Adenine DNA methylation, 3D genome organization and gene expression in the parasiteTrichomonas vaginalis

ABSTRACT Trichomonas vaginalis is a common sexually transmitted parasite that colonizes the human urogenital tract causing infections that range from asymptomatic to highly inflammatory. Recent works have highlighted the importance of histone modifications in the regulation of transcription and parasite pathogenesis. However, the nature of DNA methylation in the parasite remains unexplored. Using a combination of immunological techniques and UHPLC, we analyzed the abundance of DNA methylation in strains with differential pathogenicity demonstrating that N6-methyladenine (6mA), and not 5-methylcytosine (5mC), is the main DNA methylation mark in T. vaginalis . Genome-wide distribution of 6mA reveals that this mark is enriched at intergenic regions, with a preference for certain super-families of DNA transposable elements. We show that 6mA in T. vaginalis is associated with silencing when present on genes. Interestingly, bioinformatics analysis revealed the presence of transcriptionally active or repressive intervals flanked by 6mA-enriched regions and results from chromatin conformation capture (3C) experiments suggest these 6mA flanked regions are in close spatial proximity. These associations were disrupted when parasites were treated with the demethylation activator ascorbic acid. This finding revealed a new role for 6mA in modulating 3D chromatin structure and gene expression in this divergent member of the Excavata. SIGNIFICANCE STATEMENT Trichomonas vaginalis causes the most common non-viral sexually transmitted infection yet little is known about the regulation of gene expression in this eukaryotic parasite. We demonstrate that N6-methyladenine (6mA) is the main methylation mark in the T. vaginalis genome. 6mA is widespread in DNA of eubacterial genera, but uncommon in genomes of most eukaryotes. Examination of the genome-wide distribution of 6mA reveals a preference for intergenic regions. Transcriptionally active or repressive intervals are found to be flanked by 6mA-enriched regions and data suggest 6mA flanked regions are in close 3D spatial proximity. Our findings describe for the first time the presence of DNA methylation in T. vaginalis and reveal a new role for 6mA in modulating 3D chromatin architecture.