Base-resolution profiling of 5-glyceryl-methylcytosine in Chlamydomonas reinhardtii via deaminase-assisted sequencing [RNA-Seq]
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PROVIDER: PRJNA1287576 | ENA |
REPOSITORIES: ENA
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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
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 | GSE301797 | GEO
Project description:Base-resolution profiling of 5-glyceryl-methylcytosine in Chlamydomonas reinhardtii via deaminase-assisted sequencing [Bisulfite-Seq]
| PRJNA1287575 | ENA
Project description:5-Methylcytosine (5mC) is a crucial epigenetic modification plays a significant role in the regulation of gene expression. Accurate and quantitative detection of 5mC at single-base resolution is essential for understanding its epigenetic functions within genomes. In this study, we develop a novel NaegleriaTET-assisted deaminase sequencing (NTD-seq) method for the base-resolution and quantitative detection of 5mC in genomic DNA. The TAD-seq method utilizes a Naegleria TET-like dioxygenase (nTET) to oxidize 5mC, generating 5-methylcytosine oxidation products (5moC). We also engineered a variant of the human apolipoprotein B mRNA-editing catalytic polypeptide-like 3A (A3A), creating an A3A mutant (A3Am). Treatment with A3Am results in the conversion of cytosine to uracil, while 5moC remains unchanged. Consequently, TAD-seq enables the direct deamination of cytosine to uracil by A3Am, which is sequenced as thymine, whereas 5mC, once oxidized to 5moC by nTET, resists deamination and is sequenced as cytosine. Therefore, the cytosines that persist in the sequencing data represent the original 5mC sites. We applied NTD-seq to HEK293T cells, generating a base-resolution map of 5mC that exhibits strong concordance with maps generated by conventional BS-seq. NTD-seq emerges as a powerful, bisulfite-free approach for the single-base resolution mapping of 5mC stoichiometry in genomic DNA.
2025-04-02 | GSE263426 | GEO
Project description:5-Methylcytosine (5mC) is a crucial epigenetic modification plays a significant role in the regulation of gene expression. Accurate and quantitative detection of 5mC at single-base resolution is essential for understanding its epigenetic functions within genomes. In this study, we develop a novel nTET-assisted deaminase sequencing (TAD-seq) method for the base-resolution and quantitative detection of 5mC in genomic DNA. The TAD-seq method utilizes a Naegleria TET-like dioxygenase (nTET) to oxidize 5mC, generating 5-methylcytosine oxidation products (5moC). We also engineered a variant of the human apolipoprotein B mRNA-editing catalytic polypeptide-like 3A (A3A), creating an A3A mutant (A3Am). Treatment with A3Am results in the conversion of cytosine to uracil, while 5moC remains unchanged. Consequently, TAD-seq enables the direct deamination of cytosine to uracil by A3Am, which is sequenced as thymine, whereas 5mC, once oxidized to 5moC by nTET, resists deamination and is sequenced as cytosine. Therefore, the cytosines that persist in the sequencing data represent the original 5mC sites. We applied TAD-seq to HEK293T cells, generating a base-resolution map of 5mC that exhibits strong concordance with maps generated by conventional BS-seq. TAD-seq emerges as a powerful, bisulfite-free approach for the single-base resolution mapping of 5mC stoichiometry in genomic DNA.
2024-06-30 | GSE254203 | GEO
Project description:N4-methylcytosine is a major DNA modification integral to restriction-modification (R-M) systems in bacterial genomes. Here we describe 4mC-Tet-Assisted Bisulfite-sequencing (4mC-TAB-seq), a method that accurately and rapidly reveals the genome-wide locations of N4-methylcytosines at single-base resolution. By coupling Tet-mediated oxidation with a modified sodium bisulfite conversion reaction, unmodified cytosines and 5-methylcytosines are read out as thymines, whereas N4-methylcytosines are read out as cytosines revealing their positions throughout the genome. 4mC-TAB-seq
2015-07-11 | E-GEOD-63371 | biostudies-arrayexpress
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.
2025-07-11 | GSE287000 | GEO
Project description:Transcriptome-wide identification of 5-methylcytosine by deaminase and reader protein-assisted sequencing
| PRJNA1068978 | ENA
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.
2025-07-11 | GSE286999 | GEO
Project description:5-Methylcytosine (m5C) is one of the major post-transcriptional modifications in mRNA and is highly involved in the pathogenesis of various diseases. However, the capacity of existing assays for accurately and comprehensively transcriptome-wide m5C mapping still needs improvement. Here, we developed a deaminase and reader protein assisted RNA methylation analysis, termed DRAM, in which deaminases (APOBEC1 and TadA-8e) are fused with m5C reader proteins (ALYREF and YBX1) to identify the m5C sites through deamination events neighboring the methylation sites. This antibody-free and bisulfite-free approach provided transcriptome-wide editing regions which were highly overlapped with the publicly available BS-seq datasets. Notably, DRAM-seq even discovered a new m5C methylation locus in KAT7, a previously demonstrated key aging regulator. In addition, DRAM system even supports ultra-low input RNA (10ng) and monitor the dynamic accumulation of cellular m5C. We anticipated that the DRAM system could pave the way for uncovering further biological functions of m5C modifications.
2025-03-14 | GSE254194 | GEO