Distinct DNA binding sites contribute to the TCF transcriptional switch in C. elegans and Drosophila

Regulation of gene expression by signaling pathways often occurs through a transcriptional switch, where the transcription factor responsible for signal-dependent gene activation represses the same targets in the absence of signaling. T-cell factors (TCFs) are transcription factors in the Wnt/ß-catenin pathway, which control numerous cell fate specification events in metazoans. The TCF transcriptional switch is mediated by many co-regulators that contribute to repression or activation of Wnt target genes. It is typically assumed that DNA recognition by TCFs is important for target gene location, but plays no role in the actual switch. TCF/Pangolin (the fly TCF) and some vertebrate TCF isoforms bind DNA through two distinct domains, a High Mobility Group (HMG) domain and a C-clamp, which recognize DNA motifs known as HMG and Helper sites, respectively. Here, we demonstrate that POP-1 (the C. elegans TCF) also activates target genes through HMG and Helper site interactions. Helper sites enhanced the ability of a synthetic enhancer to detect Wnt/ß-catenin signaling in several tissues and revealed an unsuspected role for POP-1 in regulating the C. elegans defecation cycle. Searching for HMG-Helper site clusters allowed the identification of a new POP-1 target gene active in the head muscles and gut. While Helper sites and the C-clamp are essential for activation of worm and fly Wnt targets, they are dispensable for TCF-dependent repression of targets in the absence of Wnt signaling. These data suggest that a fundamental change in TCF-DNA binding contributes to the transcriptional switch that occurs upon Wnt stimulation

Schematics of the <i>ceh-22</i>, <i>psa-3</i> and <i>end-1</i> loci.

<p>For each locus, black boxes represent exons and gray boxes untranslated regions (UTRs). Start codons representing the Translation Start Site (TlSS) for each isoform are marked by ‘M’. White boxes represent the genomic region used to construct the WRE reporters and the green box the GFP variant used. The larger white boxes in the WRE reporter show the location of the HMG (red lines) and Helper sites (blue lines). Below each schematic are the genomic sequences highlighting the putative Helper sites (blue) and functional HMG sites (red) that were targeted for mutagenesis. (A) For the <i>ceh-22</i> gene (Gene ID: 179485), a transcriptional fusion of the <i>ceh-22b</i> isoform called <i>ceh-22b::VENUS </i><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004133#pgen.1004133-Lam1" target="_blank">[42]</a> was used for reporter analysis (nucleotides −1853 to −633 with the first nucleotide of the <i>ceh-22b</i> TlSS representing +1). (B) For <i>psa-3</i> (Gene ID: 181631), a translational fusion (<i>psa-3::GFP</i>) including promoter sequences (starting at -382) and the first exons of the a, b & c isoforms was used, where the <i>pqn-36</i> gene, located in the third intron was deleted, as indicated by the parentheses <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004133#pgen.1004133-Arata1" target="_blank">[43]</a>. (C) For <i>end-1</i> (Gene ID: 179893), a translational fusion containing ∼2.2 kb of promoter sequence, known as <i>end-1::GFP::H2B</i> was used <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004133#pgen.1004133-Shetty1" target="_blank">[16]</a>.</p

Helper sites and the C-clamp are not required for basal repression of Wg targets in <i>Drosophila</i>.

<p>(A–I) Confocal images of stage 16–17 embryos containing a <i>pxb::lacZ</i> WRE reporter immunostained for Wg (green) (A, D & G), lacZ (red) (B, E & H) or merged (C, F & I). The wild-type reporter shows a pattern overlapping with Wg in the second constriction of the midgut, and a non-overlapping pattern in the hindgut (A–C). Mutation of two HMG sites leads to a strong depression through the entire midgut (arrowheads), without affecting lacZ expression in the second constriction (arrow) (D–F). Mutation of two Helper sites leads to a significant decrease in the lacZ expression in the second constriction (arrow) with weak ectopic expression (arrowheads)(G–I). The hindgut expression did not vary in the different constructs and was used as an internal control. All images are representative of at least 20 embryos. (J–M) Images of adult wings containing the wing driver <i>C96-Gal4</i> crossed to wildtype (WT) (J, J′), UAS-TCF/Pan RNAi (K, K′) or UAS-TCF/Pan RNAi plus UAS-LEF1 (L, L′) or UAS-LEF1 plus the C-clamp of TCF/Pan (M, M′). Knockdown of TCF/Pan leads to notches (arrowheads) and ectopic wing margin bristles (block arrows) along the periphery of the wing (where <i>C96-Gal4</i> is active; K, K′). Expression of the human LEF1 transgene significantly rescues the ectopic bristle expression, but not the notches (L, L′). Expression of a LEF1-C-clamp chimera rescues the wing margin defects and prevents ectopic bristle formation, and causes a L5 vein defect (arrow). Details about the penetrance of these phenotypes are listed in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004133#pgen-1004133-t001" target="_blank">Table 1</a>.</p

