Supplementary Document from GKN1–miR-185–DNMT1 Axis Suppresses Gastric Carcinogenesis through Regulation of Epigenetic Alteration and Cell Cycle
Jung‐Hwan YoonYoo Jin ChoiWon Suk ChoiHassan AshktorabDuane T. SmootSuk Woo NamJung Young LeeWon Sang Park
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<p>Supplementary Document - PDF file 159K, Supplemental document including Supplemental Table, Figure legends, material & methods and refference</p>Keywords:
DNMT1
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DNMT1
DNA methyltransferase
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Dnmt3a is a de novo DNA methyltransferase that modifies unmethylated DNA. In contrast Dnmt1 shows high preference for hemimethylated DNA. However, Dnmt1 can be activated for the methylation of unmodified DNA. We show here that the Dnmt3a and Dnmt1 DNA methyltransferases functionally cooperate in de novo methylation of DNA, because a fivefold stimulation of methylation activity is observed if both enzymes are present. Stimulation is observed if Dnmt3a is used before Dnmt1, but not if incubation with Dnmt1 precedes Dnmt3a, demonstrating that methylation of the DNA by Dnmt3a stimulates Dnmt1 and that no physical interaction of Dnmt1 and Dnmt3a is required. If Dnmt1 and Dnmt3a were incubated together a slightly increased stimulation is observed that could be due to a direct interaction of these enzymes. In addition, we show that Dnmt1 is stimulated for methylation of unmodified DNA if the DNA already carries some methyl groups. We conclude that after initiation of de novo methylation of DNA by Dnmt3a, Dnmt1 becomes activated by the pre-existing methyl groups and further methylates the DNA. Our data suggest that Dnmt1 also has a role in de novo methylation of DNA. This model agrees with the biochemical properties of these enzymes and provides a mechanistic basis for the functional cooperation of different DNA MTases in de novo methylation of DNA that has also been observed in vivo.
DNMT1
DNA methyltransferase
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Abstract Background/Objectives It seems that global DNA hypomethylation in CD4+T cells is linked to the pathogenesis of systemic lupus erythematosus ( SLE ). However, the underlying mechanism by which SLE patients show hypomethylated DNA remains unclear. This study explored the relationship between DNA methylation patterns and expression levels of DNA methyltransferases ( DNMT1) and MBD2 in CD4+T cells of SLE patients. Methods CD4+T cells were obtained from 30 patients with SLE and 18 normal controls. The global DNA methylation levels in CD4+T cells were evaluated by the Methyflash DNA methylation quantification kit. The mRNA levels of DNMT1 and MBD2 were quantified by quantitative real‐time polymerase chain reaction. Results SLE patients had significantly lower global DNA methylation levels than controls, and the global DNA methylation was inversely correlated with the SLE disease activity index ( SLEDAI ). The mRNA levels of DNMT1 in SLE patients were significantly lower than that of controls and there was no correlation between DNMT1 mRNA levels and SLEDAI but there was a positive correlation between DNMT1 mRNA levels and global DNA methylation. The mRNA levels of MBD2 in SLE patients were significantly higher than in controls, and there was positive correlation between MBD2 mRNA levels and SLEDAI and an inverse correlation between MBD2 mRNA levels and global DNA methylation. Conclusions Global DNA hypomethylation may play a pivotal role in the pathogenesis of SLE . Abnormal expression levels of DNMT1 and MBD2 mRNA may be important causes of the global hypomethylation observed in CD4+T cells in SLE .
DNMT1
DNA methyltransferase
Pathogenesis
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DNA demethylation
Epigenomics
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Epigenetics describes the study of cellular modifications that can modify the expression of genes without changing the DNA sequence. DNA methylation is one of the most stable and prevalent epigenetic mechanisms. Twin studies have been a valuable model for unraveling the genetic and epigenetic epidemiology of complex traits, and now offer a potential to dissect the factors that impact DNA methylation variability and its biomedical significance. The twin design specifically allows for the study of genetic, environmental and lifestyle factors, and their potential interactions, on epigenetic profiles. Furthermore, genetically identical twins offer a unique opportunity to assess nongenetic impacts on epigenetic profiles. Here, we summarize recent findings from twin studies of DNA methylation profiles across tissues, to define current knowledge regarding the genetic and nongenetic factors that influence epigenetic variation.
