The decreased N6-methyladenine DNA modification in cancer cells

Da, Liu; Wang, Hong; Song, Wei; Xiong, Xin; Zhang, Xianhua; Hu, Zhiping; Guo, Huahu; Yang, Zhenjun; Zhai, Suodi; Zhang, Lihe; Ye, Min; Du, Quan

doi:10.1016/j.bbrc.2016.09.136

Cited by 29 publications

(31 citation statements)

References 32 publications

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“…Enzymes used to digest DNA for quantification contain unmethylated and methylated DNA Because 6mA has been reported to occur in a variety of metazoan genomes by some groups [10][11][12][13][14][15][16][17][18][19][20][21][22], but not by others [25][26][27], we developed a UHPLC-ms/ms method that can distinguish 5mC or 3mC, 4mC, and 6mA. The UHPLC-ms/ms buffers and flow rates were optimized to separate methylated nucleosides using pre-methylated standards ( Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Relatively little is known about the function of 4mC beyond its role in the restriction-modification system, which 6mA also regulates. 6mA, which was previously thought to be restricted to unicellular organisms, was recently reported to occur in multicellular organisms including fungi, Arabidopsis, worms, flies, frogs, zebrafish, and mammals, but its function remains unclear [10][11][12][13][14][15][16][17][18][19][20][21][22]. Some reports have suggested that 6mA is regulated in metazoa in response to stress [18,19,23], development [11,14,15], or cancer [24], findings which suggest a functional role.…”

Section: Introductionmentioning

confidence: 99%

“…The application of highly sensitive detection and quantification methods, including single molecule real time sequencing (SMRTseq) [28] and ultra-high performance liquid chromatography coupled with mass spectrometry (UHPLC-ms/ms), has led to widely varying estimates of 6mA abundance that range from 1 to 8000 ppm (ppm)(0.0001-0.8%) in various eukaryotic genomes [10][11][12][13][14][15][16][17][18][19][20][21][22]29]. SMRTseq measures the kinetic rate at which each new base is incorporated, which is altered by DNA modifications [28].…”

Section: Introductionmentioning

confidence: 99%

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Sources of artifact in measurements of 6mA and 4mC abundance in eukaryotic genomic DNA

et al. 2019

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Background: Directed DNA methylation on N6-adenine (6mA), N4-cytosine (4mC), and C5-cytosine (5mC) can potentially increase DNA coding capacity and regulate a variety of biological functions. These modifications are relatively abundant in bacteria, occurring in about a percent of all bases of most bacteria. Until recently, 5mC and its oxidized derivatives were thought to be the only directed DNA methylation events in metazoa. New and more sensitive detection techniques (ultra-high performance liquid chromatography coupled with mass spectrometry (UHPLC-ms/ms) and single molecule real-time sequencing (SMRTseq)) have suggested that 6mA and 4mC modifications could be present in a variety of metazoa. Results: Here, we find that both of these techniques are prone to inaccuracies, which overestimate DNA methylation concentrations in metazoan genomic DNA. Artifacts can arise from methylated bacterial DNA contamination of enzyme preparations used to digest DNA and contaminating bacterial DNA in eukaryotic DNA preparations. Moreover, DNA sonication introduces a novel modified base from 5mC that has a retention time near 4mC that can be confused with 4mC. Our analyses also suggest that SMRTseq systematically overestimates 4mC in prokaryotic and eukaryotic DNA and 6mA in DNA samples in which it is rare. Using UHPLC-ms/ms designed to minimize and subtract artifacts, we find low to undetectable levels of 4mC and 6mA in genomes of representative worms, insects, amphibians, birds, rodents and primates under normal growth conditions. We also find that mammalian cells incorporate exogenous methylated nucleosides into their genome, suggesting that a portion of 6mA modifications could derive from incorporation of nucleosides from bacteria in food or microbiota. However, gDNA samples from gnotobiotic mouse tissues found rare (0.9-3.7 ppm) 6mA modifications above background. Conclusions: Altogether these data demonstrate that 6mA and 4mC are rarer in metazoa than previously reported, and highlight the importance of careful sample preparation and measurement, and need for more accurate sequencing techniques.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Sources of artifact in measurements of 6mA and 4mC abundance in eukaryotic genomic DNA

et al. 2019

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“…[144] Later, studies reported the presence and genomic profiles of m 6 dA even in vertebrates,i ncluding zebrafish, frog,m ouse,p ig and human. [145][146][147][148][149] While the levels of m 6 dA are high in unicellular eukaryotes (ciliated protozoa and green algae), they are extremely low in all higher eukaryotes studied so far. They are so low that ar ecent ultrasensitive LC-MS 2 based approach, using an isotopically labelled m 6 dA standard, was unable to confirm the presence of m 6 dA in vertebrates.T his result questions whether m 6 dA is af requently present and epigenetically relevant base, [150] but it cannot be excluded that m 6 dA is formed as aD NA lesion or during certain developmental phases.T he idea that m 6 dA is formed as al esion could explain the requirement for an m 6 dA demethylase.…”

