Positive Feedback Of Regulating ERK Phosphorylation In MESCs Mediated By Etv5-Tet2-Fgfr2 Axis

Dynamic equilibrium of extracellular signal-regulated kinase(ERK)activity is regulated elaborately by multiple feedback loops to ensure the normal self-renewal of mouse embryonic stem cells(mESCs).Previous studies on mESCs have demonstrated that the negative feedback loops are engaged to prevent the overactivated ERK phosphorylation(pERK).It is not clear whether there is any positive feedback loop involved to maintain a minimum of pERK in mESCs.pERK entered nucleus can activate a series of transcription factors such as Etv4,Etv5,Dusp6,Spry and Sef.Dusp6,Spry and Sef play a negative feedback to regulate pERK.However,it is not clear that whether Etv4 and Etv5 can regulate pERK with negative or positive feedback? In this study,the CRISPR/Cas9 was used to generate Etv5 knockout mESCs,and the data of RNA-seq of Etv5 knockdown are also analysed.The qPCR,Western blot,bioinformatics analysis,promoter cloning,luciferase assay and methylation sequencing were performed routine detection.The main results obtained in this study are as follows:1.Construction of Etv5 KO mESCs using CRISPR/Cas9We designed double specific sgRNA targeting the seventh exon of Etv5 and cloned them into vecter of PX459 M respectively.Three clones of Etv5 KO B10,B13 and B23 were obtained.2.Etv5 regulates ERK with a positive feedbackThe expression level of pERK was downregulated in Etv5 KO mESCs compared with WT mESCs.The Etv5 KO mESCs showed slow proliferation,upregulation of Otx2 and intense signals in the nuclei with fluorescent dye Hoechst 33258.3.Positive feedback loop of regulating ERK phosphorylation in mESCs mediated by Etv5-Tet2-Fgfr2 axisTo dissect the molecular mechanism of reduced pERK caused by Etv5 KO in mESCs,we analyzed our previous RNA-seq data for Etv5 KD in mESCs and investigated the expression change of genes in FGF/ERK pathway and genes which dephosphorylate pERK.Surprisingly,we found that Fgfr2 was not only significantly downregulated in Etv5 KD mESCs when compared to nonsense control(NC)mESCs but also dominantly expressed in mESCs.In order to study how Etv5 regulated the transcription of Fgfr2 in mESCs,the predicted Fgfr2 promoter using bioinformatics analysis was cloned into PGL3.However,overexpression of Etv5 could only activate about two fold change fluorescence degree compared with control.To investigate whether Etv5 regulates Fgfr2 expression mediated by other factors,we analyzed the potential Fgfr2 promoter region by using an online ChIP database.The result showed 43 transcription factors or chromatin regulators binding to this region in which the Tet2 was significantly down-regulated caused by Etv5 KD.The mRNA and protein of Tet2 was decreased in Etv5 KO mESCs.We detected the DNA methylation of Fgfr2 promoter.Bisulfite sequencing showed the DNA methylation in Etv5 KO mESCs was 16.7% and the WT mESCs was 2.5% from the investigated region.Collectively,a positive feedback loop of regulating pERK was revealed in mESCs,which was mediated by Etv5-Tet2-Fgfr2 axis.Our findings provide a new paradigm for pERK regulation in mESCs and will be useful to understand the cell fate determination during early embryo development.