only with nuclear, nonphosphorylated Yap, indicating that the Yap/b-catenin molecular interaction is nuclear (Fig. 4C).
Because Yap associates with DNA-binding
Tead/Tef transcription factors whereas b-catenin
binds to Lef/Tcf factors (12, 13), we examined
surrounding genomic sequence of genes within
the Wnt-Hippo expression signature for Tead/Tef
and Lef/Tcf binding sites. For Sox2 and Snai2,
candidate Yap/Tead binding sites were identified
both upstream and downstream of open reading
frames (fig. S7D). Conserved Tcf/Lef binding
elements (CTTTG) were in close proximity to
Sox2 and Snai2 downstream candidate Yap/Tead
sites (fig. S7D).
Chromatin immunoprecipitation (ChIP) revealed that Yap and b-catenin were recruited to
Sox2 and Snai2 downstream regions but not
upstream sites (Fig. 4D). Sequential ChIP revealed that Yap and b-catenin concurrently occupied Sox2 and Snai2, suggesting that Yap and
b-catenin are contained within a common regulatory complex on Sox2 and Snai2 (Fig. 4D).
Transfection experiments indicated that Yap
and b-catenin transfection along with their DNA
binding co-factors induced luciferase expression
from both Sox2 and Snai2 reporter plasmids.
Luciferase reporter activity was significantly
reduced by preventing Yap or b-catenin recruit-
ment to the reporter either individually or con-
currently (Fig. 4E). These findings support the
model that Yap and b-catenin recruitment to Sox2
and Snai2 chromatin through their respective
Fig. 2. Cardiomyocyte proliferation in Salvador mutant
ventricles. E12.5 control (top) and Salv CKO (middle)
coronal sections of left ventricles: stain with TO-PRO-3,
blue; a-pHH3, red; and a-MF20, green. Arrows, pHH3/MF20-
positive cells. (Bottom) pHH3-positive cardiomyocytes
quantification. Control genotype is Nkx2.5cre; Salv f/+.
Fig. 3. Salvador deletion potentiates canonical Wnt signaling. Heat map (A)
and qRT-PCR validation (B) showing relative transcript levels of Wnt/b-catenin
target genes. Values were determined as a mean of three samples T SD with
glyceraldehyde-3-phosphate dehydrogenase control. (C) qRT-PCR of Wnt/b-
catenin target genes in b-catenin CKO hearts. (D and E) IF images with
quantification (F) of E12.5 heart sections: stain with TO-PRO-3, blue, and a-b-
catenin, green. Nuclear b-catenin identified by b-catenin/TO-PRO-3 signal
overlap (arrows). Control genotype is Nkx2.5cre; Salv f/+.