The Effect Of DNA Methylations On The Muscle Satellite Cell Myogenic Differentiation Of Myostatin Gene Edited Cattle

Myostatin(MSTN)is mostly expressed in skeletal muscle and plays a crucial role in the negative regulation of muscle mass development.Knockout of MSTN promotes muscle development but inhibits lipogenesis,which result in improved muscle and reduced fat.DNA methylation is also an important factor in the regulation of muscle development,but the interaction between MSTN and DNA methylation in muscle satellite cell differentiation has not been reported.In the present study,the effect of DNA methylation on myogenic differentiation of MSTN mutant cattle skeletal muscle satellite cells was investigated.In the first exprement,muscle tissue of MSTN mutant and the wild-type cattles were tested for transcriptome analysis,721 differentially expressed genes(DEGs)containing 647 down-regulated and 74 up-regulated genes were enriched.The most significantly enriched GO terms of biological process were related to the immune system,the positive regulation of biological processes,the developmental process,the response to stimulation,growth,and metabolic processes.The GO terms of muscle development,osteogenic differentiation,protein phosphorylation,cell proliferation,cell differentiation,regulation of gene expression,apoptosis,and fatty acid metabolism were also enriched.There were 165 down-regulated signaling pathways of which 47 were significant different(P value < 0.05),the down-regulated KEGG pathway enriched were related to muscle development,fat metabolism,osteoclast differentiation,calcium signaling pathway,longevity,growth hormone signaling pathway and cell cycle correlation.There were 21 up-regulated pathways of which 3 were significant differences(P value < 0.05),the up-regulated KEGG pathway enriched were related to pentose phosphate pathway and myocardial contraction.The differentially expressed genes also contained the DNA methylation enzyme,DNMT family and the demethylase,TET family.MSTN mutant down-regulated the DNMTs while up-regulated the TETs.Thes results showed that there was an important link between MSTN and DNA methylation.In experiment 2,the muscle satellite cells of wild-type and MSTN mutant cattle were isolated by tissue block attachment method.The cell surface antigens of the two cells were studied by flow cytometry.The marker proteins of the cells were studied by immunofluorescence.The isolated muscle satellite cells were identified by myogenic,adipogenic and osteogenic induction.The cell proliferation was detected by cell cycle detection and Edu proliferation assay.Identifications of the cells showed that the MSCs markers CD90 and CD105 were positively expressed,while the hematopoietic lineage marker CD34 was negatively expressed in both the MT and WT derived cells.Further identification of the cells by differentiation induction showed that both of the MT and WT cells positively expressed the myogenic markers of MyoD and MHC during myogenic differentiation.The presumptive satellite cells also had abilities of adipogenic and osteogenic differentiation.The expression of MSTN in both mRNA and protein levels significantly decreased in the MT derived satellite cells compared to the WT cells.These results demonstrated that the isolated cells were MSTN mutant or wild type cattle muscle satellite cells.Assay of proferliation showed that MSTN mutant promoted the cell proliferation.Then we detected the expression of cell cycle-related genes,results showed that the genes promoted cell cycle were up-regulated and the the genes inhibited cell cycle were down-regulated,of which the down-regulation of CDKN1 C was most significant.It was speculated that MSTN mutant mainly promoted the expression of CyclinA-CDK2 by inhibiting the expression of CDKN1 C,thereby promoting DNA synthesis and promoting cell cycle.Exprement 3 was designed to investigate the role of DNA methylation in myogenic differentiation of MSTN-deficient muscle satellite cells.The myotube fusion rate and the myotube length were measured during myogenesis.It was found that MSTN mutant promoted myogenic differentiation.Then the expression of myogenic genes were detected,results showed that MSTN mutant promoted the expression of these genes,the methylation levels in the promoters and gene bodies of MyoD,MyoG,PAX3 and PAX7 were significantly decreased.These results indicating that MSTN mutant demethylated the myogenic gens thus to promote their expression.Further studies found that demethylation of myogenic factors caused by MSTN mutant is achieved by up-regulating the demethylase TET1.Known that SMAD2 and SMAD3 are downstream of MSTN type II receptors,we hypothesized that the SMAD2 and SMAD3 transcription factors probably have direct interaction with TET1.Prediction of the binding site by JASPAR database showed that SMAD2/SMAD3 bind to the promoter region of TET1.The ChIP-qPCR with an anti-SMAD2/SMAD3 monoclonal antibody further proved that SMAD2/SMAD3 virtually bind to TET1 promoter region.Luciferase reporter assay indicated that overexpression of SMAD3 inhibited the activity of TET1 promoter.These results showed that that MSTN mutant demethylated myogenic-specific genes by up-regulation of TET1 which is directly controlled by SMAD2/SMAD3.Overexpression of TET1 in wild type cells promoted myogenic differentiation,myotube index increased and reduced methylations of the associated genes.In the contrary,knockdown of TET1 of the MSTN mutant cells resulted in the opposite phenomena as occurred in the overexpressed cells.In conclusion,mutant of MSTN resulted in increased activity of TET1,which induced higher levels of demethylations and improved activities of the myogenic associated genes.Signals of SMAD2/SMAD3 directly bind to the TET1 promoter region,which indicated that MSTN mutant demethylated myogenic-specific genes by up-regulation of TET1 which is directly controlled by SMAD2/SMAD3.In summary,MSTN mutant and wild-type bovine muscle satellite cells were isolated in this study.Cell cycle detection showed that MSTN mutant mainly promoted the expression of CyclinA-CDK2 by inhibiting the expression of CDKN1 C,thereby promoting DNA synthesis and cell cycle.Mutant of MSTN resulted in increased activity of TET1,which induced higher levels of demethylations and improved activities of the myogenic associated genes.Signals of SMAD2/SMAD3 directly bind to the TET1 promoter region,which indicated that MSTN mutant demethylated myogenic-specific genes by up-regulation of TET1 which is directly controlled by SMAD2/SMAD3.This study,for the first time,explored the regulatory mechanism between MSTN and DNA methylation,providing a basis for studying the relationship between MSTN and epigenetics.