Understanding the Logic of TCF Target Location in Drosophila Wnt Signaling.

Abstract

The TCF/LEF family of transcription factors are major mediators of Wnt/beta-catenin signaling in metazoans. How TCFs mediate the pleiotropic effects of Wnt signaling is poorly understood. All TCFs contain a High Mobility Group (HMG) sequence specific DNA binding domain. Many TCFs also contain a second domain, the C-clamp, which binds DNA motifs called Helper sites. Both sequences are indispensable for activation of multiple W-CRMs (Wnt-responsive cis-Regulatory Elements). However, the rules concerning what constitutes a functional HMG-Helper pair were unknown. I used in vitro binding, reporter gene analysis and bioinformatics to address this question, using Drosophila TCF/Pangolin as a model. Surprisingly, I found that TCF displays remarkable functional flexibility. A Helper site near an HMG site in all four orientations can increase in vitro binding and transcriptional response; however, spacing preferences are orientation specific. In many tissues, such as the larval imaginal discs, there is a positive correlation between in vitro binding affinity and transcriptional activation levels; and altering an HMG-Helper pair from a low to high affinity configuration dramatically increases W-CRM responsiveness. However, in other tissues, this correlation is not seen, and low affinity motifs drive robust expression. Analysis of the distribution of bipartite motif configurations in TCF bound genomic regions indicates that bipartite motifs in all four orientations are enriched at a wide range of intersite spacing, but this enrichment is most pronounced for high affinity configurations. These data suggest motif configuration plays an instructive role in setting the threshold, magnitude, and tissue-specificity of W-CRM driven transcriptional responses. Computational searches for high affinity motifs identified several novel W-CRMs. All tested elements drove reporter gene expression in patterns overlapping Wg (a fly Wnt) protein expression domains in transgenic Drosophila. Novel elements on Chromosome 3R require intact Wnt signal transduction for activation, indicating they are bona-fide W-CRMs. One of these elements drives expression in the larval prothoracic gland, a tissue not previously linked to Wnt signaling. This finding highlights the importance of W-CRM discovery in understanding the many facets of Wnt/beta-catenin signaling.