Xie Jing, Fan Chen, Zhang Jing-Long, Zhang Shi-Qiang
College of Veterinary Medicine, Northwest A&F University, Yangling 712100, China.
Yi Chuan. 2018 Mar 20;40(3):237-249. doi: 10.16288/j.yczz.17-284.
H3K4me3 is an important epigenetic modification that plays a critical role in maintaining self-renewal of mouse embryonic stem cells (mESCs). H3K4me3 is catalyzed mainly by the mixed lineage leukemia (MLL) methyl-transferase complex. ASH2L, a core subunit of the MLL complex, participates in regulating the open state of chromatin in mESCs. There are two isoforms of the ASH2L protein: ASH2L-1 (80 kDa), which only exists in mouse embryonic fibroblasts and ASH2L-2 (65 kDa), which is the predominant isoform in mESCs. The roles of Ash2l-1 and Ash2l-2 in mESCs have not yet been elucidated. In this study, we established Ash2l-1 and Ash2l-2 knockout mESCs using CRISPR/Cas9. Alkaline phosphatase (AP) staining, immunofluorescence staining, and qRT-PCR showed that there were no obvious differences on the expression level of AP and pluripotent transcription factors (Nanog, Oct4, sox2 and Klf4) among Ash2l-1 mESCs, Ash2l-2 mESCs and wild type (WT) mESCs. However, analysis of embryoid body (EB) differentiation showed that the expression level of Snai2 (ectoderm gene) and Gata4 (endoderm gene) in Ash2l-1 EBs was significantly lower than that in WT EBs (P<0.01). Western blotting assay revealed that the expression of ASH2L-2 was significantly increased (P<0.01) in Ash2l-1 mESCs and vice versa. However, there were no obvious differences on the genomic H3K4me3 level among Ash2l-1 mESCs, Ash2l-2 mESCs and WT mESCs. These results indicate that there exist compensation effects between Ash2l-1 and Ash2l-2. Bioinformatic analysis predicted that there were three and 16 potential binding sites for pluripotency transcription factors located in the promoter of Ash2l-1 and Ash2l-2, respectively. Theses transcription factors may mediate the compensation effect between Ash2l-1 and Ash2l-2. Collectively, these results indicate that the compensation effects between Ash2l-1 and Ash2l-2 may be involved in the maintenance of mESCs pluripotency and the regulation of genomic H3K4me3.
H3K4me3是一种重要的表观遗传修饰，在维持小鼠胚胎干细胞（mESCs）的自我更新中发挥关键作用。H3K4me3主要由混合谱系白血病（MLL）甲基转移酶复合物催化。ASH2L是MLL复合物的核心亚基，参与调节mESCs中染色质的开放状态。ASH2L蛋白有两种异构体：ASH2L-1（80 kDa），仅存在于小鼠胚胎成纤维细胞中；ASH2L-2（65 kDa），是mESCs中的主要异构体。Ash2l-1和Ash2l-2在mESCs中的作用尚未阐明。在本研究中，我们使用CRISPR/Cas9建立了Ash2l-1和Ash2l-2基因敲除的mESCs。碱性磷酸酶（AP）染色、免疫荧光染色和qRT-PCR显示，Ash2l-1基因敲除的mESCs、Ash2l-2基因敲除的mESCs和野生型（WT）mESCs中，AP和多能转录因子（Nanog、Oct4、Sox2和Klf4）的表达水平没有明显差异。然而，胚状体（EB）分化分析表明，Ash2l-1基因敲除的EB中Snai2（外胚层基因）和Gata4（内胚层基因）的表达水平显著低于WT EB（P<0.01）。蛋白质免疫印迹分析显示，Ash2l-1基因敲除的mESCs中ASH2L-2的表达显著增加（P<0.01），反之亦然。然而，Ash2l-1基因敲除的mESCs、Ash2l-2基因敲除的mESCs和WT mESCs之间的基因组H3K4me3水平没有明显差异。这些结果表明Ash2l-1和Ash2l-2之间存在补偿效应。生物信息学分析预测，Ash2l-1和Ash2l-2启动子中分别有3个和16个多能转录因子的潜在结合位点。这些转录因子可能介导Ash2l-1和Ash2l-2之间的补偿效应。总的来说，这些结果表明Ash2l-1和Ash2l-2之间的补偿效应可能参与了mESCs多能性的维持和基因组H3K4me3的调控。
