G9a mediates Sharp-1-dependent inhibition of skeletal muscle differentiation

Abstract

Sharp-1, a basic helix-loop-helix transcription factor, is a potent repressor of skeletal muscle differentiation and is dysregulated in muscle pathologies. However, the mechanisms by which it inhibits myogenesis are not fully understood. Here we show that G9a, a lysine methyltransferase, is involved in Sharp-1-mediated inhibition of muscle differentiation. We demonstrate that G9a directly interacts with Sharp-1 and enhances its ability to transcriptionally repress the myogenin promoter. Concomitant with a differentiation block, G9a-dependent histone H3 lysine 9 dimethylation (H3K9me2) and MyoD methylation are apparent upon Sharp-1 overexpression in muscle cells. RNA interference-mediated reduction of G9a or pharmacological inhibition of its activity erases these repressive marks and rescues the differentiation defect imposed by Sharp-1. Our findings provide new insights into Sharp-1-dependent regulation of myogenesis and identify epigenetic mechanisms that could be targeted in myopathies characterized by elevated Sharp-1 levels.

FIGURE 1:

G9a interacts with and enhances Sharp-1–mediated transcriptional repression. (A) Schematic representation of full-length G9a and a deletion mutant lacking the ankyrin repeats (G9a∆ANK). Numbers indicate amino acid residues. Cells transfected with Myc–Sharp-1, FLAG-G9a, and FLAG-G9a∆ANK were immunoprecipitated with anti–c-Myc agarose beads and immunoblotted with anti-FLAG antibody. Lysates were analyzed for Sharp-1 and G9a expression by Western blot. β-Actin was used as a loading control. (B) Schematic representation of Sharp-1 with basic (b), helix-loop-helix (HLH), and Orange (O) domains. Deletion mutants of Sharp-1 are shown. Sharp-1 was immunoprecipitated and analyzed for interaction with G9a using anti-FLAG antibody. (C) Colocalization of Myc–Sharp-1(green) and FLAG-G9a (red) was visualized by immunofluorescence in C2C12 and 10T1/2 cells. Nuclei were stained with DAPI (blue). (D) In vitro–translated G9a was used for interaction with equivalent amounts of GST–Sharp-1 and GST proteins. A 10% input was run as a control. (E–G) 10T1/2 cells were transfected with 6E-TATA-Luc along with Sharp-1 and G9a (E), with Myc-Sharp-1 and 2.5 μM of BIX-01294 (F), or with 0.25 μM UNC0638 (G). (H, I) Cells were transfected with pMyog-Luc, MyoD, Sharp-1, and G9a (H) and, in the presence of MyoD, Sharp-1 with 2.5 μM BIX-01294 or 0.25 μM UNC0638 (I). Error bars indicate mean ± SD. (J) Cells were transfected with pMyogLuc along with Sharp-1 and deletion mutants together with G9a and G9a∆ANK. Error bars indicate mean ± SD. (K) Mouse embryo sections were immunostained with anti-G9a antibody at E12, E14, and E16. Arrowheads in magnified images indicate G9a expression (brown staining) in diaphragm, tongue, and limb muscles. Negative control shows E12 embryo section stained with secondary antibody only. A magnified image of the diaphragm is shown. Scale bar, 100 μm.

FIGURE 2:

Sharp-1 inhibits differentiation with increased H3K9me2. (A) Sharp-1 expression in pBabe (control) and pBabe-Sharp-1 C2C12 cells was analyzed by Western blot. (B–D) Differentiation of control and pBabe-Sharp-1 cells was analyzed with anti-MHC antibody. Nuclei were stained with DAPI (B). The myogenic index was determined and is represented as mean ± SD (C). Lysates from undifferentiated myoblasts (day 0) and after differentiation (days 1 and 3) were examined for myogenin and troponin T (D). (E, F) ChIP assays were performed at days 0 and 2 on the myogenin promoter, using H3K9me2 and H3K9K14ac antibodies. (G) C2C12 cells were transfected with scrambled siRNA or Sharp-1 siRNA (siSharp-1). The down-regulation of endogenous Sharp-1 was analyzed by Western blot. (H–J) Differentiation in siRNA and siSharp-1 cells was quantified by immunofluorescence analysis of MHC⁺ myotubes (H), myogenic index (I), and expression of myogenin and troponin T (J). (K) H3K9me2 enrichment was analyzed by ChIP in siRNA and siSharp-1 cells in undifferentiated (day 0) and differentiated cells (day 2).

FIGURE 3:

Inhibition of G9a rescues Sharp-1–imposed differentiation block. (A) C2C12 cells overexpressing Sharp-1 were transfected with control siRNA or siG9a. G9a knockdown was determined by Western blot. (B) Cells were induced to differentiate and stained with anti-MHC antibody. Nuclei were stained with DAPI. (C) Differentiation was quantified by calculating myogenic index. (D) Myogenin and troponin T expression was determined by Western blot. (E–G) Sharp-1–overexpressing cells were incubated with DMSO (vehicle) or BIX-01294. Differentiation was assessed using anti-MHC antibody (E), myogenic index (F), and expression of myogenin and troponin T by Western blot (G). (H) Sharp-1–overexpressing cells were treated with DMSO or BIX-01294 for 0 and 2 d. ChIP assays were done using anti-H3K9me2 antibody on the myogenin promoter. (I) C2C12 cells were transfected with Myc–Sharp-1 and MyoD. MyoD was immunoprecipitated and analyzed for association with G9a and methylation in the absence and presence of UNC0638 treatment using anti-G9a and anti-Me Lys antibodies, respectively. Lysates were analyzed for expression of G9a, MyoD, and Sharp-1. (J) C2C12 cells were transfected with Myc–Sharp-1 and MyoD and G9a as indicated. The association of Sharp-1 with G9a was analyzed in the absence and presence of MyoD. (K) Endogenous MyoD methylation and association with G9a was examined by immunoprecipitation from siSharp-1 and siRNA cells using anti-Me Lys and anti-G9a antibodies. Input shows expression of G9a, Sharp-1, and MyoD in lysates. (L) MyoD occupancy on the myogenin promoter was analyzed by ChIP assays in myoblasts overexpressing pBabe, Sharp-1, or G9a. Error bars indicate mean ± SD.

FIGURE 4:

MyoD methylation is relevant in the inhibition of myogenesis by Sharp-1. (A) C2C12 cells overexpressing Sharp-1 were transfected with MyoD and MyoD(K104R). MyoD expression was analyzed by Western blot. (B-D) Control (vector), MyoD, and MyoD(K104R) cells were analyzed for differentiation with anti-MHC antibody (B), myogenic index (C), and troponin T expression by Western blot (D). (E) C3H10T1/2 cells were transfected with MyoD, MyoD(K104R), Myc-Sharp-1, and FLAG-G9a. MyoD was immunoprecipitated and probed with anti-Me Lys antibody. Expression of Sharp-1, MyoD, and G9a was analyzed in lysates. (F) ChIP assays were performed in Sharp-1–overexpressing C2C12 cells transfected with MyoD and MyoD(K104R). H3K9me2 was analyzed by ChIP assays on the myogenin promoter at D2 of differentiation. Error bars indicate mean ± SD.