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Base-resolution profiling of 5-glyceryl-methylcytosine in Chlamydomonas reinhardtii via DEA-sequencing [RNA-seq]

ABSTRACT: 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.

ORGANISM(S): Chlamydomonas reinhardtii

PROVIDER: GSE286999 | GEO | 2025/07/11

REPOSITORIES: GEO

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Project description:Transmission of 5-methylcytosine (5mC) from one cell generation to the next plays a key role in regulating cellular identity in mammalian development and diseases. While recent work has shown that the activity of DNMT1, the protein responsible for the stable inheritance of 5mC from mother to daughter cells, is imprecise; it remains unclear how the fidelity of DNMT1 is tuned in different genomic and cell state contexts. Here we describe Dyad-seq, a method that combines enzymatic detection of modified cytosines with nucleobase conversion techniques to quantify the genome-wide methylation status of cytosines at the resolution of individual CpG dinucleotides. We find that the fidelity of DNMT1-mediated maintenance methylation is directly related to the local density of DNA methylation, and for genomic regions that are lowly methylated, histone modifications can dramatically alter the maintenance methylation activity. Further, to gain deeper insights into the methylation and demethylation turnover dynamics, we extended Dyad-seq to quantify all combinations of 5mC and 5-hydroxymethylcytosine (5hmC) at individual CpG dyads to show that TET proteins preferentially hydroxymethylate only one of the two 5mC sites in a symmetrically methylated CpG dyad rather than sequentially convert both 5mC to 5hmC. To understand how cell state transitions impact DNMT1-mediated maintenance methylation, we scaled the method down and combined it with the measurement of mRNA to simultaneously quantify genome-wide methylation levels, maintenance methylation fidelity and the transcriptome from the same cell (scDyad&T-seq). Applying scDyad&T-seq to mouse embryonic stem cells transitioning from serum to 2i conditions, we observe dramatic and heterogenous demethylation and the emergence of transcriptionally distinct subpopulations that are closely linked to the cell-to-cell variability in loss of DNMT1-mediated maintenance methylation activity, with regions of the genome that escape 5mC reprogramming retaining high levels of maintenance methylation fidelity. Overall, our results demonstrate that while distinct cell states can substantially impact the DNA methylation maintenance machinery, there exists an intrinsic relationship between DNA methylation density, histone modifications and DNMT1-mediated maintenance methylation fidelity that is independent of cell state.

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Base-resolution profiling of 5-glyceryl-methylcytosine in Chlamydomonas reinhardtii via DEA-sequencing [RNA-seq]

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