Roles of TET and TDG in DNA demethylation in proliferating and non-proliferating immune cells [RNA-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. RNA-seq in BMDMs and Th2 cells. Effects of TDG or TET1-3 deletion were examined in Tdg-iKO or TET-iTKO, respectively, as compared to WT control.

TET酶（TET enzymes）可通过将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。由于这些氧化型甲基胞嘧啶（oxidized methylcytosines, oxi-mC）无法被维持性甲基转移酶DNMT1识别，因此DNA去甲基化可通过细胞分裂时的“被动”复制依赖型稀释过程实现。另一种独特的复制非依赖型（“主动”）DNA去甲基化机制，则是由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。本研究借助诱导型基因敲除小鼠开展实验，证实TET酶可同时影响复制依赖型原代T细胞分化与复制非依赖型巨噬细胞分化，而TDG无此调控作用。长期（1年）敲除Tdg的小鼠健康状态良好，存活率与造血功能均表现正常。综上，TET酶主要通过复制中的T细胞内氧化型甲基胞嘧啶的被动稀释，调控细胞分化与DNA去甲基化；而由TDG介导的主动复制非依赖型DNA去甲基化，对于免疫细胞激活与分化似乎并非必需。本研究对骨髓源性巨噬细胞（bone marrow-derived macrophages, BMDMs）与2型辅助T细胞（Th2 cells）开展了RNA测序（RNA-seq），并分别在Tdg诱导型敲除（Tdg-iKO）与TET1-3诱导型敲除（TET-iTKO）小鼠模型中检测TDG或TET1-3敲除的效应，以野生型（WT）小鼠作为对照。