The C-clamp is required for Wg activation but not basal repression in a TCF/Pan rescue assay.

<p>Two independent lines of UAS-Lef1 and UAS-Lef1-C-clamp with similar expression levels (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004133#pgen.1004133.s005" target="_blank">Figure S5B</a>) were assayed. Expression of either transgene with the <i>C96-Gal4</i> driver had little or no effect on wing development in an otherwise wild-type background. Percentages tabulated for the wing phenotypes seen upon knock down of TCF. Depletion of TCF/Pan with a UAS-driven RNAi hairpin causes mostly large notches, and leads to more than 20 ectopic bristles per wing and a high penetrance of L5 vein defects. Expression of human Lef1 (Lef1) significantly rescues the ectopic bristles, but has little effect on the size and frequency of the wing notches. In contrast, expression of Lef1 with the C-clamp of TCF/Pan (Lef1-C-clamp) rescues both ectopic bristles and the wing notch phenotype. (n) represents the number of wings examined for each genotype. Depletion of TCF/Pan and expression of Lef1 and Lef1-C-clamp also resulted in a disruption of the L5 vein (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004133#pgen-1004133-g007" target="_blank">Figure 7M</a> and data not shown). Since this phenotype has not been linked to Wg signaling, it is not considered further in this report.</p

Reduction of <i>pop-1</i> gene activity results in a prolonged defecation cycle.

<p>Eight pBocs and expulsions were observed for each individual, with the N2 and pop-1 mutants assayed at the L2 larval stage, and the RNAi fed individuals assayed as young adults.</p>*<p>P<0.05;</p>**<p>P<0.01.</p

Identification of a new POP-1 target using a computational search for Helper site-HMG site clusters.

<p>(A) Schematic depicting the <i>K08D12.3</i> locus (Gene ID: 176979) with black boxes representing exons and the gray box the flanking gene <i>pbs-1</i>. The start codon is marked by ‘M’. The white box indicates the genomic region used to construct the GFP transcriptional reporter (nucleotides −579 to +14; first nucleotide of TlSS represents +1), with the asterisk indicating where the <i>K08D12.3</i> start codon was mutated to allow GFP to be read in the correct frame. The location of the HMG and Helper sites are indicated in red and blue respectively. Fluorescence (B–D; B′–D′) and Brightfield (B″–D″) images of live late L4 larvae extrachromosomally expressing the <i>K08D12.3::VENUS</i> reporter. Strong expression was seen in the head muscles, pharyngeal muscles, posterior intestine and hindgut (arrowheads) and moderate expression in the midgut (arrows). (B) Wildtype, (C) HMG mutant and (D) Helper mutant worms were scored based on the VENUS expression in the head muscles, pharyngeal muscles and intestine. (E) Histogram showing the expression analysis of late L4 larvae from three independent lines carrying either the WT, HMG mutant or Helper mutant <i>K08D12.3::VENUS</i> reporters, grouped into strong, intermediate or weak expressers, represented by the images in panels B, C & D, respectively. (F) Competition analysis using EMSA with POP-1 protein with a 90 bp probe (sequence shown in panel A) containing the three functional Helper sites and the functional HMG site from the <i>K08D12.3</i> WRE. The POP-1 dependent shift (lane 2) is competed by an excess of unlabeled WT probe (lanes 3, 4), while unlabeled HMG mutant probe (lanes 5, 6) or the Helper3 mutant probe (lanes 7, 8) does not compete even at 200 fold excess competitor levels. Unlabeled Helper1 mutant (lanes 9, 10) and Helper2 mutant (lanes 11, 12) probes displayed a moderate level of competition. The black arrowhead represents the DNA-protein complex and the white arrowhead represents unbound probe. The data is representative of three independent experiments.</p