Probing the canonicity of the Wnt/Wingless signaling pathway

<div><p>The hallmark of canonical Wnt signaling is the transcriptional induction of Wnt target genes by the beta-catenin/TCF complex. Several studies have proposed alternative interaction partners for beta-catenin or TCF, but the relevance of potential bifurcations in the distal Wnt pathway remains unclear. Here we study on a genome-wide scale the requirement for Armadillo (Arm, <i>Drosophila</i> beta-catenin) and Pangolin (Pan, <i>Drosophila</i> TCF) in the Wnt/Wingless(Wg)-induced transcriptional response of <i>Drosophila</i> Kc cells. Using somatic genetics, we demonstrate that both Arm and Pan are absolutely required for mediating activation and repression of target genes. Furthermore, by means of STARR-sequencing we identified Wnt/Wg-responsive enhancer elements and found that all responsive enhancers depend on Pan. Together, our results confirm the dogma of canonical Wnt/Wg signaling and argue against the existence of distal pathway branches in this system.</p></div

Ubx binds to active enhancers.

<p>(A) The bar plot shows the percentage of Vienna tiles (VTs) without or with Ubx binding sites (black and red bars, respectively) that is active at any stage of embryogenesis (left) or at the indicated embryonic stages. Hypergeometric p-value: **P<10⁻¹⁰. (B) The left panel shows the fraction of all VTs (top) and the fraction of Ubx-bound VTs (bottom) that overlaps HOT regions (dark shading). The bar plot on the right shows the percentage of active tiles for the four subsets of VTs defined on the left panel. NS–not significant. Hypergeometric p-value: **P<10⁻¹⁹.</p

Mutagenesis of the <i>pan</i> gene.

<p>(A) Schematic of the <i>pan</i> gene-locus. Untranslated regions (UTR) are indicated in grey boxes, translated exons in black. HMG box in green. (B) CRISPR targeting strategy: Target sites of both sgRNAs are represented by black triangles. The PAM site is highlighted in blue, the HMG box in green. Sequences as they are present in the pan^-/--AF1AD26 (pan^-/-) cells are depicted below. (C) Wild-type (WT) and pan^-/--AF1AD26 (pan^-/-) cells were transfected with the <i>wingful</i> luciferase reporter expression vector and Renilla expression vector 24 h prior stimulation with WCM (as control CM was used). After 24h stimulation, reporter activity was analyzed. (D) Wild-type (WT) and pan^-/--AF1AD26 (pan^-/-) cells were transfected with Pangolin overexpression vector under the control of the Actin promoter together with <i>wingful</i> luciferase reporter expression vector and Renilla expression vector 24 h prior stimulation with WCM (as control CM was used). After 24h stimulation, reporter activity was analyzed.</p

Gene expression analysis of Wg/Wnt target genes in arm^-/--AFII7/8 cells.

<p>(A) Heat map of Wnt/Wg target genes showing their log2 fold change (FC) of expression after to before WCM treatment in wild-type (WT) and arm^-/--AFII7/8 (arm^-/-) cells. The genes are listed according to the strength of the induction of their expression in WT cells. Strongest up-regulated genes are on top. Up-regulated genes are shown in red, down-regulated genes in blue, no expression in white. (B) Boxplots showing the difference in gene activity for up- and down-regulated genes after WCM stimulation in wild-type (WT) and arm^-/--AFII7/8 (arm^-/-) cells. Paired t-test: * ≤ 0.05, *** ≤ 0.0001.</p

Genomic location of Ubx binding sites and recovery of known Ubx-dependent enhancers.

