Identification of target genes for DNA methylation in skeletal muscle and its medical application

骨骼肌DNA甲基化靶基因的鉴定及其医学应用

• 批准号: 23659468
• 负责人: OGAWA Yoshihiro
• 金额: $ 2.5万
• 依托单位: Tokyo Medical and Dental University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Challenging Exploratory Research
• 财政年份: 2011
• 资助国家: 日本
• 起止时间: 2011 至 无数据
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-23659468/
• 关键词: 骨格筋; DNA; メチル化; エピジェネティクス; 生活習慣病; 遺伝子発現; DNAメチル化; 骨格筋; DNA; メチル化; エピジェネティクス; 生活習慣病; 遺伝子発現; DNAメチル化

DNA methylation is essential for normal embryonic development, and altered DNA methylation patterns have been implicated in tumorigenesis. An epigenetic mechanism involving DNA methylation has also been suggested to be involved in the regulation of metabolic processes ; however, the molecular basis of this mechanism has not been clearly demonstrated. In this study, we attempted to get an insight into the role of DNA methylation in skeletal muscle, which plays important roles in exercise, energy expenditure and glucose metabolism. We made skeletal muscle-specific knockout(KO) mice with Dnmt3a(a de novo DNA methyltransferase highly expressed in skeletal muscle) by crossing Dnmt3a flox/flox mice and transgenic mice expressing Cre recombinase, driven by the skeletal muscle alpha-actin promoter. A quantitative real-time PCR analysis confirmed that Dnmt3a mRNA levels were markedly diminished in the skeletal muscle but not in other tissues of the KO mice. In this study, a genome-wide DNA methylation analysis called Microarray-based Integrated Analysis of Methylation by Isoschizomers(MIAMI) was preformed using the methylation-sensitive restriction enzyme HpaII and a genome microarray. MIAMI analysis revealed a marked decrease in DNA methylation in the KO mice, including that of several genes coding transcription factors. Despite this, the decreased DNA methylation did not correlate with the gene expression levels under the same condition. Because DNA methylation is considered a key epigenetic contributor in the maintenance of gene silencing, gene expression in the KO mice may be modified in the presence of additional metabolic stress. Therefore, analysis of metabolic phenotype of the KO mice will be essential to elucidate the epigenetic regulation of skeletal muscle gene expression and skeletal muscle-related metabolic diseases.

DNA甲基化对于正常的胚胎发育至关重要，而DNA甲基化模式的改变已与肿瘤发生有关。还建议涉及DNA甲基化的一种表观遗传机制参与代谢过程的调节。但是，尚未明确证明这种机制的分子基础。在这项研究中，我们试图深入了解DNA甲基化在骨骼肌中的作用，骨骼肌在运动，能量消耗和葡萄糖代谢中起着重要作用。我们通过跨越DNMT3A Flox/Flox小鼠和转基因小鼠，用DNMT3A（从骨骼肌肉中高表达在骨骼肌肉中高表达的从头DNA DNA甲基转移酶）制作骨骼肌肉特异性敲除（KO）小鼠。定量实时PCR分析证实，骨骼肌中DNMT3A mRNA水平显着降低，但在KO小鼠的其他组织中却没有明显降低。在这项研究中，使用对甲基化敏感的限制酶HPAII和基因组微阵列预构造了全基因组DNA甲基化分析，称为基于微阵列的基于微阵列的甲基化综合分析（迈阿密）。迈阿密分析显示，KO小鼠的DNA甲基化显着降低，包括编码转录因子的几种基因。尽管如此，DNA甲基化降低与在相同条件下的基因表达水平无关。由于DNA甲基化被认为是维持基因沉默的关键表观遗传学贡献者，因此在存在额外的代谢应激的情况下，可以改变KO小鼠中的基因表达。因此，对KO小鼠的代谢表型的分析对于阐明骨骼肌基因表达和骨骼肌相关的代谢性疾病的表观遗传调节至关重要。