LC-MS-MS quantitative analysis reveals the association between FTO and DNA methylation.
LC-MS-MS quantitative analysis reveals the association between FTO and DNA methylation.
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LC-MS-MS 定量分析揭示 FTO 与 DNA 甲基化之间的关联
DOI：
10.1371/journal.pone.0175849
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发表时间：
2017
期刊：
影响因子：
3.7
通讯作者：
Wang L
中科院分区：
文献类型：
--
作者：
Zhu Y;Zhou G;Yu X;Xu Q;Wang K;Xie D;Yang Q;Wang L
脂肪质量与肥胖相关蛋白是一种依赖于α的酮戊二酸双加氧酶，负责使单链核糖核酸中的N6-甲基腺苷(M6A)、3-甲基胸腺胺(M3T)和3-甲基尿嘧啶(M3U)去甲基化。它的其他功能尚不清楚，但数以千计的哺乳动物DNA显示5-甲基-2‘-脱氧胞苷(5mdC)修饰和5mdC去甲基酶是哺乳动物能量平衡和生育所必需的。在这里，我们的目的是确认FTO蛋白是否能使DNA中的5mdC去甲基化。然而，通过LC-MS-MS分析，我们发现FTO在体内外对5MDC没有很强的去甲基化活性。结果表明，FTO脱甲基酶具有较高的底物专一性和选择性。此外，我们还利用免疫荧光技术证明了野生型TET2的过表达，而FTO和突变型TET2在Hela细胞中的过表达导致5mdC产生更高水平的5-羟甲基-2‘-脱氧胞苷(5hmdC)。综上所述，我们的结果不仅揭示了FTO的酶活性，而且可能有助于未来发现参与表观遗传修饰功能的蛋白质。
Fat mass and obesity-associated protein (FTO) is α-ketoglutarate-dependent dioxygenase and responsible for demethylating N6-methyladenosine (m6A) in mRNA, 3-methylthymine (m3T) in single-stranded DNA (ssDNA) and 3-methyluracil (m3U) in single-stranded RNA (ssRNA). Its other function remains unknown but thousands of mammalian DNA show 5-methyl-2'-deoxycytidine (5mdC) modification and 5mdC demethylases are required for mammalian energy homeostasis and fertility. Here, we aimed to confirm whether FTO proteins can demethylate 5mdC in DNA. However, we found that FTO exhibits no potent demethylation activity against 5mdC in vitro and in vivo by using liquid chromatography-tandem mass spectrometry (LC-MS-MS). The result showed FTO demethylase has the characteristics of high substrates specificity and selectivity. In addition, we also used immunofluorescence technique to demonstrate overexpression of wild type TET2, but not FTO and mutant TET2 in Hela cells results in higher levels of 5-hydroxymethyl-2'-deoxycytidine (5hmdC) generated from 5mdC. In conclusion, our results not only reveal the enzymatic activity of FTO, but also may facilitate the future discovery of proteins involved in epigenetic modification function.