Covalent PARylation of DNA base excision repair proteins regulates DNA demethylation
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE166963 | GEO | 2023/11/08
REPOSITORIES: GEO
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Project description:Poly ADP-ribose (PAR) polymerases (PARPs) play fundamental roles in multiple DNA damage recognition and repair pathways. Persistent nuclear PARP activation causes cellular NAD+ depletion and exacerbates cellular aging. However, very little is known about mitochondrial PARP (mtPARP) and PARylation. The existence of mtPARP is controversial, and the biological roles for mtPARP induced mitochondrial PARylation are unclear. Here, we demonstrate the presence of PARP1 and PARylation in purified mitochondria. The addition of the PARP1 substrate NAD+ to isolated mitochondria induces PARylation which is suppressed by PARP inhibitor olaparib treatment. Mitochondrial PARylation was also evaluated by enzymatic labeling of terminal ADP-ribose (ELTA) labeling. To further confirm the presence of mtPARP1, we evaluated mitochondrial nucleoid PARylation by ADP ribose-chromatin affinity purification (ADPr-ChAP) . We observed that NAD+ stimulated PARylation and TFAM occupancy on the mtDNA regulatory region D-loop, inducing mtDNA transcription. These findings suggest that PARP1 is integrally involved in mitochondrial PARylation and NAD+ dependent mtPARP1 activity contributes to mtDNA transcription regulation.
2022-08-20 | GSE182533 | GEO
Project description:The A-type lamins (lamin A/C), encoded by the Lmna gene, are important structural components of the nuclear lamina. Lmna mutations lead to degenerative disorders, including the premature aging disease Hutchinson-Gilford progeria syndrome (HGPS). In addition, altered lamin A/C expression is found in various cancers. Reports indicate that lamin A/C plays a role in DNA double strand break repair, but a role in DNA base excision repair (BER) has not been described. We provide evidence for reduced BER efficiency in lamin A/C-depleted cells. The mechanism involves impairment of the APE1 and POLβ enzyme activities in BER. Also, Lmna null mouse fibroblasts displayed reduced expression of several core BER enzymes (PARP1, LIG3, and POLβ). Moreover, the robustness of APE1 and POLβ activities and the rate of BER were enhanced by lamin A/C-augmented poly(ADP-ribose) polymer formation (PARylation). Finally, we report that HGPS fibroblasts are defective in BER. Collectively, our results provide novel insights into the functional interplay between the nuclear lamina and cellular defenses against oxidative DNA damage, with implications for human cancer and aging.
2020-09-30 | GSE124465 | GEO
Project description:The A-type lamins (lamin A/C), encoded by the Lmna gene, are important structural components of the nuclear lamina. Lmna mutations lead to degenerative disorders, including the premature aging disease Hutchinson-Gilford progeria syndrome (HGPS). In addition, altered lamin A/C expression is found in various cancers. Reports indicate that lamin A/C plays a role in DNA double strand break repair, but a role in DNA base excision repair (BER) has not been described. We provide evidence for reduced BER efficiency in lamin A/C-depleted cells. The mechanism involves impairment of the APE1 and POLβ enzyme activities in BER. Also, Lmna null mouse fibroblasts displayed reduced expression of several core BER enzymes (PARP1, LIG3, and POLβ). Moreover, the robustness of APE1 and POLβ activities and the rate of BER were enhanced by lamin A/C-augmented poly(ADP-ribose) polymer formation (PARylation). Finally, we report that HGPS fibroblasts are defective in BER. Collectively, our results provide novel insights into the functional interplay between the nuclear lamina and cellular defenses against oxidative DNA damage, with implications for human cancer and aging.
