Expression Of DNA Methyltransferase Genes Research Articles

Salt Tolerance Assessment in Triticum Aestivum and Triticum Durum.

Salt stress is a multicomponent phenomenon; it includes many processes that directly or indirectly affect the plant. Attempts have been made to comprehensively consider the processes of salt stress in plants Triticum aestivum (variety Orenburgskaya 22) and Triticum durum (variety Zolotaya). The study used methods of light and fluorescence microscopy, methods of immunofluorodetection, expression of DNA methyltransferase genes, genes of the ion transporter and superoxide dismutase families, as well as biochemical determination of total antioxidant activity using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) reagent. According to morphometric indicators, the Orenburgskaya 22 variety showed greater tolerance to the action of 150 mM NaCl than the Zolotaya variety. The level of expression of genes of the HKT ion transporter family in the Orenburgskaya 22 variety is higher than in the Zolotaya variety. It was found that the expression of the DNA methyltransferase gene DRM2.1, which post-translationally methylates cytosine residues, is 22.3 times higher in Zolotaya compared to Orenburg 22 when exposed to salt. The accumulation of toxic ions is accompanied by an increase in reactive oxygen species (ROS) and increased damage to root tissue, especially in the Zolotaya variety. Using fluorescence microscopy using the Carboxy-H2DFF marker in the Orenburgskaya 22 variety at high NaCl concentrations, the highest fluorescence intensity was determined in the cap zone; in the Zolotaya variety-in the zones of the cap and root meristem. Excess ROS is more successfully removed in the Orenburgskaya 22 variety, which has a higher level of antioxidant activity (AOA), as well as the level of expression of the Cu/ZnSOD and MnSOD superoxide dismutase genes. Using programmed cell death (PCD) markers based on the release of cytochrome c from mitochondria into the cytoplasm, DNA breakage and the release of phosphatidylserine from mitochondria, the degree of damage to root cells was assessed in both wheat varieties. It has been proven that wheat cell death occurs through the mitochondrial pathway. It was noted that the salt-sensitive variety Zolotaya had a significant number of necrotic cells. Based on the data obtained, it was concluded that the Orenburgskaya 22 variety exhibits greater resistance to salinity than the Zolotaya variety. These data may be of practical importance for enhancing protective mechanisms under abiotic stress.

Prognostic value of genome-wide methylation in acute-on-chronic hepatitis B liver failure

AimMethylation status of genome varies between pre-acute-on-chronic hepatitis B liver failure (pre-ACHBLF), acute-on-chronic hepatitis B liver failure (ACHBLF), and chronic hepatitis B patients. This study aimed to find better prognostic indicators for acute-on-chronic liver failure. MethodsThe level of global genome methylation in peripheral blood mononuclear cells (PBMCs) was detected. The overall genome methylation rate was determined using MethylFlash™ Methylated DNA Quantification Kit(Colorimetric). DNMT activity were measured using DNA Methyltransferase Activity/Inhibition Assay Kit. Gene expression of DNA methyltransferases (DNMT)，methyl-CpG-binding domain (MBD) were detected by qRT-PCR. ResultsThe global genome methylation level in ACHBLF group was significantly higher than that in chronic hepatitis B group (P<0.001). There was also obvious difference of the global genome methylation level between pre-ACHBLF group and CHB group (P<0.001). Meanwhile, the activity of DNMT in ACHBLF group was significantly higher than that in chronic hepatitis B group (P<0.001). The mRNA expression level of DNMT1 was higher than that in pre-ACHBLF group (P<0.01) and CHB group (PP<0.001). The mRNA expression level of MBD1 in ACHBLF group was also higher than that in CHB group (P<0.001) and healthy controls (HCs) (P<0.01). And the mRNA expression level of MBD3 and MBD4 in ACHBLF, pre-ACHBLF and CHB group were lower than that in HCs (P<0.001). Meanwhile we observed an opposite change in the mRNA expression level of MECP2. The ROC curve suggested that global genome methylation level was a better prognostic predictor than MELD score in ACHBLF (AUC 0.950, SE 0.0237, 95%CI 0.874–0.986 VS AUC 0.863, SE 0.0439, 95%CI 0.765–0.931, P=0.0429). ConclusionsGenome methylation level can be a good biomarker in predicting the severity and prognosis of ACHBLF.

