XB-ART-53874Curr Biol August 7, 2017; 27 (15): 2357-2364.e5.
Two-Element Transcriptional Regulation in the Canonical Wnt Pathway.
PubMed ID: 28756947
PMC ID: PMC5557293
Article link: Curr Biol
Grant support: DP2 OD008471 NIH HHS , T32 GM007616 NIGMS NIH HHS
Genes referenced: axin2 cdx4 ctnnb1 en1 gsk3b nodal3.1 prl.2 sia1 tbxt tcf3 tp53
Antibodies referenced: Ctnnb1 Ab11
Article Images: [+] show captions
|Figure 1 Endogenous Genes Show Regulation Not Captured by the WRE (A) In the canonical Wnt pathway, Wnt ligand stimulation inhibits the destruction complex, resulting in the accumulation of β-catenin. Together with Tcf/Lef proteins, β-catenin binds to the WRE and activates or represses target genes. (B) Xenopus embryos were treated with LiCl for 5 min at the 32-cell stage, harvested at stage 10 for qRT-PCR assay, and scored 3–4 days later (shown here). (C) Expression of target genes, siamois (black circle) and Xnr3 (white circle). Control embryos are untreated sibling embryos. Red arrows highlight how gene expression remains wild-type despite perturbations. (D) Black circle, luciferase/renilla signal from the TopFlash reporter injected at the four-cell stage; white circle, β-catenin level in the embryo measured using western blot. Red arrows highlight how β-catenin level and TopFlash expression change with moderate perturbations. (B)–(D) are reproduced from  with permission. Data are represented as mean ± SEM from three to five biological replicates. Error bars not visible have negligible SEM.|
|Figure 2 A Suppressive 11-bp NRE Is Necessary for siamois Regulation (A) Three WREs (black) are located within 500 bp upstream in siamois promoter (pSia) (B–F and H) We built luciferase reporters using 3-kb and 848-bp pSia. The luciferase reporters were injected into each cell at the four-cell stage. Injected embryos were treated with lithium for 5 min at the 32-cell stage and harvested for dual-luciferase assay at stage 10. As an injection control, pRL-TK constitutively expressing renilla luciferase was co-injected, and the pSia-driven firefly luciferase signal was measured relative to the renilla luciferase signal. In all of the plots shown here, the luciferase/renilla signal is normalized to that in the control, untreated embryos. Data are presented as mean ± SEM from three to five biological replicates. Error bars not visible have negligible SEM. (B) Expression of 3-kb pSia-luc. (C) Expression of 848-bp pSia-luc (p value = 8.4e−4, Student’s t test). Insets below (B) and (C): Xenopus embryos were injected with 3-kb or 848-bp pSia-LacZ at the four-cell stage, treated with lithium for 5 min at the 32-cell stage, and fixed at stage 10 for X-Gal staining. In all embryos, dorsal is to the right. (D) Expression of 1.3-kb pSia-luc (black) and 963-bp pSia-luc (white). (E) Expression of 952-bp pSia-luc (white) and 888-bp pSia-luc (black). (F) Expression of pSia of various lengths in embryos treated with 150 mM LiCl (p value = 1.5e−5, Student’s t test). See also Figure S1. (G) A suppressive 11-bp NRE (blue) is located between 963 and 952 bp upstream of siamois. (H) Mutagenesis analysis of the 11-bp NRE. Data are mean luciferase/renilla signal ± SEM from two to four biological replicates.|
|Figure S1. Rescue of suppression by 11-bp NRE was observed with even shorter promoters, related to Figure 2. A 30-bp region containing the 11-bp NRE was added to the 5’-end of 848bp (orange), 638bp (white), and 385bp (grey) of siamois promoter. Xenopus embryos were injected with the constructs at 4-cell stage, treated with lithium for 5 minutes at 32- to 64-cell stage, and harvested at stage 10 for luciferase analysis.|
|Figure S2. 11-bp NREs interact with Beta-catenin and Tcf3, but not other transcription factors, related to Figure 3. (A) Known DNA binding sites of transcription factors failed to compete with the specific gel shift band. WRE probe without competition (lane 1), with 200x unlabeled 11-bp NRE (lane 2), m11 mutation competition (lane 3), and binding sites of transcription factors (lanes 4-7). (B) Purified Xenopus beta-catenin and Tcf3 proteins bound to the 11-bp NRE and WRE in dose dependent manner.|
|Figure S4. Analysis of β-catenin Chip-Seq on HCT-116 cells, related to Figure 4. (A) The 11-bp NRE motif is significantly enriched in 600-bp peaks of β-catenin Chipseq on HCT-116 cells. As a positive control, significant enrichment was also found for the WRE motif. (B) Co-enrichment analysis. Out of the 2624 total 600-bp peaks from the β-catenin Chipseq, 1428 contain the 11-bp NRE and/or WRE motifs. Out of these 1428 peaks, 421 contain both motifs.|