Similar Datasets
Project description:Dnmt1 is a key methyltransferase involved in the maintenance of DNA methylation. To determine whether developmental defects induced by hypomorphic DNA 5-methylcytosine methyltransferase activity can be offset by increasing DNA replication time, we produced fish that were mutant for both dnmt1 and DNA polymerase epsilon (pole1) and compared their whole genome bisultite sequencing and gene expression profiles to WT ones. We further determine that the observed rescue effect by pole1 is specific to dnmt1 by comparing whole-genome bisulfite sequencing and gene expression profiles of mutants for the Methionine Adenosyltransferase 2A (mat2aa) and microchromosome 10 (mcm10)
2023-11-24 | GSE181572 | GEO
Project description:Dnmt1 is a key methyltransferase involved in the maintenance of DNA methylation. To determine whether developmental defects induced by hypomorphic DNA 5-methylcytosine methyltransferase activity can be offset by increasing DNA replication time, we produced fish that were mutant for both dnmt1 and DNA polymerase epsilon (pole1) and compared their whole genome bisultite sequencing and gene expression profiles to WT ones. We further determine that the observed rescue effect by pole1 is specific to dnmt1 by comparing whole-genome bisulfite sequencing and gene expression profiles of mutants for the Methionine Adenosyltransferase 2A (mat2aa) and microchromosome 10 (mcm10)
2023-11-24 | GSE181571 | GEO
Project description:In C. elegans, mutants in the anaphase-promoting complex or cyclosome (APC/C) exhibit defects in germline proliferation, the formation of the vulva and male tail, and the metaphase to anaphase transition of meiosis I. Oocytes lacking APC/C activity can be fertilized but arrest in metaphase of meiosis I and are blocked from further development. To examine the cell cycle and developmental consequences of reducing but not fully depleting APC/C activity, we analyzed defects in embryos and larvae of mat-1/cdc-27 mutants grown at semi-permissive temperatures. Hypomorphic embryos developed to the multicellular stage but were slow to complete meiosis I and displayed aberrant meiotic chromosome separation. More severely affected embryos skipped meiosis II altogether and exhibited striking defects in meiotic exit. These latter embryos failed to produce normal eggshells or establish normal asymmetries prior to the first mitotic division. In developing larvae, extended M-phase delays in late-dividing cell lineages were associated with defects in the morphogenesis of the male tail. This study reveals the importance of dosage-specific mutants in analyzing molecular functions of a ubiquitously functioning protein within different cell types and tissues, and striking correlations between specific abnormalities in cell cycle progression and particular developmental defects.
| S-EPMC1805483 | biostudies-literature
Project description:A precise balance of DNA methylation and demethylation is required for epigenetic control of cell identity, development, and growth. DNA methylation marks are introduced by de novo DNA methyltransferases DNMT3a/b and are maintained throughout cell divisions by DNA methyltransferase 1 (DNMT1), which adds methyl groups to hemimethylated CpG dinucleotides generated during DNA replication. Ten eleven translocation (TET) dioxygenases oxidize 5-methylcytosine (mC) to 5-hydroxymethylcytosine (hmC), 5-formylcytosine (fC), and 5-carboxylcytosine (caC), a process known to induce DNA demethylation and gene reactivation. In this study, we investigated the catalytic activity of human DNMT1 in the presence of oxidized forms of mC. A mass spectrometry-based assay was employed to study the kinetics of DNMT1-mediated cytosine methylation in CG dinucleotides containing C, mC, hmC, fC, or caC across from the target cytosine. Homology modeling, coupled with molecular dynamics simulations, was used to explore the structural consequences of mC oxidation with regard to the geometry of protein-DNA complexes. The DNMT1 enzymatic activity was strongly affected by the oxidation status of mC, with the catalytic efficiency decreasing in the following order: mC > hmC > fC > caC. Molecular dynamics simulations revealed that DNMT1 forms an unproductive complex with DNA duplexes containing oxidized forms of mC as a consequence of altered interactions of the target recognition domain of the protein with the C-5 substituent on cytosine. Our results provide new structural and mechanistic insight into TET-mediated DNA demethylation.
