Figure 7. YAP1 Inhibits Wnt/β-catenin Signaling by Binding β-catenin and Inducing Dkk1.

(A) Luciferase assay of Wnt reporter in YAP1-expressing cells. C3H10T1/2 pTOP-FLASH cells were infected with control or YAP1 lentivirus and treated with 4.5 μM of CHIR 99021 for 10 hr. Firefly luciferase activity (Wnt response) was normalized to Renilla.

(B) Immunoprecipitation of β-catenin. C3H10T1/2 cells were infected with flag-YAP1 or flag-YAP1(2SA) virus and whole-cell lysates were immunoprecipitated (IP) with Flag antibody. Immune complexes and whole-cell lysates were immunoblotted (IB) as indicated.

(C) Dkk1 mRNA expression analysis by qRT-PCR. C3H10T1/2 cells were infected with the indicated lentiviral vectors and Dkk1 expression was analyzed.

(D) ChIP assay for the YAP1/TEAD-binding Dkk1 upstream region. C3H10T1/2 cells were infected with control, flag-YAP1, or flag-YAP1(2SA) and ChIP was performed with Flag antibody. Dkk1 genomic fragments were amplified by PCR with specific primers to sequences flanking YAP1/TEAD binding sites (red bars).

(E) Luciferase-reporter assay of Dkk1-promoter (Dkk1-pro) or Dkk1-pro with mutations in TEAD-binding sites (red nucleotides; Mut1/2). A 730 bp region from −1,450 to −720 upstream of the Dkk1 TSS (Dkk1-pro) was subcloned into minimal FGF4 luciferase reporter and transfected into C3H10T1/2 cells expressing YAP1 or control vectors. Luciferase activity was normalized to Renilla.

(F) Osteogenic differentiation. C3H10T1/2 cells were infected by the indicated viral vectors and an osteogenesis assay was performed. Western blot of cells expressing shDkk1 shows that the YAP1 protein level is unaffected.

(G) YAP1 inhibits Wnt during osteogenesis. C3H10T1/2-pTOP-FLASH cells were infected with control (−) or YAP1(2SA) and subjected to osteogenic differentiation for the indicated days. Firefly luciferase activity was normalized to Renilla.

*p < 0.05; error bars represent the average + SD. See also Figure S7.