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.

SUMMARYThe canonical Wnt/-catenin signaling pathway classically functions through the activation of target genes by Tcf/Lef--catenin complexes. In contrast to -catenin-dependent functions described for Tcf1, Tcf4 and Lef1, the known embryonic functions for Tcf3 in mice, frogs and fish are consistent with -catenin-independent repressor activity. In this study, we genetically define Tcf3--catenin functions in mice by generating a Tcf3N knock-in mutation that specifically ablates Tcf3--catenin. Mouse embryos homozygous for the knock-in mutation (Tcf3 N/N ) progress through gastrulation without apparent defects, thus genetically proving that Tcf3 function during gastrulation is independent of -catenin interaction. Tcf3 N/N mice were not viable, and several post-gastrulation defects revealed the first in vivo functions of Tcf3--catenin interaction affecting limb development, vascular integrity, neural tube closure and eyelid closure. Interestingly, the etiology of defects indicated an indirect role for Tcf3--catenin in the activation of target genes. Tcf3 directly represses transcription of Lef1, which is stimulated by Wnt/-catenin activity. These genetic data indicate that Tcf3--catenin is not necessary to activate target genes directly. Instead, our findings support the existence of a regulatory circuit whereby Wnt/-catenin counteracts Tcf3 repression of Lef1, which subsequently activates target gene expression via Lef1--catenin complexes. We propose that the Tcf/Lef circuit model provides a mechanism downstream of -catenin stability for controlling the strength of Wnt signaling activity during embryonic development.

Wnt signaling is intrinsic to mouse embryonic stem cell self-renewal. Therefore it is surprising that reprogramming of somatic cells to induced pluripotent stem cells (iPSCs) is not strongly enhanced by Wnt signaling. Here, we demonstrate that active Wnt signaling inhibits the early stage of reprogramming to iPSCs, while it is required and even stimulating during the late stage. Mechanistically, this biphasic effect of Wnt signaling is accompanied by a change in the requirement of all four of its transcriptional effectors: Tcf1, Lef1, Tcf3, and Tcf4. For example, Tcf3 and Tcf4 are stimulatory early but inhibitory late in the reprogramming process. Accordingly, ectopic expression of Tcf3 early in reprogramming combined with its loss-of-function late enables efficient reprogramming in the absence of ectopic Sox2. Together, our data indicate that the step-wise process of reprogramming to iPSCs is critically dependent on the stage-specific control and action of all four Tcfs and Wnt signaling.

The Glucocorticoid Receptor (GR) alters transcriptional activity in response to hormones by interacting with chromatin at GR binding sites (GBSs) throughout the genome. Our work in human breast cancer cells identifies three classes of GBSs with distinct epigenetic characteristics and reveals that BRG1 interacts with GBSs prior to hormone exposure. The GBSs pre-occupied by BRG1 are more accessible and transcriptionally active than other GBSs. BRG1 is required for a proper and robust transcriptional hormone response and knockdown of BRG1 blocks recruitment of the pioneer factors FOXA1 and GATA3 to GBSs. Finally, GR interaction with FOXA1 and GATA3 binding sites was restricted to sites pre-bound by BRG1. These findings demonstrate that BRG1 establishes specialized chromatin environments that define multiple classes of GBS. This in turn predicts that GR and other transcriptional activators function via multiple distinct chromatin-based mechanisms to modulate the transcriptional response.