Gadd45a promotes DNA demethylation through TDG
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE62496 | GEO | 2015/05/07
SECONDARY ACCESSION(S): PRJNA264295
REPOSITORIES: GEO
Similar Datasets
Project description:Growth arrest and DNA-damage-inducible protein 45 (Gadd45) family members have been implicated in DNA demethylation in vertebrates. However, it remained unclear how they contribute to the demethylation process. Here, we demonstrate that Gadd45a promotes active DNA demethylation through thymine DNA glycosylase (TDG) which has recently been shown to excise 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) generated in Ten-eleven-translocation (Tet) -initiated oxidative demethylation. The connection of Gadd45a with oxidative demethylation is evidenced by the enhanced activation of a methylated reporter gene in HEK293T cells expressing Gadd45a in combination with catalytically active TDG and Tet. Gadd45a interacts with TDG physically and increases the removal of 5fC and 5caC from genomic and transfected plasmid DNA by TDG. Knockout of both Gadd45a and Gadd45b from mouse ES cells leads to hypermethylation of specific genomic loci most of which are also targets of TDG and show 5fC enrichment in TDG-deficient cells. These observations indicate that the demethylation effect of Gadd45a is mediated by TDG activity. This finding thus unites Gadd45a with the recently defined Tet-initiated demethylation pathway. The dataset includes RRBS anlysis of 2 WT ES cell samples and 2 Gadd45a/b DKO ES cell samples.
2015-05-07 | E-GEOD-62496 | biostudies-arrayexpress
Project description:Active DNA demethylation in mammals involves TET-mediated iterative oxidation of 5-methylcytosine (5mC)/5-hydroxymethylcytosine (5hmC) and subsequent excision repair of highly oxidized cytosine bases 5-formylcytosine (5fC)/5-carboxylcytosine (5caC) by Thymine DNA glycosylase (TDG). However, quantitative and high-resolution analysis of active DNA demethylation activity remains challenging. Here we describe M.SssI methylase-assisted bisulfite sequencing (MAB-seq), a method that directly maps 5fC/5caC at single-base resolution. Genome-wide MAB-seq allows systematic identification of 5fC/5caC in Tdg-depleted embryonic stem cells, thereby generating a base-resolution map of active DNA demethylome. A comparison of 5fC/5caC and 5hmC distribution maps indicates that catalytic processivity of TET enzymes correlates with local chromatin accessibility. MAB-seq also reveals strong strand asymmetry of active demethylation within palindromic CpGs. Integrating MAB-seq with other base-resolution mapping methods enables quantitative measurement of cytosine modification states at key transitioning steps of active demethylation pathway, and reveals a regulatory role of 5fC/5caC excision repair in active DNA demethylation cascade. Analysis of 5fC/5caC excision repair-dependent active DNA demethylome by MAB-seq in mouse embryonic stem cells.
2014-11-02 | E-GEOD-62631 | 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:Background: Genome-wide methylation of cytosine can be modulated in the presence of TET and thymine DNA glycosylase (TDG) enzymes. TET enzymes are able to oxidise 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). TDG can excise the oxidative products 5fC and 5caC, initiating base excision repair. Furthermore, these modified bases are stable and detectable in the genome, raising the possibility that they could have epigenetic functions in their own right. To date, functional investigation of the genome-wide distribution of 5fC has been restricted to cell culture based systems, while its in vivo profile, in particular during development, remains unknown. Results: Here we describe the first analysis of the in vivo genome-wide profile of 5fC, across a range of dissected tissues from both wild type and Tdg-deficient E11.5 mouse embryos. Changes in the formylation profile of cytosine upon depletion of TDG suggest TET/TDG-mediated active demethylation occurs preferentially at intron-exon boundaries, and reveals a major role for TDG in shaping 5fC distribution at CpG islands. Moreover, we find enhancer regions exhibit high levels of 5fC, which accumulates at tissue-specific enhancers implicating a role in embryonic development. Conclusions: The tissue-specific distribution of 5fC can be regulated by the collective contribution of TET-mediated oxidation and excision by TDG. We show that the in vivo profile of 5fC during embryonic development resembles that of embryonic stem cells, sharing key features including enrichment of 5fC in enhancer and intragenic regions. Additionally, by investigating 5fC profiles in a tissue-specific manner from mouse embryos, we identified a targeted enrichment at active enhancers involved in tissue development. 5-formylcytosine has been mapped genomewide by pull-down and sequencing in mouse hindbrain, heart, carcass and liver. Each tissue was replicated in two different mice except for hindbrain which was replicated in four different mice.
