Total DNA Methyltransferase Activity Research Articles

Nitric oxide-mediated DNA methylation enhances cold resistance in postharvest peach fruit

Low temperature can affect DNA methylation. Since exogenous use of NO can reduce cold damage in peach fruit during cold storage, this study investigated the roles of NO on DNA methylation of peaches suffering cold stress. The results showed that exogenous NO effectively alleviated the decrease in total DNA methyltransferase (DNMT) activity and transcript levels induced by cold stress, whereas c-PTIO exacerbated the decrease in total DNMT activity and transcript levels. Further BSP analysis of the promoter regions of four cold resistance genes (PpCBF5-IS2, PpICE1-IS, PpMYC2-IS, PpCOR-IS1) in peaches showed that in peaches treated with exogenous NO, PpCBF5-IS2 and PpICE1-IS were modified by hypermethylation, PpMYC2-IS was modified by methylation, PpCOR-IS1 was modified by demethylation and insensitive to NO. It was suggested that NO could enhance the cold resistance of postharvest peaches by mediating DNA methylation.

High-throughput analyses and Bayesian network modeling highlight novel epigenetic Adverse Outcome Pathway networks of DNA methyltransferase inhibitor mediated transgenerational effects

A number of epigenetic modulating chemicals are known to affect multiple generations of a population from a single ancestral exposure, thus posing transgenerational hazards. The present study aimed to establish a high-throughput (HT) analytical workflow for cost-efficient concentration-response analysis of epigenetic and phenotypic effects, and to support the development of novel Adverse Outcome Pathway (AOP) networks for DNA methyltransferase (DNMT) inhibitor-mediated transgenerational effects on aquatic organisms. The model DNMT inhibitor 5-azacytidine (5AC) and the model freshwater crustacean Daphnia magna were used to generate new experimental data and served as prototypes to construct AOPs for aquatic organisms. Targeted HT bioassays (DNMT ELISA, MS-HRM and qPCR) in combination with multigenerational ecotoxicity tests revealed concentration-dependent transgenerational (F0-F3) effects of 5AC on total DNMT activity, DNA promoter methylation, gene body methylation, gene transcription and reproduction. Top sensitive toxicity pathways related to 5AC exposure, such as apoptosis and DNA damage responses were identified in both F0 and F3 using Gaussian Bayesian network modeling. Two novel epigenetic AOP networks on DNMT inhibitor mediated one-generational and transgenerational effects were developed for aquatic organisms and assessed for the weight of evidence. The new HT analytical workflow and AOPs can facilitate future ecological hazard assessment of epigenetic modulating chemicals.

Hydrogen sulfide mediates DNA methylation to enhance osmotic stress tolerance in Setaria italica L.

DNA methylation is an important form of epigenetic modification. It has a vital role in regulating plant growth and development, and also participates in plant response to various stresses. In recent years, hydrogen sulfide (H2S) has been shown to have similar functions as DNA methylation, but crosstalk between DNA methylation and H2S in the acquisition of drought resistance is unclear. In this study, foxtail millet (Setaria italica L.), a drought-resistant model crop, was selected as the experimental material to explore the subtle relationship between H2S and DNA methylation. The quantitative real-time (qRT)-PCR, bisulfite sequencing PCR (BSP), DNA methyltransferase (DNMT) activity detection and other techniques were used to analyze the differences of millet seedlings under osmotic stress, before and after H2S treatment. Osmotic stress induced the transcriptional expression and activity of key enzymes in H2S biosynthesis which regulated the accumulation of endogenous H2S. Physiological concentration of H2S (50 μM) can effectively alleviate the decrease of total DNMT activity and transcription level caused by osmotic stress, while the effective inhibitor of H2S biosynthesis, hydroxylamine (HA), can aggravate this change. Furthermore, transcription factors (TFs) responsive to both ‘osmotic stress’ and ‘H2S signal’ were screened. Six of them were selected to conduct further BSP analysis on seven CpG islands in their promoter regions. The results showed that, with H2S treatment, two CpG islands were hypermethylated, two were not, and three exhibited insensitivity to H2S. H2S signals may improve osmotic stress tolerance of foxtail millet by mediating DNA methylation.