The effects of thymidylate synthase 3'UTR genotype on methylation of tumor-specific genes promoter in 22 colorectal cancer patients from southern Iran.

To investigate the effects of thymidylate synthase (TS) 3'UTR genotype on promotor methylation of tumor-related genes in 22 patients with sporadic colorectal cancer (CRC) from southern Iran. We evaluated the correlations of TS 3'UTR genotype with promoter methylation of hTERT, hMLH1, MSH2, MMP2, CDH1, p14, p16, and p21 genes in CRC patients. The polymorphism of TS 3'UTR was evaluated through mutagenically specific PCR. The genes promoter methylation was determined using methylation-specific PCR. For 10 patients, the gene expression profile of epigenetic regulating enzymes, histone deacetylases (HDACs) and DNA methyltransferases (DNMTs), was also examined in both tumor and normal adjacent tissues by quantitative real time PCR. There was a significant association between the hMLH1 methylation and age of patients (P= 0.039) and also between MSH2 methylation and tumor site (P= 0.036). There was insignificant association between gene-specific methylation and TS 3'UTR genotype. However, all polymorphic genotypes of TS were associated with higher methylation of hMLH1 and CDH1 and lower methylation of MSH2. The -6bp/+6bp (heterozygous mutant) and [-6bp/+6bp, +6bp/+6bp] (homozygous mutant) genotypes resulted in higher methylation of p16, and -6bp/+6bp and [-6bp/+6bp, +6bp/+6bp] genotypes were correlated with lower methylation of MMP2. The overexpression of epigenetic enzymes, HDACs and DNMTs, was also demonstrated. There was no association between DNMTs transcript levels and gene-specific hypermethylation. The polymorphic TS genotypes, especially -6bp/+6bp, could affect methylation frequencies of studied genes. Moreover, promoter methylation status was not dependent on DNMTs gene expression. Large sample size studies may contribute to validate these findings.

MODL-41. LOSS OF DISTAL CHROMOSOME 6Q AND ASSOCIATED PDE10A INDUCES AN AGGRESSIVE GLIOMA PHENOTYPE BY FACILITATING PRONEURAL TO MESENCHYMAL TRANSITION

Abstract Somatic copy number alterations (SCNAs) are common in glioblastoma, with some SCNAs having prognostic implications. Identifying and characterizing SCNA-related genes acting as oncogenes or tumor suppressors may potentially provide new therapeutic avenues for gliomas. In this study, we sought to uncover novel SCNA-related genes by employing an unbiased log rank analysis screen of genomic regions associated with glioblastoma survival. We identified 6q27 loss as an independent poorly-prognostic SCNA across three separate glioblastoma cohorts. An in vitro CRISPR screen using human neural stem cells identified five genes in the 6q27 region that caused increased cellular proliferation. One of these genes, phosphodiesterase 10A (PDE10A), had a gene dosage effect on expression and decreased expression in human and mouse glioma tumor tissue compared to normal brain, which are both common tumor suppressor phenotypes. Knockdown of Pde10a in mouse neural stem cells in vitro validated the human cell CRISPR screen phenotype. Suppression of Pde10a in RCAS/Tva mouse glioma in vivo caused decreased survival with increased tumor grade, mitotic activity, necrosis, Vegfa expression, and AKT/pS6 signaling. Single cell RNA-sequencing of mouse gliomas revealed that Pde10a suppression results in a shift from proneural to mesenchymal-like state. In human glioma tissue, PDE10A loss is also associated with an increased mesenchymal signature. Top activated pathways in Pde10a suppressed gliomas involved cell-cell interaction, including upregulation of cell adhesion molecules and downregulation of focal adhesion complex disassembly genes. Functional analysis in vitro confirmed that Pde10a suppression results in increased extracellular matrix adhesion and decreased migration. Relevant to standard glioblastoma treatment, PDE10A loss was associated with decreased MGMT promoter methylation, decreased DNA methyltransferase gene expression, and it potentiated radiation resistance in vitro. In summary, we leveraged several multiomic and experimental platforms to reveal PDE10A as a tumor suppressor in glioblastoma, predominantly by inducing a proneural to mesenchymal transition phenotype.

