Fig. 7
The Wnt antagonist, Sfrp1, is directly repressed by JARID2 and the PRC2 complex. a Sfrp1 mRNA is upregulated in JARID2 depleted cells as assayed by qRT-PCR. Error bars are S.E.M. ***p value < 0.001 versus scr. n ≥ 3. b Activation of canonical Wnt signaling pathway suppresses Sfrp1 expression. Scr- or shJarid2-expressing cells were differentiated for 2 days and shJarid2 cells were treated with either 10 mM NaCl or 10 mM LiCl while differentiating. mRNA expression of Sfrp1 was assayed by qRT-PCR. Fold change in expression of mRNA was calculated relative to undifferentiated scramble control. Data are plotted as means with S.E.M. and analyzed with one-way ANOVA followed by Tukey’s HSD post hoc test. Means not sharing same letter are statistically significant (p < 0.001). n ≥ 3. c, d JARID2 directly represses Sfrp1. Scr or shJarid2 cells were used for ChIP assays using antibodies against JARID2 (c), trimethylation of histone 3 lysine 27 (H3K27me3) (d) and nonspecific antibody (IgG) with primers specific to the Sfrp1 promoter. Error bars are S.E.M. ***p value < 0.001. n ≥ 3. e–h SFRP1 expression inhibits muscle gene expression. An expression construct for Sfrp1 (cDNA) was transiently expressed in C2C12 cells and RNA harvested 48 h post-transfection (UD) and following an additional 36 h of differentiation conditions (D1.5). mRNA expression was assayed by qRT-PCR for Sfrp1 (e) Myod1 (f), Myog (g) and Tnni2 (h). Error bars are S.E.M. ***p value < 0.001 versus time-matched vector. n ≥ 3. i Inhibition of MYOD and MYOG expression by SFRP1 was confirmed by western blot assays. SFRP1 was transiently expressed in C2C12 cells and protein harvested 48 h post-transfection (UD) and following an additional 36 h of differentiation conditions (D1.5). Data were quantified and normalized to GAPDH (right side of panel). j SFRP1 inhibits Wnt-mediated transcriptional activity. Reporter assays were performed using TOPFlash and FOPFlash luciferase reporters in C2C12 cells transfected with Sfrp1 (cDNA) or vector control. Growth medium was replaced with differentiation medium 24 h post-transfection, and luciferase activity was detected 48 h post-transfection. Error bars are S.E.M. n ≥ 3. k SFRP1 inhibits translocation of β-catenin to the nucleus. Nuclear and cytoplasmic fractions of proteins from cells as in i were probed for ß-catenin, LAMIN A/C (loading control for nucleus) and tubulin (loading control for cytoplasm). Data were quantitated, normalized to respective loading controls, and plotted as a stacked graph (right side of panel). Error bars are S.E.M. n ≥ 3