| S-EPMC6310613 | biostudies-literature
Project description:Fast, early embryonic cell cycles have correspondingly fast S phases. In early Drosophila embryos, forks starting from closely spaced origins replicate the whole genome in 3.4 min, ten times faster than in embryonic cycle 14 and a hundred times faster than in a wing disc. It is not known how S phase duration is regulated. Here we examined prolongation of embryonic S phases, its coupling to development, and its relationship to the appearance of heterochromatin.Imaging of fluorescent nucleotide incorporation and GFP-PCNA gave exquisite time resolution of S phase events. In the early S phases, satellite sequences replicated rapidly despite a compact chromatin structure. In S phases 11-13, a delay in satellite replication emerged in sync with modest and progressive prolongation of S phase. In S phase 14, major and distinct delays ordered the replication of satellites into a sequence that occupied much of S phase. This onset of late replication required transcription. Satellites only accumulated abundant heterochromatin protein 1 (HP1) after replicating in S phase 14. By cycle 15, satellites clustered in a compact HP1-positive mass, but replication occurred at decondensed foci at the surface of this mass.The slowing of S phase is an active process, not a titration of maternal replication machinery. Most sequences continue to replicate rapidly in successive cycles, but increasing delays in the replication of satellite sequences extend S phase. Although called constitutively heterochromatic, satellites acquire the distinctive features of heterochromatin, compaction, late replication, HP1 binding, and aggregation at the chromocenter, in successive steps coordinated with developmental progress.
| S-EPMC3108027 | biostudies-literature
Project description:The xenotopic expression of the alternative oxidase AOX from the tunicate Ciona intestinalis in diverse models of human disease partially alleviates the phenotypic effects of mitochondrial respiratory chain defects. AOX is a non-proton pumping, mitochondrial inner membrane-bound, single-subunit enzyme that can bypass electron transport through the cytochrome segment, providing an additional site for ubiquinone reoxidation and oxygen reduction upon respiratory chain overload. We set out to investigate whether AOX expression in Drosophila could counteract the effects of mitochondrial DNA (mtDNA) replication defects caused by disturbances in the mtDNA helicase or DNA polymerase ?. We observed that the developmental arrest imposed by either the expression of mutant forms of these enzymes or their knockdown was not rescued by AOX. Considering also the inability of AOX to ameliorate the phenotype of tko25t, a fly mutant with mitochondrial translation deficiency, we infer that this alternative enzyme may not be applicable to cases of mitochondrial gene expression defects. Finding the limitations of AOX applicability will help establish the parameters for the future putative use of this enzyme in gene therapies for human mitochondrial diseases.
| S-EPMC6052043 | biostudies-literature
Project description:DNA replication is initiated at replication origins on chromosomes at their scheduled time during S phase of the cell cycle. Replication timing control is highly conserved among eukaryotes but the underlying mechanisms are not fully understood. Recent studies have revealed that some telomere-binding proteins regulate replication timing at late-replicating origins throughout the genome. To investigate the molecular basis of this process, we analyzed the effects of excessive elongation of telomere DNA on replication timing by deleting telomere-associated shelterin proteins in Schizosaccharomyces pombe. We found that rap1∆ and poz1∆ cells showed abnormally accelerated replication at internal late origins but not at subtelomere regions. These defects were suppressed by removal of telomere DNA and by deletion of the telomere-binding protein Taz1. Furthermore, Sds21-a counter protein phosphatase against Dbf4-dependent kinase (DDK)-accumulated at elongated telomeres in a Taz1-dependent manner but was depleted at internal late origins, indicating that highly elongated telomeres sequester Sds21 at telomeres and perturb replication timing at internal regions. These results demonstrate that telomere DNA length is an important determinant of replication timing at internal regions of chromosomes in eukaryotes.
