Fig 2: Characterization of Pik3r1 enhancer-promoter communication. (A) Quantitative local 4C-seq signal for the Pik3r1 viewpoint in skeletal muscle. GR binding sites (GR) are depicted with blue lines, GREs with blue stars, Ctcf binding sites identified by ChIP-seq with cyan arrowheads, indicating convergent motif orientation. H3K4me1 and Myod1 ChIP-seq profiles are presented in green and red, respectively. Enhancer-promoter contact regions are highlighted in orange on the 4C-seq chart. Ctcf-bound promoter and enhancer regions are boxed in cyan. (B, C) Quantitative local 4C-seq signal for the Pik3r1 viewpoint in skeletal muscle of control (upper panels in B and C) mice compared with skeletal muscle GR(i)skm-/- mice (B, lower panel), or with double positive (DP) thymocytes (C, lower panel). Enhancer-promoter contact regions lost upon GR ablation and absent in DP thymocytes are highlighted in red. Contact regions present in both control and GR(i)skm-/- mice are highlighted in light blue. Contact regions present in both control and DP thymocytes are highlighted in light green. Contact regions that are specific for GR(i)skm-/- mice and DP thymocytes are highlighted in dark blue and dark green, respectively. GR binding sites (GBS) are depicted with black lines and GREs with black stars.

Fig 3: SGI-110 inhibits cell proliferation more effectively in aRMS than eRMS cells (A) Cell lines were exposed to the indicated concentrations of SGI-110 and cellular proliferation rate was monitored in an IncuCyte S3 live cell analysis system for 8–9 days. Data represent the mean ± SEM of a representative experiment. ***P < 0.001 versus control (i.e., DMSO). (B) Representative images of DMSO, 500 nM and 700 nM SGI-110 treated RMS cells at day 8. Scale bar = 700 μm. (C) Immunoblot of the total RH30 and RH41 cell extracts treated with the indicated concentrations of SGI-110 or DMSO (control) for 5 days, probed with antibodies against FGFR4, FOXO1, IGF-1R and MYOD1. β-Actin used as a loading control. (D) Densitometric analysis of the immunoblot in C using iBright Analysis Software. Results are the means ± SD pooled from three independent experiments, ** P < 0.01 versus untreated controls (i.e., DMSO).

Fig 4: Characterization of Myod1 and GR binding at the Eif4ebp2 and Pik3r1 loci. (A) HOMER known motif analysis on Myod1 peaks located at GR-bound enhancer regions. BG refers to estimated background. (B, C) Tag density map of H3K27ac, H3K4me1, H3K4me3 and GR in skeletal muscles, and Myod1 in C2C12 myotubes, ± 5 kb from the GR peak center (B) and corresponding average tag density profiles of cluster 2 (C). (D) Overlap between genes bound by GR in skeletal muscle and by Myod1 in C2C12 myotubes obtained from 3 merged datasets, and genes that are down regulated in GR(i)skm-/- mice. (E) Western blot analysis of skeletal muscle nuclear extracts immunoprecipitated with anti-GR or anti-Myod1 antibodies. Membranes were decorated with anti-GR and anti-Myod1 antibodies. rIgG served as a control for immunoprecipitation. Non-immunoprecipitated extracts (10% input) were also analyzed. (F) ChIP-qPCR analysis performed at indicated locations with anti-Myod1 antibodies or rIgG in skeletal muscle of wild-type mice. (G, H) ChIP-qPCR analysis performed at indicated locations with anti-GR (G) or anti-Myod1 (H) antibodies in C2C12 myotubes transfected with siRNA directed against Myod1 (siMyod1) (G), GR (siGR) (H) or scrambled siRNA (siCtrl). (I) Relative Eif4ebp2 and Pik3r1 transcript levels determined in C2C12 myotubes transfected with siCtrl, siGR or siMyod1. (F-I): n = 3 independent experiments in triplicate. Mean + SEM. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig 5: Characterization of tumorigenicity and differentiation potential of WT and Myod1 gene KO cells. a, b Soft agar colony formation assay of WT and KO cells during a 10-day period (a). Significance in difference of colony formation (b) was calculated based on experiments in triplicate using two-tailed Student’s t-test. **p < 0.01. Colonies larger than 25 μm in diameter were counted. c Difference in invasion and migration of WT and KO cells during time periods as indicated. d–f Tumor formation of WT and KO cells in nude mice. (D) shows tumor formation in five of five mice injected with KO cells, and e, f show difference in tumor volume (e) and weight (f) between WT and KO cells. Significance of difference in tumor volume between two groups of mice was calculated using two-way ANOVA-Bonferroni/Dunn test, and significance of difference in tumor weight was calculated using two-tailed Student’s t-test. Data are shown as mean ± SEM. **p < 0.01, ***p < 0.001. g–i Tissue differentiation in tumor formed by KO cells, as examined by tissue-specific gene expression (g, h) and marker expression (i). g, h show difference in expression of genes representing neuronal differentiation and neural stemness (g), and mesodermal and endodermal tissues (h) between KO cells and KO cell xenograft tumors, as detected with RT-qPCR. Significance in expression change was calculated based on experiments in triplicate using two-tailed Student’s t-test. Data are shown as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. NS: not significant. i IHC detection of cell/tissue markers in tumor sections. Below the panels for marker detection are corresponding sections stained with HE. Objective magnification for sections: 20×; insets: 40×