H3K4me3是一种重要的表观遗传修饰,主要由MLL(mixed lineage leukemia)甲基转移酶复合体催化,对小鼠胚胎干细胞(mouse embryonic stem cells, mESCs)自我更新能力的维持具有重要作用。ASH2L是MLL复合体中一个重要的核心亚单位,参与调控mESCs中染色质的开放状态。ASH2L在mESCs中有2个异构体：ASH2L-1(80 kDa)和ASH2L-2(65 kDa),且以ASH2L-2的表达为主;而在小鼠胚胎成纤维细胞(mouse embryonic fibroblast, MEF)中,只有ASH2L-1表达。目前,Ash2l-1和Ash2l-2在mESCs中的作用尚不清楚。本文利用CRISPR/Cas9基因组编辑技术,建立了Ash2l-1 ^-/-和Ash2l-2 ^-/-mESCs。通过碱性磷酸酶染色、免疫荧光染色和qRT-PCR发现,Ash2l-1 ^-/-和Ash2l-2 ^-/-mESCs在碱性磷酸酶、多能性调控转录因子(Oct4、Nanog、Sox2和Klf4)的表达与野生型对照无显著差异。通过拟胚体分化实验,发现Ash2l-1 ^-/-mESCs诱导的拟胚体在Snai2(外胚层标记基因)和Gata4(内胚层标记基因)的表达上显著低于野生型mESCs诱导的拟胚体(P<0.01)。通过Western blotting,发现Ash2l-1 ^-/-mESCs中ASH2L-2的表达显著上调(P<0.01),Ash2l-2 ^-/-mESCs中ASH2L-1的表达显著上调(P<0.01),而Ash2l-1 ^-/-和Ash2l-2 ^-/-mESCs中,基因组H3K4me3的表达与野生型对照并无显著差异。这表明Ash2l-1和Ash2l-2之间存在补偿效应。利用JASPAR和KEGG预测分析发现,Ash2l-1和Ash2l-2启动子区分别具有3个和16个潜在的多能性转录因子结合位点,这些转录因子可能介导实现Ash2l-1和Ash2l-2之间的补偿效应。以上结果表明,Ash2l-1和Ash2l-2之间的补偿效应可能参与mESCs多能性的维持和基因组H3K4me3的调控。

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.