Wang Min, Xie Neng-Bin, Gang Fang-Yin, Zhang Shan, Zeng Li, Ji Tong-Tong, Xiong Jun, Guo Xia, Hao Ying, Liu Yu, Yuan Bi-Feng
Department of Occupational and Environmental Health, School of Public Health, Wuhan University, Department of Radiation and Medical Oncology, Zhongnan Hospital of Wuhan University, Wuhan, 430071, China.
Hubei Key Laboratory of Biomass Resource Chemistry and Environmental Biotechnology, College of Chemistry and Molecular Sciences, Wuhan University, Wuhan, 430072, China.
Sci China Life Sci. 2025 May 22. doi: 10.1007/s11427-024-2702-8.
5-Methylcytosine (5mC) is a crucial epigenetic modification which plays a significant role in the regulation of gene expression. Accurate and quantitative detection of 5mC at single-base resolution is essential for understanding its epigenetic functions within genomes. In this study, we develop a novel Naegleria TET-assisted deaminase sequencing (NTD-seq) method for the base-resolution and quantitative detection of 5mC in genomic DNA. The NTD-seq method utilizes a Naegleria TET-like dioxygenase (nTET) to oxidize 5mC, generating 5-methylcytosine oxidation products (5moC). We also engineered a variant of the human apolipoprotein B mRNA-editing catalytic polypeptide-like 3A (A3A), creating an A3A mutant (A3Am). Treatment with A3Am results in the conversion of cytosine to uracil, while 5moC remains unchanged. Consequently, NTD-seq enables the direct deamination of cytosine to uracil by A3Am, which is sequenced as thymine, whereas 5mC, once oxidized to 5moC by nTET, resists deamination and is sequenced as cytosine. Therefore, the cytosines that persist in the sequencing data represent the original 5mC sites. We applied NTD-seq to HEK293T cells, generating a base-resolution map of 5mC that exhibits strong concordance with maps generated by conventional BS-seq. NTD-seq emerges as a powerful, bisulfite-free approach for the single-base resolution mapping of 5mC stoichiometry in genomic DNA.
5-甲基胞嘧啶（5mC）是一种关键的表观遗传修饰，在基因表达调控中发挥着重要作用。在单碱基分辨率下准确且定量地检测5mC对于理解其在基因组中的表观遗传功能至关重要。在本研究中，我们开发了一种新型的纳氏虫TET辅助脱氨酶测序（NTD-seq）方法，用于在基因组DNA中对5mC进行碱基分辨率和定量检测。NTD-seq方法利用一种类似纳氏虫TET的双加氧酶（nTET）氧化5mC，生成5-甲基胞嘧啶氧化产物（5moC）。我们还对人载脂蛋白B mRNA编辑催化多肽样3A（A3A）进行了改造，创建了一个A3A突变体（A3Am）。用A3Am处理会导致胞嘧啶转化为尿嘧啶，而5moC保持不变。因此，NTD-seq能够通过A3Am将胞嘧啶直接脱氨为尿嘧啶，尿嘧啶在测序时被识别为胸腺嘧啶，而5mC一旦被nTET氧化为5moC，就会抵抗脱氨作用，并在测序时被识别为胞嘧啶。所以，测序数据中持续存在的胞嘧啶代表了原始的5mC位点。我们将NTD-seq应用于HEK293T细胞，生成了一个5mC的碱基分辨率图谱，该图谱与传统亚硫酸氢盐测序（BS-seq）生成的图谱具有高度一致性。NTD-seq成为一种强大的、无需亚硫酸氢盐的方法，用于在基因组DNA中对5mC化学计量进行单碱基分辨率定位。
