Wei Alex, Zhang Hongjie, Qiu Qi, Fabyanic Emily B, Hu Peng, Wu Hao
Department of Genetics, University of Pennsylvania, Philadelphia, PA 19104.
Penn Epigenetics Institute, University of Pennsylvania, Philadelphia, PA 19104.
bioRxiv. 2023 Sep 27:2023.09.26.559662. doi: 10.1101/2023.09.26.559662.
Enzymatic erasure of DNA methylation in mammals involves iterative 5-methylcytosine (5mC) oxidation by the ten-eleven translocation (TET) family of DNA dioxygenase proteins. As the most abundant form of oxidized 5mC, the prevailing model considers 5-hydroxymethylcytosine (5hmC) as a key nexus in active DNA demethylation that can either indirectly facilitate replication-dependent depletion of 5mC by inhibiting maintenance DNA methylation machinery (UHRF1/DNMT1), or directly be iteratively oxidized to 5-formylcytosine (5fC) and 5-carboxycytosine (5caC) and restored to cytosine (C) through thymine DNA glycosylase (TDG)-mediated 5fC/5caC excision repair. In proliferative somatic cells, to what extent TET-dependent removal of 5mC entails indirect DNA demethylation via 5hmC-induced replication-dependent dilution or direct iterative conversion of 5hmC to 5fC/5caC is unclear. Here we leverage a catalytic processivity stalling variant of human TET1 (TET1.var: T1662E) to decouple the stepwise generation of 5hmC from subsequent 5fC/5caC generation, excision and repair. By using a CRISPR/dCas9-based epigenome-editing platform, we demonstrate that 5fC/5caC excision repair (by wild-type TET1, TET1.wt), but not 5hmC generation alone (by TET1.var), is requisite for robust restoration of unmodified cytosines and reversal of somatic silencing of the methylation-sensitive, germline-specific gene promoter. Furthermore, integrated whole-genome multi-modal epigenetic sequencing reveals that hemi-hydroxymethylated CpG dyads predominantly resist replication-dependent depletion of 5mC on the opposing strand in TET1.var-expressing cells. Notably, TET1.var-mediated 5hmC generation is sufficient to induce similar levels of differential gene expression (compared to TET1.wt) without inducing major changes in unmodified cytosine profiles across the genome. Our study suggests 5hmC alone plays a limited role in driving replication-dependent DNA demethylation in the presence of functional DNMT1/UHRF1 mechanisms, but can regulate gene expression as a epigenetic mark in proliferative somatic cells.
哺乳动物中DNA甲基化的酶促消除涉及由DNA双加氧酶蛋白的10-11易位（TET）家族对5-甲基胞嘧啶（5mC）进行的迭代氧化。作为氧化5mC的最丰富形式，普遍的模型认为5-羟甲基胞嘧啶（5hmC）是活性DNA去甲基化的关键节点，它既可以通过抑制维持DNA甲基化机制（UHRF1/DNMT1）间接促进依赖复制的5mC消耗，也可以直接被迭代氧化为5-甲酰基胞嘧啶（5fC）和5-羧基胞嘧啶（5caC），并通过胸腺嘧啶DNA糖基化酶（TDG）介导的5fC/5caC切除修复恢复为胞嘧啶（C）。在增殖性体细胞中，依赖TET的5mC去除在多大程度上需要通过5hmC诱导的依赖复制的稀释进行间接DNA去甲基化，或者将5hmC直接迭代转化为5fC/5caC尚不清楚。在这里，我们利用人TET1的催化持续性停滞变体（TET1.var：T1662E）将5hmC的逐步生成与随后的5fC/5caC生成、切除和修复解耦。通过使用基于CRISPR/dCas9的表观基因组编辑平台，我们证明5fC/5caC切除修复（由野生型TET1，即TET1.wt进行），而不是单独的5hmC生成（由TET1.var进行），是未修饰胞嘧啶的强劲恢复以及甲基化敏感的种系特异性基因启动子的体细胞沉默逆转所必需的。此外，整合的全基因组多模态表观遗传测序显示，在表达TET1.var的细胞中，半羟甲基化的CpG二联体主要抵抗互补链上依赖复制的5mC消耗。值得注意的是，TET1.var介导的5hmC生成足以诱导相似水平的差异基因表达（与TET1.wt相比），而不会在全基因组未修饰的胞嘧啶谱中引起重大变化。我们的研究表明，在存在功能性DNMT1/UHRF1机制的情况下，单独的5hmC在驱动依赖复制的DNA去甲基化方面作用有限，但可以作为增殖性体细胞中的一种表观遗传标记来调节基因表达。
