Division of Animal and Nutritional Sciences, West Virginia University, Morgantown, WV, 26506-6108, USA.
Department of Biological Sciences, University of Delaware, Newark, DE, USA.
Mar Biotechnol (NY). 2017 Aug;19(4):321-327. doi: 10.1007/s10126-017-9756-6. Epub 2017 Jun 3.
MyoD is an important myogenic transcription factor necessary for the differentiation of myogenic precursor cells (MPC) to form mature myotubes, a process essential for muscle growth. Epigenetic markers such as CpH methylation are known gene regulators that are important for the differentiation process. In this study, we investigated whether DNA methylation is a potential mechanism associated with the ability of 17β-estradiol (E2) to reduce MyoD gene expression and muscle growth in rainbow trout. Rainbow trout received a single intraperitoneal injection of E2 or the injection vehicle (control). Skeletal muscle was collected 24 h post injection and analyzed for DNA methylation within the MyoD gene and the expression of DNA methyltransferases. CpG islands of the MyoD gene were predicted using MethPrimer software, and these regions were PCR amplified and analyzed using bisulfite sequencing. The percent methylation of the targeted CpG did not differ between control and E2-treated fish. However, percent CpH methylation in the MyoD exon 1 region was elevated with E2 treatment. Two of the methylated CpH sites were located in conserved transcription factor binding motifs, estrogen response element (ERE), and Myc binding site. Quantitative real-time PCR analysis revealed a significant increase in expression of DNA methyltransferases, Dnmt3a and Dnmt3b, in E2-treated muscle, suggesting an increased genome methylation. Differential CpH methylation in MyoD gene of control and E2-treated fish suggests an epigenetic mechanism through which E2 decreases MyoD gene expression and contributes to reduced muscle growth.
MyoD 是一种重要的肌肉生成转录因子,对于肌原细胞(MPC)分化为成熟的肌管至关重要,这是肌肉生长的关键过程。已知表观遗传标记如 CpH 甲基化是重要的基因调控因子,对于分化过程至关重要。在这项研究中,我们研究了 DNA 甲基化是否是与 17β-雌二醇(E2)降低虹鳟鱼 MyoD 基因表达和肌肉生长能力相关的潜在机制。虹鳟鱼接受了单次腹腔内注射 E2 或注射载体(对照)。注射后 24 小时收集骨骼肌,分析 MyoD 基因内的 DNA 甲基化和 DNA 甲基转移酶的表达。使用 MethPrimer 软件预测 MyoD 基因的 CpG 岛,这些区域通过亚硫酸氢盐测序 PCR 扩增并进行分析。靶 CpG 的甲基化百分比在对照组和 E2 处理组的鱼之间没有差异。然而,MyoD 外显子 1 区域的 CpH 甲基化百分比随着 E2 处理而升高。两个甲基化的 CpH 位点位于保守的转录因子结合基序中,即雌激素反应元件(ERE)和 Myc 结合位点。定量实时 PCR 分析显示,E2 处理的肌肉中 DNA 甲基转移酶 Dnmt3a 和 Dnmt3b 的表达显著增加,表明基因组甲基化增加。MyoD 基因中对照组和 E2 处理组的差异 CpH 甲基化表明,E2 通过表观遗传机制降低 MyoD 基因表达并导致肌肉生长减少。