Figure 6. Pou3f1 represses BMP and Wnt signaling at the transcriptional level.

(A) Expression levels of BMP signaling target genes in control and Pou3f1-knockdown ESCs differentiated in serum-free medium. (B) Expression levels of BMP signaling target genes in control and Pou3f1-overexpressing ESCs in unbiased differentiation. (C) Luciferase assays using BRE-luc in control and Pou3f1-shRNA vector-transfected ESCs with or without BMP4 treatment in N2B27 medium. (D) Luciferase assays using BRE-luc in control and Pou3f1-expressing vector-transfected ESCs with or without BMP4 treatment in N2B27 medium. (E) Pou3f1 ChIP assays in control, Pou3f1-full length, or in Pou3f1-ΔHOMEO lentivirus-transfected P19 cells. Pou3f1 enrichment at the Id1-BRE was normalized to the Id1 3′ UTR region. (F) pSmad1 ChIP assay in control and Pou3f1-full length lentivirus-transfected P19 cells with or without BMP4 treatment. A pSmad1/5/8-specific antibody was used in the assay. pSmad1 enrichment at the Id1-BRE and control 3′ UTR region were analyzed. (G) Dose-dependent inhibitory effect of Pou3f1 on the BRE-luc reporter activities. P19 cells were transfected with increasing amounts of Pou3f1-expressing vector and treated with or without BMP4 in N2B27 medium. (H) Expression levels of Wnt signaling target genes in control and Pou3f1-knockdown ESCs differentiated in serum-free medium. (I) Expression levels of Wnt signaling target genes in control and Pou3f1-overexpressing ESCs in unbiased differentiation. (J) Luciferase assays using TOPflash in control and Pou3f1-shRNA vector-transfected ESCs with or without stimulation of Wnt3a in N2B27 medium. (K) Luciferase assays using TOPflash in control and Pou3f1-expressing vector-transfected ESCs with or without stimulation of Wnt3a in N2B27 medium. (L) Dose-dependent inhibitory effect of Pou3f1 on the TOPflash luciferase reporter activities. P19 cells were transfected with increasing amounts of Pou3f1-expressing vector and treated with or without CHIR99021 in N2B27 medium. The values represent the mean ± SD. (*p<0.05; **p<0.01).

DOI: http://dx.doi.org/10.7554/eLife.02224.015

Figure 6—figure supplement 1. Pou3f1 interferes with BMP and Wnt signaling pathways at the transcriptional level.

(A) In situ hybridization of cId1 expression (blue) in chick embryos that were electroporated with IRES-GFP (control vector, a and a′) or with Pou3f1-IRES-GFP (b and b′). GFP expression (brown) was detected in a′ and b′ by immunohistochemical assay. (B) Luciferase assays using BRE-luc in P19 cells that were transfected with control, Pou3f1-full length or with each of the Pou3f1-deletion mutant vectors shown in Figure 5—figure supplement 1 with or without BMP4 stimulation in N2B27 medium. (C) Luciferase assays using TOPflash-luc in P19 cells that were transfected with control, Pou3f1-full length expression or with each Pou3f1-deletion mutant vector shown in Figure 5—figure supplement 1 with or without Wnt3a stimulation in N2B27 medium. The values represent the mean ± SD for B and C.

DOI: http://dx.doi.org/10.7554/eLife.02224.016