Kavoosi Sam, Sudhamalla Babu, Dey Debasis, Shriver Kirsten, Arora Simran, Sappa Sushma, Islam Kabirul
Department of Chemistry , University of Pittsburgh , Pennsylvania 15260 , USA . Email:
Chem Sci. 2019 Sep 30;10(45):10550-10555. doi: 10.1039/c9sc02629k. eCollection 2019 Dec 7.
Ten-eleven translocation (TET) enzymes oxidize C-H bonds in 5-methylcytosine (5mC) to hydroxyl (5hmC), formyl (5fC) and carboxyl (5caC) intermediates en route to DNA demethylation. It has remained a challenge to study the function of a single oxidized product. We investigate whether alkyl groups other than methyl could be oxidized by TET proteins to generate a specific intermediate. We report here that TET2 oxidizes 5-ethylcytosine (5eC) only to 5-hydroxyethylcytosine (5heC). In biochemical assays, 5heC acts as a docking site for proteins implicated in transcription, imbuing this modification with potential gene regulatory activity. We observe that 5heC is resistant to downstream wild type hydrolases, but not to the engineered enzymes, thus establishing a unique tool to conditionally alter the stability of 5heC on DNA. Furthermore, we devised a chemical approach for orthogonal labeling of 5heC. Our work offers a platform for synthesis of novel 5-alkylcytosines, provides an approach to 'tame' TET activity, and identifies 5heC as an unnatural modification with a potential to control chromatin-dependent processes.
10-11易位（TET）酶将5-甲基胞嘧啶（5mC）中的C-H键氧化为羟基（5hmC）、甲酰基（5fC）和羧基（5caC）中间体，从而实现DNA去甲基化。研究单一氧化产物的功能一直是一项挑战。我们研究了除甲基以外的烷基是否能被TET蛋白氧化以生成特定中间体。我们在此报告，TET2仅将5-乙基胞嘧啶（5eC）氧化为5-羟乙基胞嘧啶（5heC）。在生化分析中，5heC作为与转录相关蛋白的结合位点，赋予这种修饰潜在的基因调控活性。我们观察到5heC对下游野生型水解酶具有抗性，但对工程酶没有抗性，从而建立了一种独特的工具来有条件地改变5heC在DNA上的稳定性。此外，我们设计了一种对5heC进行正交标记的化学方法。我们的工作为新型5-烷基胞嘧啶的合成提供了一个平台，提供了一种“调控”TET活性的方法，并将5heC鉴定为一种具有控制染色质依赖性过程潜力的非天然修饰。