2016-05-31 | E-GEOD-77447 | 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
Project description:Active DNA demethylation in mammals involves TET-mediated iterative oxidation of 5-methylcytosine (5mC)/5-hydroxymethylcytosine (5hmC) and subsequent excision repair of highly oxidized cytosine bases 5-formylcytosine (5fC)/5-carboxylcytosine (5caC) by Thymine DNA glycosylase (TDG). However, quantitative and high-resolution analysis of active DNA demethylation activity remains challenging. Here we describe M.SssI methylase-assisted bisulfite sequencing (MAB-seq), a method that directly maps 5fC/5caC at single-base resolution. Genome-wide MAB-seq allows systematic identification of 5fC/5caC in Tdg-depleted embryonic stem cells, thereby generating a base-resolution map of active DNA demethylome. A comparison of 5fC/5caC and 5hmC distribution maps indicates that catalytic processivity of TET enzymes correlates with local chromatin accessibility. MAB-seq also reveals strong strand asymmetry of active demethylation within palindromic CpGs. Integrating MAB-seq with other base-resolution mapping methods enables quantitative measurement of cytosine modification states at key transitioning steps of active demethylation pathway, and reveals a regulatory role of 5fC/5caC excision repair in active DNA demethylation cascade.
2014-11-02 | GSE62631 | GEO
Project description:Background: Genome-wide methylation of cytosine can be modulated in the presence of TET and thymine DNA glycosylase (TDG) enzymes. TET enzymes are able to oxidise 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). TDG can excise the oxidative products 5fC and 5caC, initiating base excision repair. Furthermore, these modified bases are stable and detectable in the genome, raising the possibility that they could have epigenetic functions in their own right. To date, functional investigation of the genome-wide distribution of 5fC has been restricted to cell culture based systems, while its in vivo profile, in particular during development, remains unknown. Results: Here we describe the first analysis of the in vivo genome-wide profile of 5fC, across a range of dissected tissues from both wild type and Tdg-deficient E11.5 mouse embryos. Changes in the formylation profile of cytosine upon depletion of TDG suggest TET/TDG-mediated active demethylation occurs preferentially at intron-exon boundaries, and reveals a major role for TDG in shaping 5fC distribution at CpG islands. Moreover, we find enhancer regions exhibit high levels of 5fC, which accumulates at tissue-specific enhancers implicating a role in embryonic development. Conclusions: The tissue-specific distribution of 5fC can be regulated by the collective contribution of TET-mediated oxidation and excision by TDG. We show that the in vivo profile of 5fC during embryonic development resembles that of embryonic stem cells, sharing key features including enrichment of 5fC in enhancer and intragenic regions. Additionally, by investigating 5fC profiles in a tissue-specific manner from mouse embryos, we identified a targeted enrichment at active enhancers involved in tissue development.
2016-05-31 | GSE77447 | GEO
Project description:TET enzymes mediate DNA demethylation by oxidizing 5-methylcytosine (5mC) in DNA to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). Because these oxidized methylcytosines (oxi-mC) are not recognized by the maintenance methyltransferase DNMT1, DNA demethylation can occur through “passive”, replication-dependent dilution as cells divide. A distinct, replication-independent (“active”) mechanism of DNA demethylation involves excision of 5fC and 5caC by the DNA repair enzyme thymine DNA glycosylase (TDG), followed by base excision repair. Here we used inducible gene-disrupted mice to show that TET enzymes influence both replication-dependent primary T cell differentiation and replication-independent macrophage differentiation, whereas TDG has no effect. Mice with long-term (1 year) deletion of Tdg are healthy and show normal survival and hematopoiesis. In summary, TET enzymes regulate differentiation and DNA demethylation primarily through passive dilution of oxidized methylcytosines in replicating T cells, and active, replication-independent DNA demethylation mediated by TDG does not appear to be essential for immune cell activation or differentiation.
2021-05-13 | GSE163894 | GEO