Repressive Cytosine Methylation is a marker of Viral Gene Transfer across distant eukaryotes
Cytosine DNA methylation patterns vary widely across eukaryotes, with its ancestral roles being understood to have had roles in both transposable element silencing and host gene regulation. To further explore these claims, in this study, we reevaluate the evolutionary origins of DNA methyltransferases and characterise the roles of cytosine methylation on underexplored lineages, including the amoebozoan Acanthamoeba castellanii, the glaucophyte Cyanophora paradoxa, and the heterolobosean Naegleria gruberi. Our analysis of DNA methyltransferase evolution reveals a complex ancestral eukaryotic repertoire, with several eukaryotic lineages encoding enzymes likely acquired through lateral gene transfer (LGT). In the three species examined, DNA methylation is enriched on young transposable elements and silenced genes, suggesting an ancestral repressive function, without the transcriptionally linked gene body methylation of plants and animals. Notably, the closest homologues of many of the silenced, methylated genes in diverse eukaryotes belong to viruses, including giant viruses. Given the widespread occurrence of this pattern across diverse eukaryotic groups, we propose that cytosine methylation was a mechanism originally acquired from bacterial donors to mitigate the expression of both transposable and viral elements, and that this function may persist in creating a permissive atmosphere for LGT in diverse eukaryotic lineages. These findings further highlight the importance of epigenetic information to annotate eukaryotic genomes, as it helps delimit potentially functional LGTs from silenced parasitic elements, less likely to be actively contributing to organism adaptations. Overall design: We generated Enzymatic Methyl-seq for 3 distantly related microbial eukaryotes grown axenically in laboratory conditions, including the amoebozoan Acanthamoeba castellanii, the heterolobosean Naegleria gruberi and the glaucophyte Cyanophora paradoxa. Moreover, both A. castellanii and N. gruberi were treated with cytidine analogues (5-Azacytidine, Decitabine, Zebularine) known to interfere with 5mC deposition, and compared to DMSO treated control samples. Effects on 5mC levels were measured by EM-seq and RNA-seq was used to characterise the transcriptional response. A. castellanii and N. gruberi were also sequenced with Whole Genome Bisulphite Sequencing.