Section: Discovery Of Oxidized Bases Beyond Hmdcmentioning

confidence: 99%

Non‐canonical Bases in the Genome: The Regulatory Information Layer in DNA

et al. 2018

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Multicellular organisms developed the concept of specialized cells that perform specific functions. Examples are neurons and fibroblast to name just two out of more than 200. These cellular differences are established based on the same sequence information stored in the cell nucleus of all cells of an organism. The sequence information needs consequently different interpretations by the different cell types. During cellular development this interpretation of the genetic code has to be tightly regulated in space and time. Interpretation of the sequence information involves the controlled activation and silencing of specific genes so that certain proteins are made in one cell type but not in others. This involves an additional regulatory information layer beyond the pure base sequence. One aspect of this regulatory information layer relies on functional groups that are attached to the C(5) position of the canonical base dC. Currently four regulatory, non-canonical bases with a methyl (CH )-, a hydroxymethyl (CH OH)-, a formyl (CHO)- and a carboxyl (COOH)- group are known. While 5-methyl-cytidine is long recognised to be a regulatory base in the genome, the other three bases and the enzymes responsible for generating them, were just recently discovered.

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“…Vor kurzem wurden genomische Profile von m 6 dA in der Grünalge Chlamydomonas reinhardtii , im Fadenwurm Caenorhabditis elegans und in der Fruchtfliege Drosophila melanogaster erstellt. Später wurden auch Studien veröffentlicht, in denen das Vorkommen und genomische Profile von m 6 dA in Wirbeltieren wie Zebrafischen, Fröschen, Mäusen, Schweinen und Menschen gezeigt wurden . Während der Anteil an m 6 dA in einzelligen Eukaryoten relativ hoch ist (Wimpertierchen und Grünalge), sind die gefundenen Mengen dieser Modifikation in höheren Eukaryoten extrem gering.…”

Section: Die Phylogenetische Verteilung Der Modifizierten Dna‐base N6unclassified

Nichtkanonische Basen im Genom: die regulative Informationsebene in der DNA

et al. 2018

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In multizellulären Organismen findet man spezialisierte Zellen, die jeweils eine bestimmte Funktion wahrnehmen. Diese zellulären Unterschiede beruhen alle auf der identischen Sequenzinformation, die im Zellkern gespeichert ist. Daher muss diese Sequenzinformation von den verschiedenen Zelltypen unterschiedlich interpretiert werden. Während des frühen Entwicklungsstadiums einer Zelle muss diese Übersetzung räumlich und zeitlich streng kontrolliert werden. Die jeweilige Interpretation der Sequenzinformation umfasst die kontrollierte Aktivierung und Stilllegung spezifischer Gene, sodass ein Protein nur in bestimmten Zellen exprimiert wird. Dafür ist eine weitere Informationsebene vonnöten, die sich von der reinen Basensequenz abhebt. Ein Aspekt dieser regulatorischen Informationsebene beruht auf funktionellen Gruppen an der C5‐Position der kanonischen Base dC. Heute kennt man vier dieser regulatorischen, nichtkanonischen Basen, die eine CH3‐, CH2OH‐, CHO‐ oder COOH‐Gruppe aufweisen. Während 5‐Methylcytidin schon lange als eine der Basen im Genom mit dieser Funktion bekannt ist, wurden die anderen drei Basen – und die für die Entstehung verantwortlichen Enzyme – erst kürzlich entdeckt.

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The decreased N6-methyladenine DNA modification in cancer cells

Cited by 29 publications

References 32 publications

Sources of artifact in measurements of 6mA and 4mC abundance in eukaryotic genomic DNA

Sources of artifact in measurements of 6mA and 4mC abundance in eukaryotic genomic DNA

Non‐canonical Bases in the Genome: The Regulatory Information Layer in DNA

Nichtkanonische Basen im Genom: die regulative Informationsebene in der DNA

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