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Functionally distinct roles for TET-oxidized 5-methylcytosine bases in somatic reprogramming to pluripotency [ATAC-seq]

ABSTRACT: Active DNA demethylation via Ten-eleven Translocation (TET) family enzymes is essential for epigenetic reprogramming in cell state transitions. TET enzymes catalyze up to three successive oxidations of 5-methylcytosine (5mC), generating 5- hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), or 5-carboxycytosine (5caC). Although these bases are known to contribute to distinct demethylation pathways, the lack of tools to uncouple these sequential oxidative events has constrained our mechanistic understanding of TET’s role in reprogramming. Here, we employ biochemically engineered TET mutants to examine their effects on the reprogramming efficiency of mouse embryonic fibroblasts (MEFs). We show that only TET mutants proficient for oxidation to 5fC/5caC are able to rescue the reprogramming potential of Tet2 -deficient MEFs. This effect correlated with an increased capacity to drive DNA demethylation at reprogramming enhancers, ultimately accelerating chromatin opening in these regions. Together, these experiments demonstrate that DNA demethylation through 5fC/5caC intermediates is critical for iPSC reprogramming.

ORGANISM(S): Mus musculus

PROVIDER: GSE150964 | GEO | 2021/12/21

REPOSITORIES: GEO

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Functionally distinct roles for TET-oxidized 5-methylcytosine bases in somatic reprogramming to pluripotency [RNA-Seq]

Project description:Active DNA demethylation via Ten-eleven Translocation (TET) family enzymes is essential for epigenetic reprogramming in cell state transitions. TET enzymes catalyze up to three successive oxidations of 5-methylcytosine (5mC), generating 5- hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), or 5-carboxycytosine (5caC). Although these bases are known to contribute to distinct demethylation pathways, the lack of tools to uncouple these sequential oxidative events has constrained our mechanistic understanding of TET’s role in reprogramming. Here, we employ biochemically engineered TET mutants to examine their effects on the reprogramming efficiency of mouse embryonic fibroblasts (MEFs). We show that only TET mutants proficient for oxidation to 5fC/5caC are able to rescue the reprogramming potential of Tet2 -deficient MEFs. This effect correlated with an increased capacity to drive DNA demethylation at reprogramming enhancers, ultimately accelerating chromatin opening in these regions. Together, these experiments demonstrate that DNA demethylation through 5fC/5caC intermediates is critical for iPSC reprogramming.

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Single-base resolution analysis of active DNA demethylation using methylase-assisted bisulfite sequencing

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Bisulfite-free and Base-resolution Analysis of 5-formylcytosine at Whole-genome Scale

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Bisulfite-free and Base-resolution Analysis of 5-formylcytosine at Whole-genome Scale

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.

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Single-base resolution analysis of active DNA demethylation using methylase-assisted bisulfite sequencing

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Distinct and stage-specific contributions of TET1 and TET2 to stepwise cytosine oxidation in the transition from naive to primed pluripotency

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

Roles of TET and TDG in DNA demethylation in proliferating and non-proliferating immune cells [RNA-seq]

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.

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Roles of TET and TDG in DNA demethylation in proliferating and non-proliferating immune cells [CMS-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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