Department of Cell and Developmental Biology, University of Pennsylvania, Philadelphia, PA 19104, USA.
Department of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.
Mol Cell. 2021 Feb 18;81(4):859-869.e8. doi: 10.1016/j.molcel.2020.11.045. Epub 2020 Dec 21.
Active DNA demethylation via ten-eleven translocation (TET) family enzymes is essential for epigenetic reprogramming in cell state transitions. TET enzymes catalyze up to three successive oxidations of 5-methylcytosine (5mC), generating 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), or 5-carboxycytosine (5caC). Although these bases are known to contribute to distinct demethylation pathways, the lack of tools to uncouple these sequential oxidative events has constrained our mechanistic understanding of the role of TETs in chromatin reprogramming. Here, we describe the first application of biochemically engineered TET mutants that unlink 5mC oxidation steps, examining their effects on somatic cell reprogramming. We show that only TET enzymes proficient for oxidation to 5fC/5caC can rescue the reprogramming potential of Tet2-deficient mouse embryonic fibroblasts. This effect correlated with rapid DNA demethylation at reprogramming enhancers and increased chromatin accessibility later in reprogramming. These experiments demonstrate that DNA demethylation through 5fC/5caC has roles distinct from 5hmC in somatic reprogramming to pluripotency.
通过 TET 家族酶的主动 DNA 去甲基化对于细胞状态转变中的表观遗传重编程至关重要。TET 酶催化 5-甲基胞嘧啶（5mC）的三个连续氧化反应，生成 5-羟甲基胞嘧啶（5hmC）、5-甲酰胞嘧啶（5fC）或 5-羧基胞嘧啶（5caC）。尽管这些碱基已知有助于不同的去甲基化途径，但缺乏分离这些连续氧化事件的工具限制了我们对 TET 在染色质重编程中的作用的机制理解。在这里，我们描述了首次应用生化工程化的 TET 突变体来分离 5mC 氧化步骤，以检查它们对体细胞重编程的影响。我们表明，只有能够氧化为 5fC/5caC 的 TET 酶才能挽救 Tet2 缺陷型小鼠胚胎成纤维细胞的重编程潜力。这种效应与重编程增强子处的快速 DNA 去甲基化以及重编程后期染色质可及性增加相关。这些实验表明，在体细胞重编程为多能性过程中，通过 5fC/5caC 的 DNA 去甲基化与 5hmC 具有不同的作用。
