miR-302 cluster miRNAs are downregulated in inducible β-catenin knockout ESCs.

<p>A–D, β-catenin^{<i>flox/-</i>} or β-catenin^{<i>flox/-</i>}: <i>CreER</i>^<i>T2</i> ESCs were treated with 4-hydroxy-tamoxifen (Tam) or control EtOH for 3 days. A, protein levels of β-catenin were determined by Western blot analysis. B, Tam treatment of the parental cell line did not alter gene expression as revealed by qRT-PCR experiments. Bars represent the mean ± SEMof two independent experiments. C, D, Four hours before harvesting, Wnt3a (PeproTech; 100 ng/ml) or control BSA/PBS was administered to β-catenin^{<i>flox/-</i>}: <i>CreER</i>^<i>T2</i> ESCs. Gene expression was analyzed using qRT-PCR experiments, and results were normalized to EtOH + BSA/PBS treated cells (set to 1). Data represent the mean of four independent experiments ± SEM C, statistically significant changes in <i>pri-302</i> and <i>Axin2</i> gene expression upon Wnt-3a or Tam treatment are indicated by asterisks. D, expression of pluripotency-associated genes was analyzed. No statistically significant changes in gene expression could be observed upon Wnt3a treatment. Asterisks above the bars indicate statistical significance of Tam + BSA/PBS treated cells compared to EtOH + BSA/PBS treated cells; Student’s t test *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001, ns: not significant.</p

The Wnt/β-Catenin Pathway Regulates the Expression of the miR-302 Cluster in Mouse ESCs and P19 Cells

<div><p>MicroRNAs of the miR-302 cluster are involved in early embryonic development and somatic cell reprogramming. Expression of the miR-302 gene is regulated by the binding of the pluripotency factors Oct4, Sox2 and Nanog to the miR-302 promoter. The specific expression pattern of the miR-302 gene suggested that additional transcription factors might be involved in its regulation. Here, we show that the miR-302 promoter is a direct target of the Wnt/β-catenin signaling pathway. We found that the miR-302 promoter contains three different functional Tcf/Lef binding sites. Two of the three sites were located within the cluster of Oct4/Sox2/Nanog binding sites and were essential for Wnt/β-catenin-mediated regulation of the miR-302 gene. Tcf3, the only Tcf/Lef factor that bound to the miR-302 promoter, acted as a repressor of miR-302 transcription. Interestingly, mutations in the two Tcf/Lef binding sites and the Oct4/Nanog binding sites abolished miR-302 promoter responsiveness to Wnt signaling, suggesting that the Tcf/Lef and the Oct4/Nanog sites interact genetically.</p> </div

Tcf3 binds to the miR-302 promoter <i>in</i><i>vivo</i> and represses its activity.

<p>A,B, ChIP experiments were performed in mESCs (A) or P19 cells (B) with antibodies specific for Tcf1, Tcf3, Tcf4 and Lef1. Goat IgG served as a negative control. Precipitated DNA was used to amplify different genomic regions as indicated by qPCR. Data represent the mean of two independent experiments ± SD. C, luciferase assays were performed to examine the activities of different pGL4.10-miR-302 promoter constructs upon Tcf3 knockdown. mESCs were transfected with Tcf3 specific siRNAs. Twenty-four hours after the transfection of siRNAs, the indicated plasmids were transfected, and luciferase assays were performed 24 hours later. Luciferase activity was normalized to the solvent control (BSA/PBS) for each construct. Data represent the mean of four independent experiments ± SD. Statistical significance is indicated; Student’s t test *<i>p</i><0.05, ns: not significant.</p

Wnt signaling regulates expression of the miR-302 cluster.

<p>A–D, mESCs were incubated with murine Wnt3a (A, B; R&D Systems; 150 ng/ml) or Dkk-1 (C-E; R&D Systems; 100 ng/µl) for 8 hours prior to harvesting and total RNA (A,C) or miRNA (B,D) isolation. A,C, qRT-PCR analysis was performed to determine the expression levels of the primary <i>miR-302</i> transcript (pri-302), <i>Axin2</i> and <i>Actb</i>. B,D: expression levels of mature miRNAs were analyzed by qRT-PCR. Data represent mean values ± SEM from 4–5 independent experiments. Statistical significance over solvent control (BSA/PBS) is shown; Student’s t test *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001.</p

Active Wnt signaling in mESCs and P19 cells.

<p>Wnt/β-catenin responsive reporters (SuperTOPflash and pGL3B-Axin2) and their respective corresponding controls (SuperFOPflash and pGL3 Basic) were transfected into (A) mESCs or (B) P19 cells. Cells were either treated with Wnt-3a (Peprotech; 100 ng/ml) or GSK-3 inhibitor (GSK-3i; 10 µM). Luciferase activity was normalized to the respective control plasmid and solvent (set to 1). Data represent the average of three (wt ESCs) or two (P19) replicates ± standard deviation (SD) nd: not determined.</p

The responsiveness of the <i>miR-302</i> promoter to Wnt/β-catenin signaling depends on Tcf BS 5 and Tcf BS 6 and the Oct4/Nanog BS.

<p>Luciferase assays were carried out to examine the activities of different pGL4.10-miR-302 promoter constructs upon β-catenin knockdown (A) or Wnt3a treatment (B,C) (Peprotech; 100 ng/ml). Luciferase activity was normalized to the solvent control (BSA/PBS) for each construct. A, mESCs were transfected with β-catenin specific siRNAs. Twenty-four hours after the transfection of siRNAs, the indicated plasmids were transfected and luciferase assays were performed 24 hours later. B,C, mESCS (B) or P19 cells (C) were transfected with the indicated plasmids and treated with Wnt3a. Twenty-four hours later, luciferase assays were performed. Results of three (A,B) or two (C) independent experiments are shown. Error bars indicate standard error of the mean (SEM). Statistical significance is indicated; Student’s t test *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001, ns: not significant. BS: binding site.</p

Treatment of ARβ1 <i>(βcat</i>^del/flox :Cre-ER-T2) ES cells with 4-OHT induces Cre-mediated recombination of the <i>β-catenin</i> genomic locus leading to ablation of β-catenin.

<p>(A) Immunocytochemical staining for β-catenin protein on cultured ARβ1 cells reveals loss of β-catenin protein already at day two and appears to be absent at day three. (B) Western-immunoblotting against β-catenin demonstrates the almost complete loss of protein within 3 days of culturing. (C) <i>β-catenin</i> mRNA levels drop already 24 h after 4-OHT administration as revealed by quantitative RT-PCR (n = 3). ARβ1 ES cells do not respond to Wnt3a mediated canonical Wnt signaling after treatment with 4-OHT. (D) SR1 (<i>βcat</i>^del/flox)) and ARβ1 ES cells were treated for three days with 4-OHT and Wnt3a protein to induce canonical Wnt-signaling. Quantitative RT-PCR analysis of Wnt-target genes revealed that in contrast to SR1 cells ARβ1 cells are do not up-regulate significantly <i>Axin 2</i>, <i>T-Brachyury</i> and Cdx 1 (n = 3). *P<0,05, **P<0,01; student’s t-test. Error bars = s.e.m.; scale bar 10 µm.</p