Institute of Biotechnology, Life Sciences Center, Vilnius University, Vilnius 10257, Lithuania.
Institute of Biotechnology, Life Sciences Center, Vilnius University, Vilnius 10257, Lithuania; Institute of Chemistry, Faculty of Chemistry and Geosciences, Vilnius University, Vilnius 03225, Lithuania.
Mol Cell. 2022 Mar 3;82(5):1053-1065.e8. doi: 10.1016/j.molcel.2022.02.008.
Enzymatic methylation of cytosine to 5-methylcytosine in DNA is a fundamental epigenetic mechanism involved in mammalian development and disease. DNA methylation is brought about by collective action of three AdoMet-dependent DNA methyltransferases, whose catalytic interactions and temporal interplay are poorly understood. We used structure-guided engineering of the Dnmt1 methyltransferase to enable catalytic transfer of azide tags onto DNA from a synthetic cofactor analog, Ado-6-azide, in vitro. We then CRISPR-edited the Dnmt1 locus in mouse embryonic stem cells to install the engineered codon, which, following pulse internalization of the Ado-6-azide cofactor by electroporation, permitted selective azide tagging of Dnmt1-specific genomic targets in cellulo. The deposited covalent tags were exploited as "click" handles for reading adjoining sequences and precise genomic mapping of the methylation sites. The proposed approach, Dnmt-TOP-seq, enables high-resolution temporal tracking of the Dnmt1 catalysis in mammalian cells, paving the way to selective studies of other methylation pathways in eukaryotic systems.
DNA 中胞嘧啶的酶促甲基化形成 5-甲基胞嘧啶是涉及哺乳动物发育和疾病的基本表观遗传机制。DNA 甲基化是由三种依赖 AdoMet 的 DNA 甲基转移酶的集体作用引起的，其催化相互作用和时间相互作用知之甚少。我们使用 Dnmt1 甲基转移酶的结构指导工程，使在体外从合成辅因子类似物 Ado-6-叠氮化物上将叠氮化物标签催化转移到 DNA 上。然后，我们通过 CRISPR 在小鼠胚胎干细胞中编辑 Dnmt1 基因座，以安装经过工程改造的密码子，该密码子在通过电穿孔内化 Ado-6-叠氮化物辅因子后，允许在细胞内选择性地对 Dnmt1 特异性基因组靶标进行叠氮标记。所沉积的共价标记被用作“点击”处理，用于读取毗邻的序列和精确的甲基化位点的基因组图谱。所提出的方法 Dnmt-TOP-seq 能够在哺乳动物细胞中对 Dnmt1 催化进行高分辨率的时间跟踪，为在真核系统中选择性地研究其他甲基化途径铺平了道路。