2019-10-02 | GSE120389 | GEO
Project description:Ten-eleven translocation (TET) proteins oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytsosine (5fC), and 5-carboxylcytosine (5caC). 5fC/5caC can be excised and repaired by the base excision repair (BER) pathway, implicating 5mC oxidation in active DNA demethylation. Genome-wide DNA methylation is erased in the transition from metastable states to ground state of embryonic stem cells (ESCs) and in migrating primordial germ cells (PGCs), while some resistant regions become demethylated only in gonadal PGCs. Understanding the mechanisms underlying global hypomethylation in naïve ESCs and developing PGCs will be useful for realizing cellular pluripotency and totipotency. In this study, we found that PRDM14, the PR-domain-containing transcriptional regulator, accelerates the TET-BER cycle, resulting in the promotion of active DNA demethylation in ESCs. Induction of PRDM14 expression rapidly removed the 5mC associated with transient elevation of 5hmC at pluripotency-associated genes, germline-specific genes, and imprinted loci but not across the entire genome, which resemble second wave of DNA demethylation in gonadal PGCs. PRDM14 physically interacts with TET1/TET2 and enhances the recruitment of TET1/TET2 at target loci. Knockdown of Tet1/Tet2 impaired transcriptional regulation and DNA demethylation by PRDM14. The repression of the BER pathway by administration of pharmacological inhibitors against APE1 and PARP1 and the knockdown of thymine DNA glycosylase (TDG) also impaired DNA demethylation by PRDM14. Furthermore, DNA demethylation induced by PRDM14 normally takes place in the presence of aphidicolin, which is an inhibitor of G1/S progression. Together, our analysis provides mechanistic insight into DNA demethylation in naive pluripotent stem cells and developing PGCs. To investigate the function of TET1/TET2 in transcriptional regulation by PRDM14 in ESCs, we exploited microarray analysis using total mRNA derived from Scramble, Scramble + PRDM14, Tet1/Tet2 KD, Tet1/Tet2 KD + PRDM14 mouse ESC.
2013-12-31 | E-GEOD-52598 | biostudies-arrayexpress
Project description:The transition of oxidized 5-methylcytosine (5mC) intermediates into base excision repair (BER) pipeline to complete DNA demethylation remains enigmatic. We report here that UHRF2, the only paralog of UHRF1 in mammals that is neither able to maintain 5mC nor to rescue Uhrf1-/- phenotype, is physically and functionally associated with BER. UHRF2 is allosterically activated by 5-hydroxymethylcytosine (5hmC) and acts as a ubiquitin E3 ligase to catalyze K33-linked polyubiquitination of XRCC1, a core subunit of the BER complex. This nonproteolytic action stimulates the interaction of XRCC1 with the ubiquitin binding domain-bearing RAD23B, leading to the formation of the TDG-RAD23B-BER complex amenable to complete DNA demethylation. Integrative epigenomic analysis in mouse embryonic stem cells reveals that the Uhrf2-coordinated TDG-RAD23B-BER complex functionally links to the completion of DNA demethylation at promoters, and that during neuronal commitment, Uhrf2-associated and H3K4me1-marked latent enhancers undergo poised-to-active transition accompanied by H3K27ac acquisition. Accordingly, Uhrf2 ablation impairs DNA demethylation and abolishes neuronal differentiation. Together, these observations highlight the essentiality of 5hmC-switched UHRF2 E3 ligase activity in commissioning the accomplishment of active DNA demethylation.
2022-11-26 | GSE141297 | GEO
Project description:Formation of a repair enzyme complex is beneficial to DNA repair. Despite cellular studies showed that mitochondrial DNA polymerase (Pol ) and poly(ADP-ribose) polymerase 1 (PARP1) were found in the same complex along with other mitochondrial DNA repair enzymes and mitochondrial PARP1 level is correlated with mtDNA integrity, the molecular basis for the functional connection between Pol and PARP1 has not been illustrated, because cellular functions of PARP1 in DNA repair are intertwined with metabolism via NAD+ (nicotinamide adenosine dinucleotide), the substrate of PARP1 and a metabolic cofactor. To dissect the direct effect of PARP1 on mtDNA from the secondary perturbation metabolism, we report here biochemical studies that recapitulated Pol PARylation observed in cells, showed that PARP1 regulates Pol activity during DNA repair in a metabolic cofactor NAD+ (nicotinamide adenosine dinucleotide)-dependent manner. In the absence of NAD+, PARP1 completely inhibits Pol, while increasing NAD+ level to physiological concentration enables Pol to resume maximum repair activity. Because cellular NAD+ levels are linked to metabolism and to ATP production via oxidative phosphorylation, our results suggest that mtDNA damage repair is correlated with cellular metabolic state and integrity of the respiratory chain.
