Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), Department of Chemistry , Wuhan University , Wuhan 430072 , People's Republic of China.
Anal Chem. 2018 Dec 18;90(24):14622-14628. doi: 10.1021/acs.analchem.8b04833. Epub 2018 Dec 4.
The report of the existence of 5-hydroxymethylcytosine (hmC) in mammalian genomes is a milestone discovery. hmC is now generally viewed as the sixth base of DNA with important functions on epigenetic regulation. The in-depth investigation of the biological functions of hmC requires elucidating the distribution patterns of hmC in genomes, better in single-nucleotide resolution. It was reported that the cytosine deaminases of the APOBEC (apolipoprotein B mRNA-editing catalytic polypeptide-like) family are nucleic acid editing enzymes and can deaminate cytosine (C) to form uracil (U). Particularly, a subfamily of APOBEC (APOBEC3A) can efficiently deaminate both C and 5-methylcytosine (mC). In the current study, we identified that APOBEC3A protein can effectively deaminate C, mC, and hmC but shows no observable deamination activity toward glycosylated hmC (β-glucosyl-5-hydroxymethyl-2'-deoxycytidine, ghmC) by using the restriction enzyme-based assay and liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) analysis. By virtue of the differential deamination activity of APOBEC3A toward C, mC, and ghmC in conjugation with sequencing, we developed the single-nucleotide resolution analysis of hmC in DNA. In this analytical strategy, the original C and mC in DNA will be deaminated by APOBEC3A to form U and thymine (T), both of which will read as T during sequencing, while ghmC is resistant to deamination and will read as C during sequencing. Therefore, the remaining C in the sequence context only could come from original hmC, which offers the single-nucleotide resolution analysis of hmC in DNA. This APOBEC3A-mediated deamination sequencing (AMD-seq) is straightforward and involves no bisulfite treatment, which avoids the substantial degradation of DNA. Future application of this strategy can be performed for the reliable mapping of hmC in genome-wide scale at the single-nucleotide resolution.
羟甲基胞嘧啶（hmC）存在于哺乳动物基因组中的报告是一项里程碑式的发现。现在，hmC 通常被视为 DNA 的第六种碱基，在表观遗传调控中具有重要功能。深入研究 hmC 的生物学功能需要阐明 hmC 在基因组中的分布模式，最好达到单核苷酸分辨率。据报道，载脂蛋白 B mRNA 编辑酶催化多肽样（APOBEC）家族的胞嘧啶脱氨酶是核酸编辑酶，可以将胞嘧啶（C）脱氨形成尿嘧啶（U）。特别是 APOBEC 的一个亚家族（APOBEC3A）可以有效地脱氨 C 和 5-甲基胞嘧啶（mC）。在本研究中，我们通过基于限制酶的测定和液相色谱-电喷雾电离-串联质谱（LC-ESI-MS/MS）分析鉴定出 APOBEC3A 蛋白可以有效地脱氨 C、mC 和 hmC，但对糖苷化 hmC（β-葡萄糖基-5-羟甲基-2'-脱氧胞苷，ghmC）没有可观察到的脱氨活性。由于 APOBEC3A 对 C、mC 和 ghmC 的差异脱氨活性以及与测序相结合，我们开发了 DNA 中 hmC 的单核苷酸分辨率分析。在这种分析策略中，DNA 中的原始 C 和 mC 将被 APOBEC3A 脱氨形成 U 和胸腺嘧啶（T），在测序过程中两者都将读为 T，而 ghmC 对脱氨具有抗性，在测序过程中读为 C。因此，序列背景中仅剩下的 C 只能来自原始 hmC，从而提供了 DNA 中 hmC 的单核苷酸分辨率分析。这种 APOBEC3A 介导的脱氨酶测序（AMD-seq）方法简单直接，不涉及亚硫酸氢盐处理，避免了 DNA 的大量降解。未来可以在全基因组范围内以单核苷酸分辨率可靠地对 hmC 进行映射。
