| Epigenetics is the study of stable alternation in gene expression and function without changing DNA sequence and leading to inheritable phenotype, mainly including DNA methylation, histone modification, chromatin remodeling and non-coding RNA-mediated regulation. Histone modifications including histone methylation, acetylation, phosphorylation and ubiquitination play crucial roles in gene activation and inactivation.MyoD, one of bHLH protein, play a crucial role in muscle differentiation. In our experiments, Low serum medium was used to induce C2C12 cells to differentiate, and the differentiation mechanism and the binding of associated transcription factors and chromatin modifying enzymes, especially protein arginine methyltransferase 1 to the myogenin promoter were investigated.The morphology of C2C12 cells are triangular or spindly, and fewer are polygonal. After transferred to low serum medium, the C2C12 cells align regularly and acquire typical myogenic modality gradually, such as a more elongated shape, and multinucleated myofiber-like structure. when the C2C12 cells are treated with adenosine dialdehyde (AdOx) to block the PRMT, Low serum medium induced C2C12 cells differentiation was inhibit. The morphology of C2C12 cells are more big and don't present a multinucleated myofiber-like structure. Moreover, the increase of protein level and mRNA of myogenin was prevented by AdOx during C2C12 cells differentiation. In contrast, the protein level of PRMT1, PCAF and CARM1 was not changed during C2C12 cells differentiation in the presence of AdOx. These data indicate that arginie methylation is involved in muscle differentiation.Dual-luciferase reporter assay showed that PRMT1 and CARM1 can enhanced the myogenin gene reporter activity when we transfected the C2C12 cells with each member of PRMTs. We further co-transfected C2C12 cells with PRMT1, CARM1 and PCAF, the myogenin gene reporter activity assays showed that PRMT1 synergistically coactivates together with CARM1 and PCAF MyoD-mediated transcription at the myogenin promoter. Chromatin Immunoprecipitation (ChIP) assay was developed in recent years to detect protein factors that bind to chromatin. The binding of MyoD, PRMT1, PCAF and CARM1 to the myogenin promoter in control or differentiated C2C12 cells induced by low serum medium was studied by this method. We found that these factors were recruited on the myogenin gene promoter after induced for 3 hours in the low serum medium.In vitro GST-pulldown assays showed that MyoD and PRMT1 exist in a complex, And the binding domain required for the interaction between MyoD and PRMT1 was determined, while the C-terminal domain of MyoD don't interact with PRMTl. We further subcloned MyoD into pEGFP-C1 and PRMT1 into pmCherry-C1, which express GFP-MyoD and Cherry-PRMT1 respectively. When C2C12 were co-transfected with these fusion protein plasmids, MyoD and PRMT1 were visualized by autofluorescence (green) of GFP and (red) of Cherry with a confocal microscopy, respectively. Green fluorescence of GFP-MyoD and red fluorescence of Cherry-PRMT1 merge well (yellow) in nucleus.To demonstrate the direct interaction between CARM1 with PCAF, the in vitro and in vivo binding assays were done. And the binding domain required for the interaction between CARM1 and PCAF was determined. C-terminal domain of CARM1 (CARM11-141) can not interacts with PCAF, and PCAF HAT (histone acetyltransferase) domain interacts with CARM1.In vitro methylation reaction assay and autoradiography indicated that, among the members of protein arginine methyltransferase family, only PRMT1 methylates MyoD. To investigate the occurrence of in vivo methylation of MyoD, we performed metabolical labeling of HEK293T cells with L-[methy-3H]-methionine. Cells were transfected with Flag-tagged MyoD in the absence and presence of AdOx and simultaneously metabolical labeling was performed for 1h in the presence of translational inhibitors. Flag-MyoD protein was immunopurified from the cell extracts, resolved by SDS-PAGE and methylation was detected by autoradiography. The data indicate that in vivo methylation of MyoD occurs and was inhibited in the presence of AdOx. In order to map the methylation site(s) in MyoD, we expressed several N-and C-terminal deletion constructs of MyoD as GST-tagged fusions in bacteria. In vitro methylation in the presence of GST-PRMT1 revealed that the methylation site(s) of PRMT1 in MyoD are located between amino acid 102 and 318, and which encompass the bHLH domain and the C-terminal domain.To identify the exact site(s) of PRMT1 methylation in MyoD we methylated MyoD in the presence of GST-PRMT1 in vitro. The ESI mass spectrum revealed a doubly charged peak at m/z 910.36 corresponding to the dimethylated sequence of the tryptic MyoD fragment RQNGYDTAYYSEAVR (amino acids 221-235). Fragment ion (MS/MS) spectrum resulting from this doubly charged precursor clearly indicated that the first arginine within the peptide (R221 in the full-length context of MyoD) is dimethylated. In conclusion, PRMTs play an positive role in regulating the low serum medium induced C2C12 cells differentiation. Histone arginine methylation by PRMTs along with other histone modifications and the activation of transcription factors that jointly exert an increased expression of myogenin gene and result in the C2C12 cells to differentiation. We found for the first time that PRMT1 methylate MyoD in vitro and in vivo. Further studies on the regulatory functions of PRMT1 in C2C12 cell differentiation shed lights on our understanding of muscle differentiation.
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