Quantitative sequencing of 5-formylcytosine in DNA at single-base resolution
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
SUBMITTER: Giovanni Marsico
PROVIDER: E-GEOD-56572 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Similar Datasets
Project description:Bisulphite (BS) converted DNA from 2 paternal uniparental diploidies (pUPDs), one maternal (mUPD) and 5 control leukocytes samples were hybridized to the Infinium HumanMethylationEPIC BeadChip (Illumina), obtaining the BS DNA methylation profiles across approximately 850,000 CpGs. In addition, the 5 control leukocyte samples were also coverted using oxidative bisulphite (oxBS) treatment. The selective chemical oxidation of 5-hydroxymethylcytosine (5hmC) to 5-formylcytosine (5fC) and the deamination of the latter to uracil during the BS conversion allowed the quantification of independent 5-methylcytosine (5mC) and 5hmC methylation levels at every single CpG.
2018-07-11 | GSE103738 | GEO
Project description:Bisulphite (BS) and oxidative bisulphite (oxBS) converted DNA from 4 normal human placentas were hybridized to the Illumina HumanMethylation450 Beadchip v1.2, obtaining the BS and oxBS DNA methylation profiles across approximately 450,000 CpGs. By using the oxBS treatment, the selective chemical oxidation of 5-hydroxymethylcytosine (5hmC) to 5-formylcytosine (5fC) and the deamination of the latter to uracil during the BS conversion allowed the quantification of independent 5-methylcytosine (5mC) and 5hmC methylation levels at every single CpG. In consequence, this data set characterizes the genome-wide distribution of 5hmC in the human placenta offerring a high-confidence list of intervals enriched for 5hmC in this tissue.
2017-09-12 | GSE93429 | GEO
Project description:Cytosine DNA bases can be methylated by DNA methyltransferases and subsequently oxidized by TET proteins. The resulting 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) are considered demethylation intermediates as well as stable epigenetic marks. To dissect the contribution of these cytosine modifying enzymes, we generated combinations of Tet knockout (KO) embryonic stem cells (ESCs) and systematically measured protein and DNA modification levels at the transition from naive to primed pluripotency. Whereas the increase of genomic 5-methylcytosine (5mC) levels during exit from pluripotency correlated with an upregulation of the de novo DNA methyltransferases DNMT3A and DNMT3B, the subsequent oxidation steps turned out to be far more complex. The strong increase of oxidized cytosine bases (5hmC, 5fC, and 5caC) was accompanied by a drop in TET2 levels, yet the analysis of KO cells suggested that TET2 is responsible for most 5fC formation. The comparison of modified cytosine and enzyme levels in Tet KO cells revealed distinct and differentiation-dependent contributions of TET1 and TET2 to 5hmC and 5fC formation arguing against a processive mechanism of 5mC oxidation. The apparent independent steps of 5hmC and 5fC formation suggest yet to be identified mechanisms regulating TET activity and may constitute another layer of epigenetic regulation.
2020-07-24 | PXD019514 | Pride
Project description:To characterize the largely unknown functions of oxidised methylcytosines (oxi-mC; 5hmC/5fC/5caC) in DNA, several sequencing protocols have been recently developed. Quantitative analysis is complicated because DNA methylation modifications need to be deconvoluted from the data which is affected by several experimental parameters, including e.g. imperfect bisulphite conversion, oxidation efficiencies, various chemical labeling and protection steps, and sequencing errors. Here, we present a hierarchical generative model, Lux, for integrative analysis of any combination of BS-seq and âoxi-mCâ-seq (BS-seq/oxBS-seq/TAB-seq/fCAB-seq/CAB-seq/redBS-seq/MAB-seq) data. We show that Lux improves quantification and comparison of methylation levels over existing methods and that Lux can easily process any oxi-mC-seq data sets to quantify all cytosine modifications simultaneously together with their experimental parameters. Application of Lux to targeted data from Tet2 knockdown ESCs and T cells during development demonstrates DNA modification quantification at unprecedented detail, quantifies active demethylation pathway and reveals 5hmC localization in putative regulatory regions. Examine the distribution of C, 5mC, and 5hmC in ES and Tet2 knock-down cells within selected loci. Half of the samples are measured using the traditional bisulphite-seq protocol but the other half is measured using the oxidative bisulphite-seq protocol (oxBS-seq).
