Department of Chemistry and Institute for Biophysical Dynamics, University of Chicago, Chicago, IL 60637, USA.
Cell. 2013 Apr 25;153(3):678-91. doi: 10.1016/j.cell.2013.04.001. Epub 2013 Apr 18.
TET proteins oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). 5fC and 5caC are excised by mammalian DNA glycosylase TDG, implicating 5mC oxidation in DNA demethylation. Here, we show that the genomic locations of 5fC can be determined by coupling chemical reduction with biotin tagging. Genome-wide mapping of 5fC in mouse embryonic stem cells (mESCs) reveals that 5fC preferentially occurs at poised enhancers among other gene regulatory elements. Application to Tdg null mESCs further suggests that 5fC production coordinates with p300 in remodeling epigenetic states of enhancers. This process, which is not influenced by 5hmC, appears to be associated with further oxidation of 5hmC and commitment to demethylation through 5fC. Finally, we resolved 5fC at base resolution by hydroxylamine-based protection from bisulfite-mediated deamination, thereby confirming sites of 5fC accumulation. Our results reveal roles of active 5mC/5hmC oxidation and TDG-mediated demethylation in epigenetic tuning at regulatory elements.
TET 蛋白将 5-甲基胞嘧啶（5mC）氧化为 5-羟甲基胞嘧啶（5hmC）、5-甲酰胞嘧啶（5fC）和 5-羧基胞嘧啶（5caC）。哺乳动物 DNA 糖苷酶 TDG 切除 5fC 和 5caC，表明 5mC 氧化参与 DNA 去甲基化。在这里，我们通过化学还原与生物素标记相结合，确定了 5fC 的基因组位置。在小鼠胚胎干细胞（mESCs）中的全基因组 5fC 图谱显示，5fC 优先出现在其他基因调控元件中的 poised 增强子中。在 Tdg 缺失的 mESCs 中的应用进一步表明，5fC 的产生与 p300 协调重塑增强子的表观遗传状态。这一过程不受 5hmC 影响，似乎与 5hmC 的进一步氧化以及通过 5fC 实现去甲基化有关。最后，我们通过基于羟胺的保护避免亚硫酸氢盐介导的脱氨作用，在碱基分辨率上解析了 5fC，从而证实了 5fC 积累的位点。我们的结果揭示了活跃的 5mC/5hmC 氧化和 TDG 介导的去甲基化在调节元件的表观遗传调节中的作用。
