Detection of N⁶-Methyladenine in Eukaryotes

DNA N⁶-methyladenine (6mA) is a chemical modification at the N⁶-positon of adenine. In the last decades, 6mA had been found in genome from numerous prokaryotic species, but only existed in a few lower eukaryotes. In prokaryotes, 6mA plays an important role in restriction-modification, DNA replication, and DNA mismatch repair. Because of the too low abundance of 6mA, it was long-stalled whether 6mA existed in multicellular eukaryotes and playing any functions, particularly in mammals. In recent years, partially benefitting from the advances in analytical methods, 6mA was found in the genomes from Drosophila melanogaster, Chlamydomonas algae, Caenorhabditis elegans, zebrafish, Xenopus laevis and mouse embryonic stem cells and even in the human genome. The 6mA was dynamic changed in early embryonic development of fly and zebrafish and much more enriched in gene body of transposons in fly, repetitive regions in zebrafish, around the transcription start sites in Chlamydomonas, and widespread distribution in C. elegans, indicating 6mA probably playing different functions in different species. Meanwhile, 6mA methylases and demethylases were found in fly, worm, and Chlamydomonas. In this chapter, we will briefly review the distribution, regulation, and function of 6mA in eukaryotes and focus on the advances of 6mA analysis methods, especially LC-MS/MS, immunoprecipitation, next-generation sequencing, and single-molecule real-time sequencing technology.