Abstract
BackgroundDirected 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.ResultsHere, 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.ConclusionsAltogether 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.
Highlights
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
Enzymes used to digest DNA for quantification contain unmethylated and methylated DNA Because N6adenine methylation (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 C5-cytosine methylation (5mC) or 3mC, N4-cytosine methylation (4mC), and 6mA
Because bacteria have relatively high levels of DNA methylation, and the enzymes used to digest gDNA are purified from bacteria, we were concerned about possible DNA contamination of the digestion enzymes
Summary
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. Directed DNA methylation by specific methyltransferase enzymes occurs in both prokaryotes and eukaryotes. These modifications can mark regions of the genome for control of a variety of processes, including base pairing, duplex stability, replication, repair, transcription, nucleosome positioning, X-chromosome inactivation, imprinting, and epigenetic memory [1,2,3]. Other groups have been unable to detect 6mA in metazoa [25,26,27], raising the question of whether detection techniques are reliable
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