There’s no denying that we love DNA methylation, so we are delighted to learn of another form of DNA methylation that has come on the scene as an epigenetic mark. N6-Methyladenosine (6mA or m6A) refers to the methylation of adenosine at its nitrogen-6 position, as opposed to 5mC and related oxidative marks (5hmC, 5fC, 5caC) that occur on the carbon-5 position of Cytosine. While 6mA was discovered in DNA long ago and characterized in prokaryotes, 6mA’s eukaryotic function has only just been figured out with researchers from University of Chicago, Harvard University, and the Chinese Academy of Sciences showing that m6A functions as as a true epigenetic mark in three diverse eukaryotes.
A notable contributor was Chuan He whose lab was involved in all three papers and previously showed that 6mA is a mRNA modification in human cells, with a RNA demethylase known as FTO that is involved in obesity. Others have a shown that m6A mRNA methylation has a role in stem cell differentiation, is present at an omic level, and has a role in miRNA processing. However, this set of papers now takes on the role of m6A in DNA.
Manel Esteller shares “It was known for years that bacteria, evolutionarily very distant living organisms of us, had 6mA in its genome with a protective function against the insertion of genetic material from other organisms. But it was believed that this was a phenomenon of primitive cells and … very static.”
6mA Becomes Epigenomic and Dynamic
In order to establish m6A as an epigenetic mark, the groups looked at the biology of this methylation in three different eukaryotic organisms.
Fu and team used the green algae Chlamydomonas to show that 6mA:
- is present in 84% of genes
- is located at ApT dinucleotides around transcription start sites (TSS)
- is present in a bimodal distribution
- marks active genes
- is associated with nucleosome position.
Greer and colleagues characterized 6mA in C. elegans and showed:
- there is a 6mA DNA demethylase (NMAD-1)
- there is also a possible 6mA DNA methyltransferase (DAMT-1)
- 6mA crosstalks with histone H3K4 methylation
- 6mA has a role in transgenerational inheritance.
Zhang and coworkers went into Drosophila and found that 6mA is:
- regulated by a TET homolog (during embryogenesis) that is known as DNA Methyl Adenosine Demethylase (DMAD).
- catalyzed in vitro by DMAD
- essential for development, where DMAD mainly removes 6mA from transposons, possibly to suppress transposons in the ovary.
The Future of 6mA and DNA
Heyn and Esteller have provided a lay of the land by overviewing the 3 Cell papers, noting the similarities between the recent maturation of 6mA and 5hmC as true epigenetic marks, despite being known as DNA modifications for so long. The review also covers the tech needed to examine 6mA in DNA, including:
- ultra-high performance liquid chromatography-triple quadrupole mass spectrometry coupled with multiple-reaction monitoring (UHPLC-MRM-MS/MS)
- 6mA ChIP-seq (6mA-IPseq)
- restriction enzyme guided sequencing (6mA-REseq)
- single-molecule real-time (SMRT) technology, aka third gen seq, which discriminates based on enzyme kinetics.
Manel Esteller shares that “These studies suggest that algae, worms, and flies possess 6mA and it acts to regulate the expression of certain genes, thus constituting a new epigenetic mark. This work has been possible thanks to the development of analytical methods with high sensitivity because levels of 6mA in described genomes are low. In addition it seems that 6mA would play a specific role in stem cells and early stages of development. Now the challenge we face is to confirm this data and find out whether mammals, including humans …have this… and consider what its role is.”
While Chuan He touches on how presence of this mark in these three organisms suggest a wide functional role: “The conservation of this modification from simple unicellular eukaryotes to vastly different worms and flies indicates its wide presence and functional roles. All three studies together uncover a potential new epigenetic mark on eukaryotic DNA. They open a new field of biology and chemical biology.”