Wnt target enhancer regulation by a CDX/TCF transcription factor collective and a novel DNA motif

Abstract

Transcriptional regulation by Wnt signalling is primarily thought to be accomplished by a complex of β-catenin and TCF family transcription factors (TFs). Although numerous studies have suggested that additional TFs play roles in regulating Wnt target genes, their mechanisms of action have not been investigated in detail. We characterised a Wnt-responsive element (WRE) downstream of the Wnt target gene Axin2 and found that TCFs and Caudal type homeobox (CDX) proteins were required for its activation. Using a new separation-of-function TCF mutant, we found that WRE activity requires the formation of a TCF/CDX complex. Our systematic mutagenesis of this enhancer identified other sequences essential for activation by Wnt signalling, including several copies of a novel CAG DNA motif. Computational and experimental evidence indicates that the TCF/CDX/CAG mode of regulation is prevalent in multiple WREs. Put together, our results demonstrate the complex nature of cis- and trans- interactions required for signal-dependent enhancer activity.

SOX9 binds TCFs to mediate Wnt/β-catenin target gene activation

“…We then tested the contribution of SOX sites for WRE activation by mutating them in the c-Myc-335-luc reporter. Previously, we found that mutating the 4 TCF sites caused a reduction in enhancer activity ( 9 ). Mutating either one of the SOX sites caused a reduction in enhancer activity comparable to mutating the 4 TCF binding sites.…”

“… F) Cartoon of the c-Myc-335 enhancer showing positions of the newly identified SOX binding sites (purple) along with previously characterised TCF, CDX, and CAG sites ( 9 ).…”

“…Previous work from our group had generated a luciferase reporter driven by c-Myc-335 which showed Wnt-dependent activity ( 9 ). In LS174T cells, we found that RNAi-mediated SOX9 depletion reduced c-Myc-335 reporter activity (Fig.…”

“…To understand how SOX9 was recruited to this enhancer, we searched its sequence for potential SOX9 binding sites. Our previous work on the cis -regulatory logic of c-Myc-335 had identified 4 TCF binding sites, 2 sites bound by CDX proteins, and 5 repeated motifs we named CAG sites, all of which contributed to enhancer activity ( 9 ). Using SOX9 binding site data from the JASPAR database, we scanned the c-Myc-335 sequence for putative clusters of SOX9 binding sites.…”

“…One well-studied example is TFs of the CDX family, which bind to several WREs and recruit TCFs to them ( 7, 8 ). Their ability to form complexes with TCFs is essential for the activation of several Wnt target genes ( 9 ).…”