Expression Specificity And Compensation Effect Of Ash2l-1/Ash2l-2 In Mouse Embryonic Stem Cells

H3K4me3 is an important epigenetic modification that plays a critical role in maintaining self-renewal of mouse embryonic stem cells?m ESCs?.H3K4me3 is catalyzed mainly by the mixed lineage leukemia?MLL?methyl-trans-ferase 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: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^-/-mESCs using CRISPR/Cas9.Alkaline phosphatase?AP?staining,immunofluorescence staining,qRT-PCR,and Embryoid body?EB?differentiation test revealed that Ash2l-1^-/-and Ash2l-2^-/-mESCs still have pluripotency,while Ash2l-1^-/-mESCs may have developmental defects in the form of ectoderm and endoderm derived organs.Western blotting,transmission electron microscope and prediction of JASPAT and KEGG showed that there exists compensation effects between Ash2l-1 and Ash2l-2,which may mediated by some pluripotency transcription factors.The main results obtained in this study are as follows:1.The expression of specific Ash2l-1/Ash2l-2 in mESCsUsing Western blotting,the expression of ASH2L was detected in mESCs and MEF.Results showed that there are two isoforms of the ASH2L:ASH2L-1,which only exists in mouse embryonic fibro-blasts and ASH2L-2,which is the predominant isoform in mESCs.The comparison of ASH2L-1 and ASH2L-2 structure showed that ASH2L-1 and ASH2L-2have common sequence,except ASH2L-1 has a length of 89 amino acid sequence in N terminal.ASH2L-1 and ASH2L-2 have PHD,WH and SPRY domain.However,the expression of ASH2L-1 and ASH2L-2 were drove by different promoter:the expression of ASH2L-1 was drove by CpG islands promoter,while the expression of ASH2L-2 was drove by long terminal repeat RLTR12B promoter.2.Establishing Ash2l-1^-/-and Ashl2-2^-/-mESCsUsing the online website,we designed gRNA targeting Ash2l-1-CpG islands and Ash2l-2-RLTR12B respectively,and constructed the gRNA/Cas9 targeting vector.The PCR reaction and sequencing identification revealed that we established Ash2l-1^-/-and Ash2l-2^-/-m ESCs.The results showed the Ash2l-2-RLTR12B promoter was knockout in Ash2l-2^-/-m ESCs,and the Ash2l-1-CpG islands promoter was knockout in Ash2l-1^-/-mESCs.3.The identification of pluripotency of Ash2l-1^-/-and Ash2l-2^-/-mESCsAP staining showed that there were no obvious differences on the expression level of AP among Ash2l-1^-/-mESCs,Ash2l-2^-/-mESCs and wild type?WT?m ESCs.Immunofluorescence staining,and qRT-PCR showed that there were no obvious differences on the expression level of pluripotent transcription factors?Nanog,Oct4,sox2 and Klf4?among Ash2l-1^-/-mESCs,Ash2l-2^-/-mESCs and WT mESCs.EB differentiation test 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?.4.The complementary effect of Ash2l-1 and Ash2l-2Western blotting 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.The observation of transmission electron microscope showed that there were no obvious differences on the hetero-chromatin level among Ash2l-1^-/-mESCs,Ash2l-2^-/-mESCs and WT m ESCs.The prediction of JASPAT and KEGG showed that there were three and 16 potential binding sites for pluripotency transcription factors located in the promoter of Ash2l-1 and Ash2l-2,respectively.In conclusion,this study identified that there are two isoforms of the ASH2L in mESCs:ASH2L-1 and ASH2L-2,and ASH2L-2 has a mESCs specific expression.Using CRISPR/Cas9,we established Ash2l-1^-/-and Ash2l-2^-/-mESCs and found that they still have pluripotency,and their developmental defects may be reflected in the process of cell differentiationin.The compensation effects between Ash2l-1 and Ash2l-2 may be involved in the maintenance of mESCs pluripotency,the regulation of genomic H3K4me3 and hetero-chromatin,which laid the foundation for the study on the mechanisms of Ash2l-1 and Ash2l-2 on mESCs further.