Glycolic Acid Silences Inflammasome Complex Genes, NLRC4 and ASC, by Inducing DNA Methylation in HaCaT Cells.

AHAs (α-hydroxy acids), including glycolic acid (GA), have been widely used in cosmetic products and superficial chemical peels. Inflammasome complex has been shown to play critical roles in inflammatory pathways in human keratinocytes. However, the anti-inflammatory mechanism of GA is still unknown. The aim of this study is to investigate the relationship between the expression of the inflammasome complex and epigenetic modification to elucidate the molecular mechanism of the anti-inflammatory effect of GA in HaCaT cells. We evaluated NLRP3, NLRC4, AIM2, and ASC inflammasome complex gene expression on real-time polymerase chain reaction (PCR). Methylation changes were detected in these genes following treatment with DNA methyltransferase (DNMT) inhibitor 5-aza-2'-deoxycytidine (5-Aza) with or without the addition of GA using methylation-specific PCR (MSP). GA inhibited the expressions of these inflammasome complex genes, and the decreases in the expressions of mRNA were reversed by 5-Aza treatment. Methylation was detected in NLRC4 and ASC on MSP, but not in NLRP3 or AIM2. GA decreased NLRC4 and ASC gene expression by increasing not only DNA methyltransferase 3B (DNMT-3B) protein level, but also total DNMT activity. Furthermore, silencing of DNMT-3B (shDNMT-3B) increased the expressions of NLRC4 and ASC. Our data demonstrated that GA treatment induces hypermethylation of promoters of NLRC4 and ASC genes, which may subsequently lead to the hindering of the assembly of the inflammasome complex in HaCaT cells. These results highlight the anti-inflammatory potential of GA-containing cosmetic agents in human skin cells and demonstrate for the first time the role of aberrant hypermethylation in this process.

Abstract POSTER-BIOL-1306: DNA methylation changes during epithelial-to-mesenchymal transition in ovarian cancer cells

Abstract Epithelial-to-mesenchymal transition (EMT) is an important step in the process of metastasis and has been linked to cancer cell stemness. We hypothesize that aside from defined genetic changes associated with EMT, epigenetic mechanisms are involved and have not been fully elucidated. We determined DNA methylation in SKOV3 ovarian cancer (OC) cells during EMT induced by TGF-ß using the Infinium HumanMethylation450 array. Methylation levels of CpG sites were expressed as ß-values ranging from 0 (totally unmethylated) to 1 (totally methylated). Methylation of 468 and 390 sites changed (P<.01, ANOVA and FDR<0.05) after 48h and 120h of TGF-ß stimulation. The majority of changes (81%) reflected TGF-ß-induced increase in methylation. Methylation of 160 sites changed similarly at 48 and 120 h indicating that 34% of initial changes were sustained. The number of methylation changes decreased significantly after TGF-ß removal from the cell cultures, demonstrating that removal of TGF-ß stimulation reversed the methylation changes at most sites and restituted the baseline methylome. These results were consistent with those of the principal component and hierarchical clustering analyses. TGF-ß increased overall (average of all CpG sites) DNA methylation, total DNA methyltransferase activity, and amounts of DNMT1 protein and mRNA. The average methylation of CpG islands located in or near promoters increased in 193 genes at 24h and 494 at 120h, while no changes were observed after TGF-ß removal. Pathway analysis identified methylation changes in genes belonging to functional networks relevant to EMT and cancer progression, including: cellular movement, cell cycle, organ morphology, cellular development, and cell death and survival. Treatment of OC cells with the DNA methylation inhibitor SGI-110 prevented TGF-ß-induced EMT in association with an inhibition of the TGF-ß-induced downregulation of E-Cadherin. Results demonstrate that DNA methylation is a dynamic process involved in regulation of genes implicated in EMT and metastasis. Citation Format: Horacio Cardenas, Edyta Vieth, Jiyoon Lee, Yunlong Liu, Kenneth P. Nephew, Daniela Matei. DNA methylation changes during epithelial-to-mesenchymal transition in ovarian cancer cells [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-BIOL-1306.

