Opposing effects of Tcf3 and Tcf1 control Wnt stimulation of embryonic stem cell self-renewal

Abstract

The observation that Tcf3 (MGI name: Tcf7l1) bound the same genes as core stem cell transcription factors, Oct4 (MGI name:Pou5f1), Sox2 and Nanog, revealed a potentially important aspect of the poorly understood mechanism whereby Wnts stimulate self renewal of pluripotent mouse embryonic stem (ES) cells. Although the conventional view of Tcf proteins as the β-catenin-binding effectors of Wnt signaling suggested Tcf3-β-catenin mediated activation of target genes would stimulate ES cell self renewal, here we show that an antagonistic relationship between Wnt3a and Tcf3 on gene expression is important for regulating ES cell self renewal. Genetic ablation of Tcf3 replaced the requirement for exogenous Wnt3a or GSK3-inhibition for self renewal of ES cells, demonstrating that inhibition of Tcf3-repressor is the necessary downstream effect of Wnt signaling. Interestingly, the molecular mechanism underlying Wnt’s effects required both Tcf3-β-catenin and Tcf1-β-catenin interactions, as they each contributed to Wnt stimulation of self renewal and gene expression. Finally, the combination of Tcf3 and Tcf1 was necessary to recruit Wnt-stabilized β-catenin to Oct4 binding sites in ES cell chromatin. These results elucidate the molecular link between the effects of Wnt and the regulation of the Oct4/Sox2/Nanog network.

Wnt Pathway Regulation of Embryonic Stem Cell Self-Renewal

“…This model is directly supported by several recent observations: Overexpression of Tcf3 inhibited stem cell gene expression (Nishiyama et al 2009) and stimulated rapid differentiation of mESCs (Yi et al 2011). Treating Tcf3-overexpressing mESCs with Wnt3a effectively blocked differentiation and restored self-renewal (Yi et al 2011).…”

“…This model is directly supported by several recent observations: Overexpression of Tcf3 inhibited stem cell gene expression (Nishiyama et al 2009) and stimulated rapid differentiation of mESCs (Yi et al 2011). Treating Tcf3-overexpressing mESCs with Wnt3a effectively blocked differentiation and restored self-renewal (Yi et al 2011). Tcf3 2/2 mESCs self-renewed in serum-free conditions without the addition of LIF or a GSK3 inhibitor, that is, in the presence of ERK inhibitor alone (Wray et al 2011;Yi et al 2011), indicating that ablation of Tcf3 effectively replaced the GSK3 inhibitor.…”

“…Transcriptional profiling of Tcf3 2/2 ESCs showed that Tcf3 ablation caused strongly opposite effects on gene expression compared with Oct4 knockdown and Nanog knockdown (Loh et al 2006;Yi et al 2008). It is notable that the effect on Oct4-regulated genes was measured in Tcf3 2/2 cells, which did not display elevated Oct4 protein levels (Pereira et al 2006;Yi et al 2008), and that knockdown of Nanog in Tcf3 2/2 cells did not reduce the levels of stem cell genes examined (Yi et al 2011). Thus, the effects of Tcf3 ablation went beyond just Nanog and Oct4 stimulation and appeared to directly affect the genes co-occupied by Tcf3/Oct4/Sox2/Nanog.…”

“…Treating Tcf3-overexpressing mESCs with Wnt3a effectively blocked differentiation and restored self-renewal (Yi et al 2011). Tcf3 2/2 mESCs self-renewed in serum-free conditions without the addition of LIF or a GSK3 inhibitor, that is, in the presence of ERK inhibitor alone (Wray et al 2011;Yi et al 2011), indicating that ablation of Tcf3 effectively replaced the GSK3 inhibitor. Thus, the pro-selfrenewal effects of Wnt/b-catenin signaling in mESCs appear to function by inhibiting Tcf3-repressor activity.…”

Modeling heterogeneity in the pluripotent state: A promising strategy for improving the efficiency and fidelity of stem cell differentiation

“…LIF cause the activation of the LIF/JAK/Stat3 signaling pathway, through the activation of some Janus tyrosine kinases (JAK), triggering the phosphorylation of Stat3 and the activation of the expression of several genes required for mESCs and hESCs self‐renewal 46, 47, 48, 49. The combination of inhibitors in 2i and 3i culture mediums, which act on some specific genes from the Wnt/β‐catenin signaling pathway, promote pluripotency in mESCs through the stabilization of β‐catenin that override the inhibitory effect of Tcf3 on some important pluripotency regulators, such as Oct4 and Nanog 50, 51. The TGF‐β/SMAD signaling pathway maintains self‐renewal in mESC through the BMP/SMAD signalling activation of Id family genes 52, while in hESCs is the Activin/Nodal/SMAD2/3 cascade the one responsible for promoting pluripotency 53.…”