Supplementary Figure 4: PRC2–Ezh1 complex is redistributed on muscle-specific targets that become repressed; validations and controls.
From: A cytosolic Ezh1 isoform modulates a PRC2–Ezh1 epigenetic adaptive response in postmitotic cells
a. Ezh1, Eed and Suz12 ChIP-qPCR analyses on Myogenin, Myh3, Desmin and NeuroG1 promoters in control (MT) and H2O2-treated myotubes (MT-H2O2). Results are represented as fold enrichment over the mock. Mean and s.e.m. (Independent experiments: n=4, except for Eed, n=3), One-tailed paired t-test: for Ezh1 ChIP: Myogenin MT versus MT-H2O2 p=0.014, Myh3 MT versus MT-H2O2 p=0.021; for Eed ChIP: Myh3 MT versus MT-H2O2 p=0.003; for Suz12 ChIP: Myog MT versus MT-H2O2 p=0.006. b. Ezh2 ChIP-qPCR analysis on Myogenin, Myh3, Desmin and NeuroG1 promoters in control (MT) and H2O2-teated myotubes (MT-H2O2). Results are represented as fold enrichment over the mock. Mean and s.e.m. (Independent experiments: n=6). c. Left, western blots detecting HA-Ezh1 and Myogenin protein levels in nuclear (Nuc) and cytosolic (Cyt) extracts of C2C12 stable line overexpressing HA tagged-Ezh1. (Independent experiments: n=1). Right, HA ChIP-qPCR analyses on Myogenin, Myh3, Desmin and NeuroG1 promoters in control and H2O2-treated myotubes overexpressing HA tagged Ezh1. Results are represented as fold enrichment over the mock. Mean is indicated (Independent experiments: n=2, plotted with single values). d. H3K27me3 ChIP-qPCR analyses on Myogenin, Myh3, Desmin and NeuroG1 promoters in control (MT), H2O2-treated (MT-H2O2) and Ezh1 depleted myotubes treated with H2O2 (MT-H2O2 Ezh1 kd). ChIP results are represented as percentage of immunoprecipitated H3. Mean and s.e.m. (Independent experiments: n=4 in MT and MT-H2O2, n=2 in MT-H2O2 Ezh1 kd, plotted with single values). One-tailed paired t-test: Myh3, MT versus MT-H2O2 p=0.041. e. Expression levels of Myogenin, Myh3, Desmin and NeuroG1 genes in control (MT) and FeSO4 treated (MT-FeSO4) myotubes. Data were normalized on Gapdh expression. Mean and s.e.m. (Independent experiments: n=3). Two-tailed paired t-test: Myogenin p=0.018. f. Expression levels of Myogenin, Myh3, Desmin, and NeuroG1 genes in control (MT) and 43°C heat-shock-treated (MT-43°C) myotubes. Data were normalized on Gapdh expression. Mean and s.e.m. (Independent experiments: n=4 for Myog, n=5 for Myh3 and Des, n=2 for NeuroG1, plotted with single values). Two-tailed paired t-test: Myh3, p=0.022. g. Expression levels of Myogenin and Myh3 genes, in control (MT), H2O2-treated (MT-H2O2) and recovered myotubes (MT Recovery). Data were normalized on Gapdh expression. Mean is indicated (Independent experiments: n=2, plotted with single values). h. Jmjd3 ChIP-qPCR analyses on Myogenin, Myh3, Desmin and NeuroG1 promoters in control (MT) and H2O2 treated myotubes (MT-H2O2). ChIP results are represented as fold enrichment over the mock. Mean and s.e.m. (Independent experiments: n=3).