Epigenetic Reprogramming of Mitochondrial Dysfunction in hyperhomocysteinemia

An elevated level of homocysteine (Hyperhomocysteinemia) is associated with increased risk factor of neurological disorder. Homocysteine (Hcy) modulates cellular methylation reactions. Previously we have demonstrated that HHcy caused significant metabolic changes and altered mitochondrial function in mouse brain endothelial cells (bEND3). However, the mechanisms behind such alterations remain unclear. The present study examined if the effects of Hcy on mitochondria dysfunction are mediated through an epigenetic mechanism. Following exposure to Hcy for 72 h, global DNA methylation and histone H3 lysine 9 (H3K9) acetylation were examined using flow cytometric analysis. Total DNA methyltransferase activity (DNMT) and protein levels of DNMT 3 were measured. Mitochondrial respiratory capacity and mitochondrial permeability transition (MPT) were measured by fluorescence dye. The result suggested that HHcy caused global DNA hypomethylation and histone hyperacetylation. Total DNMT activity significantly decreased which was accompanied by a significant reduction in protein levels of DNMT 3 and loss of MPT. Treatment of bEND3 with the DNMT inhibitor, 5‐aza‐2′‐deoxycytidine, mimicked the functional changes induced by Hcy. This study suggests that the Hcy related metabolic changes including altered mitochondrial function may be mediated through an epigenetic mechanism.

Homocysteine levels impact directly on epigenetic reprogramming in astrocytes

Although the neurotoxic effects of homocysteine have been well elucidated, the effects of homocysteine in astrocytes have received little attention until recently. Previously we have demonstrated that elevated levels of homocysteine caused significant metabolic changes and altered mitochondrial function in primary cultures of astrocytes. However, the mechanisms behind such alterations remain unclear. As homocysteine is a key metabolite in one-carbon metabolism the present study examined if the effects of homocysteine on astrocyte function are mediated through an epigenetic mechanism. Following exposure to homocysteine for 72 h, global DNA methylation and H3K9 acetylation were examined using flow cytometric analysis. Total DNA methyltransferase activity and protein levels of DNA methyltransferase 3B were measured. Exposure to homocysteine resulted in global DNA hypomethylation ( p < 0.05) and histone hyperacetylation ( p < 0.05). Total DNA methyltransferase activity significantly decreased following exposure to homocysteine (from 11.5 ± 3.9 to 6.0 ± 1.7 OD/h/mg protein, p < 0.01) which was accompanied by a significant reduction in protein levels of DNA methyltransferase 3B ( p < 0.05). Treatment of astrocytes with the DNA methyltransferase inhibitor, 5-aza-2′-deoxycytidine, mimicked the functional changes induced by homocysteine. In conclusion, the results demonstrate significant epigenetic modifications following exposure to homocysteine in astrocytes and these changes seem to mediate functional alterations.

Reduction of pancreatic cancer cell viability and induction of apoptosis mediated by siRNA targeting DNMT1 through suppression of total DNA methyltransferase activity

Aberrant methylation leads to epigenetic changes in human genes that may cause carcinogenesis. DNA methyltransferase 1 (DNMT1) plays an important role in maintaining DNA methylation patterns during genomic DNA replication. To understand the role of this protein in pancreatic cancer cell growth and apoptosis, small interfering RNA (siRNA) oligonucleotides were used to knockdown DNMT1 expression in pancreatic cancer PaTu8988 cells. We found that the DNMT1 siRNA markedly decreased DNMT1 expression and total DNA methyltransferase activity in the cells. Upon the inhibition of DNMT1 expression, the proliferation of the tumor cells was inhibited. Tumor cell growth was arrested in the S-phase of the cell cycle and cells underwent apoptosis. The expression of p21 was up-regulated and the ratio of Bax/Bcl-2 expression was increased after DNMT1 knockdown in PaTu8988 cells. Furthermore, DNMT1 siRNA caused demethylation of the tumor suppressor gene hMLH1, resulting in its re-expression in PaTu8988 cells. The results of this study suggest that DNMT1 siRNA oligonucleotides are candidates for further evaluation as therapeutic tools for the clinical control of pancreatic cancer.