<p>(A) Genomic distribution of Ubx peaks (right) in comparison to the genome (left). (B-H) UCSC Genome Browser screenshots [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161997#pone.0161997.ref083" target="_blank">83</a>] of Ubx (blue), mock (green) ChIP-seq fragment density tracks and the Ubx peak calls at known Ubx-dependent enhancers (red bars) (see the main text for references). Panel (B) also contains the fragment density tracks for the two input samples (grey). (I) UCSC Genome Browser view of the <i>hth</i> locus and examples of Ubx-bound embryonic enhancers and their activity patterns [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161997#pone.0161997.ref022" target="_blank">22</a>]. (J) UCSC Genome Browser view of the <i>Con</i> locus and Ubx-bound embryonic enhancer [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161997#pone.0161997.ref022" target="_blank">22</a>]. Vienna tiles (VT) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161997#pone.0161997.ref022" target="_blank">22</a>] are marked by slate blue boxes and their ID numbers are indicated.</p

Creation of an epitope-tagged Ubx allele by homologous recombination.

<p>(A) Design of the targeting construct (top), the integration to the endogenous Ubx locus and the location of the probe for Southern blotting (middle) and the locus after the cassette removal (bottom). (B) The eye color and haltere morphology for candidate flies before (left) and after cassette removal (white-eyed fly) (right). (C) Southern blot confirming the correct integration for two independently recombined <i>Drosophila</i> lines #11 and #12 (heterozygous for the insert). w¹¹¹⁸ flies were used as a control.</p

Identification of Wnt/Wg-responsive enhancers.

<p>(A) Schematic overview of the STARR-seq experimental setup (adapted from [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006700#pgen.1006700.ref045" target="_blank">45</a>]). (B) Cartoon representing STARR-seq peaks that were induced or repressed after activation of the Wnt/Wg pathway and the number of such peaks. (C) Distribution of induced peaks according to their fold induction. (D) UCSC genome browser screenshot of STARR-seq tracks in the <i>nkd</i> gene loci. (E) PWM logos for the Pan and Helper motif. (F) Fold induction of normalized luciferase signal for induced enhancers near <i>odd</i>, <i>how</i> and <i>lbe</i> genes. In red: wild-type sequences; in grey: sequences with mutated Pan motif. All DNA sequences are listed in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006700#pgen.1006700.s010" target="_blank">S3 Table</a>. Paired t-test: *p-value = 0.01; **p-value≤10–4. Data are shown as mean ± SD of two experiments.</p

Wnt/Wg-induced enhancers depend on Pan.

<p>(A) Scatterplots show signal fold induction at induced enhancers for STARR-seq replicates in wild-type (WT) cells (left) and for comparison of WT versus pan^-/--AF1AD26 (pan^-/-) cells (right). Grey dots indicate non-significant induction (p-value>0.001). (B) UCSC browser screenshot of STARR-seq tracks in WT and pan^-/--AF1AD26 (pan^-/-) cells for <i>nkd</i> gene locus.</p

Identification of Wnt/Wg-responsive genes.

<p>(A) Volcano plot showing the log2 fold change (x-axis) and statistical significance (y-axis; -log10 p-value) of Wnt/Wg-responsive genes. 51 genes were significantly differently expressed after WCM-treatment (red dots), 40 genes showed an up-regulation (positive targets), 11 genes were down-regulated (negative targets) after WCM treatment. Black dots represent all genes that showed an altered expression profile. (B) Heatmap showing the log2 fold change (FC) of expression of the 51 Wnt/Wg-responsive genes in wild-type cells (WT). The expression profile of positive target genes is depicted in red, the expression profile of negative targets is in blue. (C) Confirming of candidate genes using qRT-PCR. <i>Drosophila</i> Kc cells were stimulated with WCM or CM for 24 h. Analysis of expression profiles of candidate target genes (7 positive and 5 negative) after treatment versus control confirmed their induction after WCM stimulation.</p

Clustered Ubx binding sites at the genomic loci of important developmental regulators.

<p>(A, B) UCSC Genome Browser screenshots at BX-C and <i>hth</i> gene loci show ChIP-seq fragment density tracks and Ubx peak calls, revealing many clustered binding sites. (C, D) The number of Ubx peaks per 100 kb on chromosomes 3R and 2L (each 100 kb window starts at a Ubx peak, covering all possible windows that contain at least one Ubx peak). The plots for chromosomes 2R, 3L, X and 4 are in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161997#pone.0161997.s001" target="_blank">S1 Fig</a> Representative genes for all windows containing ≥25 Ubx peaks are labeled.</p