DNA N(6)-methyladenine: a new epigenetic mark in eukaryotes?

Abstract

DNA N(6)-adenine methylation (N(6)-methyladenine; 6mA) in prokaryotes functions primarily in the host defence system. The prevalence and significance of this modification in eukaryotes had been unclear until recently. Here, we discuss recent publications documenting the presence of 6mA in Chlamydomonas reinhardtii, Drosophila melanogaster and Caenorhabditis elegans; consider possible roles for this DNA modification in regulating transcription, the activity of transposable elements and transgenerational epigenetic inheritance; and propose 6mA as a new epigenetic mark in eukaryotes.

Figure 1. Methods to detect 6mA in genomic DNA

(Left) An antibody against N⁶-methyladenine can sensitively recognize 6mA and enrich 6mA-containing DNA for subsequent next generation sequencing (NGS); 6mA-sensitive restriction enzymes can specifically recognize either methylated or unmethylated adenines in their recognition sequence motifs, and this can be captured by sequencing to determine the exact locations of 6mA. (Right) Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) can differentiate methylated adenine from unmodified adenine and quantify the 6mA/A ratio by normalization to a standard curve; single molecule real time (SMRT) sequencing can detect modified nucleotides by measuring the rate of DNA base incorporation (dashed arrow) during sequencing.

Figure 2. DNA methyltransferases and demethylases

a | Three families of DNA methyltransferases and two families of demethylases in 10 different model organisms are shown in a simplified phylogenetic tree. Color codes represent the reported presence of 6mA and/or 5mC in the corresponding organism (not the activity of the mentioned enzymes). The MT-A70 family proteins are widely conserved in eukaryotes but are missing in E. coli. They function as the RNA methyltransferases in plants and animals but appear to serve as the DNA 6mA methyltransferase in C. elegans. The Tet family proteins are DNA cytosine demethylases but have been proposed to demethylate adenines in D. melanogaster. The AlkB family proteins are conserved in all the organisms shown, and are DNA 6mA demethylases in C. elegans. The black dot indicates at least one member of each protein family exists in the corresponding organism. * The D. melanogaster genome contains a low level of 5mC (~0.03% of total cytosines). b | Two members — METTL3 and METTL14 — of the MT-A70 family are known to catalyze N⁶-adenine methylation in mammalian mRNA, while FTO and ALKBH5, members of the AlkB family, have been found to demethylate N⁶-methyladenine in mRNA. DAMT-1 is an MT-A70 family member that potentially mediates DNA 6mA methylation in C. elegans. An AlkB family member, NMAD-1, was identified as a demethylase in C. elegans. In D. melanogaster, the potential 6mA demethylase DMAD is a member of the Tet family proteins.

Figure 3. 6mA genomic distribution and function

a | Single-base resolution maps of 6mA in Chlamydomonas reinhardtii reveal a periodic distribution pattern, with depletion at active transcription start sites (TSS) and enrichment around them. Portrayed are the nucleosomes, which are mutually exclusive with 6mA. The phasing level of nucleosomes varies from highly phased near the TSS to randomly positioned ~1 kb from the TSS, where the 6mA methylation level also drops. 6mA sites are found in the linker regions. b | In C. elegans spr-5 mutants, 6mA and H3K4me2 levels increase progressively from generation to generation in conjunction with a decline of H3K9me3 levels and fertility. c | DNA 6mA methylation is correlated with increased transposon expression in D. melanogaster. Loss of the demethylase DMAD leads to elevated 6mA levels and increased expression of transposons.