Mechanism Study Of The Role Of Tfcp211 In Inducing Esrrb Expression To Promote Self-renewal Of Mouse Embryonic Stem Cells

The development of the preimplantation embryo and lineage commitment of stem cells,derived from early blastocyst,are highly regulated.Three stem cell types can be isolated from the mouse blastocyst and cultured in vitro,including the trophectoderm derived trophoblast stem(TS)cells,the primitive endoderm derived stem-cell-like extraembryonic endoderm(XEN)cells and the inner cell mass(ICM)derived embryonic stem cells(ESCs).Among these three types of stem cells,the most popular research is mouse embryonic stem cells(mESCs).Under appropriate in vitro culture conditions,ESCs are able to proliferate without differentiation,termed’self-renewal’,and at the same time retain the capacity to differentiation into the cells of all three primary germ layers,termed ’pluripotency’.To date,although ESC-like cells from many species have been established,only ESCs derived from mice and rats possess the ability to generate germline-competent chimeric offspring and thus represent a’naive’pluripotent state.Interestingly,the available human ESCs(hESCs)are more similar to mouse post-implantation epiblast-derived stem cells(mEpiSCs)than to mouse ESCs(mESCs)in their self-renewal requirements and morphology,and thus represent ’primed’ pluripotency state.Notably,the primed state ESCs could be reprogrammed into naive pluripotency state through overexpression of single gene,such as Stat3,Tfcp211 and Klf4.mESCs and mEpiSCs require different culture conditions for maintaining their pluripotency.mEpiSCs can maintain self-renewal through addition of basic fibroblast growth factor(bFGF)and recombinant human Activin-A(ActivinA),while mESCs can maintain the undifferentiated state through two different culture systems:serum medium,supplemented with leukaemia inhibitory factor(LIF),or serum-free medium N2B27,supplemented with two small molecule inhibitors(2i),CHIR99021 and PD0325901.CHIR99021 and PD0325901 maintain self-renewal through inhibition of glycogen synthase kinase 3(GSK3)and mitogen-activated protein kinase kinase(MEK),respectively.LIF supports self-renewal by inducing activation of signal transducer and activator of transcription3(STAT3),and further activates some pluripotency genes in the downstream of STAT3.However,the mechanism of these genes in promoting the self-renewal of mESCs is still unclear.It has been previously reported that transcription factor CP2-like protein 1(Tfcp211)play an important role in establishing and maintaining naive pluripotency as an important downstream target of LIF/2i.Therefore,overexpression of Tfcp211 is able to replace LIF and 2i to maintain the sternness of ESCs.In addition,upregulation of Tfcp211 is capable of reprogramming EpiSCs to naive pluripotency.However,it remains unclear that how Tfcp211 maintains mESC self-renewal.To solve this problem,we screened and identified the target gene downstream of Tfcp211 and performed a series of validations.According to previous literature report(doi:10.1126/science.1248882),estrogen-related receptor beta(Esrrb)and human salian-like gene(Sall4)may be the two downstream target genes of Tfcp211.To confirm this prediction,we overexpressed Tfcp211 in mESCs and detected the expression of Esrrb and Sall4 by real-time quantitative PCR(qRT-PCR).The results showed that overexpression of Tfcp211 only induced Esrrb transcript.Therefore,we preliminarily demonstrated that Esrrb is a downstream target gene of Tfcp211.To further validate that Esrrb is a downstream target gene of Tfcp211,we performed the following four experiments.First,we used an inducible system to induce Tfcp211 expression and then analyzed the expression of Esrrb by qRT-PCR.The results showed that the longer the doxycycline treatment time,the higher the expression of Esrrb.Second,we downregulated the expression of Tfcp211 and then detected the Esrrb transcript.The qRT-PCR results showed that the expression level of Esrrb was also decreased.Third,we analyzed the binding sites of Tfcp211 on the Esrrb promoter from the JASPAR database and performed chromatin immunoprecipitation experiments(ChIP).The results showed that Tfcp211 has binding sites on the Esrrb promoter.Finally,in order to further verify the interaction between Tfcp211 and Esrrb,we mutated the binding site and performed a luciferase reporter gene assay.The results showed that the fluorescence intensity of the wild type was 2.7 times that of the mutant.The above data indicates that Esrrb is a direct target gene of Tfcp211.Given that Esrrb is a direct target gene of Tfcp211,we thus want to investigate whether Esrrb can mediate the function Tfcp211.Therefore,we designed four experiments.First,we decreased Esrrb in Tfcp211 overexpressed mESCs,the alkaline phosphatase staining(AP)and immunofluorescence experiments showed that Tfcp211 overexpressed mESCs differentiated.Second,Tfcp211 was overexpressed in mEpiSCs and Esrrb expression was reduced.The results showed that downregulation of Esrrb could impair the ability of Tfcp211 to reprogram mEpiSCs enter naive pluripotency state.Taken together,our results demostrated that Tfcp211 maintains self-renewal by inducing expression of Esrrb in mESCs.Our findings not only enrich the regulatory network of stem cell pluripotency,but also lay a theoretical foundation for future clinical applications of stem cells.