Molecular Mechanism Of IWR1 In Maintaining The Self-renewal Of Human Embryonic Stem Cells

Human embryonic stem cells(hESCs)can self-renew and maintain pluripotency in vitro.They are grown on inactivated mouse fibroblasts(ie,feeder layers)supplemented with serum substitutes(Knockout Serum Replacement,KSR)and various cytokines(such as ActivinA and bFGF).hESCs grow slowly and can not tolerate single cell digestion.Although commercially available Matrigel can partially replace feeder cells,hESCs cultured on Matrigel easy to differentiate and apoptosis.Besides,Matrigel is expensive.Therefore,the new methods for culturing hESCs have become one of the hot research directions of regenerative medicine.In order to improve the current status of hESCs culture,our previous study found a small molecule inhibitor of Wnt/?-catenin signaling pathway,IWR1,which is an inhibitor of Tankyrases.The function of Tankyrases is mainly to promote the degradation of Axin protein.Axin protein is the main component of ?-catenin degradation complex.Therefore,small IWR1-treated cells can stabilize Axin protein to degrade ?-catenin protein.?-catenin-mediated signaling has been extensively studied in regard to its role in the regulation of hESCs.It has been reported that inhibition of Wnt/?-catenin signaling pathway can promote self-renewal of hESCs,but the specific mechanism of action is still unclear.?-catenin enters the nucleus mainly interacts with the TCF famil to induce the expression of target,we thus want to make sure which and how TCF mediates this event,and these results will get the molecular mechanism by which IWR1 maintains self-renewal of hESCs.This topic is mainly divided into the following parts:First,previous literature reports that overexpression of ?-catenin(Ctnnb1)induces rapid hESCs differentiation.To confirm the function of Ctnnbl in hESCs,a Flag-tagged Ctnnbl,containing an activating mutation at codon 33(S33Y,a serine-totyrosine missense mutation)was overexpressed in hESCs using a PiggyBac vector(PB-Ctnnbl)in which Ctnnbl expression was efficiently enhanced.PB-Ctnnbl cells differentiated and exhibited high levels of differentiation associated genes Cxcr4,FoxA2 and Mixl1 after 6 days,while empty vector(PB)hESCs retained typical hESCs morphology.positive alkaline phosphatase activity,and high expression levels of the pluripotency markers Oct4,TRA1-81 and Nanog.These results suggest that overexpression of Ctnnbl induces rapid hESCs differentiation;Second,to examine whether Ctnnbl induces hESCs differentiation via its transcriptional activity,we generated a series of deletion mutants lacking specific domains based on the CtnnbIs33Y gene.The Ctnnb1S33Y/?N48-217:lacking the N-terminal amino acids 48 to 217,Ctnnb1S33Y/?N218-467:lacking the amino acids at the N-terminus 218 to 467,Ctnnb1S33Y/?C468:lacking the C-terminal amino acids 468 to 781,and Ctnnb1S33Y/?C694:Ctnnb1 protein deleted C-terminal amino acids 694 to 781,were Flag tagged and were overexpressed in HES2 hESCs.After 2 passages in normal hESCs Culture media,the Ctnnb1S33Y and Ctnnb1S33Y/?N48-217 induced complete hESCs differentiation.Most hESCs still maintained pluripotency in PB and Ctnnb1S33Y/?N48-217 cells.The results of TOPFlash reporter activity showed that Ctnnb1S33Y and Ctnnb1S33Y/?N48-217 strongly activate TCF-dependent transcription,but the effect of the S33Y mutation on TCF transcription was largely abrogated by deletion of certain Ctnnbl domains,such as the C terminus(S33Y/AC468 and C694)or armadillo repeats 3-8(S33Y/?N218-467).Our data are thus consistent with a previous model in which Ctnnbl binds to TCF through armadillo repeats 3-8 and activates transcription via its C-terminus and also indicate that Ctnnbl induces hESCs differentiation mainly by interacting with LEF1/TCF family members;Third,LEF1/TCF family transcription factors contain four paralogues(LEF1,TCF1,TCF3 and TCF4)and are all able to interact with Ctnnb1.To determine which TCF interacts with Ctnnbl to induce hESCs differentiation,we overexpressed each Flag-tagged TCF member in hESCs and found that only elevated Tcfl expression led to hESCs differentiation.Tcfl-overexpressing hESCs became flat,lost AP activity and expressed lower levels of the pluripotency gene Oct4 and Nanog compared with PB control cells,while showing enhanced expression of differentiation genes,such as definitive endoderm(Gata4 and Gata6),mesoderm(T and Mixll)and ectoderm(Fgf5)markers.Consistent with this,immunostaining revealed that the majority of Tcfl transgenic cells were positive for Gata6.These results indicate that Ctnnbl induces hESCs differentiation mainly by interacting with Tccf1;Fina1,to further define the mechanism by which Tcfl induces hESCs differentiation,we performed RNA-sequence to assess the gene expression pattern of hESCs expressing PB vector or PB-TCF1.We found that Gata6 was significantly induced.RNA interference was then used to downregulate the expression levels of Gata6 in TCF1 overexpressed hESCs.Strikingly,we found that knockdown of Gata6 efficiently eliminated the differentiation-induced effect of Tcfl.These cells expressed higher levels of Oct4 and Nanog,but lower levels of Gata6,Gata4 and FoxA2,compared with scramble control cells transfected with a Tcfl transgene after two passages.The results indicated that Gata6 is the major downstream target of TCF1 in inducing hESCs differentiation.In summary,through the reverse thinking,we conculd that IWR1 promote hESCs self-renewal mainly by blocking the interaction between ?-catenin and TCF1,and further leading to the low expression level of differnetation genes,such as gata6.These results expands our understanding of human embryonic stem cells.