| Background and objective:The application of pluripotent stem cells depends on our understanding ofthe molecular mechanism of their differentiation process.In the process of differentiation and development,normal embryos must undergo lineage-specific differentiation to differentiate the inner cell mass cells into three germ layers:mesoderm,endoderm,and ectoderm.The specialization of the three germ layers is a key process during early development.Mouse embryonic stem cells are derived from the inner cell mass of the blastocyst stage and have the characteristics of pluripotency and self-renewal.Mouse embryonic stem cells have an open chromatin structure,and their selfrenewal and differentiation are largely regulated by histone modifications and chromatin remodeling.Rifl(RAP1 interacting factor 1)was first identified in budding yeast as a telomereassociated protein that negatively regulates telomere length.In mammals,in addition to regulating telomere length homeostasis,Rifl has also been shown to inhibit direct post-break resection after DN A double-strand break damage,promote non-homologous end recombination(NHEJ)in the G1 phase,and by binding to G4-quadruplex and 3D Chromatin changes are involved in the global regulation of the Replication Timing program.Rifl highly expresses in early embryonic development in vivo and in mouse embryonic stem cells in vitro,but hardly expresses in fully differentiated cells except germ cells.Furthermore,deletion of Rifl leads to early embryonic lethality in C57BL/6J mice,suggesting that Rifl is critical for early embryo development,but whether Rifl regulates early mESCs differentiation and its mechanism remain unclear.This study aims to understand the role and molecular mechanism of Rifl in the germ layer differentiation of embryonic stem cells,and lay the foundation for applications in developmental research,disease models,drug screening,and regenerative medicine.Methods:First,a mouse embryonic stem cell line with stable inducible knockout of Rifl was constructed.And then we detect the changes of pluripotency,cell cycle progression and proliferation ability of Rif1 knockout mouse embryonic stem cells(mESCs)by real-time quantitative reverse transcriptase polymerase chain reaction(RT-qPCR),immunofluorescence(IF),Western blot,Flow cytometry(FACS)and other techniques;Secondly,the embryoid body was used as the model of mESCs differentiation in vitro,and we detected the expression of D2,D4,D6 germ layer-specific genes at different time points of mESCs differentiation;Then,teratoma was used as the model of mESCs differentiation in vivo,and the development of the three germ layers was observed by pathological section staining;In addition,RNA highthroughput sequencing(RNA-Seq)was used to determine which gene expression was affected by Rif1 deletion,and to analyze the published key signaling pathway members of cell differentiation ERK1/2,compare the RNA-seq data of ERK1/2 knockdown and Rif1 knockdown to observe the similarity of their effect on gene expression changes;Chromatin immunoprecipitation followed high-throughput sequencing(ChIP-Seq)experiment was performed with RIF 1 antibody to determine the genes directly bound by RIF 1.Combined with RNA-seq data,the genes directly bound by RIF1 and whose expression was affected by Rif1 deletion were analyzed and the functional classification and downstream biological behavior of Rif1 target genes were predicted;Finally,the published ChIP-Seq data of the H3K27 methyltransferase EZH2,ERK2 in wild-type mESCs were analyzed to observe the similarity of their binding sites in the genome and which genes H3K27me3 binding peaks were changed by Rif1 deletion.In order to find out whether Rif1 controls the expression of differentiation-related genes by regulating histone methylation modification,the RIF1 antibody coimmunoprecipitation(co-IP)experiment confirmed that Rif1 controls gene expression by regulating H3K27me3 through EZH2.The results of ChIP-seq were verified by chromatin coimmunoprecipitation quantitative PCR(ChIP-qPCR)to determine the changes in the binding of EZH2,H3K27me3,and p-ERK1/2 on mesendoderm-specific genes.ResultsWe use CRISPR gene editing technology to establish a stable conditional knockout of Rif1 mouse embryonic stem cell line,loss of Rif1 in mESC cells leads to decreased expression of pluripotency genes Oct4,Esrrb,Klf2,Sox2,and inhibition of cell cycle G1/S transition,the weaker proliferation ability which proves that Rif1 is essential for maintaining the pluripotency and self-renewal of mESCs;RNA-seq results show that the up-regulated genes after Rif1 knockout are significantly enriched in processes or signals related to cell differentiation and development pathways,genes whose expression was downregulated were mainly enriched for cell cycle-related processes,which is consistent with our observed phenotype.Three differentiation models in vitro showed that the expression of Rif1 gradually decreased with the process of differentiation,which revealed that Rif1 was highly related to cell differentiation and development.During the free differentiation of embryoid bodies in vitro,the expression of germ layer-specific genes after Rifl knockout was detected.The expression of endoderm-specific genes was significantly increased(P<0.05),and the results of H&E staining in teratoma also showed that Rifl knockout promoted mesendoderm differentiation.To identify the direct downstream target genes of Rifl,we clarified that RIF1 represses genes by directly binding to the promoter regions of differentiation-related genes by combined analysis of chromatin immunoprecipitation sequencing(ChIP-seq)and RNA-seq results of Rifl in mESCs expression.RIF1,EZH2,and H3K27me3 co-localize extensively in the genome,and all bind to the promoter regions of mesendoderm-specific genes.After Rifl knockout,H3K27me3 modification is specifically reduced in gene regions related to cell differentiation and development.Motif analysis showed that the transcription factors Sox17,Prdm15 and Foxe1,which regulate differentiation,were involved in the regulation of H3K27me3.Further analysis of the mechanism of knockout on the regulation of germ layer-specific genes showed that the H3K27me3 occupancy in the promoter region of mesendoderm-specific genes decreased after Rifl knockout.ChIP-qPCR verified that EZH2 and H3K27me3 were in the promoter regions of germ layer-specific genes.As a result of the reduced occupancy,co-IP experiments confirmed that RIF1 can directly bind to the methyltransferase EZH2 to regulate the trimethylation of H3K27,thereby regulating the expression of differentiation genes.Early embryonic development is closely related to the MAPK signaling pathway.RNAseq results showed that the expression of genes activated by MAPK signaling pathway was upregulated after Rifl knockout,and Western blot results showed that the protein levels of pERK1/2 were significantly higher Rifl-KO mESCs.up-regulated in s.ERK2 ChIP-seq results showed that ERK2 specifically binds to genes related to cell differentiation and embryonic development,and also binds to the promoter regions of germ layer-specific genes.The results of ChIP-qPCR showed that the binding of p-ERK1/2 to the promoter regions of germ layerspecific genes was significantly increased after Rifl knockout(P<0.05).In order to verify that the promotion of differentiation is related to the activation of ERK1/2,PD0325901,a specific inhibitor of p-ERK1/2,was added,and the results showed that Rifl knockout had an inhibitory effect on the promotion of mesendoderm differentiation.Conclusion:This study not only reveals the regulatory role of Rifl in the early differentiation of mESCs,but also elucidates the mechanism by which RIF1 binds to EZH2 to promote H3K27me3 modification in the promoter region of germ layer genes to inhibit gene expression.The research further helps us understand how chromatin modification regulates the process of cell differentiation.The synergistic effect of signaling pathway and chromatin modification is proposed for cell fate determination,which provides a new theoretical basis for understanding the molecular mechanism of early embryonic development and the mature regulation of cell differentiation in regenerative medicine. |
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