The Mechanistic Study Of Nanog In ESC Differentiation

The balance between pluripotency and self-renewal of embryonic stem cells is maintained by a series of regulatory network composed of core transcription factors—Oct4,Sox2,and Nanog.Among them,Nanog has been considered as a central player in the specification of pluripotent cell identity and thoroughly studied upon mechanism of pluripotency and reprogramming.However,the regulatory mechanism of Nanog in mESCs differentiation remains largely unknown.Therefore,this article focuses on exploring the molecular mechanism how Nanog regulates the differentiation of mESCs To define this scientific question,we performed embryoid body experiments with Nanog conditional knockout mESCs and found loss of Nanog promoted the differentiation of three germ layers.It is noteworthy that the absence of Nanog results in the up-regulation of transcription factor Tbx3,which has been reported to be involved in ESCs differentiation.In order to verify whether Nanog acts through Tbx3,we knocked out Tbx3 through CRISPR/Cas9 system and found the differentiation phenotype caused by Nanog knockout can be rescued in the absence of Tbx3.Furthermore,utilizing ChIP-Seq and Luciferase assay,we found several potential binding sites of Nanog.Moreover,we confirmed that Nanog bound to the promoter region of Tbx3,thereby inhibiting the expression of Tbx3 through ChIP-qPCR and CRISPR/dCas9 system.In conclusion,our research demonstrated that the loss of Nanog didn't markedly alter the pluripotent markers of mESCs but elevated the expression of Tbx3 through binding small regions of the Tbx3 promoter,thus promoting the differentiation of mESCs into three germ layers.This study is conducive to enriching the molecular mechanism of Nanog upon regulating mouse embryonic stem cell differentiation,and provides a great theoretical basis for understanding the process and mechanism of early embryo development.