Genome-wide analysis of H3K27me3 in Ezh2-conditional iMEFs

We analyzed the genomic distribution of H3K27me3 in a clone of c-Myc iMEFs (clone C2) either i) wild-type for Ezh2, ii) in the presence of overexpressed exogenous Ezh2, iii) Ezh2-mutant, and iv) Ezh1/Ezh2 pre-deletion (Ezh1/Ezh2 introduced before deletion of endogenous Ezh2) and Ezh2 post-deletion rescue (Ezh2 re-introduced in Ezh2-mutant cells). Overall design: Experiment 1: H3K27me3 ChIP-seq in C2 iMEFs either Ezh2 WT, Ezh2-mutant or Ezh2 post-rescue condition. Experiment 2: H3K27me3 ChIP-seq in C2 iMEFs either Ezh2 WT, Ezh2-mutant, in the presence of overexpressed Ezh2, in Ezh1 pre-rescue condition and in Ezh2 pre-rescue condition.

本研究针对c-Myc永生化小鼠胚胎成纤维细胞（c-Myc immortalized mouse embryonic fibroblasts, iMEFs）克隆C2（clone C2），分析了组蛋白H3赖氨酸27三甲基化（H3K27me3）的基因组分布，所纳入的细胞组别包含以下四类：① 增强子之锌指同源物2（Enhancer of zeste homolog 2, Ezh2）野生型细胞；② 过表达外源性Ezh2的细胞；③ Ezh2突变型细胞；④ Ezh1/Ezh2预敲除（即内源性Ezh2敲除前先引入Ezh1/Ezh2）以及Ezh2敲除后回补（即在Ezh2突变细胞中重新引入Ezh2）的细胞。整体实验设计如下：实验1：对克隆C2的iMEFs，分别在Ezh2野生型、Ezh2突变型及Ezh2敲除后回补条件下，开展H3K27me3染色质免疫沉淀测序（Chromatin Immunoprecipitation sequencing, ChIP-seq）。实验2：对克隆C2的iMEFs，分别在Ezh2野生型、Ezh2突变型、过表达Ezh2、Ezh1预回补以及Ezh2预回补条件下，开展H3K27me3 ChIP-seq。