Genome-wide mapping of <i>N</i><sup>4</sup>-methylcytosine at single-base resolution by APOBEC3A-mediated deamination sequencing

Xiong, Jun; Wang, Ping; Shao, Wen-Xuan; Li, Gaojie; Ding, Jiang-Hui; Xie, Neng-Bin; Wang, Min; Cheng, Qing-Yun; Xie, Conghua; Ci, Weimin; Yuan, Bi-Feng; Feng, Yun

doi:10.1039/d2sc02446b

Cited by 15 publications

(12 citation statements)

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“…Previous studies reported that APOBEC3 family proteins can deaminate cytosine to form uracil in DNA. , Along this line, we demonstrated that APOBEC3A (A3A) could efficiently deaminate cytosine and 5mC but not N 4 -methylcytosine (4mC) . By virtue of this unique property of the A3A protein, we developed an APOBEC3A-mediated deamination sequencing (4mC-AMD-seq) method for mapping 4mC at the single-base resolution .…”

Section: Resultsmentioning

confidence: 83%

“…25,26 Along this line, we demonstrated that APOBEC3A (A3A) could efficiently deaminate cytosine and 5mC but not N 4methylcytosine (4mC). 27 By virtue of this unique property of the A3A protein, we developed an APOBEC3A-mediated deamination sequencing (4mC-AMD-seq) method for mapping 4mC at the single-base resolution. 27 However, A3A could not distinguish cytosine and 5mC.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

“…27 By virtue of this unique property of the A3A protein, we developed an APOBEC3A-mediated deamination sequencing (4mC-AMD-seq) method for mapping 4mC at the single-base resolution. 27 However, A3A could not distinguish cytosine and 5mC.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

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Engineered APOBEC3C Sequencing Enables Bisulfite-Free and Direct Detection of DNA Methylation at a Single-Base Resolution

et al. 2022

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DNA methylation (5-methylcytosine, 5mC) is the most important epigenetic modification in mammals. Deciphering the roles of 5mC relies on the quantitative detection of 5mC at the single-base resolution. Bisulfite sequencing (BS-seq) is the most often employed technique for mapping 5mC in DNA. However, bisulfite treatment may cause serious degradation of input DNA due to the harsh reaction conditions. Here, we engineered the human apolipoprotein B mRNA-editing catalytic polypeptide-like 3C (A3C) protein to endow the engineered A3C (eA3C) protein with differential deamination activity toward cytosine and 5mC. By the virtue of the unique property of eA3C, we proposed an engineered A3C sequencing (EAC-seq) method for the bisulfitefree and quantitative mapping of 5mC in DNA at the single-base resolution. In EAC-seq, the eA3C protein can deaminate C but not 5mC, which is employed to differentiate C and 5mC in sequencing. Using the EAC-seq method, we quantitatively detected 5mC in genomic DNA of lung cancer tissue. In contrast to the harsh reaction conditions of BS-seq, which could lead to significant degradation of DNA, the whole procedure of EAC-seq is carried out under mild conditions, thereby preventing DNA damage. Taken together, the EAC-seq approach is bisulfite-free and straightforward, making it an invaluable tool for the quantitative detection of 5mC in limited DNA at the single-base resolution.

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Section: Resultsmentioning

confidence: 83%

Section: ■ Results and Discussionmentioning

confidence: 99%

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Engineered APOBEC3C Sequencing Enables Bisulfite-Free and Direct Detection of DNA Methylation at a Single-Base Resolution

et al. 2022

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“…Previous studies demonstrated that A3A protein could efficiently deaminate C and 5mC in DNA to form U and T, respectively. , However, the oxidative derivative of 5mC, i.e., 5-carboxylcytosine (5caC), was resistant to deamination by A3A due to the presence of the carboxy group in 5caC . On the other hand, one recent study found that the mutant of CG-specific methyltransferase M.MpeI, i.e., M.MpeI-N374K, can specifically transfer a carboxymethyl group from caSAM to cytosine to form 5camC.…”

Section: Resultsmentioning

confidence: 99%

Bisulfite-Free and Single-Base Resolution Detection of Epigenetic DNA Modification of 5-Methylcytosine by Methyltransferase-Directed Labeling with APOBEC3A Deamination Sequencing

