Modular oxidation of cytosine modifications and their application in direct and quantitative sequencing of 5-hydroxymethylcytosine
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ABSTRACT:
ORGANISM(S): Mus musculus Homo sapiens
PROVIDER: GSE214006 | GEO | 2023/03/15
REPOSITORIES: GEO
Similar Datasets
Project description:Although various methods have been developed for sequencing cytosine epigenetic modifications, specific and quantitative sequencing of the two major epigenetic modifications of cytosines, 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) at base-resolution is still challenging. Most times it requires subtraction of two methods to obtain both the true 5mC and 5hmC information, which increases noise and requires high sequencing depth. Recently we developed TET assisted pyridine borane sequencing (TAPS) for bisulfite-free direct sequencing of DNA methylation, which provides the sum of 5mC and 5hmC. Here we extend it to two sister methods, TAPSβ and CAPS (Chemical-Assisted Pyridine borane Sequencing), for whole-genome subtraction-free and specific sequencing of 5mC and 5hmC, respectively. We also demonstrated Pyridine borane Sequencing (PS) of whole-genome 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC), the further oxidized derivatives of 5mC and 5hmC. This completes the versatile borane reduction chemistry-based methods as a comprehensive suite for direct and quantitative sequencing of all four individual cytosine epigenetic modifications.
2020-12-14 | GSE155613 | 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. 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:We report a new bisulfite-free 5mC and 5hmC base-resolution sequencing method: TET Assisted Pyridine borane Sequencing (TAPS). TAPS relies on mild reactions and detects DNA modifications directly, without affecting unmodified cytosines. We applied this method for the first time to whole genome sequencing in E14 mESC cell line. For comparsion we prepared whole-genome bisulfite sequencing in the same cell line. Compared with bisulfite sequencing, TAPS results in higher mapping rates, more even coverage and lower sequencing cost, enabling more informative and cheaper methylome analyses. We expect TAPS to become the new standard in epigenetic DNA sequencing.
2018-12-24 | GSE112520 | GEO
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: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
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.
2015-09-07 | GSE66144 | GEO
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:Ten-eleven translocation (Tet) family of DNA dioxygenases converts 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5- carboxylcytosine (5caC) through iterative oxidation reactions. While 5mC and 5hmC are relatively abundant, 5fC and 5caC are at very low levels in the mammalian genome. Thymine DNA glycosylase (TDG) and base excision repair (BER) pathways can actively remove 5fC/5caC to regenerate unmethylated cytosine, but it is unclear to what extent and at which part of the genome such active demethylation processes take place. Here, we have performed high-throughput sequencing analysis of 5mC/5hmC/5fC/5caC- enriched DNA using modification-specific antibodies and generated genome-wide distribution maps of these cytosine modifications in wild-type and Tdg-deficient mouse embryonic stem cells (ESCs). We observe that the steady state 5fC and 5caC are preferentially detected at repetitive sequences in wild-type mouse ESCs. Depletion of TDG causes marked accumulation of 5fC and 5caC at a large number of distal gene regulatory elements and transcriptionally repressed/poised gene promoters, suggesting that Tet/TDG-dependent dynamic cycling of 5mC oxidation states may be involved in regulating the function of these regions. Thus, comprehensive mapping of 5mC oxidation and BER pathway activity in the mammalian genome provides a promising approach for better understanding of biological roles of DNA methylation and demethylation dynamics in development and diseases. Refer to individual Series
2013-04-18 | E-GEOD-42250 | biostudies-arrayexpress
Project description:Ten-eleven translocation (Tet) family of DNA dioxygenases converts 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5- carboxylcytosine (5caC) through iterative oxidation reactions. While 5mC and 5hmC are relatively abundant, 5fC and 5caC are at very low levels in the mammalian genome. Thymine DNA glycosylase (TDG) and base excision repair (BER) pathways can actively remove 5fC/5caC to regenerate unmethylated cytosine, but it is unclear to what extent and at which part of the genome such active demethylation processes take place. Here, we have performed high-throughput sequencing analysis of 5mC/5hmC/5fC/5caC- enriched DNA using modification-specific antibodies and generated genome-wide distribution maps of these cytosine modifications in wild-type and Tdg-deficient mouse embryonic stem cells (ESCs). We observe that the steady state 5fC and 5caC are preferentially detected at repetitive sequences in wild-type mouse ESCs. Depletion of TDG causes marked accumulation of 5fC and 5caC at a large number of distal gene regulatory elements and transcriptionally repressed/poised gene promoters, suggesting that Tet/TDG-dependent dynamic cycling of 5mC oxidation states may be involved in regulating the function of these regions. Thus, comprehensive mapping of 5mC oxidation and BER pathway activity in the mammalian genome provides a promising approach for better understanding of biological roles of DNA methylation and demethylation dynamics in development and diseases. In this dataset, we include the DIP-seq data of 5mC, 5hmC, 5fC and 5caC in both control and Tdg knockdown mouse embryonic stem cells.
2013-04-18 | E-GEOD-46111 | biostudies-arrayexpress
Project description:Ten-eleven translocation (Tet) family of DNA dioxygenases converts 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5- carboxylcytosine (5caC) through iterative oxidation reactions. While 5mC and 5hmC are relatively abundant, 5fC and 5caC are at very low levels in the mammalian genome. Thymine DNA glycosylase (TDG) and base excision repair (BER) pathways can actively remove 5fC/5caC to regenerate unmethylated cytosine, but it is unclear to what extent and at which part of the genome such active demethylation processes take place. Here, we have performed high-throughput sequencing analysis of 5mC/5hmC/5fC/5caC- enriched DNA using modification-specific antibodies and generated genome-wide distribution maps of these cytosine modifications in wild-type and Tdg-deficient mouse embryonic stem cells (ESCs). We observe that the steady state 5fC and 5caC are preferentially detected at repetitive sequences in wild-type mouse ESCs. Depletion of TDG causes marked accumulation of 5fC and 5caC at a large number of distal gene regulatory elements and transcriptionally repressed/poised gene promoters, suggesting that Tet/TDG-dependent dynamic cycling of 5mC oxidation states may be involved in regulating the function of these regions. Thus, comprehensive mapping of 5mC oxidation and BER pathway activity in the mammalian genome provides a promising approach for better understanding of biological roles of DNA methylation and demethylation dynamics in development and diseases. Gene expression comparison of control and Tdg knockdown mouse embryonic stem cells.
2013-04-18 | E-GEOD-46112 | biostudies-arrayexpress