Research On The Mechanisms Of Tet2 Regulating Skeletal Myoblast Differentiation In Vitro

Methylation at the 5 position of cytosine (C) in DNA is an important epigenetic modification, which is involved in regulation of a diverse range of biological processes, such as genomic imprinting, X chromosome inactivation, chromatin modification, and regulation of gene expression. The recent discovery that ten-eleven translocation (TET) family enzymes, including Tetl, Tet2 and Tet3, can oxidize 5-methylcytosine, which has greatly advanced our understanding of DNA demethylation. The three mammalian Tet proteins are Fe2+ and 2-oxoglutarate-dependent dioxygenases that successively oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) in DNA. Tet proteins have roles in diverse biological processes, including epigenetic regulation of gene transcription, embryonic development, stem cell function and cancer, but the mechanisms on regulation of skeletal myogenesis are still poorly defined. Skeletal muscle is essential for whole-body to produce force and motion,but many aspects of the regulatory mechanisms underlying skeletal myogenesis are unclear. Our research focuses on exploring the function of Tet and vitamin C in skeletal myoblast differentiation.Firstly, we found, by immunostaining method, that after differentiation induction of C2C12 myoblasts, the levels of 5-hmC was extremely higher in differentiated myotubes than in undifferentiated C2C12 myoblasts, while 5mC level was generally lower in differentiated cells than in undifferentiated cells. Meanwhile, both Tetl and Tet2 expression were upregulated after differentiation. Immunostaining indicated that Tet2 protein was localized, in dotted patterns, in the nuclei of C2C12 myoblasts and differentiated myotubes.Next, we investigated the fuction of Tet in myoblast differentiation. We constructed a vector that expresses the catalytic domain of Tetl or Tet2.Overexpression of Tet2 in C2C12 cells led to significantly increased expression of myoblast differentiation-associated genes (Myog, Myf6 and myomaker), while the expression of these genes was not significantly altered by overexpression of Tetl. We then examined the influence of Tetl-or ret2-knockdown by siRNA on expression of myogenesis-associated genes. The results showed that knockdown of Tet2, but not Tetl, significantly downregulated the expression of myoblast differentiation-associated genes. we used short hairpin RNA (shRNA)-expressing plasmid to achieve prolonged silence of Tet2 expression. We found that shRNA-mediated Tet2-knockdown impaired C2C12 differentiation, and decreased the expression of Myog, Myf6 and myomaker in either myoblasts or myotubes. To further understand the regulation of expression of these genes, the DNA methylation status of Myog, Myf6 and myomaker were determined by bisulfite sequencing PCR. Methylation levels of Myog and myomaker promoters were significantly increased in Tet2-knockdown cells as compared to the non-knockdown cells.. Together, our results suggest that Tet2 involves in DNA demethylation of some specific gene loci, regulates the expression of relevant genes, such as Myog and myomaker, and play an important role on C2C12 differentiation.It was demonstrated recently that vitamin C can enhance the 5hmC generation through promoting Tet activity. In our study, we found that vitamin C treatment significantly increased the 5hmC levels in a concentration-dependent manner, induced demthylation of Myog and myomaker promoters and improved gene expression in C2C12 cells, we examined the function of vitamin C in Tet2-knockdown cells. The results showed that knockdown of Tet2 dramatically decreased the efficacy of vitamin C in enhancing the 5hmC generation, Myog/myomaker expression and myotube formation. These results demonstrate that the effect of vitamin C is dependent on Tet2-involved pathway.In conclusion, Tet2 expression are upregulated during myoblast differentiation. Tet2 mediated DNA demethylation is an important mechanism for promoting myogenic genes expression and accelerating differentiation of skeletal muscle cells. Besides, vitamin C promotes skeletal myoblast differentiaion in a Tet2-dependent manner.