Roles of TET and TDG in DNA demethylation in proliferating and non-proliferating immune cells [CMS-Seq]

TET enzymes mediate DNA demethylation by oxidizing 5-methylcytosine (5mC) in DNA to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). Because these oxidized methylcytosines (oxi-mC) are not recognized by the maintenance methyltransferase DNMT1, DNA demethylation can occur through “passive”, replication-dependent dilution as cells divide. A distinct, replication-independent (“active”) mechanism of DNA demethylation involves excision of 5fC and 5caC by the DNA repair enzyme thymine DNA glycosylase (TDG), followed by base excision repair. Here we used inducible gene-disrupted mice to show that TET enzymes influence both replication-dependent primary T cell differentiation and replication-independent macrophage differentiation, whereas TDG has no effect. Mice with long-term (1 year) deletion of Tdg are healthy and show normal survival and hematopoiesis. In summary, TET enzymes regulate differentiation and DNA demethylation primarily through passive dilution of oxidized methylcytosines in replicating T cells, and active, replication-independent DNA demethylation mediated by TDG does not appear to be essential for immune cell activation or differentiation.

TET酶（TET enzymes）通过将DNA中的5-甲基胞嘧啶(5-methylcytosine, 5mC)氧化为5-羟甲基胞嘧啶(5-hydroxymethylcytosine, 5hmC)、5-甲酰基胞嘧啶(5-formylcytosine, 5fC)与5-羧基胞嘧啶(5-carboxylcytosine, 5caC)，介导DNA去甲基化过程。由于此类氧化型甲基胞嘧啶(oxidized methylcytosines, oxi-mC)无法被维持性DNA甲基转移酶DNMT1(maintenance methyltransferase DNMT1)识别，细胞分裂时可通过依赖复制的“被动”稀释途径完成DNA去甲基化。另一种不依赖复制的DNA去甲基化“主动”机制，则由DNA修复酶胸腺嘧啶DNA糖苷酶(thymine DNA glycosylase, TDG)切除5fC与5caC，随后启动碱基切除修复通路完成修复。本研究借助诱导性基因敲除小鼠模型证实：TET酶可同时调控依赖复制的原代T细胞分化，以及不依赖复制的巨噬细胞分化，而TDG并无此类调控功能。长期（1年）敲除Tdg的小鼠状态健康，存活率与造血功能均无明显异常。综上，TET酶主要通过在复制型T细胞中对氧化型甲基胞嘧啶进行被动稀释，实现对细胞分化与DNA去甲基化的调控；而由TDG介导的主动、不依赖复制的DNA去甲基化通路，对于免疫细胞的激活与分化并非必需。