Muscle Biology Research Unit, Division of Animal Products Research, NARO Institute of Livestock and Grassland Science, Tsukuba, Japan.
Physiol Genomics. 2021 Feb 1;53(2):69-83. doi: 10.1152/physiolgenomics.00099.2020. Epub 2021 Jan 18.
Skeletal muscles are comprised of two major types of myofibers, fast and slow. It is hypothesized that once myofiber type is determined, muscle fiber-type specificity is maintained by an epigenetic mechanism, however, this remains poorly understood. To address this, we conducted a comprehensive CpG methylation analysis with a reduced representation of bisulfite sequencing (RRBS). Using GFP- mouse, we visually distinguished and separately pooled slow-type and -negative fast-type fibers for analyses. A total of 31,967 and 26,274 CpGs were hypermethylated by ≥10% difference in the fast- and slow-type fibers, respectively. Notably, the number of promoter-hypermethylated genes with downregulated expression in the slow-type fibers was 3.5 times higher than that in the fast-type fibers. Gene bodies of the fast-type-specific myofibrillar genes , , , , and were hypermethylated in the slow-type fibers, whereas those of the slow-type-specific genes , , and were hypermethylated in the fast-type fibers. Each of the instances of gene hypermethylation was associated with the respective downregulated expression. In particular, a relationship between CpG methylation sites and the transcription variant distribution of was observed, suggesting a regulation of alternative promoter usage by gene body CpG methylation. An association of hypermethylation with the regulation of gene expression was also observed in the transcription factors and . These results suggest not only a myofiber type-specific regulation of gene expression and alternative promoter usage by gene body CpG methylation but also a dominant effect of promoter-hypermethylation on the gene expressions in slow myofibers.
骨骼肌由两种主要类型的肌纤维组成，即快肌和慢肌。人们假设，一旦肌纤维类型确定，肌肉纤维类型特异性就会通过表观遗传机制来维持，但这一点仍知之甚少。为了解决这个问题，我们使用亚硫酸氢盐测序的代表性降低（RRBS）进行了全面的 CpG 甲基化分析。使用 GFP-小鼠，我们通过视觉区分并分别汇集慢型和-阴性快型纤维进行分析。在快型和慢型纤维中，分别有 31967 个和 26274 个 CpG 被≥10%的差异高度甲基化。值得注意的是，在慢型纤维中下调表达的启动子高度甲基化基因数量是快型纤维的 3.5 倍。快型特异性肌球蛋白基因 、 、 、 和 的基因体在慢型纤维中被高度甲基化，而慢型特异性基因 、 和 的基因体在快型纤维中被高度甲基化。每种基因的高度甲基化都与相应的下调表达有关。特别是，观察到 CpG 甲基化位点与 转录变体分布之间存在关系，这表明基因体 CpG 甲基化对 替代启动子使用的调节。在转录因子 和 中也观察到了高度甲基化与基因表达调节之间的关联。这些结果不仅表明肌纤维类型特异性通过基因体 CpG 甲基化调节基因表达和替代启动子使用，而且表明启动子高度甲基化对慢肌纤维中基因表达的主导作用。
