ABSTRACT: Methylation of cytosines at the 5th carbon position of the aromatic ring has long been regarded as the predominant type of DNA base modification in eukaryotes. Often called “the fifth base”, C5-methylcytosine (5mC) plays an important role in genome defense against mobile genetic elements, and is mostly associated with transcriptional silencing, establishment of the closed chromatin configuration, and repressive histone modifications. Recently, another type of DNA modification, N6-methyladenine (6mA), has been added to the repertoire of modified bases in eukaryotic DNA, and was mostly linked to elevated transcription levels. In prokaryotes, 5mC and 6mA typically constitute components of restriction-modification (R-M) systems, along with N4-methylcytosine (4mC), which so far has been confined to bacteria. Here we report the first case of 4mC occurrence in eukaryotic DNA. We find that bdelloid rotifers, small freshwater invertebrates known for their ability to reproduce clonally and to acquire genes from non-metazoan sources, lack the canonical eukaryotic C5-methyltransferases but instead encode an amino-methyltransferase of bacterial origin, N4CMT, which is present in all bdelloid families separated by tens of millions of years of evolution. The recombinant N4CMT introduces 4mC into genomic DNA in vivo and in vitro. Using SMRT-seq (PRJNA558051), MeDIP-seq, ChIP-seq, and RNA-seq, we examined genome-wide distribution of non-canonical base modifications over annotated genomic features and observed an excess of 4mC in silenced transposable elements and certain tandem repeats, while 6mA tends to associate with transcribed genes and active chromatin. The presence of the chromodomain in N4CMT explains its affinity for repressive histone marks, H3K9me3 and especially H3K27me3. Our results expand the known repertoire of eukaryotic base modifications, shed light on the process of recruitment of methyl groups as epigenetic marks in DNA, and highlight the role of horizontal gene transfer as an important driver of evolutionary innovation in eukaryotes.
