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

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

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 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

POP-1 consensus HMG and Helper sites and models for the TCF transcriptional switch.

<p>(A) Genomic sequences of the functional HMG and Helper sites, with the box indicating a HMG-Helper site pair with similar orientation in each WRE. (B) Sequence logos showing the consensus of HMG and Helper sites, based on the functional sites used in this study. (C, D) Model to explain the differential requirement of HMG and Helper sites in the TCF transcriptional switch, without (C) and with (D) Wnt/ß-catenin signaling . We propose that the DNA binding properties of TCF are influenced by co-regulators, with ß-catenin stabilizing the HMG-Helper site interaction. It is suggested that POP-1 may recognize HMG sites surrounded by two Helper sites as a dimer. This model does not preclude the existence of addition DNA-binding co-factors for POP-1 in either the absence or presence of signaling.</p

HMG and Helper sites contribute differentially to the regulation of <i>end-1</i> during early embryogenesis.

<p>Deconvolved (A–C) and Nomarski (A′–C′) images showing expression of a stably integrated <i>end-1::GFP::H2B</i> reporter in the endodermal (E) and/or mesodermal (MS) daughters of live embryos at the 2E stage. The wild type (WT) reporter shows strong GFP expression in the E cell daughters (A, A′). Mutation of the HMG site leads to a significant reduction of GFP expression in the E daughters and a significant derepression of <i>end-1::GFP::H2B</i> in the MS daughters (B, B′). Mutation of two Helper sites leads to a significant reduction of GFP in the E daughters (C, C′), but little or no depression in the MS daughters (C, C′). (D) Histograms summarizing the results from over 100 embryos from three independent lines for each construct, grouped by strong, weak or no expression in the E (upper graph) and MS (lower graph) cells.</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

A synthetic HMG-Helper site reporter reveals a novel POP-1 function in rhythmic defecation behaviour.

<p>(A–F) Nomarski images of animals with stably integrated <i>POPHHOP</i> (<i>6× HMG-Helper::GFP</i>) and <i>POPTOP</i> (<i>7× HMG::mCherry</i>) reporters showing GFP (A, D) and mCherry (B, E) fluorescence. Live L1 larvae have overlapping expression of GFP and mCherry in some tail neurons (A–C) and live L3 larvae display overlapping DTC expression (D–F). In addition, POPHHOP displayed strong expression in the int9 intestinal cells of early L1 Larvae (A) onward through adulthood (not shown). (G–H) Stably integrated <i>POPHHOP</i> animals in a wild-type (G) or <i>pop-1(hu9)</i> background (H). The reporter expression seen in the int9 cells, tail neurons, and occasionally in the VC neurons is low or undetectable in the <i>pop-1</i> mutants. Scale bars = 10 µm. (I) Box-whisker plot showing the median (line inside the box), third quartile (upper box), first quartile (lower box), longest pBoc cycle time (upper whisker limit) and shortest pBoc cycle time (lower whisker limit) for N2 controls and two <i>pop-1</i> alleles at the L2 stage. A statistically significant increase was seen in the pBoc cycle time based on a Student's two-tailed <i>t</i> test (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004133#pgen-1004133-t001" target="_blank">Table 1</a>). (J) 8 individual pBocs (X-axis) were monitored (n = 26, each color representing one larva) for each genotype and plotted against time between each pBoc (y-axis). <i>pop-1(q645)</i> mutants have greater variability between pBocs than the wild-type N2 control. (K) Box-whisker plot showing the pBoc period of <i>pop-1</i> depleted worms compared to ctrl RNAi worms using the OLB11 strain, which allows intestine-specific RNAi <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004133#pgen.1004133-McGhee1" target="_blank">[65]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004133#pgen.1004133-Pilipiuk1" target="_blank">[66]</a>. Animals were assayed at the young adult stage. A statistically significant increase was seen in the pBoc cycle time based on a Student's two-tailed <i>t</i> test (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004133#pgen-1004133-t001" target="_blank">Table 1</a>). (L) 8 individual pBocs (X-axis) were monitored in young adults (n = 24, each color representing one adult) for each genotype and plotted against time between each pBoc (y-axis). <i>pop-1 RNAi</i> leads to a high variability in the cycle time in <i>pop-1</i> depleted adults compared to controls.</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