Fig 2: Myoblast differentiation induces SET7-dependent methylation of the SRF MADS-box.a Immunoprecipitation-based in vivo cellular methylation assay using anti-methylated lysine (anti-a-MeK) antibody revealed that SRF was methylated during C2C12 differentiation. Notably, methylation was increased in the cells cultured in differentiation medium (DM). b HA-tagged truncated mutants of SRF (SRF-A, SRF-AB, and SRF-C) were transfected into C2C12 cells, and then, the cells were subjected to serum deprivation (DM). The SRF MADS-box region was methylated, as determined with a a-MeK immunoprecipitation-based assay. c Transfection of SET7 induced the methylation of SRF in the C2C12 cells. d In vitro methyltransferase assay in complete cell-free conditions showing that the chimeric protein GST-SET7 induced the methylation of GST-SRF. GST protein served as a control. Left: Coomassie blue staining was used to determine the expression of GST proteins. Right: Autoradiograph. e Enzymatically dead SET7 (SET7 H297A) failed to induce the methylation of SRF. f Set7 siRNA attenuated the binding of SRF to the proximal SRE in the Acta1 gene promoter. ChIP analysis. g Expression of SET7 was increased during C2C12 differentiation. h Association between endogenous SET7 and SRF was enhanced in DM.

Fig 3: KDM2B inhibits myoblast differentiation.a Forced expression of KDM2B decreased the expression levels of skeletal muscle genes in C2C12 cells. Treatment with DM for 3 days induced the expression of myosin heavy chain (MHC), MyoD, and muscle creatinine kinase (MCK). These increases were attenuated when KDM2B was overexpressed. The overexpression of KDM2B was performed through the transfection of pCMV-3xFlag-KDM2B. The empty vector pCMV-3xFlag served as a mock control. b qRT-PCR analysis showing that the increased transcript levels of skeletal a-actin (Acta1), Mck, Myogenin, and MyoD were downregulated by the overexpression of KDM2B. c Treatment with DM for 3 days induces elongation and multinucleation of C2C12 cells, which was attenuated by the forced expression of KDM2B. Immunocytochemistry was performed with an anti-MHC antibody. d Direct cell count after immunocytochemical analysis with MHC showing that MHC-positive multinucleated cell count was reduced when KDM2B was transfected. Images from 20–24 different fields were randomly obtained. Both MHC-positive and multinucleated cells, as well as total cells (stained with DAPI) were counted. e Knockdown of Kdm2b with Kdm2b siRNA #1 enhanced skeletal muscle gene expression induced by treatment with DM for 1 day. f qRT-PCR results for Mck and Acta1. g Immunocytochemical analysis with an anti-MHC antibody showed that elongation and multinucleation were enhanced by transfection with Kdm2b siRNA. *p < 0.05; **p < 0.01.

Fig 4: SRF K165 is a biologically active methylation site involved in muscle differentiation.a Promoter analysis using the Acta1 promoter revealed that, among the peptides tested, only SRF K165A lost its basal transcriptional activity. Notably, SRF K154A retained sufficient promoter activity, which was comparable to that of the wild-type peptide. b DM induces Acta1 mRNA expression, which was further potentiated by the transfection with SRF WT. However, this enhancement was not observed when SRF K165A was transfected. c Myogenin mRNA level. d ChIP analysis showing that SRF K165A loses its ability to bind to two SREs (CArG-proximal and CArG-distal) in the Acta1 promoter. To pull down either SRF WT or SRF K165A, an anti-HA antibody was used in the ChIP analysis. e Gel shift assay showing that SRF K165A loses its binding affinity for the distal SRE. f Immunoprecipitation-based in vivo cellular methylation assay showing that the transfection of SET7 induced the methylation of SRF WT, whereas it failed to methylate SRF K165A. g DM induced the methylation of SRF WT, whereas it failed to methylate SRF K165A. h Elongation and tube formation induced by treatment with DM for 1 day was enhanced by transfection with pCGN-HA-SRF WT (SRF WT), whereas this enhancement is not seen with transfection with pCGN-HA-SRF K165A (SRF K165A). Immunocytochemical analysis with anti-MHC antibody. i MHC-positive multinucleated cell count. j Transfection of SET7 enhanced SRF WT-induced transactivation of the Acta1 promoter but failed to activate SRF K165A. * and @p < 0.05; ** and @@p < 0.01; NS, not significant.

Fig 5: KDM2B demethylates SRF WT to mimic SRF K165A.a GST-KDM2B reduced the methylation of SRF in a dose-dependent fashion as indicated by an in vitro methylase assay. b In vitro methylase assay with SRF synthetic peptide. GST-SET7 induced the methylation of the SRF K165 peptide, an effect partially attenuated by the addition of GST-KDM2B. c Immunoprecipitation-based in vivo methylation assay. The DM-induced methylation of SRF was reduced by the transfection of KDM2B in C2C12 cells. d Transfection with pCMV-3x-Flag-KDM2B reduced the pCGN-HA-SRF WT-induced transactivation of the Acta1 promoter, whereas it failed to affect pCGN-HA-SRF K165A. * and @p < 0.05; **p < 0.01; NS, not significant.