TET-mediated oxidation of 5-mC participates in both passive and active DNA demethylation, which exerts a significant influence on diverse biological processes. Mass spectrometry has identified multiple phosphorylation sites of TET2. However, the functions of these phosphosites and their corresponding kinases are mostly unknown. Here, we showed that AMP-activated protein kinase (AMPK) phosphorylates murine TET2 at the serine residue 97 (S97), and the phosphorylation enhances TET2 stability through promoting its binding to 14-3-3β. AMPK ablation resulted in decreased global 5-hmC levels at the myotube stages, severe differentiation defects of C2C12 cells and significantly, total loss of expression of Pax7. Genome-wide analyses revealed increased DNA methylation at genic and enhancer regions of AMPK-null myoblasts and myotubes. Using CRISPR/Cas9 technology, we showed that a novel enhancer, which is hypermethylated in AMPK-null cells, regulates Pax7 expression. The phospho-mimicking mutant, TET2-S97E, could partly rescue the differentiation defect in AMPK-ablated C2C12 cells. Together, our data demonstrated that AMPK is a critical regulator of myogenesis, partly through phosphorylating TET2.

中文翻译：

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AMPK 对 TET2 的磷酸化在肌原性分化中是必不可少的。

TET 介导的 5-mC 氧化参与被动和主动 DNA 去甲基化，这对多种生物过程产生重大影响。质谱法已鉴定出 TET2 的多个磷酸化位点。然而，这些磷酸位点及其相应激酶的功能大多是未知的。在这里，我们展示了 AMP 活化蛋白激酶 (AMPK) 在丝氨酸残基 97 (S97) 处磷酸化鼠 TET2，并且磷酸化通过促进其与 14-3-3β 的结合来增强 TET2 的稳定性。AMPK 消融导致肌管阶段的整体 5-hmC 水平降低、C2C12 细胞的严重分化缺陷和显着的 Pax7 表达完全丧失。全基因组分析显示 AMPK 无效的成肌细胞和肌管的基因和增强子区域的 DNA 甲基化增加。使用 CRISPR/Cas9 技术，我们展示了一种在 AMPK 无效细胞中高度甲基化的新型增强剂，可调节 Pax7 表达。磷酸化模拟突变体 TET2-S97E 可以部分挽救 AMPK 消融的 C2C12 细胞的分化缺陷。总之，我们的数据表明 AMPK 是肌生成的关键调节剂，部分是通过磷酸化 TET2。