Structure-function analysis of the C-clamp of TCF/Pangolin in Wnt/ß-catenin signaling.

The evolutionarily conserved Wnt/ß-catenin (Wnt/ß-cat) pathway plays an important role in animal development in metazoans. Many Wnt targets are regulated by members of the TCF/LEF1 (TCF) family of transcription factors. All TCFs contain a High Mobility Group (HMG) domain that bind specific DNA sequences. Invertebrate TCFs and some vertebrate TCF isoforms also contain another domain, called the C-clamp, which allows TCFs to recognize an additional DNA motif known as the Helper site. While the C-clamp has been shown to be important for regulating several Wnt reporter genes in cell culture, its physiological role in regulating Wnt targets is less clear. In addition, little is known about this domain, except that two of the four conserved cysteines are functionally important. Here, we carried out a systematic mutagenesis and functional analysis of the C-clamp from the Drosophila TCF/Pangolin (TCF/Pan) protein. We found that the C-clamp is a zinc-binding domain that is sufficient for binding to the Helper site. In addition to this DNA-binding activity, the C-clamp also inhibits the HMG domain from binding its cognate DNA site. Point mutations were identified that specifically affected DNA-binding or reduced the inhibitory effect. These mutants were characterized in TCF/Pan rescue assays. The specific DNA-binding activity of the C-clamp was essential for TCF/Pan function in cell culture and in patterning the embryonic epidermis of Drosophila, demonstrating the importance of this C-clamp activity in regulating Wnt target gene expression. In contrast, the inhibitory mutation had a subtle effect in cell culture and no effect on TCF/Pan activity in embryos. These results provide important information about the functional domains of the C-clamp, and highlight its importance for Wnt/ß-cat signaling in Drosophila

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

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

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

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

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

The C-clamp of POP-1 facilitates binding to DNA containing Helper sites.

<p>(A–D) EMSAs showing binding of wild-type recombinant POP-1 and a POP-1 C-clamp mutant to the <i>ceh-22b</i> WRE probe (1.5 femtomoles/reaction) described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004133#pgen-1004133-g001" target="_blank">Figure 1A</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004133#pgen-1004133-g002" target="_blank">2N</a> (A), the <i>psa-</i>3 probe (3 femtomoles/reaction) described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004133#pgen-1004133-g001" target="_blank">Figure 1B</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004133#pgen.1004133.s001" target="_blank">S1B</a> (B), the <i>K08D12.3</i> probe (4 femtomoles/reaction) described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004133#pgen-1004133-g003" target="_blank">Figure 3A and 3F</a> (C) and the <i>ceh-22b</i> probe (3 femtomoles/reaction) with both Helper sites mutated (D). The <i>ceh-22b</i>, <i>psa-3</i> and <i>K08D12.3</i> WT probes show strong binding with increasing amounts (0.4 and 0.8 µg/reaction) of POP-1 WT protein (lanes 2 and lane 3 respectively) but not with the POP-1 C-clamp mutant (lane 4 and lane 5 respectively). Under conditions designed to detect lower affinity binding (0.75 and 1.5 µg of POP-1; 3 femtomoles of probe and longer exposure times), binding to the <i>ceh-22b</i> probe lacking Helper sites (containing only the HMG2 site) was similar with WT and mutant POP-1. The data are representative of three independent experiments.</p