A β-catenin/XTcf-3 complex binds to thesiamois promoter to regulate dorsal axis specification in Xenopus

Abstract

The Wnt pathway regulates the early dorsal-ventral axis in Xenopus through a complex of ␤-catenin and HMG box transcription factors of the Lef/Tcf family. We show that the promoter of the dorsalizing homeo box gene siamois is a direct target for the ␤-catenin/XTcf-3 complex, establishing a link between the Wnt pathway and the activation of genes involved in specifying the dorsal axis. By injecting siamois reporter constructs into the animal pole of Xenopus embryos, we show that a 0.8-kb fragment of the siamois promoter is strongly activated by ␤-catenin. The proximal 0.5 kb, which is also activated by ␤-catenin, contains three Lef/Tcf-binding sites. Mutations in these sites eliminate the ␤-catenin-mediated activation of siamois and show that siamois is regulated by the ␤-catenin/XTcf-3 complex, in combination with additional transcriptional activators. When expressed at the equator of the embryo, the siamois promoter is activated to much higher levels on the dorsal side than the ventral side. Ectopic ventral expression of ␤-catenin raises the ventral expression of the siamois promoter to the dorsal levels. Conversely, ectopic dorsal expression of dominant-negative XTcf-3 abolishes the dorsal activation of the siamois promoter. Furthermore, elimination of the Lef/Tcf sites elevates the ventral expression of siamois, revealing a repressive role for XTcf-3 in the absence of ␤-catenin. Finally, we find that the endogenous siamois activator, although present throughout the dorsal side of the embryo, is most potent in the dorsal vegetal region. We propose that the dorsal activation of siamois by the ␤-catenin/XTcf-3 complex combined with the ventral repression of siamois by XTcf-3 results in the restriction of endogenous siamois expression to the dorsal side of Xenopus embryos.

PAWS 1 controls Wnt signalling through association with casein kinase 1α

“…Levels of endogenous and active β‐catenin also increased following injection of xPAWS1 mRNA into naïve animal caps (Fig 3B and D). Finally, animal caps expressing xPAWS1 activated the direct Wnt target Siamois 20, 21, 22 and the dorsalising factor Chordin 18, confirming that PAWS1 can activate the canonical Wnt pathway (Fig 3F). We mapped the domain of PAWS1 that is required for Wnt signalling by testing the ability of different PAWS1 fragments to induce secondary axes in Xenopus and to activate Siamois expression in animal caps (Fig 3G–I).…”

MicroRNA-199a is induced in dystrophic muscle and affects WNT signaling, cell proliferation, and myogenic differentiation

“…The TOPFLASH (8xTCF/LEF-binding sites) and FOPFLASH (8xTCF/LEF sites mutated) have been used in WNT reporter assays to characterize WNT signaling levels in various cell lines. 37,38 To further validate miR-199a-5p as a regulator of WNT signaling, we overexpressed miR-199a-5p along with the TOPFLASH or the control FOPFLASH luciferase reporters in either HEK293T cells or normal human primary MB. Overexpression of miR-199a-5p inhibited the TOPFLASH (8xTCF/LEF-binding sites) but not the FOPFLASH reporter in both HEK293T cells and human primary MB (Figures 7d and e).…”

Wnt signaling in disease and in development

“…This is regulated by the β-catenin interactions with the TCF/LEF DNA binding proteins. In the absence of the Wnt signal, TCF acts as a repressor of Wnt/Wg target genes [23], by forming a complex with Groucho [24]. The repressing effect of Groucho is mediated by interactions with Histone Deacetylases (HDAC), which are thought to make DNA refractive to transcriptional activation [25].…”