MSTN Mutant Promotes Myogenic Differentiation by Increasing Demethylase TET1 Expression via the SMAD2/SMAD3 Pathway

Abstract

Myostatin (MSTN) is mostly expressed in skeletal muscle and plays crucial roles in the negative regulation of muscle mass development. The methylation and demethylation of myogenesis-specific genes are major regulatory factors in muscle satellite cell differentiation. The present study was designed to investigate the mechanism of myogenic differentiation regulated by MSTN mutation (MT) and the methylation/demethylation state of downstream genes. The results showed that, in the MSTN-/+ satellite cells, a higher myotube fusion index and a larger myotube length were observed compared to the wild type controls; the genes associated with myogenesis were all up-regulated compared to the WT controls. The methylation of the promoters and gene bodies of PAX3, PAX7, MyoD, and MyoG were all down-regulated, while the expression of the key demethylase TET1 was significantly promoted. ChIP-qPCR was used to demonstrate that the SMAD2/SMAD3 complex combined with the promoter of TET1 to inhibit the activity of TET1 promoter, indicating that MSTN may regulate TET1 via SMAD2/SMAD3. The overexpression of TET1 in wild type cells promoted myogenic differentiation, increased the myotube index, and reduced the methylation of the associated genes. On the contrary, the knockdown of TET1 in the MSTN mutant cells resulted in the opposite phenomena as in the overexpressed cells. In conclusion, the myostatin mutant showed an increased transcriptional activity of TET1, inducing higher levels of demethylation and improving the transcriptional activity levels of myogenic differentiation-associated genes. The binding of SMAD2/SMAD3 directly to the TET1 promoter region indicated that the MSTN mutant demethylated the myogenesis-specific genes by up-regulating TET1, which is directly controlled by SMAD2/SMAD3.

Myostatin site‐directed mutation and simultaneous PPARγ site‐directed knockin in bovine genome

“…Many studies have shown that MSTN has a negative regulatory effect on skeletal muscle growth and inhibits the formation and differentiation of muscle cells. The mutation or deletion of MSTN can promote the proliferation and hypertrophy of muscle cells and fibers (Clop et al, 2006; L. Gao et al, 2020; Kishioka et al, 2008; Zhang et al, 2019). The overexpression of MSTN activates the Smad2/3 and p38MAPK pathways, reduces CDK2 protein and cyclin D expression (W. Yang, Zhang, Li, Wu, & Zhu, 2007), increases the expression of its inhibitor p21, reduces the active CDK2 cyclin complex, inhibits the phosphorylation of the Rb protein, achieves the arrest of myoblasts in the G1 phase, and inhibits the proliferation of myocytes.…”

MSTN mutant promoted bovine muscle satellite cell proliferation by regulating the binding of SMAD2/SMAD3 with CDKN1C

“…Western blotting was performed according to reference of our previously reported [16]. Brie y, the protein of cell samples were extracted using Radio Immunoprecipitation Assay (RIPA) buffer, then boiled and mixed with the loading buffer in proportion, electrophoresed in a 10% SDS-polyacrylamide gel and transferred onto a polyvinylidene di uoride membrane by electroblotting.…”

“…Muscle satellite cells (MSCs) cultured from cattle of MSTN mutant (MT) and wild type (WT) were obtained and identi ed as our previously reported [16]. MSCs of both MT and WT were cultured in DMEM supplemented with 10% horse serum (HS) and 20% fetal bovine serum (FBS) with 5% CO 2 at 38.5°C.…”

“…The primers used for PCR are listed in Table S1. DNA ampli cation was performed as the previously described program: 95°C for 30 s, followed by 40 cycles at 95°C for 5 s, 60°C for 34 s, and a nal melting curve stage [16]. The cycle threshold (Ct) values of housekeeping gene GAPDH were used to normalized the targeted genes using the 2 -ΔΔCt method [17].…”

“…But the further mechanism of MSTN regulating cell cycles were not well known. In this study, we used MSTN mutant bovine muscle satellite cells (MSCs) described in the previous study [16], detected the effect of MSTN mutant to cell cycles and the regulating pathway in the MSTN mutant bovine muscle satellite cells we obtained.…”