21. Among genes with highly altered mRNA expression, 23.1% switch between H3K4me3 and H3K27me3 marks, and those point to the master EMT targets and regulators CDH1, PDGFRA and ESRP1. Strikingly, Twist1 increases the number of bivalent genes by more than two fold. Inhibition of the H3K27 methyltransferases EZH2 and EZH1, which form part of the PRC2 complex, results in blocking EMT and stemness properties. Conclusion: Our findings demonstrate that the EMT program requires epigenetic remodeling by the Polycomb/Trithorax complexes leading to increased cellular plasticity which suggests that its inhibition will prevent EMT, and the associated breast cancer metastasis. Overall design: RNAseq profiles of human mammary epithelial cells before (HMLE_parental) and after Twist1 transfection (HMLE_Twist) were generated in monolayer (HMLE_Twist2D) and sphere culture by deep sequencing using SOLID

研究目的：表征Twist1介导的上皮间质转化（Epithelial-Mesenchymal Transition, EMT）后人类细胞的表观遗传变化。
研究方法：以永生化人乳腺上皮细胞及其经Twist1诱导发生上皮间质转化的细胞为研究对象，采用全基因组转录组RNA测序（RNA-seq）、数字限制性酶甲基化分析（digital restriction enzyme analysis of methylation, DREAM）检测DNA甲基化水平，同时通过染色质免疫沉淀测序（Chromatin Immunoprecipitation sequencing, ChIP-seq）分析H3K4me3与H3K27me3两种组蛋白修饰，以此全面表征细胞的表观遗传与转录组图谱。
研究结果：上皮间质转化过程伴随局灶性高甲基化与广泛的全基因组DNA低甲基化，且该表观遗传改变主要富集于转录沉默的基因区。在染色质层面，带有H3K4me3修饰标记的基因启动子数量增加超过五分之一；H3K27me3则发生动态的基因组重分布，表现为半数基因启动子区域的H3K27me3修饰丢失，且其测序峰的整体大小缩减近一半，这一变化与丝氨酸21位点磷酸化水平升高的EZH2蛋白表达变化相平行。在mRNA表达发生显著改变的基因中，23.1%的基因在H3K4me3与H3K27me3两种修饰标记之间发生切换，其中涵盖核心上皮间质转化靶标与调控因子CDH1、PDGFRA及ESRP1。值得注意的是，Twist1可使二价基因的数量增加两倍以上。抑制属于PRC2复合物组分的H3K27甲基转移酶EZH2与EZH1，可阻断上皮间质转化进程与干细胞干性特征。
研究结论：本研究结果证实，上皮间质转化程序需要多梳/三胸组蛋白复合物（Polycomb/Trithorax complexes）介导的表观遗传重塑，进而提升细胞可塑性，这提示靶向抑制该通路可阻断上皮间质转化及其相关的乳腺癌转移。
实验整体设计：采用SOLID高通量深度测序技术，对永生化人乳腺上皮细胞（HMLE_parental）及其经Twist1转染的细胞（HMLE_Twist）分别在单层培养（HMLE_Twist2D）与球体培养体系中进行转录组RNA测序，以获取其转录组表达谱。