Figure 9. Set7 antagonizes Suv39h1 to promote MyoD-mediated skeletal muscle myoblast differentiation. (A) C2C12 myoblasts were transfected with control siRNA (Ctrl.) or siRNA against Set7. Cells were transferred to differentiation medium for the indicated times, and Western blotting was performed with cell extracts using antibodies that recognize the indicated proteins. β-Tubulin was used as a loading control. (B) Methyltransferase gene expression during C2C12 differentiation. Quantitative RT-PCR analysis of Ash1l, myogenin (Myog), Set7, and Prmt5 gene expression at day 0 (D0) and day 3 (D3) of differentiation with and without Set7 siRNA treatment (siSet7). Data represent the means + SD from three independent experiments. *, P < 0.05. (C) IPs were performed with anti–H3-K4, anti–H3-K9, and anti-Suv39h1 antibodies as indicated. The amount of DNA in each sample (input) is shown. IPs performed with IgG were used as controls. GAPDH was used as a loading control. (D) MEF cells from wild type (+/+) or Suv39h1/h2 double knockout (−/−) were transiently transfected with an expression plasmid for MyoD. Myogenic conversion was scored by positive staining for MHC expression (red) 6 d after differentiation induction. DAPI counter stains the nucleus (blue). Bar, 40 µm. The result of quantitative analysis, which was presented as the percentage of nuclei in myotubes from 10 randomly chosen microscopic fields, is presented on the right. Data represent the means ± SD from at least three independent experiments. P = 0.002. (E) RT-PCR analyses of the expression of Suv39h1, Suv39h2, and Set7 transcripts using total RNA isolated from wild-type (+/+) or Suv39h1/h2 double knockout (−/−) MEFs. GAPDH was used as a loading control. (F) Western blot analyses using antibodies that recognize Set7 and H3-K4 proteins from cell extracts of wild-type (+/+) or Suv39h1/h2 double knockout (−/−) MEFs. β-Tubulin was used as a loading control. (G) A working model to account for the molecular regulation of MyoD-mediated skeletal muscle differentiation, which is modulated by Set7 and Suv39h1. In undifferentiated myoblasts, MyoD is associated with Suv39h1/2 on the promoters/enhancers of myogenic differentiation genes. Suv39h1-mediated H3-K9 methylation resulted in condensation of chromatin and transcriptional repression. During skeletal muscle differentiation, increased Set7 expression will compete with Suv39h1 for MyoD association and H3-K4 methylation. This will consequently lead to the occupation of MyoD at the promoters/enhancers of myogenic differentiation genes, resulting in myogenic activation.
Figure 9.

Set7 antagonizes Suv39h1 to promote MyoD-mediated skeletal muscle myoblast differentiation. (A) C2C12 myoblasts were transfected with control siRNA (Ctrl.) or siRNA against Set7. Cells were transferred to differentiation medium for the indicated times, and Western blotting was performed with cell extracts using antibodies that recognize the indicated proteins. β-Tubulin was used as a loading control. (B) Methyltransferase gene expression during C2C12 differentiation. Quantitative RT-PCR analysis of Ash1l, myogenin (Myog), Set7, and Prmt5 gene expression at day 0 (D0) and day 3 (D3) of differentiation with and without Set7 siRNA treatment (siSet7). Data represent the means + SD from three independent experiments. *, P < 0.05. (C) IPs were performed with anti–H3-K4, anti–H3-K9, and anti-Suv39h1 antibodies as indicated. The amount of DNA in each sample (input) is shown. IPs performed with IgG were used as controls. GAPDH was used as a loading control. (D) MEF cells from wild type (+/+) or Suv39h1/h2 double knockout (−/−) were transiently transfected with an expression plasmid for MyoD. Myogenic conversion was scored by positive staining for MHC expression (red) 6 d after differentiation induction. DAPI counter stains the nucleus (blue). Bar, 40 µm. The result of quantitative analysis, which was presented as the percentage of nuclei in myotubes from 10 randomly chosen microscopic fields, is presented on the right. Data represent the means ± SD from at least three independent experiments. P = 0.002. (E) RT-PCR analyses of the expression of Suv39h1, Suv39h2, and Set7 transcripts using total RNA isolated from wild-type (+/+) or Suv39h1/h2 double knockout (−/−) MEFs. GAPDH was used as a loading control. (F) Western blot analyses using antibodies that recognize Set7 and H3-K4 proteins from cell extracts of wild-type (+/+) or Suv39h1/h2 double knockout (−/−) MEFs. β-Tubulin was used as a loading control. (G) A working model to account for the molecular regulation of MyoD-mediated skeletal muscle differentiation, which is modulated by Set7 and Suv39h1. In undifferentiated myoblasts, MyoD is associated with Suv39h1/2 on the promoters/enhancers of myogenic differentiation genes. Suv39h1-mediated H3-K9 methylation resulted in condensation of chromatin and transcriptional repression. During skeletal muscle differentiation, increased Set7 expression will compete with Suv39h1 for MyoD association and H3-K4 methylation. This will consequently lead to the occupation of MyoD at the promoters/enhancers of myogenic differentiation genes, resulting in myogenic activation.

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