Evaluation of epigenetic-related gene expression (DNMT, HDAC1) in Iranian patients with systemic lupus erythematosus

BackgroundSystemic lupus erythematosus (SLE) is an autoimmune disease in which the immune system abnormally reacts against cells and tissues leading to inflammation. Epigenetic alterations, including DNA methylation and histone modification, have critical effects on autoimmune disease and SLE pathogenesis via dysregulation of critical genes. AimsThe purpose of this study was to evaluate the epigenetic-related gene expression of DNA methyltransferase (DNMT) and histone deacetylase 1 (HDAC1) in Iranian patients with SLE. MethodsThis matched case–control study included 16 people with SLE and 16 healthy people who were referred to the Rafsanjani rheumatology clinic, in southeast Iran. The expression of DNMT and HDAC1 genes was measured through a real-time PCR assay of blood samples. ResultsDNMT gene expression did not differ significantly between SLE and healthy groups (P=0.21). In contrast, HDAC1 gene expression was enhanced in the SLE group, but this enhancement failed to reach statistical significance (P=0.94). ConclusionThe results of this study suggest that overexpression of HDAC1 could serve as a diagnostic for SLE disease. Additional studies with larger sample sizes are required to confirm our findings. Evaluation of other genes related to SLE disease is essential and may help to make an accurate diagnosis of the disease.

DNA methyltransferase expression (DNMT1, DNMT3a and DNMT3b) as a potential biomarker for anti-VEGF diabetic macular edema response.

DNA methylation is involved in Diabetic Retinopathy progression showing a metabolic memory mechanism. However, the association of DNA methyltransferase with diabetic macular edema is still unknown. We aimed to describe the differences in DNA methyltransferase gene expression in patients with different diabetic macular edema responses. A total of 27 diabetic patients, aged 59-90 years, were prospectively enrolled in this cross-sectional study. The participants were classified into control group (CG, n = 11), diabetic macular edema responders (rDME, n = 9) and non-responder diabetic macular edema (nrDME, n = 7) after anti-vascular endothelial growth factor (anti-VEGF) treatment. Only cases with a complete ophthalmological examination, digital 133° color fundus, and SD-OCT assessments were used. After RNA extraction and first-strand cDNA synthesis, quantitative real-time PCR was performed with specific primers on the CFX Connect™ Real-Time PCR Detection System to assess differential transcriptional expression patterns. The DNMT1 gene showed a positive correlation (r = 0.617; p = 0.043) with Best Corrected Visual Acuity (BCVA) in CG, a positive correlation (r = 0.917; p = 0.010) with HbA1c in nrDME and a negative correlation (r = -0.659; p = 0.049) with GCL-IPL thickness in rDME. DNMT3A gene showed a positive correlation (r = -0.890; p = 0.001) with Sub-foveal Choroidal thickness in rDME whereas DNMT3b gene showed a negative correlation (r = -0.815; p = 0.007) with HbA1c and RNFL (r = -0.664; p = 0.026) in CG. Patients with similar metabolic profile risk factors showed associated DNA methyltransferase transcriptional expression patterns differences fitting with the anti-VEGF diabetic macular edema response. Further studies are needed to clarify if these results (1) reflect disease evolution, (2) translate the therapeutic impact, (3) or can help to predict the therapeutic resistance profile.

Global DNA methylation, DNA methyltransferase and stress-related gene expression in ovine oocytes and embryos after exposure to metabolic stressors.

DNA methylation, considered the most prominent epigenetic mark was important for the gene regulation in embryonic development. The present study aimed at evaluating the effects of metabolic stressors [Non-esterified fatty acid (NEFA), β-hydroxy-butyric acid (BHB), ammonia and urea] exposure during the in vitro ovine oocyte maturation, global DNA methylation, DNA methyltransferase and stress-related gene expression. Colorimetric analysis of global DNA methylation and the expression of the DNA methyltransferase genes (DNMT1, DNMT3A, and DNMT3B) were assessed in the matured oocytes, 2-cell embryos and blastocysts produced in vitro from oocytes exposed with the metabolic stressors during 24 h of the in vitro maturation (IVM). Further, the mRNA expression of the stress-related genes (SOD1, SOD2) in the matured oocytes, 2-cell embryos and blastocysts produced was assessed. Significant difference in global DNA methylation levels between all the treatments tested was observed when compared with control in oocytes, two-cell embryos and blastocysts. Elevated concentration of metabolic stressors resulted in increased expressions of several stress-related genes, i.e., SOD1, SOD2 and in mRNA expression of DNA methyltransferase genes. The present study is the first to report that the DNA methylation was sensitive to the effects of the metabolic stressors in ovine oocytes/embryos. The aberrant expressions of genes during oocyte development targeted in the present study can provide evidence for the early embryo developmental arrest and blastocysts quality. These results highlighted the sensitivity of the early embryogenesis and more precisely of the reprogramming period to metabolites challenges, in a realistic situation of elevated concentration of metabolic stressors.