Reduced genomic cytosine methylation and defective cellular differentiation in embryonic stem cells lacking CpG binding protein.

Cytosine methylation at CpG dinucleotides is a critical epigenetic modification of mammalian genomes. CpG binding protein (CGBP) exhibits a unique DNA-binding specificity for unmethylated CpG motifs and is essential for early murine development. Embryonic stem cell lines deficient for CGBP were generated to further examine CGBP function. CGBP(-)(/)(-) cells are viable but show an increased rate of apoptosis and are unable to achieve in vitro differentiation following removal of leukemia inhibitory factor from the growth media. Instead, CGBP(-)(/)(-) embryonic stem cells remain undifferentiated as revealed by persistent expression of the pluripotent markers Oct4 and alkaline phosphatase. CGBP(-)(/)(-) cells exhibit a 60 to 80% decrease in global cytosine methylation, including hypo-methylation of repetitive elements, single-copy genes, and imprinted genes. Total DNA methyltransferase activity is reduced by 30 to 60% in CGBP(-)(/)(-) cells, and expression of the maintenance DNA methyltransferase 1 protein is similarly reduced. However, de novo DNA methyltransferase activity is normal. Nearly all aspects of the pleiotropic CGBP(-)(/)(-) phenotype are rescued by introduction of a CGBP expression vector. Hence, CGBP is essential for normal epigenetic modification of the genome by cytosine methylation and for cellular differentiation, consistent with the requirement for CGBP during early mammalian development.

Inhibition of DNA methyltransferase reverses cisplatin induced drug resistance in murine neuroblastoma cells

Background: Acquired drug resistance is a major obstacle to the successful treatment of neuroblastoma by chemotherapy. Recent studies from our laboratory have demonstrated that drug-induced alterations in DNA methylation play an important role in this process. Methods: The reversal of resistance to cisplatin in murine neuroblastoma (MNB) was induced by inhibition of DNA methyltransferase activity. MNB cells overexpressing DNA methyltransferase activity (Dnmt3a or Dnmt3b) were established by stable co-transfection of wild type MNB cells with plasmids containing Dnmt3a or Dnmt3b cDNA. Cytotoxic response (IC 50), total DNA methyltransferase activity and expression of Dnmt3a or Dnmt3b methyltransferase were determined in Dnmt3a or Dnmt3b transfected MNB cells, respectively. Results: These data demonstrated that total DNA methyltransferase activity was increased to 3-fold above controls ( P < 0.001) in cisplatin resistant MNB cells, 3-fold in Dnmt3a and 4-fold in Dnmt3b transfected MNB cells. Western blot and RT-PCR data confirmed a corresponding increase in Dnmt3a and 3b expression in cisplatin resistant and transfected cells when compared with control MNB cells ( P < 0.001). MNB clones overexpressing Dnmt3a or Dnmt3b proteins were resistant to cisplatin treatment (10 −6 M). Incubation of cisplatin resistant, Dnmt3a or Dnmt3b overexpressing MNB cells with 5′-azacytidine (5′-azaC), a methylation inhibitor (2.5 μM) significantly decreased DNA methyltransferase activity, expression of Dnmt3a and Dnmt3b proteins and mRNA levels of cisplatin resistant, Dnmt3a and Dnmt3b transfected MNB cells. Cytotoxicity studies using the MTT assay demonstrated that the sensitivity of cisplatin resistant, Dnmt3a and Dnmt3b overexpressing MNB cells to cisplatin was increased 10-fold ( P < 0.001) following 5′-azaC treatment. Conclusions: These findings suggest that the overexpression of DNA methyltransferase is associated with a cisplatin resistant phenotype in MNB cells that may or may not be true in animal studies or in the clinical setup. Thus, DNA methylation plays a central role in onset of drug resistance in cisplatin resistant neuroblastoma cells in vitro.