Extensive N4 Cytosine Methylation is Essential forMarchantiaSperm Function

Abstract 4-methylcytosine (4mC) is an important DNA modification in prokaryotes, but its relevance, and even presence in eukaryotes have been mysterious. Here we show that spermatogenesis in the liverwort Marchantia polymorpha involves two waves of extensive DNA methylation reprogramming. First, 5-methylcytosine (5mC), a well-known eukaryotic DNA modification, expands from transposons to the entire genome. Notably, the second wave installs 4mC throughout genic regions, covering over 50% of CG sites in sperm. 4mC is catalyzed by two novel methyltransferases (MpDN4MT1a and MpDN4MT1b) specifically expressed during late spermiogenesis. Deletion of Mp DN4MT1s eliminates 4mC, alters the sperm transcriptome, and produces sperm with swimming defects. Our results reveal extensive 4mC in a eukaryote and define a new family of eukaryotic methyltransferases, thereby expanding the repertoire of functional eukaryotic DNA modifications.