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. 2020 Oct 26;8:587778. doi: 10.3389/fcell.2020.587778

FIGURE 6.

FIGURE 6

The murine Pitx3 promoter is dose-dependently repressed by LEF1-mediated WNT/b-catenin signaling. (A) Alignments of conserved LEF1/TCF BSs/WREs (yellow boxes) in Pitx3 promoter regions of mouse, rat, human, rhesus monkey, and chimp (WRE 1/2, minus strand), or mouse and rat (WRE 3, plus strand). (B) Position of the three conserved LEF1/TCF BSs/WREs (WRE 1-3) in the mouse Pitx3 (mPitx3) gene promoter relative to the transcription start site (TSS), and of the six primer pairs used in ChIP-PCR analysis. (C) Sheared genomic DNA (gDNA) isolated from the VM of E11.5 CD-1 embryos. M, DNA ladder. (D) Representative ChIP-PCR analysis of LEF1 binding to the mPitx3 promoter in the E11.5 mouse VM using six overlapping primer pairs (Primer 1-6). H2O, negative PCR control; rIgG, negative ChIP control; Pol II, positive ChIP control; LEF11, LEF1 mAb C12A5; LEF12, LEF1 mAb C18A7; and input, sheared E11.5 mouse VM gDNA. (E,F) Fold change of luciferase (LUC) activity in HEK-293 cells (relative to Pitx3 promoter/reporter only-transfected cells, set as 1) after transfection of decreasing amounts of S33Y-b-catenin or LEF1 cDNA alone or together with LEF1 or S33Y-b-catenin plasmids, respectively, [n = 3 (LEF1 gradient) and 4 (b-catenin gradient) independent experiments; statistical testing for significance was done between mPitx3 promoter/reporter only-transfected cells (white bars) and Pitx3 promoter + S33Y-b-catenin or LEF1-transfected cells (gray bars), and between the latter and cells transfected with decreasing amounts of S33Y-b-catenin or LEF1 plasmids in the presence of LEF1 or S33Y-b-catenin (black bars), respectively, using one-way ANOVA followed by Bonferroni’s multiple comparisons post hoc tests; F(7,23/16) = 8.0/12.0, P < 0.0001]. (G) Alignment of mutant LEF1/TCF BSs/WREs (WRE 1-3) and wt counterparts in the mPitx3 promoter. (H–J) Fold change of luciferase (LUC) activity in HEK-293 cells [relative to wt Pitx3 promoter/reporter only-transfected cells, set as 1; white bar in (H)] after transfection of mutant WRE 1-3 Pitx3 promoter/reporter alone [gray bars in (H), n = 4 independent experiments] or together with S33Y-b-catenin (I) or LEF1 cDNA (J) [n = 4 (+S33Y-b-catenin) and 7 (+LEF1) independent experiments; statistical testing for significance was done between wt and mutant WRE 1-3 Pitx3 promoter/reporter-transfected cells in each condition using Wilcoxon–Mann–Whitney test]. *P < 0.05; **P < 0.005; ***P < 0.0001; and ns, not significant.