The Role Of Asxl2 In The Differentiation Of Mouse Embryonic Stem Cell Into Cardiomyocyte And Identification Of Its Binding Partners

Background:Drosophila Asx(Additional sex combs)gene is one of the ETP(Enhancers of trithorax and Polycomb)gene, whose encoded protein functions in regulating or recruiting Polycomb-group repressor complex and trithorax-group activator complex. Asxl2, one of the mammalian Asx homologs, was first identified in 2003. And then, ASXL2 mutationswas found in diverse human tumors suchas breast cancers, prostate cancers, bladder cancers and pancreatic cancers. Recently, Asxl2 is found with high level expression in mammal heart and requied for the normal functionof mammal heart, Asxl2-/- mice have impairedheart functionis.Even so, its fuction and molecule mechanism research in dieases is still not in-depth. Research has found that Asxl2 is in a complex with Bap1(BRCA1 associated protein 1), an ubiquitin carboxy-terminal hydrolase, and deubiquitylates H2AK119ub1.The relationship between the catalytic activity of Asxl2-Bap1 complex and the function of Asxl2 needs further research.Objective:In the current research, we tried to investigate the functions of Asxl2 in m ESC(mouse embryonic stem cells)to cardiocytes differentiation. Next, the binding proteins of Asxl2 were found, helping to clarify the molecole mechanism.Methods:Establish m ESC lines with Asxl2 knocked outby using CRISPR/Cas9 n system. The genomic DNA of the monoclonal cell lines was extracted and a DNA fragment flanked the target site was amplified by genotyping PCR then sequenced.Western blotting were applied to confirm whether Asxl2 was successfully knocked out.Pendant-drop methodwas utilized to cultrue wide type m ESC and Asxl2 knocked out m ESC to ardiocytes differentiation.Observe thephenotype at day 2 to day 9, count the ratio of the beating cell clusters at day 9 and detect the expression of cardiac developmental transcription factors Mef2c(myocyte enhancer factor2) and Hand1(heart and neural crest derivatives expressed transcript 1) at day 0, day 2 and day 5. Co-immunoprecipitation(Co-IP) was used to identifiy the binding proteins of Asxl2.Results:Part I : Construction of Asxl2 gene knock out stable mESC cell line with CRISPR/Cas9 n system.1. CRISPR/Cas9 n plasmids that targeted Asxl2 gene were successfully constructed.2. mESC were co-transfected with the two recombinant constructs. After puromycin selection,subclonal cell lines were obtained and DNA fragments flanked the target site were amplified by genotyping PCR then sequenced, confirmed that one of them had Asxl2 gene mutation.3. Western blotting confirmed that Asxl2 was completely depleted in the monoclonal m ESC cell line.Part II:Asxl2 is required in the differentiation of m ESC into cardiomyocytes.1. Compared to wide type m ESC,the Asxl2 knock out subclonal cell line had different phenotype in the differentiation into cardiomyocytes.2. Compared to wide type m ESC,the Asxl2 knock out subclonal cell line had lower ratio of the beating cell clusters at day 9.3. Compared to wide type m ESC, the expression of cardiac developmental transcription factors Mef2 c and Hand1 was decrease in the Asxl2 knock out subclonal cell line.Part III:Asxl2 can interact with Bap1,but not Ezh2(enhancer of zeste 2 polycomb repressive complex 2 subunit)in m ESC.Conclusion:1. Asxl2 can regulate the differentiation of m ESC into cardiomyocytes.2. Asxl2 can bind with Bap1,but not Ezh2.