Lyu Cong, Niu Yamei, Lai Weiyi, Wang Yu, Wang Yaning, Dai Peibin, Ma Chunhui, Chen Shaokun, Li Yao, Jiang Guibin, Liang Zhiyong, Ma Wenbin, Gao Zhengliang, Tong Wei-Min, Wang Hailin
The State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing, China.
University of Chinese Academy of Sciences, Beijing, China.
Cell Discov. 2022 Apr 30;8(1):39. doi: 10.1038/s41421-022-00399-x.
The entity of DNA N-methyladenine (6mA) in mammals remains elusive and subsequently its roles in diseases are poorly understood. Here we exploited a bacterial DNA contamination-free and ultrasensitive UHPLC-MS/MS assay to reassess DNA 6mA in human glioblastomas and unveiled that DNA 6mA (~0.08 ppm) is extremely rare. By the use of two independent heavy stable isotope-labeling strategies, we further prove that the observed 6mA is solely generated by DNA polymerase-mediated misinocorporation. In vitro experiments point toward that the generation of misincorporated DNA 6mA is associated with the cellular stresses-caused release of RNA N-methyladenine (mA) nucleoside, which is profoundly inhibited by hypoxia milieu. Consistently, compared with normal brain tissues, DNA 6mA decreases in hypoxic human gliomas. Our data also strongly support that rare DNA 6mA rather than relatively abundant DNA 5-methylcytosine and 5-hydroxymethylcytosine is a hallmark of poor prognosis of IDH1/2 mutation-absent glioblastoma patients, reflecting the incidence of cytotoxic stresses and subsequent release of mA nucleoside. The released mA nucleoside may selectively preserve a subset of the glioblastoma cells and stimulate their stemness and proliferation. Noteworthily, demethylation-inhibiting IDH1 mutation increases the DNA 6mA content in human gliomas, but the depletion of the demethylase candidate ALKBH1 fails to do so, together suggesting the presence of other unknown 6mA demethylase for erasing misincorporated DNA 6mA. This is the first report on the identification of the misincorporated 6mA together with its origin and roles in diseases.
哺乳动物中DNA N-甲基腺嘌呤（6mA）的实体仍然难以捉摸，因此其在疾病中的作用也知之甚少。在这里，我们利用一种无细菌DNA污染的超灵敏UHPLC-MS/MS检测方法，重新评估了人类胶质母细胞瘤中的DNA 6mA，发现DNA 6mA（约0.08 ppm）极其罕见。通过使用两种独立的重稳定同位素标记策略，我们进一步证明观察到的6mA完全是由DNA聚合酶介导的错误掺入产生的。体外实验表明，错误掺入的DNA 6mA的产生与细胞应激导致的RNA N-甲基腺嘌呤（mA）核苷的释放有关，而缺氧环境会强烈抑制这种释放。一致的是，与正常脑组织相比，缺氧的人类胶质瘤中DNA 6mA减少。我们的数据还有力地支持，罕见的DNA 6mA而非相对丰富的DNA 5-甲基胞嘧啶和5-羟甲基胞嘧啶是IDH1/2突变缺失的胶质母细胞瘤患者预后不良的标志，反映了细胞毒性应激的发生率以及随后mA核苷的释放。释放的mA核苷可能会选择性地保留一部分胶质母细胞瘤细胞，并刺激它们的干性和增殖。值得注意的是，抑制去甲基化的IDH1突变会增加人类胶质瘤中的DNA 6mA含量，但去甲基化酶候选物ALKBH1的缺失却没有这种作用，这共同表明存在其他未知的6mA去甲基化酶来消除错误掺入的DNA 6mA。这是关于错误掺入的6mA及其起源和在疾病中的作用的首次报道。
