N6-methyladenine is an epigenetic marker of mammalian early life stress

Abstract

Recent evidence described 6-methyladenine (6 mA) as a novel epigenetic regulator in a variety of multicellular species, including rodents; however, its capacity to influence gene expression in the mammalian brain remains unknown. We examined if 6 mA is present and regulated by early life stress associated with predator odor exposure (POE) within the developing rat amygdala. Our results provide evidence that 6 mA is present in the mammalian brain, is altered within the Htr2a gene promoter by early life stress and biological sex, and increased 6 mA is associated with gene repression. These data suggest that methylation of adenosine within mammalian DNA may be used as an additional epigenetic biomarker for investigating the development of stress-induced neuropathology.

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

“…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.…”

“…These results taken together suggest that 6mA is a rare modification in more recently evolved eukaryotic genomes under basal conditions. Although we cannot be certain that it exists or is the product of directed DNA methylation, it may be present and become elevated at specific loci or specific developmental stages [50], or plays roles under certain stress conditions [18,19,23]. It will be critical, in the coming years, to identify biological conditions where 6mA becomes appreciably elevated.…”

“…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. However, other groups have been unable to detect 6mA in metazoa [25][26][27], raising the question of whether detection techniques are reliable.…”

“…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].…”

MutT homologue 1 (MTH1) removes N6-methyl-dATP from the dNTP pool

“…Recently, the development of SMRT sequencing of chromatin immunoprecipitation enriched DNA has enabled the detection of N6-methyl-dA in vertebrate DNA (30,37) and several studies suggest that N6methyl adenine modification in DNA constitutes a second layer of epigenetic regulation, alongside cytosine methylation (38). Changes in N6methylation levels in genomic DNA have been shown to have a major impact on diverse processes such as tumorigenesis (30,32), development (25), cell differentiation, maintenance of cell type and stress response (25,33,37,39,40). The association between changes in N6-methyl-dA levels and disease suggests that N6-methyl-dA is a biologically relevant DNA modification despite its evidently low abundance in eukaryotes.…”