Two-Element Transcriptional Regulation in the Canonical Wnt Pathway

The canonical Wnt pathway regulates numerous fundamental processes throughout development and adult physiology and is often disrupted in diseases [1-4]. Signal in the pathway is transduced by β-catenin, which in complex with Tcf/Lef regulates transcription. Despite the many processes that the Wnt pathway governs, β-catenin acts primarily on a single cis element in the DNA, the Wnt-responsive element (WRE), at times potentiated by a nearby Helper site. In this study, working with Xenopus, mouse, and human systems, we identified a cis element, distinct from WRE, upon which β-catenin and Tcf act. The element is 11 bp long, hundreds of bases apart from the WRE, and exhibits a suppressive effect. In Xenopus patterning, loss of the 11-bp negative regulatory elements (11-bp NREs) broadened dorsal expression of siamois. In mouse embryonic stem cells, genomic deletion of the 11-bp NREs in the promoter elevated Brachyury expression. This reveals a previously unappreciated mechanism within the Wnt pathway, where gene response is not only driven by WREs but also tuned by 11-bp NREs. Using electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP), we found evidence for the NREs binding to β-catenin and Tcf-suggesting a dual action by β-catenin as a signal and a feedforward sensor. Analyzing β-catenin ChIP sequencing in human cells, we found the 11-bp NREs co-localizing with the WRE in 45%-71% of the peaks, suggesting a widespread role for the mechanism. This study presents an example of a more complex cis regulation by a signaling pathway, where a signal is processed through two distinct cis elements in a gene circuitry.