Base-resolution profiling of 5-glyceryl-methylcytosine in Chlamydomonas reinhardtii via DEA-sequencing [WGBS]
5gmC (5-glyceryl-methylcytosine), a vitamin C-derived hypermodified base, was identified in the genome of Chlamydomonas reinhardtii. Despite its potential involvement in active DNA demethylation, the global distribution of 5gmC and its function as a stable epigenetic mark remain poorly understood. In this study, we employed a commercial DNA deaminase to distinguish 5gmC from 5mC (5-methylcytosine) and C, enabling precise profiling of 5gmC across the entire genome. This DEA-sequencing method demonstrates superior performance compared to the previously proposed approach, which relies on TET dioxygenase-coupled bisulfite treatment. Using both methods, we identified numerous confident 5gmC sites. Unlike 5mC, which predominantly occurs at CpG sites, 5gmC is more evenly distributed across both CpG and non-CpG contexts. Remarkably, 5gmC and 5mC are largely mutually exclusive, with less than 2% of the sites overlapping between the two modifications. Additionally, 5gmC shows significant enrichment at transcriptional start sites (TSS) and transcriptional end sites (TES), contrasting with the more extensive localization of 5mC in intergenic and gene body regions. Importantly, 5gmC levels are positively correlated with transcription, while 5mC typically exhibits an inverse relationship with gene expression. Together, these findings suggest that 5gmC is not only an intermediate in active DNA demethylation but also functions as a stable epigenetic mark, potentially influencing transcriptional regulation independently of 5mC. Overall design: WGBS of 5mC (BS) and 5gmC (DEA/TET-BS) in the C. reinhardtii strains (CC125 and CC5325).