2020-12-16 | MSV000086606 | MassIVE
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. Gene expression comparison of control and Tdg knockdown mouse embryonic stem cells.
2013-04-18 | E-GEOD-46112 | biostudies-arrayexpress
Project description:Neurons harbor high levels of endogenous single strand DNA breaks (SSBs) that are targeted to neuronal enhancers and correlate with marks of DNA demethylation. To determine the source of SSBs at neuronal enhancers, we depleted the thymidine DNA glycosylase TDG, which excises TET-mediated oxidized methylcytidines 5fC and 5caC, to produce unmodified C. In differentiating neurons, induced degradation of TDG led to the disappearance of SSBs, demonstrating the existence of ongoing TET‑mediated oxidation. Using an independent model of macrophage differentiation from reprogrammed pre-B cells, we demonstrate that TET/TDG-mediated active demethylation may be a general mechanism underlying post-mitotic lineage specification. We find that macrophage differentiation prefers short patch base excision repair (SP-BER) to fill-in single nucleotide gaps, whereas neurons also frequently utilize the long-patch (LP-BER) sub-pathway. By measuring the distribution of SSBs relative to sites of oxidized cytosine, we observed that stretches of 2-30 bases are synthesized distal from the methylated CpG site during repair. Disrupting gap-filling using anti-neoplastic nucleoside analogs resulted in continuous DNA damage/repair events at enhancers each resolving within 1-2 hours, but ultimately triggering neuronal cell death. This DNA damage response and toxicity was dependent on TDG activity. Thus, TET-mediated active DNA demethylation promotes endogenous DNA damage at regulatory elements, a process which normally contributes to cell identity but can also provoke neurotoxicity following anti-cancer treatments.
2022-12-01 | GSE210314 | GEO
Project description:Neurons harbor high levels of endogenous single strand DNA breaks (SSBs) that are targeted to neuronal enhancers and correlate with marks of DNA demethylation. To determine the source of SSBs at neuronal enhancers, we depleted the thymidine DNA glycosylase TDG, which excises TET-mediated oxidized methylcytidines 5fC and 5caC, to produce unmodified C. In differentiating neurons, induced degradation of TDG led to the disappearance of SSBs, demonstrating the existence of ongoing TET‑mediated oxidation. Using an independent model of macrophage differentiation from reprogrammed pre-B cells, we demonstrate that TET/TDG-mediated active demethylation may be a general mechanism underlying post-mitotic lineage specification. We find that macrophage differentiation prefers short patch base excision repair (SP-BER) to fill-in single nucleotide gaps, whereas neurons also frequently utilize the long-patch (LP-BER) sub-pathway. By measuring the distribution of SSBs relative to sites of oxidized cytosine, we observed that stretches of 2-30 bases are synthesized distal from the methylated CpG site during repair. Disrupting gap-filling using anti-neoplastic nucleoside analogs resulted in continuous DNA damage/repair events at enhancers each resolving within 1-2 hours, but ultimately triggering neuronal cell death. This DNA damage response and toxicity was dependent on TDG activity. Thus, TET-mediated active DNA demethylation promotes endogenous DNA damage at regulatory elements, a process which normally contributes to cell identity but can also provoke neurotoxicity following anti-cancer treatments.
2022-12-01 | GSE210313 | GEO