Fig. 8: JARID2 and EZH2 directly repress TBX3 in skeletal muscle.

a, b C2C12 cells were transfected with shEzh2 (a) or shJarid2 (b) and scr control. Stable isolates were assayed by qRT-PCR for mRNA expression of Tbx2 and Tbx3. Error bars, S.E. and, ***p ≤ 0.001 versus scr. c Methylation of the Tbx3 promoter is dependent on JARID2. ChIP assays were performed on C2C12 cell lines with scr or shJarid2 using antibodies against H3K27^me3 and primers to the Tbx3 promoter. Error bars S.E. and ***p ≤ 0.001 as indicated. d TBX3 binds the Tbx2 promoter. ChIP assays were performed on C2C12 cell lines with scr or shJarid2 using antibodies against TBX3 and primers to the Tbx2 promoter. Error bars S.E. and *p ≤ 0.05, **p ≤ 0.01 as indicated. e Tbx3 KO cells analyzed by the western blot assay. Cells transfected with EV without Tbx3 guide RNA are labeled EV. f Tbx2 is upregulated upon Tbx3 deletion as assayed by qRT-PCR. Error bars S.E. and ***p ≤ 0.001. g Cells transfected with EV without Tbx3 guide RNA (EV) were treated with 1.25 μM GSK126 24 h post seeding for 48 h before assaying gene expression by qRT-PCR. Error bars S.E. and ***p ≤ 0.001 as indicated. h Inhibition of EZH2 does not lead to repression of Tbx2 in Tbx3 depleted cells. Tbx3 KO cells and control (EV) were treated and assayed for gene expression as in (g). Error bars S.E. and ***p ≤ 0.001 versus EV. n.s. represents not statistically significant. i Inhibition of EZH2 does not alter Tbx2 expression in Tbx3 depleted cells. Tbx3 KO cells were treated and assayed as in (g). n.s. represents not statistically significant. j Model of the PRC2-TBX3-TBX2 genetic axis in rhabdomyosarcoma and skeletal muscle. The PRC2 complex is highly expressed during proliferation in myoblasts and RMS (in orange) which represses TBX3 and allows TBX2 expression, which represses p21/p14/PTEN that are required for differentiation (in green). A decline in PRC2 expression derepresses TBX3 which represses TBX2 and promotes differentiation and myotube formation in skeletal muscle