et al. 2022

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DNA methylation (5-methylcytosine, 5mC) is the most prevalent epigenetic modification that is predominantly found in CG dinucleotides in mammalian genomes. In-depth investigation of the functions of 5mC heavily relies on the quantitative measurement of 5mC at single-base resolution in genomes. Here, we proposed a methyltransferase-directed labeling with APOBEC3A (A3A) deamination sequencing (MLAD-seq) method for the single-base resolution and quantitative detection of 5mC in DNA. In MLAD-seq, a mutant of DNA methyltransferase, M.MpeI-N374K, is utilized to selectively transfer a carboxymethyl group to the 5 position of cytosine in the CG dinucleotide to form 5-carboxymethylcytosine (5camC) using carboxy-S-adenosyl-l-methionine (caSAM) as the cofactor. After A3A treatment, 5camC is resistant to the deamination and base pairs with guanine. Thus, the cytosines in CG sites are read as C in sequencing. On the contrary, the methyl group in 5mC inhibits its carboxymethylcytosine by M.MpeI-N374K and therefore is readily deaminated by A3A to produce thymine that pairs with adenine and is read as T in sequencing. The differential readouts from C and 5mC in the MLAD-seq enable the single-base resolution mapping of 5mC in CG sites in DNA. With the developed MLAD-seq method, we observed the hypermethylation in the promoter region of retinoic acid receptor β (RARB) gene from human nonsmall cell lung tumor tissue. Compared to harsh reaction conditions in bisulfite sequencing that could lead to significant degradation of DNA, the whole procedure of MLAD-seq is carried out under mild conditions, which will avoid DNA damage. Thus, MLAD-seq is more suitable in the scenario where only limited input DNA is available. Taken together, the MLAD-seq offers a valuable tool for bisulfite-free, single-base resolution and quantitative detection of 5mC in limited DNA.

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“…Resistance of 4mC to enzymatic deamination by APOBEC3A (4mC‐AMD‐seq) was used to discriminate 4mC from C and 5mC in the radioresistant bacterium Deinococcus radiodurans . [ 66 ] This method avoids excessive DNA degradation and can be applied to low‐input samples. Further improvement of 4mC detection methods in eukaryotes will be essential for establishment of validated 4mC reference datasets for training of bioinformatic pipelines, and for eventual elucidation of its biological functions.…”

Section: Methodological Challenges In Identifying Non‐canonical Base ...mentioning

confidence: 99%

Shaping eukaryotic epigenetic systems by horizontal gene transfer

2023

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DNA methylation constitutes one of the pillars of epigenetics, relying on covalent bonds for addition and/or removal of chemically distinct marks within the major groove of the double helix. DNA methyltransferases, enzymes which introduce methyl marks, initially evolved in prokaryotes as components of restriction‐modification systems protecting host genomes from bacteriophages and other invading foreign DNA. In early eukaryotic evolution, DNA methyltransferases were horizontally transferred from bacteria into eukaryotes several times and independently co‐opted into epigenetic regulatory systems, primarily via establishing connections with the chromatin environment. While C5‐methylcytosine is the cornerstone of plant and animal epigenetics and has been investigated in much detail, the epigenetic role of other methylated bases is less clear. The recent addition of N4‐methylcytosine of bacterial origin as a metazoan DNA modification highlights the prerequisites for foreign gene co‐option into the host regulatory networks, and challenges the existing paradigms concerning the origin and evolution of eukaryotic regulatory systems.

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Genome-wide mapping of N⁴-methylcytosine at single-base resolution by APOBEC3A-mediated deamination sequencing

Abstract: N4-methylcytosine (4mC) is a natural DNA modification occurring in thermophiles and plays important roles in restriction-modification (R-M) systems in bacterial genomes. However, the precise location and sequence context of 4mC...

Cited by 15 publications

References 49 publications

Engineered APOBEC3C Sequencing Enables Bisulfite-Free and Direct Detection of DNA Methylation at a Single-Base Resolution

Engineered APOBEC3C Sequencing Enables Bisulfite-Free and Direct Detection of DNA Methylation at a Single-Base Resolution

Bisulfite-Free and Single-Base Resolution Detection of Epigenetic DNA Modification of 5-Methylcytosine by Methyltransferase-Directed Labeling with APOBEC3A Deamination Sequencing

Shaping eukaryotic epigenetic systems by horizontal gene transfer

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Genome-wide mapping of N4-methylcytosine at single-base resolution by APOBEC3A-mediated deamination sequencing

Abstract: N4-methylcytosine (4mC) is a natural DNA modification occurring in thermophiles and plays important roles in restriction-modification (R-M) systems in bacterial genomes. However, the precise location and sequence context of 4mC...

Cited by 15 publications

References 49 publications

Engineered APOBEC3C Sequencing Enables Bisulfite-Free and Direct Detection of DNA Methylation at a Single-Base Resolution

Engineered APOBEC3C Sequencing Enables Bisulfite-Free and Direct Detection of DNA Methylation at a Single-Base Resolution

Bisulfite-Free and Single-Base Resolution Detection of Epigenetic DNA Modification of 5-Methylcytosine by Methyltransferase-Directed Labeling with APOBEC3A Deamination Sequencing

Shaping eukaryotic epigenetic systems by horizontal gene transfer

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Genome-wide mapping of N⁴-methylcytosine at single-base resolution by APOBEC3A-mediated deamination sequencing