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Previous studies have demonstrated a dynamic epigenetic regulation of genes expression in placenta trophoblasts and a dynamic imbalance of DNA methylation and hydroxymethylation. Reduced IGF-1 has been observed in preeclampsia. This study was to investigate the interactive roles between IGF-1 and the global DNA methylation/hydroxymethylation, and the status of DNA methylation/hydroxymethylation and associated enzymes such as DNMTs and TETs in peeeclamptic placentas and hypoxic trophoblasts. It was found that IGF-1 was decreased in preeclamptic placentas and hypoxic trophoblasts when compared to the control group using immunohistochemisty, western blot, qRT-PCR and ELISA. Pyrophosphate sequencing showed IGF-1 promoter was significantly hypermethylated in preeclamptic placentas, which was responsible for reduced IGF-1 expression. Preeclamptic placentas and hypoxic trophoblasts were hypermethylated and hypohydroxymethylated accompanied by remarkably higher 5mC, DNMT1 and DNMT3b, and lower DNMT3a, 5hmC, TET1, TET2 and TET3 detected by immunohistochemisty, western blot, qRT-PCR and ELISA. Pearson's correlation confirmed a statistically significant negative correlation between IGF-1 and DNMT1. Furthermore, both treatment with 5-Aza-dc and DNMT1-siRNA significantly increased the expression of IGF-1 in HTR8 cells, indicating the potential mechanism of DNMT1-mediated DNA methylation in IGF-1 regulation. However, IGF-1 didn't change DNA methylation or hydroxymethylation. These findings suggest that preeclampsia is associated with hypermethylation of IGF-1 promoter mediated by DNMT1 and provide new insights into the diagnosis and treatment of preeclampsia.
DNMT1
DNMT3B
DNA methyltransferase
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TOBACCO AND DNA METHYLATION THE CASE FOR EPIGENETIC ALTERATIONS The mechanisms of the long-term impacts of exposure to chemical substances remain poorly understood. While genotoxic and mutagenic effects have been well characterized, epigenetic mechanisms such as DNA methylation could also account for the delayed effects of exposures. It is in the case of tobacco that the strongest arguments for a role of these mechanisms have been obtained in human populations. This text presents recent data on this issue demonstrating the plausibility of epigenetic mechanisms to explain the persistence of biological signals long after stopping exposure.
Epigenesis
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Carcinogenesis is a stepwise process of accumulation of genetic and epigenetic abnormalities that can lead to cellular dysfunction. It has become clear that epigenetic changes are equally important for this multistep process to produce its results. This article describes the different roles that epigenetic modulation may play during carcinogenesis and how an early detection and chemopreventive intervention strategy that takes both sides of the equation into account would be advantageous.
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Abstract Background DNA methylation plays an important role in regulating gene expression in mammals. The covalent DNMT1 inhibitors 5-azacytidine and decitabine are widely used in research to reduce DNA methylation levels, but they impart severe cytotoxicity which limits their demethylation capability and confounds interpretation of experiments. Recently, a non-covalent inhibitor of DNMT1 called GSK-3484862 was developed by GlaxoSmithKline. We sought to determine whether GSK-3484862 can induce demethylation more effectively than 5-azanucleosides. Murine embryonic stem cells (mESCs) are an ideal cell type in which to conduct such experiments, as they have a high degree of DNA methylation but tolerate dramatic methylation loss. Results We determined the cytotoxicity and optimal concentration of GSK-3484862 by treating wild-type (WT) or Dnmt1/3a/3b triple knockout (TKO) mESC with different concentrations of the compound, which was obtained from two commercial sources. Concentrations of 10 µM or below were readily tolerated for 14 days of culture. Known DNA methylation targets such as germline genes and GLN-family transposons were upregulated within 2 days of the start of GSK-3484862 treatment. By contrast, 5-azacytidine and decitabine induced weaker upregulation of methylated genes and extensive cell death. Whole-genome bisulfite sequencing showed that treatment with GSK-3484862 induced dramatic DNA methylation loss, with global CpG methylation levels falling from near 70% in WT mESC to less than 18% after 6 days of treatment with GSK-3484862. The treated cells showed a methylation level and pattern similar to that observed in Dnmt1 -deficient mESCs. Conclusions GSK-3484862 mediates striking demethylation in mESCs with minimal non-specific toxicity.
DNMT1
Decitabine
DNA demethylation
Bisulfite sequencing
Reprogramming
Demethylation
DNA methyltransferase
Azacitidine
CpG site
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Transient nutritional exposures during critical ontogenic periods can cause persistent changes in gene expression, metabolism, and risk of various diseases. We have been investigating whether such ‘developmental programming’ occurs via nutritional influences on developmental epigenetics. Our studies in agouti viable yellow and axin-fused mice showed that developmental establishment of DNA methylation at ‘metastable epialleles’ is especially sensitive to maternal nutritional status around the time of conception. At metastable epialleles, DNA methylation is established stochastically in the early embryo and subsequently maintained during differentiation of diverse lineages, resulting in systemic interindividual epigenetic variation that is not genetically mediated. Lately, using a multiple-tissue screen for interindividual variation in DNA methylation, we have identified human genomic regions that appear to be metastable epialleles. Stochastic establishment of DNA methylation at these loci is affected by maternal nutrition around the time of conception, consistent across multiple tissues, and stable for many years. Most recently, our studies using genome-wide bisulfite sequencing have identified candidate metastable epialleles that are associated with human disease, providing exciting opportunities for epigenetic epidemiology.
Epigenesis
Epigenomics
Genomic Imprinting
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