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Base-resolution profiling of 5-glyceryl-methylcytosine in Chlamydomonas reinhardtii via deaminase-assisted sequencing [RNA-Seq]

ABSTRACT: 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 (DEA-seq) 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, 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 loss of 5gmC at repressive H3K9me1 sites. 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.

ORGANISM(S): Chlamydomonas reinhardtii

PROVIDER: GSE301797 | GEO | 2025/09/05

REPOSITORIES: GEO

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Base-resolution profiling of 5-glyceryl-methylcytosine in Chlamydomonas reinhardtii via deaminase-assisted sequencing [Bisulfite-Seq]

Project description: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 (DEA-seq) 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, 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 loss of 5gmC at repressive H3K9me1 sites. 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.

2025-09-05 | GSE301796 | GEO

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

Project description: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.

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

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Base-resolution maps of 5-formylcytosine and 5-carboxylcytosine reveal genome-wide DNA demethylation dynamics

Project description:5-methylcytosine (5mC), the predominant epigenetic modification on DNA, plays critical roles in mammalian development and is dysregulated in various human pathologies. In mammals, the TET family of dioxygenases can oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) in a stepwise manner. 5fC and 5caC are selectively recognized and excised by mammalian thymine DNA glycosylase (TDG), and restored to normal cytosine through base excision repair (BER). Once 5mC/5hmC is converted to 5fC and/or 5caC, the modified cytosine is committed to demethylation through BER. Thus 5fC and 5caC most likely mark active demethylation in the mammalian genome. Here we introduce a genome-wide approach to obtain single-base resolution maps of 5fC and 5caC, respectively. We show that, in mouse embryonic stem cells (mESCs), 5fC and 5caC are preferentially generated at highly hypomethylated regions and more active enhancers. Moreover, 5caC-marked regions are characterized by the lowest methylation and highest enhancer activity among all modification sites associated with 5hmC, 5fC and 5caC, and are enriched adjacent to pluripotency transcription factor (TF)-binding motifs. These observations, together with the surprising lack of overlap between 5fC and 5caC sites, highlight a gradient of Tet-mediated 5mC oxidation activity at regulatory elements in tuning epigenetic dynamics11. DNA immunoprecipitation coupled chemical-modification assisted bisulfite sequencing (DIP-CAB-Seq) for Tdg fl/fl and Tdg-/- mESCs

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Whole-genome mapping of 5-hydroxymethylcytosine at single-base resolution by double-stranded DNA deamination sequencing

Project description:5-Hydroxymethylcytosine (5hmC) is a crucial epigenetic mark that plays a key role in regulating various physiological and pathological processes. Although several methods have been developed to detect 5hmC, direct genome-wide mapping at single-base resolution remains highly desirable. Herein, we developed a double-stranded deamination sequencing (DDD-seq) method for genome-wide mapping of 5hmC at single-base resolution. This method utilizes the double-stranded DNA deaminase SsdAcat from Pseudomonas syringae, which efficiently deaminates cytosine (C), 5-methylcytosine (5mC), 5hmC, 5-formylcytosine (5fC), and 5-carboxycytosine (5caC) in duplex DNA, but not glycosylated 5hmC (5gmC). In DDD-seq, C, 5mC, 5fC, and 5caC in dsDNA are deaminated by SsdAcat and read as T during sequencing, while 5gmC is resistant to deamination, allowing its identification by detecting the remaining C in sequence reads. The map of 5hmC generated by DDD-seq in mouse cerebellum tissue closely aligns with results obtained from the ACE-seq method. Additionally, applying DDD-seq to cerebellum tissue of mouse subjected to chronic sleep deprivation revealed significant global changes in 5hmC distribution on genomic DNA. In contrast to previous single-stranded deaminase APOBEC3A-based mapping methods that require denaturation of dsDNA into ssDNA, DDD-seq eliminates this step, reducing risks associated with incomplete denaturation and simplifying sequencing library construction. Additionally, SsdAcat demonstrates superior thermostability and activity across a wide range of pH values and temperatures, making DDD-seq applicable in broader scenarios with more accessible conditions. Collectively, the DDD-seq method is straightforward, bisulfite-free, and eliminates the need for DNA denaturation step, making it a valuable tool for direct and quantitative detection of 5hmC in genomes at single-base resolution.

