The Function And Mechanism Of Tbx3 In Maintenance Of MESCs Na(?)ve State

Mouse embryonic stem cells(mESCs)are pluripotent cells derived from the inner cell mass(ICM)of blastocysts,which have delicate pluripotency regulation network.In culture condition with serum,some pluripotent genes are heterogeneously expressed,leading to the interchange between naive and primed pluripotent state in mESCs.Study on heterogeneous expression of pluripotent genes not only is important for understanding the molecular regulation of pluripotency in mESCs,but also provides theoretical basis for the study of naive human embryonic stem cells(hESCs).Tbx3 belongs to T-box family,playing crucial roles in the development of mammary gland and limbs,besides,Tbx3 can improve the quality of induced pluripotent stem cells.Recent studies demonstrate that Tbx3 is heterogeneously expressed in mESCs,but the mechanism underlying heterogeneous expression of Tbx3 requires clarification.For purifying mESCs with differential expression level of Tbx3,CRISPR/Cas9 targeting site was designed just upstream the stop codon of Tbx3 and green fluorescent protein(GFP)cassette was introduced at this site(Tbx3-C-GFP).For identifying expression pattern of Tbx3 in vivo,we generated Tbx3-2A-GFP mice,in which the self-cleaving 2A peptide was used to mediate the fusion between Tbx3 and GFP in order to protect the integrity of Tbx3 protein.Tbx3high and Tbx3low/no mESCs were purified by fluorescence activated cell sorting(FACS)of cells that did or did not express GFP.There were obvious differences between Tbx3high and Tbx3low/no mESCs.Tbx3high mESCs showed higher expression of genes associated with naive pluripotency(Klf4,Prdm14,Rex1,Esrrb),while Tbx3low/no mESCs showed higher expression of genes associated with lineage development(Fgf5,Foxa2,T,Dnmt3b).Colony-forming efficiency of single Tbx3low/no mESC was lower compared to that of single Tbx3high mESC.It is noted that the rate of proliferation of Tbx3low/no mESCs was reduced compared to that of Tbx3high mESCs.Besides,Tbx3high mESCs had a shorter G0/G1 phase and showed higher expression of key genes for G1-S transition(Cdk2,Ccnd3).In treatment with retinoic acid or removal of LIF,Tbx3low/no mESCs were more sensitive to signal of differentiation.When introduced back into blastocyst,Tbx3high mESCs had high potency of contributing to embryonic tissues in chimeras,in contrast,Tbx3low/no mESCs showed poor ability.Morever,Tbx3high and Tbx3low/no mESCs had the ability to convert into each other’s state.These results demonstrated that Tbx3high mESCs resembled ICM-like cells,while Tbx3low/no mESCs resembled primitive ectoderm-like cells.It suggested that Tbx3 was a specific naive pluripotent marker of mESCs.To determine the mechanism underlying the role of Tbx3 playing in the maintenance of naive pluripotency in mESCs,we compared the global transcription profiles of Tbx3high mESCs to those of Tbx3low/no mESCs by RNA sequencing analysis.The population of upregulated genes in Tbx3high mESCs was enriched with genes associated with maintenance of pluripotency in mESCs.On the other hand,the population of upregulated genes in Tbx3low/no mESCs was enriched with genes associated with lineage commitment.For identifying genes directly regulated by Tbx3 in the maintenance of naive pluripotent state in mESCs,we reanalyzed the published ChIP-seq(Chromatin immunoprecipitation followed by sequencing)data of Tbx3 and found that Tbx3 could directly activate expression of Suz12,which is required for polycomb repressive complex 2(PRC2)enzymatic activity.Loss of Suzl2 could lead to an upregulation of genes associated with early lineage commitment,such as Fgf5,Fgf15 and Otx2.Further experiments indicated that Tbx3 suppressed phosphorylation level of Smad2 by regulating the expression of Nodal.The increase of phosphorylation level of Smad2 was the main reason for higher expression of genes associated with mesendoderm development in Tbx3low/no mESCs.In addition,inhibition of TGF-β signaling pathway could facilitate the maintenance of pluripotency in mESCs.In summary,our results demonstrated that Tbx3 is a specific naive marker of mESCs and safeguards naive pluripotency by a dual mechanism.On the one hand,Tbx3 suppressed expression of genes associated with early lineage commitment by activating expression of Suz12.On the other hand,Tbx3 suppressed expression of genes associated with mesendoderm development by regulation of TGF-β signaling pathway.