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國家生物信息中心和武漢大學(xué)聯(lián)合在Anal. Chem. 發(fā)文使用PuriMag G-strep磁珠

Whole-Genome Mapping of Epigenetic Modification of 5-Formylcytosine at Single-Base Resolution by Chemical Labeling Enrichment and Deamination Sequencing

Jiang-Hui Ding, Gaojie Li, Jun Xiong, Fei-Long Liu, Neng-Bin Xie, Tong-Tong Ji, Min Wang, Xia Guo, Yu-Qi Feng, Weimin Ci*, and Bi-Feng Yuan*
Cite this: Anal. Chem. 2024, 96, 11, 4726–4735
Publication Date:March 7, 2024
https://doi.org/10.1021/acs.analchem.4c00425


Abstract

DNA cytosine methylation (5-methylcytosine, 5mC) is a predominant epigenetic modification that plays a critical role in a variety of biological and pathological processes in mammals. In active DNA demethylation, the 10-11 translocation (TET) dioxygenases can sequentially oxidize 5mC to generate three modified forms of cytosine, 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). Beyond being a demethylation intermediate, recent studies have shown that 5fC has regulatory functions in gene expression and chromatin organization. While some methods have been developed to detect 5fC, genome-wide mapping of 5fC at base resolution is still highly desirable. Herein, we propose a chemical labeling enrichment and deamination sequencing (CLED-seq) method for detecting 5fC in genomic DNA at single-base resolution. The CLED-seq method utilizes selective labeling and enrichment of 5fC-containing DNA fragments, followed by deamination mediated by apolipoprotein B mRNA-editing catalytic polypeptide-like 3A (APOBEC3A or A3A) and sequencing. In the CLED-seq process, while all C, 5mC, and 5hmC are interpreted as T during sequencing, 5fC is still read as C, enabling the precise detection of 5fC in DNA. Using the proposed CLED-seq method, we accomplished genome-wide mapping of 5fC in mouse embryonic stem cells. The mapping study revealed that promoter regions enriched with 5fC overlapped with H3K4me1, H3K4me3, and H3K27ac marks. These findings suggest a correlation between 5fC marks and active gene expression in mESCs. In conclusion, CLED-seq is a straightforward, bisulfite-free method that offers a valuable tool for detecting 5fC in genomes at a single-base resolution.


DNA胞嘧啶甲基化（5-甲基胞嘧啶，5mC）是主要的表 觀 遺傳 修改這在哺乳動物的各種生物學(xué)和病理過程中起著關(guān)鍵作用。在活性 DNA 去甲基化中，10-11 易位 （TET） 雙加氧酶可以依次氧化 5mC 以產(chǎn)生三種修飾形式的胞嘧啶，即 5-羥甲基胞嘧啶 （5hmC），5-甲?；奏ぃ?fC）和5-羧基胞嘧啶（5caC）。除了作為去甲基化中間體外，最近的研究表明，5fC在基因表達和染色質(zhì)組織中具有調(diào)節(jié)功能。雖然已經(jīng)開發(fā)了一些方法來檢測全基因組的 5fC映射基數(shù)為 5fC分辨率仍然非?？扇　Ｔ诖?，我們提出了一個化學(xué)的 標(biāo)簽 富 集和脫氨基作用 測 序（CLED-seq）檢測基因組DNA中5fC的方法單-基礎(chǔ) 分辨率.CLED-seq 方法利用選擇性標(biāo)簽和富 集含 5fC 的 DNA 片段，然后是脫氨基作用由載脂蛋白 B mRNA 編輯催化多肽樣 3A（APOBEC3A 或 A3A）介導(dǎo)，以及測 序.在 CLED-seq 過程中，所有 C、5mC 和 5hmC 在測 序，5fC 仍被讀取為 C，從而能夠精確檢測 DNA 中的 5fC。使用所提出的CLED-seq方法，我們完成了全基因組映射小鼠胚胎干細胞中的5fC。這映射研究表明，富含 5fC 的啟動子區(qū)域與 H3K4me1、H3K4me3 和 H3K27ac 標(biāo)記重疊。這些發(fā)現(xiàn)表明 5fC 標(biāo)記與 mESC 中的活性基因表達之間存在相關(guān)性?？傊?，CLED-seq是一種簡單明了的、不含亞硫酸氫鹽的方法，為檢測基因組中的5fC提供了一種有價值的工具。單-基礎(chǔ) 分辨率.


Experimental Section
Library Construction for CLED-seq

10 μg of genomic DNA (in 500 μL of water) from mESCs was fragmented with a JY92-II N Ultrasonic Homogenizer (Scientz) to obtain 300–500 bp DNA fragments. The fragmented DNA was lyophilized to dryness and then reacted with 5 μM biotin-ONH2 and 100 mM MES buffer (pH 5.0) at 37 °C for 4 h. The resulting DNA was purified by ethanol precipitation to remove the excess biotin-ONH2. The biotin-labeled DNA fragments were isolated by using PuriMag G-streptavidin magnetic beads (PuriMag Biotech. Xiamen, China) according to the manufacturer's recommended procedure. The mixture (50 μL) of enriched 5fC-containing DNA fragments and spike-ins (395-bp 5mC-DNA, 10 pg) were end-repaired and dA-tailed using Hieff NGS Fast-Pace End Repair/dA-Tailing Module kit (YEASEN Biotechnology Co., Ltd., Shanghai, China) according to the manufacturer’s recommended protocol. As for the adapter ligation, 60 μL of end-repaired DNA and 5 μL of phosphorylated adapter (10 μM) were ligated using Hieff NGS Ultima DNA Ligation Module kit (YEASEN Biotechnology Co., Ltd., Shanghai, China) at 20 °C for 6 h. 

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