Faculty of Chemistry and Chemical Biology, TU Dortmund University, Otto-Hahn-Straße 6, 44227 Dortmund, Germany.
J Am Chem Soc. 2020 Apr 22;142(16):7289-7294. doi: 10.1021/jacs.0c01193. Epub 2020 Apr 14.
Ten-eleven-translocation (TET) dioxygenases catalyze the oxidation of 5-methylcytosine (5mC), the central epigenetic regulator of mammalian DNA. This activity dynamically reshapes the epigenome and transcriptome by depositing oxidized 5mC derivatives and initiating active DNA demethylation. However, studying this dynamic is hampered by the inability to selectively activate individual TETs with temporal control in cells. We report activation of TETs in mammalian cells by incorporation of genetically encoded 4,5-dimethoxy-2-nitrobenzyl-l-serine as a transient active-site block, and its subsequent deprotection with light. Our approach enables precise insights into the impact of cancer-associated TET2 mutations on the kinetics of TET2 catalysis , and allows time-resolved monitoring of target gene activation and transcriptome reorganization. This sets a basis for dissecting the order and kinetics of chromatin-associated events triggered by TET catalysis, ranging from DNA demethylation to chromatin and transcription regulation.
十 - 十一易位 (TET) 双加氧酶催化 5-甲基胞嘧啶 (5mC) 的氧化，这是哺乳动物 DNA 中重要的表观遗传调控因子。这种活性通过沉积氧化的 5mC 衍生物和启动活性 DNA 去甲基化，动态重塑表观基因组和转录组。然而，由于无法在细胞中具有时间控制的选择性激活单个 TET，因此研究这种动态受到了阻碍。我们通过将遗传编码的 4,5-二甲氧基-2-硝基苄基-L-丝氨酸掺入作为瞬时活性位点阻断物来报告哺乳动物细胞中 TET 的激活，并用光进行后续的保护。我们的方法能够精确地了解与癌症相关的 TET2 突变对 TET2 催化动力学的影响，并允许对靶基因激活和转录组重排进行时间分辨监测。这为剖析 TET 催化引发的染色质相关事件的顺序和动力学奠定了基础，范围从 DNA 去甲基化到染色质和转录调控。