2016-07-03 | E-GEOD-68574 | biostudies-arrayexpress
Project description:TET proteins oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). 5fC and 5caC are excised by mammalian DNA glycosylase TDG, implicating 5mC oxidation in DNA demethylation. Here we show that the genomic locations of 5fC can be determined by coupling chemical reduction with biotin tagging. Genome-wide mapping of 5fC in mouse embryonic stem cells (mESCs) reveals that 5fC preferentially occurs at poised enhancers among other gene regulatory elements. Application to Tdg null mESCs further suggests that 5fC production coordinates with p300 in remodeling epigenetic states of enhancers. This process, which is not influenced by 5hmC, appears to be associated with further oxidation of 5hmC and commitment to demethylation through 5fC. Finally, we resolved 5fC at base-resolution by hydroxylamine-based protection from bisulfite-mediated deamination, thereby confirming sites of 5fC accumulation. Our results reveal roles of active 5mC/5hmC oxidation and TDG-mediated demethylation in epigenetic tuning at regulatory elements. We report here a chemical labeling method that effectively differentiates 5fC from 5mC, 5hmC, and 5caC in genomic DNA. First, we quantitatively protect endogenous 5hmC with a regular glucose using b-glucosytransferase-catalyzed 5hmC glucosylation. Then, we selectively reduce 5fC with NaBH4 to 5hmC, and chemically label the resulting 5hmC (from 5fC) with an azide-modified glucose. Biotin can be installed subsequently for specific enrichment of 5fC. Our method thereby provides an effective tool of general utility for the genomic localization of 5fC. Here we provide genome-wide profiles of 5hmC, 5fC, and p300 in Tdg fl/fl and Tdg-/- mESCs as well as a 5fC control (Non-NaBH4) and polyA RNA-Seq expression data. Genome-wide profiles of 5hmC and 5fC in mESCs differentiated to embryoid bodies are also included. We also report the development and application of a single-base resolution method for the detection of 5fC in genomic DNA by hydroylamine mediated protection of 5fC from deamination during bisulfite treatment, or 5fC Chemical Assisted Bisulfite Sequencing (fCAB-Seq). We applied this method in parallel with conventional ChIP-Methyl-Seq to H3K4me1 ChIP enriched DNA from Tdg fl/fl and Tdg-/- mice.
2013-04-18 | E-GEOD-41545 | biostudies-arrayexpress
Project description:To characterize the largely unknown functions of oxidised methylcytosines (oxi-mC; 5hmC/5fC/5caC) in DNA, several sequencing protocols have been recently developed. Quantitative analysis is complicated because DNA methylation modifications need to be deconvoluted from the data which is affected by several experimental parameters, including e.g. imperfect bisulphite conversion, oxidation efficiencies, various chemical labeling and protection steps, and sequencing errors. Here, we present a hierarchical generative model, Lux, for integrative analysis of any combination of BS-seq and âoxi-mCâ-seq (BS-seq/oxBS-seq/TAB-seq/fCAB-seq/CAB-seq/redBS-seq/MAB-seq) data. We show that Lux improves quantification and comparison of methylation levels over existing methods and that Lux can easily process any oxi-mC-seq data sets to quantify all cytosine modifications simultaneously together with their experimental parameters. Application of Lux to targeted data from Tet2 knockdown ESCs and T cells during development demonstrates DNA modification quantification at unprecedented detail, quantifies active demethylation pathway and reveals 5hmC localization in putative regulatory regions. Examine the distribution of C, 5mC, and 5hmC in DP, CD4 SP, and naïve CD4 T cells within selected loci. Half of the samples are measured using the traditional bisulphite-seq protocol but the other half is measured using the oxidative bisulphite-seq protocol (oxBS-seq).
2016-07-03 | E-GEOD-68575 | biostudies-arrayexpress
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
2015-01-20 | E-GEOD-56429 | biostudies-arrayexpress
Project description:Active DNA demethylation in mammals involves TET-mediated oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxycytosine (5caC). However, genome-wide detection of 5fC at single-base resolution remains challenging. Here we present a bisulfite-free method for the whole-genome analysis of 5fC, based on a selective chemical labeling of 5fC and subsequent C-to-T transition during PCR. Base-resolution 5fC maps reveal limited overlap with 5hmC, with 5fC-marked regions more active than 5hmC-marked ones. Utilization of cyclization-enabled C-to-T transition of 5fC (hence “fC-CET”) to obtain genome-wide map of 5fC at single-base resolution WT and TdgKO mES cell lines. Two non-enriched input DNAs (Input: preAI), two AI labeled samples (Input: AI), two pull-down output samples.
2015-09-07 | E-GEOD-66144 | biostudies-arrayexpress
Project description:Cytosine base modifications 5-methylcytosine (5mC), 5-hydroxymethylcytosine (5hmC) and 5-formylcytosine (5fC) are present in mammalian DNA. Here, reduced bisulfite sequencing is developed for quantitatively sequencing 5fC at single-base resolution. This method is then applied with oxidative bisulfite sequencing to gain a map of 5mC, 5hmC and 5fC in mouse embryonic stem cells.
2014-04-08 | GSE56572 | GEO
Project description:TET proteins oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). 5fC and 5caC are excised by mammalian DNA glycosylase TDG, implicating 5mC oxidation in DNA demethylation. Here we show that the genomic locations of 5fC can be determined by coupling chemical reduction with biotin tagging. Genome-wide mapping of 5fC in mouse embryonic stem cells (mESCs) reveals that 5fC preferentially occurs at poised enhancers among other gene regulatory elements. Application to Tdg null mESCs further suggests that 5fC production coordinates with p300 in remodeling epigenetic states of enhancers. This process, which is not influenced by 5hmC, appears to be associated with further oxidation of 5hmC and commitment to demethylation through 5fC. Finally, we resolved 5fC at base-resolution by hydroxylamine-based protection from bisulfite-mediated deamination, thereby confirming sites of 5fC accumulation. Our results reveal roles of active 5mC/5hmC oxidation and TDG-mediated demethylation in epigenetic tuning at regulatory elements.
2013-04-18 | GSE41545 | GEO