Tributyltin causes generational neurodevelopmental toxicity and the protective effect of folic acid in zebrafish

Tributyltin (TBT), a common organotin environmental pollutant, may pose a threat to human development during critical early-life periods. We aimed to assess the neurodevelopmental intergenerational toxicity of early-life exposure to TBT and the protective effect of DNA methyl donor folic acid (FA). Specifically, after early-life exposure (1–21 days post-fertilization, dpf) to TBT (0, 1, 10 and 100 ng/L), zebrafish (Danio rerio) were cultured in clean medium until sexual maturity. The exposed females were mated with unexposed males to produce embryos (F1). The F1 generation were cultured (4–120 hours post-fertilization, hpf) with and without 1 mmol/L FA. The neurotoxic effects of early-life TBT exposure for zebrafish and their offspring (F1) were significantly enhanced anxiety and reduced aggression, decreased gene expression of DNA methyltransferase in the brain and increased serotonin levels in the body. Moreover, the intergenerational neurodevelopmental toxicity, as manifested in the F1 generation, was attenuated by FA supplementation. In summary, early-life TBT exposure led to intergenerational neurodevelopmental deficits in zebrafish, and DNA methyl donors had a protective effect on F1 neurodevelopment, which can inform the prevention and treatment of intergenerational neurotoxicity due to organotin pollutants.

DNA methylation variation is crucial to restore adventitious rooting ability during in vitro shoot culture-induced rejuvenation in apple rootstock.

In vitro shoot culture has been widely used for restoring adventitious rooting ability in rooting recalcitrant woody perennial species for the past few decades, but its molecular mechanism is largely uncovered. DNA methylation is an essential epigenetic mark that participates in many biological processes. Recent reports suggested a role of DNA methylation in vitro culture in plants. In this study, we characterized the single-base resolution DNA methylome and transcriptome of adult and in vitro shoot culture-induced rejuvenation cuttings of apple rootstock M9T337. We found a global decrease in DNA methylation during rejuvenation, which may be correlated with increased expression of DNA demethylase genes and decreased expression of DNA methyltransferase genes. We additionally documented DNA hypomethylation in 'T337'_R in gene protomer associated with higher transcript levels of several adventitious rooting-related genes. The application of a DNA methylation inhibitor (5-azacytidine) enhanced the adventitious rooting ability and the expression level of adventitious rooting-related genes, such as, MdANT, MdMPK3, MdABCB21, MdCDC48, MdKIN8B, pri-MdMIR156a5 and pri-MdMIR156a12. Together, the DNA hypomethylation is critical for the rejuvenation-dependent adventitious rooting ability in apple rootstock. In addition, increased DNA methylation was also found in thousands of genes in 'T337'_R. We additionally documented that DNA hypermethylation is required for inhibition of adventitious rooting-repressed genes, such as MdGAD5a, encoding glutamate decarboxylase, which can catalyze glutamate decarboxylated to form γ-aminobutyric acid (GABA). Our results revealed that in vitro shoot culture-dependent DNA methylation variation plays important roles in adventitious rooting in apple rootstock.

Global and Regional DNA methylation silencing of PPARγ Associated with Glioblastoma Multiforme Pathogenesis.

