Base-resolution profiling of 5-glyceryl-methylcytosine in <i>Chlamydomonas reinhardtii</i> via deaminase-assisted sequencing

5gmC (5-glyceryl-methylcytosine), a vitamin C-derived hypermodified base, has been identified in the genome of Chlamydomonas reinhardtii. However, the global distribution of 5gmC and its role as an epigenetic mark remain poorly understood. In this study, we employed a DNA deaminase to distinguish 5gmC from 5mC (5-methylcytosine) and cytosines, enabling precise profiling of 5gmC across the genome. This deaminase-assisted sequencing demonstrates superior performance compared to the previously proposed TET-coupled bisulfite sequencing. Using both methods, we identified numerous confident 5gmC sites. Unlike 5mC, which predominantly occurs at CpG sites, 5gmC is preferentially located in CHH contexts. Remarkably, over half of 5gmC sites are mutually exclusive from 5mC, with the remaining sites inversely correlated with 5mC levels, suggesting a role in active DNA demethylation. Additionally, 5gmC is enriched within introns, contrasting with the more extensive localization of 5mC in intergenic and promoter regions. Importantly, 5gmC levels are positively correlated with transcription, while 5mC typically exhibits an inverse relationship with gene expression, consistent with the enrichment of 5mC but lack of enrichment of 5gmC at H3K9me1-marked repressive chromatin. Collectively, these findings suggest that 5gmC is not only an intermediate for active DNA demethylation but also functions as a stable epigenetic mark, potentially influencing transcriptional regulation independently of 5mC.