Single-Nucleotide Resolution Analysis of 5-Hydroxymethylcytosine in DNA by Enzyme-Mediated Deamination in Combination with Sequencing,Analytical Chemistry

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Single-Nucleotide Resolution Analysis of 5-Hydroxymethylcytosine in DNA by Enzyme-Mediated Deamination in Combination with Sequencing
Analytical Chemistry ( IF 6.7 ) Pub Date : 2018-11-20 00:00:00 , DOI: 10.1021/acs.analchem.8b04833
Qiao-Ying Li ₁ , Neng-Bin Xie ₁ , Jun Xiong ₁ , Bi-Feng Yuan ₁ , Yu-Qi Feng ₁

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The report of the existence of 5-hydroxymethylcytosine (hm⁵C) in mammalian genomes is a milestone discovery. hm⁵C 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 hm⁵C requires elucidating the distribution patterns of hm⁵C 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 (m⁵C). In the current study, we identified that APOBEC3A protein can effectively deaminate C, m⁵C, and hm⁵C but shows no observable deamination activity toward glycosylated hm⁵C (β-glucosyl-5-hydroxymethyl-2′-deoxycytidine, ghm⁵C) 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, m⁵C, and ghm⁵C in conjugation with sequencing, we developed the single-nucleotide resolution analysis of hm⁵C in DNA. In this analytical strategy, the original C and m⁵C in DNA will be deaminated by APOBEC3A to form U and thymine (T), both of which will read as T during sequencing, while ghm⁵C is resistant to deamination and will read as C during sequencing. Therefore, the remaining C in the sequence context only could come from original hm⁵C, which offers the single-nucleotide resolution analysis of hm⁵C 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 hm⁵C in genome-wide scale at the single-nucleotide resolution.

中文翻译：

酶介导的脱氨结合测序法测定DNA中5-羟甲基胞嘧啶的单核苷酸分辨率

哺乳动物基因组中存在5-羟甲基胞嘧啶（hm ⁵ C）的报道是一个里程碑式的发现。hm ⁵ C现在通常被视为DNA的第六个碱基，在表观遗传调控中具有重要功能。深入研究hm ⁵ C的生物学功能需要阐明hm ⁵ C在基因组中的分布模式，单核苷酸分辨率更好。据报道，APOBEC（载脂蛋白B mRNA编辑催化多肽样）家族的胞嘧啶脱氨酶是核酸编辑酶，可以使胞嘧啶（C）脱氨基形成尿嘧啶（U）。特别是，APOBEC的一个亚家族（APOBEC3A）可以有效地将C和5-甲基胞嘧啶脱氨（m ⁵C）。在当前的研究中，我们发现APOBEC3A蛋白可以有效地使C，m ⁵ C和hm ⁵ C脱氨基，但是对糖基化的hm ⁵ C（β-葡萄糖基-5-羟甲基-2'-脱氧胞苷，ghm ⁵ C）通过基于限制性酶的分析和液相色谱-电喷雾电离-串联质谱分析（LC-ESI-MS / MS）进行分析。凭借APOBEC3A的差动脱氨酶活性朝向C，M的⁵ ℃，GHM ⁵与测序共轭C，我们开发了HM的单核苷酸分辨率分析⁵中DNA℃。在这种分析策略中，原始的C和m ⁵DNA中的C将被APOBEC3A脱氨基形成U和胸腺嘧啶（T），两者在测序过程中均读为T，而ghm ⁵ C具有抗脱氨性，在测序过程中均读为C。因此，序列上下文中的其余C只能来自原始的hm ⁵ C，它提供了DNA中hm ⁵ C的单核苷酸分辨率分析。该APOBEC3A介导的脱氨测序（AMD-seq）非常简单，不涉及亚硫酸氢盐处理，从而避免了DNA的大量降解。这个策略的未来的应用程序可以为HM的可靠映射来执行⁵在全基因组规模的单核苷酸分辨率℃。

更新日期：2018-11-20

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