A new hydroxymethylcytocine-like DNA modification linked to virulence gene expression, is the predominant cytosine modification in malaria parasites. hmC-like in Plasmodium

DNA cytosine modifications are key epigenetic regulators of cellular processes in mammalian cells, with their misregulation leading to varied disease states. In the human malaria parasite Plasmodium falciparum, a unicellular eukaryotic pathogen, little is known about the predominant cytosine modifications, cytosine methylation and hydroxymethylation. Here, we report the first identification of a hydroxymethylcytosine-like (hmC-like) modification in P. falciparum using a suite of biochemical methods. For genome-wide mapping, to discriminate between cytosine hydroxymethylation and the previously reported methylation (5mC), we combined bisulfite sequencing and oxidative bisulfite sequencing. The resulting data challenge previously reported 5mC levels for P. falciparum: hmC-like is the predominant cytosine modification in P. falciparum (0.2% of all cytosines and over 90% of all modified cytosines), and is enriched at genic regions in a CHH context. Levels of the hmC-like base in gene bodies (i.e., exons and introns) negatively correlate to transcript levels, with more than 900 genes stably marked with this modification throughout asexual development: these include genes involved in pathogenesis, drug binding and motor activity. Our work highlights the existence of a new epigenetic pathway in P. falciparum that could be exploited for the development of new antimalarials.