| Mammalian development begins with the combination of sperm and egg,both of which form fertilized eggs and then start the developmental process.After that,the fertilized egg undergoes a series of cleavages,the number of cells increases,and the differentiation of cell function forms.In 1981,mouse embryonic stem cells(mESCs)were successfully derived from pre-implanted mouse embryo blastocysts by Evans and Kaufinan,and the earliest embryonic stem cell lines were established.After that,people gradually isolated the embryonic stem cells from human,rat and et al.Since embryonic stem cells have the biological characteristics of self-renewal and multi-directional differentiation,they have very important research value both in biological development and in clinical application.After a great deal of work by the predecessors,people as far have found ways to maintain the state of self-renewal of some ES cells in vitro in the long term.However,since embryonic stem cells maintaining self-renewal is an extremely complicated biological process,it is of great significance to study the regulation mechanism of embryonic stem cells maintaining self-renewal and differentiation in an all-round way,which is beneficial to its extensive and safe application.Currently there are two main culture medium for embryonic stem cells in vitro:one is LIF/serum medium and the other is serum-free medium N2B27 added two small molecule inhibitors(2i)CHIR and PD03.Different medium maintain the self-renewal of embryonic stem cells through different signal pathways.LIF/serum medium is mainly through the LIF/STAT3 signal pathway.Our previous study found that Tfcp211,the only target gene in many LIF/STAT3 signaling pathways,was the only gene that resulted in the differentiation of mouse embryonic stem cells under LIF/serum culture.This shows that Tfcp211 occupies a very important position in the process of self-renewal of mESCs by LIF/STAT3 signaling pathway.And later it was found that over-expression of both Tfcp211 and klf2 could replace 2i to maintain self-renewal of mouse embryonic stem cells.Tfcp211 plays a very important role as a common target gene of many pathways.However,it is not very clear how Tfcp211 regulate self-renewal of mESCs.To investigate the specific mechanism by which Tfcp211 regulates self-renewal in mESCs,we firstly down-regulated the expression of Tfcp211 in cells and examined the gene expression of each germ layer to see which germ layers differentiate.The results showed that endoderm,mesoderm and trophoblast genes were significantly increased.To validate this result from the reverse direction,we performed Embryoid body(EB)differentiation using the i-Tfcp211 cell line in which the expression of Tfcp211 is inducibly increased when the Doxcycline was added.The results showed that overexpression of Tfcp211 during EB formation inhibited the differentiation of mESCs towards endoderm,mesoderm and trophectoderm.Overal,these data indicate that Tfcp211 is able to block the formation of endoderm,mesoderm and trophectoderm in mESCs.The structure of TFCP2L1 protein is the basis of its function,so we experimentally analyzed the role of the different domains of TFCP2L1 protein in maintaining embryonic stem cell self-renewal.First,we divided the TFCP2L1 protein into four domains:N-terminus(aa 1-20),CP2-like domain(aa 21-242),SAM domain(aa 310-376)and C-terminus(aa 377-480).Then we constructed four mESCs cell lines overexpressed with these deletion mutants(?NT??CP2?? SAM??CT).We obseved that ? NT and ?CP2 could not maintain mESC self-renewal without LIF.Moerover,ANT failed to reprogramm of mEpiSCs into the naive state,and the reprograming-efficiency of ACP2 is extremely low.The above results demonstrate that the N-terminus and CP2-like domains are important for Tfcp211 to sustain and induce naive pluripotency.In the process of cultivating mutant cell lines,we noted that the ANT and ACP2 cell lines differentiated under normal LIF/serum culture condition.qRT-PCR detection revealed that the ACP2 cell line had higher expression of endoderm genes(Gata4 and Gata6),while the ANT cell lines expressed higher mesoderm(T and Mixl)and trophoblast genes(Cdx2 and Gata3),when compared with the empty vector transfected cells.To get insight into howTfcp211 inhibits the differentiation of mesoderm and trophectoderm.After a series of experiments,we revealed that the differentiation of ANT cell lines towards mesoderm and trophectoderm is due to the upregulation of Lefl expression.Thus,Tfcp211 represses mesoderm and trophoblast genes mainly through suppression of Lefl transcript.Finally,we want to explore the mechanism by which Tfcp211 regulates the endoderm?We found that MTA family genes might associated with this event.However,only down-regulation of MTAl,but not other MTA family members,led to the differentiation of mESCs overexpressed Tfcp211 toward endoderm.On the contrary,overexpression of MTA 1 is able to delay mESC differentiation.The Co-IP experiments showed that there is a protein interaction between them.Together,we can conclude that TFCP2L1 inhibits the expression of endoderm genes through interaction with MTA1 protein. |
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