| S-EPMC6617677 | biostudies-literature
Project description:DNA methyltransferase 1 (DNMT1) is an important component of the epigenetic machinery and is responsible for copying DNA methylation patterns during cell division. Coordination of DNA methylation and DNA replication is critical for maintaining epigenetic programming. Knockdown of DNMT1 leads to inhibition of DNA replication, but the mechanism has been unclear. Here we show that depletion of DNMT1 with either antisense or small interfering RNA (siRNA) specific to DNMT1 activates a cascade of genotoxic stress checkpoint proteins, resulting in phosphorylation of checkpoint kinases 1 and 2 (Chk1 and -2), gammaH2AX focus formation, and cell division control protein 25a (CDC25a) degradation, in an ataxia telangiectasia mutated-Rad3-related (ATR)-dependent manner. siRNA knockdown of ATR blocks the response to DNMT1 depletion; DNA synthesis continues in the absence of DNMT1, resulting in global hypomethylation. Similarly, the response to DNMT1 knockdown is significantly attenuated in human mutant ATR fibroblast cells from a Seckel syndrome patient. This response is sensitive to DNMT1 depletion, independent of the catalytic domain of DNMT1, as indicated by abolition of the response with ectopic expression of either DNMT1 or DNMT1 with the catalytic domain deleted. There is no response to short-term treatment with 5-aza-deoxycytidine (5-aza-CdR), which causes demethylation by trapping DNMT1 in 5-aza-CdR-containing DNA but does not cause disappearance of DNMT1 from the nucleus. Our data are consistent with the hypothesis that removal of DNMT1 from replication forks is the trigger for this response.
| S-EPMC1636877 | biostudies-literature
Project description:DNA methyltransferase 1 (DNMT1) has been reported to interact with a wide variety of factors and to contain intrinsic transcriptional repressor activity. When a conservative point mutation was introduced at the key catalytic residue, mutant DNMT1 failed to rescue any of the phenotypes of Dnmt1-null embryonic stem (ES) cells, which indicated that the biological functions of DNMT1 are exerted through the methylation of DNA. ES cells that expressed the mutant protein did not survive differentiation. Intracisternal A-particle family retrotransposons were no longer methylated and were transcribed at high levels. The proper localization of DNMT1 depended on normal genomic methylation, and we discuss the implications of this finding for epigenetic dysregulation in cancer.
| S-EPMC1900033 | biostudies-literature
Project description:BackgroundMethylation at C-5 (5-mdC) of CpG base pairs, the most abundant epigenetic modification of DNA, is catalyzed by 3 essential DNA methyltransferases (Dnmt1, Dnmt3a and Dnmt3b). Aberrations in DNA methylation and Dnmts are linked to different diseases including cancer. However, their role in alcoholic liver disease (ALD) has not been elucidated.Methodology/principal findingsDnmt1 wild type (Dnmt1(+/+)) and hypomorphic (Dnmt1(N/+)) male mice that express reduced level of Dnmt1 were fed Lieber-DeCarli liquid diet containing ethanol for 6 weeks. Control mice were pair-fed calorie-matched alcohol-free liquid diet, and Dnmtase activity, 5-mdC content, gene expression profile and liver histopathology were evaluated. Ethanol feeding caused pronounced decrease in hepatic Dnmtase activity in Dnmt1(+/+) mice due to decrease in Dnmt1 and Dnmt3b protein levels and upregulation of miR-148 and miR-152 that target both Dnmt1 and Dnmt3b. Microarray and qPCR analysis showed that the genes involved in lipid, xenobiotic and glutathione metabolism, mitochondrial function and cell proliferation were dysregulated in the wild type mice fed alcohol. Surprisingly, Dnmt1(N/+) mice were less susceptible to alcoholic steatosis compared to Dnmt1(+/+) mice. Expression of several key genes involved in alcohol (Aldh3b1), lipid (Ppara, Lepr, Vldlr, Agpat9) and xenobiotic (Cyp39a1) metabolism, and oxidative stress (Mt-1, Fmo3) were significantly (P<0.05) altered in Dnmt1(N/+) mice relative to the wild type mice fed alcohol diet. However, CpG islands encompassing the promoter regions of Agpat9, Lepr, Mt1 and Ppara were methylation-free in both genotypes irrespective of the diet, suggesting that promoter methylation does not regulate their expression. Similarly, 5-mdC content of the liver genome, as measured by LC-MS/MS analysis, was not affected by alcohol diet in the wild type or hypomorphic mice.Conclusions/significanceAlthough feeding alcohol diet reduced Dnmtase activity, the loss of one copy of Dnmt1 protected mice from alcoholic hepatosteatosis by dysregulating genes involved in lipid metabolism and oxidative stress.
| S-EPMC3414497 | biostudies-literature