The study aims to characterise the genome-wide distribution of 5-hydroxymethylcytosine during asexual stages of the malaria parasite Plasmodium falciparum.

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 and genome-wide distribution map of 5-hydroxymethylcytosine (5hmC) in P. falciparum. For genome-wide mapping, to discriminate between 5hmC and the previously reported 5-methylcytosine (5mC), we used a combination of bisulfite sequencing and oxidative bisulfite sequencing. Our results challenge previously reported 5mC levels for P. falciparum and 5mC/5hmC ratios and patterns in mammalian cells: 5hmC is the predominant cytosine modification in P. falciparum (0.2% of all cytosines and 98% of all modified cytosines), and is enriched at genic regions in a CHH context. Gene body 5hmC levels negatively correlate to transcript levels, with 580 genes stably marked with 5hmC throughout asexual development: these include genes involved in pathogenesis 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.