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A vitamin C-derived DNA modification catalyzed by a TET homolog in green alga [Bisulfite-seq]

Project description:Methylation of cytosine to 5-methylcytosine (5mC) is a prevalent DNA modification found in many organisms. Sequential oxidation of 5mC by TET dioxygenases results in a cascade of additional epigenetic marks and promotes DNA demethylation in mammals1,2. However, the enzymatic activity and the function of TET homologs present in lower eukaryotes remains largely unexplored. In our study of TET homologs in the green alga Chlamydomonas reinhardtii (C. reinhardtii), we have found a 5mC-modifying enzyme (CMD1) that catalyzes conjugation of a glyceryl moiety onto the methyl group of 5mC through a carbon-carbon bond, resulting in two novel stereoisomeric nucleotide products. The catalytic activity of CMD1 requires Fe(II) and the integrity of its His-x-Asp (HxD) binding motif, which is conserved in Fe-dependent oxygenases3. However, unlike all previous described TET enzymes which utilize 2-oxoglutarate (2-OG) as a co-substrate4, CMD1 utilizes L-ascorbic acid (vitamin C, VC) as an essential co-substrate. VC itself is the source of the glyceryl moiety that modifies 5mC, with concurrent formation of glyoxylic acid and CO2. The VC-derived DNA modification is present in the genome of C. reinhardtii and its level decreases significantly in a CMD1 mutant strain. The fitness of CMD1 mutant cells to high light exposure is reduced, mainly due to deficient expression of the critical non-photochemical quenching (NPQ) effector gene LHCSR3, which is hypermethylated in the mutant cells. Our study thus reveals a new eukaryotic DNA base modification, and its involvement in a functionally conserved but mechanistically divergent DNA demethylation pathway for the epigenetic regulation of photosynthesis.

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A vitamin C-derived DNA modification catalyzed by a TET homolog in green alga [RNA-seq]

Project description:Methylation of cytosine to 5-methylcytosine (5mC) is a prevalent DNA modification found in many organisms. Sequential oxidation of 5mC by TET dioxygenases results in a cascade of additional epigenetic marks and promotes DNA demethylation in mammals. However, the enzymatic activity and the function of TET homologs present in lower eukaryotes remains largely unexplored. In our study of TET homologs in the green alga Chlamydomonas reinhardtii (C. reinhardtii), we have found a 5mC-modifying enzyme (CMD1) that catalyzes conjugation of a glyceryl moiety onto the methyl group of 5mC through a carbon-carbon bond, resulting in two novel stereoisomeric nucleotide products. The catalytic activity of CMD1 requires Fe(II) and the integrity of its His-x-Asp (HxD) binding motif, which is conserved in Fe-dependent oxygenases. However, unlike all previous described TET enzymes which utilize 2-oxoglutarate (2-OG) as a co-substrate, CMD1 utilizes L-ascorbic acid (vitamin C, VC) as an essential co-substrate. VC itself is the source of the glyceryl moiety that modifies 5mC, with concurrent formation of glyoxylic acid and CO2. The VC-derived DNA modification is present in the genome of C. reinhardtii and its level decreases significantly in a CMD1 mutant strain. The fitness of CMD1 mutant cells to high light exposure is reduced, mainly due to deficient expression of the critical non-photochemical quenching (NPQ) effector gene LHCSR3, which is hypermethylated in the mutant cells. Our study thus reveals a new eukaryotic DNA base modification, and its involvement in a functionally conserved but mechanistically divergent DNA demethylation pathway for the epigenetic regulation of photosynthesis.

2019-05-04 | GSE122717 | GEO

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