PARP inhibition induces cytotoxicity in mESCs by activating endogenous retroviruses [ATAC-seq]
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE166962 | GEO | 2026/05/11
REPOSITORIES: GEO
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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.
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Project description:Genome-wide DNA demethylation is a unique feature of mammalian development and naïve pluripotent stem cells. So far, it was unclear how mammals specifically achieve global DNA hypomethylation, given the high conservation of the DNA (de-)methylation machinery among vertebrates. We found that DNA demethylation requires TET activity but mostly occurs at sites where TET proteins are not bound suggesting a rather indirect mechanism. Among the few specific genes bound and activated by TET proteins was the naïve pluripotency and germline marker Dppa3 (Pgc7, Stella), which undergoes TDG dependent demethylation. The requirement of TET proteins for genome-wide DNA demethylation could be bypassed by ectopic expression of Dppa3. We show that DPPA3 binds and displaces UHRF1 from chromatin and thereby prevents the recruitment and activation of the maintenance DNA methyltransferase DNMT1. We demonstrate that DPPA3 alone can drive global DNA demethylation when transferred to amphibians (Xenopus) and fish (medaka), both species that naturally do not have a Dppa3 gene and exhibit no post-fertilization DNA demethylation. Our results show that TET proteins are responsible for active and - indirectly also for - passive DNA demethylation; while TET proteins initiate local and gene-specific demethylation in vertebrates, the recent emergence of DPPA3 introduced a unique means of genome-wide passive demethylation in mammals and contributed to the evolution of epigenetic regulation during early mammalian development.
2020-10-12 | PXD019794 | Pride
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.
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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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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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Project description:RUNX1 (also known as AML1) is a key transcription factor for definitive hematopoietic stem cell development and following hematopoietic cell linage specifications, in which chromatin- or epigennome-mediated regulation by RUNX1, particularly regulation of DNA methylation status, is proposed to be involved in addition to its direct gene expression regulation. However, how RUNX1 regulates DNA methylation status and its role in the hematopoiesis remain to be elucidated. Here we first demonstrated that RUNX1 induces RUNX1 binding site directed DNA demethylation across the whole genome. HaloTag-based pull-down assay revealed associations of RUNX1 with active DNA demethylation related proteins such as TET, TDG or GADD45A, suggested that the RUNX1-mediated DNA demethylation is active DNA demethylation mechanism. Additional combinatorial overexpression of TET and TDG enlarged the RUNX1-mediated DNA demethylation, supporting what RUNX1-mediated DNA demethylation is active DNA demethylation. These results strongly suggested that RUNX1-mediated DNA demethylation is achieved by recruiting those proteins involved in active DNA demethylation. Finally, we found that the RUNX1-mediated demethylation predominately targets and activates hematopoietic genes whose promoter regions are demethylated during hematopoiesis. Collectively, our insight suggested that RUNX1-mediated binding site directed DNA demethylation is a novel mechanism of hematopoietic gene activation.
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Project description:Tet-mediated DNA oxidation is a new type of epigenetic modification in mammals and its role in the regulation of cell fate transition remains poorly understood. Here, we derive mouse embryonic fibroblasts (MEFs) deleted in all three Tet genes and examine their capability to be reprogrammed into iPS cells. We demonstrate that these Tet-deficient MEFs cannot be reprogrammed due to a blockage in the mesenchymal-to-epithelial transition (MET). Reprogramming of MEFs deficient in TDG is similarly blocked. The blockage is caused by impaired activation of crucial microRNAs, which depends on oxidative demethylation promoted by Tet and TDG. Reintroduction of either miR-200c or catalytically active Tet and TDG restores reprogramming to the respective knockout MEFs. Thus, oxidative demethylation is essential for somatic cell reprogramming. These findings provide mechanistic insights into the operation of epigenetic barriers in cell lineage conversion.
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Project description:Thymine DNA Glycosylase (TDG) is a versatile protein involved in DNA mismatch repair, DNA demethylation, and transcriptional regulation. Here, we identify TDG as a synthetic essential effector in p53-deficient tumours. We found that deletion of TDG inhibits cell proliferation specifically in p53-deficient cancer cells. Using a genetically engineered mouse model of lung adenocarcinoma (LUAD), we demonstrate that depletion of TDG suppresses tumour growth in the p53-deficient context. Notably, A novel and selective inhibitor developed to disrupt the DNA binding activity of TDG exhibits therapeutic efficacy against p53-deficient tumours in both cellular and xenograft models. Mechanistically, TDG coordinates with p53 to orchestrate the transcription of RNA helicase DHX9, which functions to eliminate dsRNA. Depletion of TDG in p53-deficient cells results in the downregulation of DHX9 and aberrant cytoplasmic double-strand (dsRNA) accumulation derived from Alu elements, subsequently activating the RIG-I/MDA5-MAVS mediated antiviral response. Moreover, single-cell RNA-sequencing analysis revealed the depletion of TDG in p53-deficient tumours facilitates the recruitment of tumour-infiltrating lymphocytes. We observed that TDG inhibition combined with immune checkpoint blockade (ICB) achieved a strong synergistic anti-tumour effect in p53-deficient tumours. This study highlights the function of TDG in maintaining p53-deficient tumour growth and provides an alternative therapeutic target for p53-deficient cancers.
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