Phosphorylation of TET2 by AMPK is indispensable in myogenic differentiation [MeDIP-seq]

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), 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. MeDIP sequencing of WT and AMPK-/- C2C12 cells at day 0 and day 8 of differentiation. Two clones of AMPK-/- cells were used and input DNA controls were included for all samples.

TET介导的5-甲基胞嘧啶(5-mC)氧化过程同时参与被动与主动DNA去甲基化，对多种生物学过程具有显著调控作用。质谱技术已鉴定出TET2的多个磷酸化位点，但这些磷酸化位点的功能及其对应的激酶大多尚未明确。本研究证实，AMP活化的蛋白激酶(AMP-activated protein kinase, AMPK)可在丝氨酸残基97位(S97)磷酸化小鼠TET2，并通过促进其与14-3-3β的结合增强TET2的稳定性。AMPK敲除会导致肌管阶段全局5-羟甲基胞嘧啶(5-hmC)水平下降，C2C12细胞出现严重的分化缺陷，且尤为关键的是Pax7基因表达完全丧失。全基因组分析显示，AMPK敲除的成肌细胞与肌管的基因区域及增强子区域的DNA甲基化水平升高。本研究利用CRISPR/Cas9技术发现，一个在AMPK敲除细胞中呈高甲基化状态的新型增强子可调控Pax7的表达。磷酸化模拟突变体TET2-S97E可部分挽救AMPK敲除C2C12细胞的分化缺陷。综上，本研究数据证实AMPK是肌发生过程的关键调控因子，其部分作用机制通过磷酸化TET2实现。本研究针对分化第0天与第8天的野生型(Wild Type, WT)及AMPK敲除(AMPK-/-) C2C12细胞开展了甲基化DNA免疫沉淀测序(MeDIP sequencing)；实验使用了两株AMPK敲除细胞克隆，并为所有样本设置了输入DNA对照。