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-related homeodomain (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.

Time-resolved analysis of Wnt-signaling reveals β-catenin temporal genomic repositioning and cell type-specific plastic or elastic chromatin responses

“…As examples, we performed two analyses that exemplify the versatility of our dataset. First, orthogonal motif analysis that considers both time- and cell-resolved data points (Figure 2E) permitted us to draw the following conclusions: i) the predominant, if not unique, response of Wnt/β-catenin to GSK3 inhibition in HEK293T is mediated by TCF/LEF transcription factors, in agreement with previous studies (Schuijers et al, 2014); ii) contributions of other transcription factors, such as the CDX homeobox proteins (Ramakrishnan et al, 2021), could fine-tune this response; iii) in hESCs, the TCF/LEF footprint is accompanied by motifs for OCT4 (Kelly et al, 2011) and those of other pluripotency factors (Cole et al, 2008), followed by a gradual appearance of GATA consensus sequences that mark mesoderm differentiation (Molkentin et al, 1997). As a second analysis, we searched for the biological processes underlying β-catenin time-course cell-specific behavior by generating a peak-to-gene(s) annotation based both on proximity and functional characterization of regulatory regions using GREAT (McLean et al, 2010), to then assign cell-specific biological processes that emerge across time after stimulation (Figure 2F, G, Figure S2).…”

“…HEK293T cells appear to modulate their response in a manner that is almost exclusively executed by the b-catenin-TCF/LEF complex, while no statistical enrichment for SOX and other pluripotency factors was observed. Of note, in HEK293T, the presence of the caudal-related homeodomain (CDX) motifs supports the identification by the Cadigan lab of a TCF/CDX module within a recently identified potent enhancer downstream of AXIN2 (Ramakrishnan et al, 2021) (Figure 1D). We propose that the association with cell-specific transcription factors will explain in part the differential distribution of b-catenin in different models.…”

“…We could consistently observe b-catenin peaks in correspondence of the AXIN2 promoter, starting already at 90 minutes after CHIR treatment and proceeding for 3 days. We confirmed the binding of b-catenin on a downstream enhancer, that we term "Cadigan enhancer", identified by(Ramakrishnan et al, 2021). We further report a hESCs-specific b-catenin binding event approximately 30 kb upstream of the AXIN2 TSS, which could indicate a putative hESCs-specific regulatory region.…”

Pathogenesis of neural tube defects: The regulation and disruption of cellular processes underlying neural tube closure

“…Furthermore, the spatial restriction of PAX3 within the dorsal NE highlights the importance of regulated proliferation, even within similarly fated cells. This restriction of PAX3 dorsally is accomplished in part by the CDX family of proteins which interact with posteriorizing WNT signals and act as inducers for Pax3 transcription, the loss of which results in a truncated anteroposterior axis, fused somites, and craniorachischisis (Ferras et al, 2012; Ramakrishnan et al, 2021; Savory et al, 2011). CDX2, which localizes with PAX3 dorsally, seems to be particularly important as it also regulates PTK7 which interacts with multiple WNT receptors to fine‐tune canonical and non‐canonical WNT signaling (Berger et al, 2017; Hayes et al, 2013).…”