Combined double knockout of Ezh2 and G9a leads to expression change in murine retinal ganglion cells

Purpose: The goal of this study is to evaluate H3K27me3 histone marks in retinal ganglion cells in Ezh2 (Math5Cre, Ezh2-/-) and combined G9a-Ezh2 (Math5Cre; G9a+/-Ezh2-/-) knockout mutant mice at P1. Both Ezh2 and G9a are major epigenetic silencing machineries. Ezh2 mediates the trimethylation of lysine 27 on histone 3. Emerging evidences show an interdependence between Ezh2 and G9a to facilitate K27 trimethylation. Methods: P1 retinal ganglion cells were collected and chromatin immunoprecipitation was performed with EZ- CHIP kit from Millipore. Library preparation was done with Rubicon Genomics Thruplex DNAseq. Results: Here, we find a pool of common K27me3 target genes of Ezh2 and G9a in double knockout that leads to dysfunction in double mutant RGCs. Conclusion: This study provides the first Chip-seq analysis of K27me3 in murine RGCs. Our data supports that an interaction between Ezh2 and G9a is also evident in murine retinal ganglion cells. This work should be a framework for further study of retinal ganglion cells and retinal diseases. Examination of H3K27me3 marks in single Ezh2 (Math5Cre; Ezh2-/-) and combined G9a-Ezh2 (Math5Cre; G9a+/-Ezh2-/-) knockout murine retinal ganglion cells

### 研究目的
本研究旨在评估出生后第1天（P1）的Ezh2敲除（Math5Cre，Ezh2-/-）及G9a-Ezh2联合敲除（Math5Cre；G9a+/-Ezh2-/-）突变小鼠视网膜神经节细胞（retinal ganglion cells）中的H3K27me3组蛋白修饰（H3K27me3 histone marks）。Ezh2与G9a均为核心表观遗传沉默调控因子，其中Ezh2可介导组蛋白H3第27位赖氨酸的三甲基化。已有研究证据显示，二者存在相互依赖的协同机制，共同促进K27三甲基化修饰。

### 实验方法
收集出生后第1天的视网膜神经节细胞，采用Millipore公司的EZ-CHIP试剂盒完成染色质免疫共沉淀（chromatin immunoprecipitation, ChIP）实验；文库构建使用Rubicon Genomics公司的Thruplex DNAseq试剂盒进行。

### 实验结果
本研究发现，在双敲除小鼠中存在一批由Ezh2与G9a共同调控的K27me3靶基因，该靶基因集合的异常与双突变视网膜神经节细胞的功能障碍密切相关。

### 研究结论
本研究首次完成了小鼠视网膜神经节细胞中H3K27me3的染色质免疫共沉淀测序（ChIP-seq）分析。实验数据证实，Ezh2与G9a之间的相互作用在小鼠视网膜神经节细胞中同样存在。本研究可为后续视网膜神经节细胞及视网膜疾病的相关研究提供理论框架。本研究同时对单敲除Ezh2（Math5Cre；Ezh2-/-）及G9a-Ezh2联合敲除（Math5Cre；G9a+/-Ezh2-/-）的小鼠视网膜神经节细胞的H3K27me3修饰进行了检测。