Towards molecular evolutionary epigenomics with an expanded nucleotide code involving methylated bases

In molecular evolution analyses, genomic DNA sequence information is usually represented in the form of 4 bases (ATGC). However, research since the turn of the century has revealed the importance of epigenetic genome modifications, such as DNA base methylation, which can now be decoded using advanced sequence technologies. Here we provide an integrated framework for analyzing molecular evolution of nucleotide substitution, methylation, and demethylation using an expanded nucleotide code that incorporates different types of methylated bases. As a first attempt, we analyzed substitution rates between bases, both unmethylated and methylated ones. As the model methylomes, we chose those of Helicobacter pylori, an unicellular bacterium with the largest known repertoire of sequence-specific DNA methyltransferases. We found that the demethylation rates are remarkably high while the methylation rates are comparable with the substitution rates between unmethylated bases. We found that the ribosomal proteins known for sequence conservation showed high methylation and demethylation frequencies, whereas the genes for DNA methyltransferases themselves showed low methylation and demethylation frequencies compared to base substitution. This work represents the first step towards molecular evolutionary epigenomics, which, we expect, would contribute to understanding epigenome evolution.