N6-methyladenine (6mA) was recently identified as a new base modification in metazoan genomic DNA. Methylation on adenine was previously thought to be restricted to unicellular organisms. The identification of this mark in metazoans has raised questions about the role of 6mA in metazoan biology. Some studies suggest that 6mA may correlate with developmental regulation, active transcription start sites, or transposon expression, and potentially carry non-genetic information across generations. However, 6mA is a rare DNA modification in eukaryotes and its role in regulating biological processes is still unclear. Because of the low level of 6mA in eukaryotes, some researchers have questioned whether it plays a role in regulating biological processes or is an evolutionary holdover from bacteria. Studies of 6mA have been slowed by a lack of practical methods for accurately examining 6mA locations. Current epigenomic mapping techniques are either prohibitively expensive or do not provide sufficient resolution and accuracy to map the rare 6mA occurrences in metazoan genomes. Here we propose to develop a new technique for identifying sites of adenine methylation genome-wide at nucleotide resolution in a cost-effect manner. We will determine the utility of this technique first in bacteria and then apply it to examine 6mA distribution during development in zebrafish, where we have found that it changes dramatically. This technique will provide a new tool for scientists to define 6mA sites in metazoans to enable the study of its potential role in epigenetics, imprinting and development.
6-methyladenine (6mA) is a newly identified DNA base modification in metazoans. However, little is known about 6mA?s effect on chromatin structure or its function in biology. This proposal aims to develop an affordable nucleotide resolution sequencing technique to examine 6mAs distribution across eukaryotic genomes so we can better understand this novel bases effect in eukaryotes.!