N6-methyladenine: a potential epigenetic mark in eukaryotes

Methylation modifications in DNA in the forms of 5-methylcytosine (5mC) and N 6-methyladenine (6mA) are one of the most important epigenetic marks that have been proposed to regulate gene expression and control numerous cellular and biological processes. The prevailing view is that, while 5mC serves as the predominant type, if not the only type, of methylated base in mammals to regulate gene expression and maintain chromatin architecture [1], 6mA is important in bacteria to control a number of biological functions, such as DNA replication and repair, gene expression, and host-pathogen interactions. In particular, 6mA is essential for the viability of some bacterial strains [2]. Previous studies have reported that 6mA is not only present at considerable levels in genomic DNA from a number of unicellular eukaryotes, but also is detected in some plant DNA. However, 6mA has been shown to be present at extremely low levels in most higher eukaryotes and particularly in mammals. Thus, whether 6mA occurs widely and what its potential roles are in higher eukaryotic cells remain unknown [2]. We hypothesize that, if 6mA does play a role, the potential introduction of this modification by methyltransferases could be reversed by a demethylase-mediated demethylation process, given the low levels of 6mA detected in higher eukaryote DNA. Thus, the detection of 6mA demethylases in the genomes of higher eukaryotes is critical to test the model we have hypothesized. We and others recently provided compelling evidence showing that 6mA modification occurred in DNA from three different eukaryotes, Chlamydomonas reinhardtii, Caenorhabditis elegans, and Drosophila melanogaster, and that it played conserved epigenetic roles in regulating gene expression [3-5]. In Drosophila, we found that 6mA was highly present at the very early embryonic stage, but very low levels were present at the late embryonic stage, suggesting that 6mA modification may be dynamic during embryogenesis. Indeed, further evidence revealed that the dynamics of 6mA modification were tightly regulated by a DNA 6mA demethylase (DMAD), the Drosophila Tet-like protein. Genetic analyses revealed that loss of DMAD led to a significant increase in levels of 6mA in multiple adult tissues, and the adult mutant fly displayed strong developmental defects, supporting the notion that DMAD suppressed DNA 6mA modification in vivo, and DMAD-mediated 6mA demethylation was essential for development and tissue homeostasis. Moreover, 6mA DNA immunoprecipitation sequencing (MeDIP-seq) analyses revealed that DMAD determined 6mA distribution in transposon regions in Drosophila ovary DNA and that the regulation of …