The relationship between peroxisome proliferator-activated receptor gamma (PPARγ) expression level and epigenetic modifications occurring in glioblastoma multiforme (GBM) pathogenesis is largely unknown. Herein, we examine the association of PPARγ expression with its promoter and genomic global DNA methylation status, as well as DNA methyltransferases (DNMTs) gene expression in GBM patients. We examined the patterns of promoter methylation and PPARγ expression in 26 GBM tissues and 13 adjacent non-tumor tissues by methylation-specific PCR (MSP), real-time PCR, and ELISA, respectively. Also, we examined the genomic global 5-methyl cytosine levels and DNMTs gene expression using ELISA and real-time PCR methods, respectively. We found that hypermethylation on a specific region of the PPARγ promoter is significantly associated with the downregulation of the PPARγ gene and protein level in GBM patients. Interestingly, the amount of 5-methyl cytosine level was significantly reduced in GBM patients and positively correlated with PPARγ protein expression. Furthermore, the expression level of DNMT1, DNMT3A, and 3B were upregulated in GBM patients and the average expression level of all three DNMTs was positively correlated with tumor area. Also, we found that tumors from cortical regions exhibited a higher global DNA hypomethylation and PPARγ hypermethylation was related to the increase in GBM risk. Our study demonstrated that global DNA methylation and PPARγ epigenetic silencing is associated with the GBM risk. Our data provide a novel molecular mechanistic insight into epigenetic silencing of PPARγ in GBM patients that may be relevant as a key tumor marker for GBM pathogenesis.

A history of childhood maltreatment is associated with altered DNA methylation levels of DNA methyltransferase 1 in maternal but not neonatal mononuclear immune cells.

Childhood maltreatment (CM) is associated with alterations in DNA methylation (DNAm) especially in stress response genes. Due to the higher risk of overall health complications of individuals with a parental history of CM, intergenerational transmission of CM-associated DNAm changes has been investigated but remains unclear. In this study, we investigated if different severities of CM have any influence on the DNAm of DNA methyltransferase 1 (DNMT1), an important enzyme of the DNAm machinery, in immune and buccal cells of mother-newborn dyads. DNAm was assessed by mass spectrometry using immune cell DNA from mothers (N = 117) and their newborns (N = 113), and buccal cell DNA of mother-newborn dyads (N = 68 each). Mothers with a history of CM had lower mean methylation of DNMT1 in immune cells compared to the mothers without a CM history. CM status only influenced maternal DNMT1 gene expression when at least moderate CM was reported. Buccal cell DNAm was not associated with CM status. Maternal history of CM was not linked to any alterations in DNMT1 mean DNAm in any of the cell types studied in newborns. We conclude that the CM-associated alterations in DNMT1 DNAm might point to allostatic load and can be physiologically relevant, especially in individuals with more severe CM experiences, resulting in an activated DNA methylation machinery that might influence stress response genes. Our lack of significant findings in buccal cells shows the tissue-specific effects of CM on DNAm. In our sample with low to moderate maternal CM history, there was no intergenerational transmission of DNMT1 DNAm in newborns.

Distinct global DNA methylation and NF-κB expression profile of preimplantation biopsies from ideal and non-ideal kidneys.

Epigenetic mechanisms may affect the ideal and non-ideal kidneys selected for transplantationand their inflammatory gene expression profile differently and may contribute to poor clinical outcomes. Study the Global DNA methylation and the expression profiles of the DNA methyltransferases (DNMTs) and nuclear factor kappa B (NF-κB) in preimplantation kidney biopsies from ideal and non-ideal kidneys (expanded criteria donor (ECD) and with KDPI > 85%). In a sample consistingof45 consecutive pre-implantation biopsies,global DNA methylation levels were detected by LINE-1 repeated elements using bisulfite pyrosequencing. DNMT gene expression was assessed by real-time quantitative polymerase chain reaction, and NF-κB protein expression by immunofluorescence. ECD kidneys displayed increased methylation levels in LINE-1, and DNMT1 and DNMT3B expression was upregulated when comparing ECD to standard criteria donor kidneys. Similarly, kidneys with KDPI > 85% exhibited increased LINE-1 methylation and DNMT1 upregulation when compared to a KDPI ≤ 85%. NF-κB protein expression levels were greatly increased in both types of non-ideal kidneys compared to ideal kidneys. Moreover, hypermethylation of LINE-1 was associated with cold ischemia time > 20h and ECD kidney classification. This study shows that global DNA hypermethylation and high expression of NF-κB occurred in both types of non-ideal kidneys and were associated with prolonged cold ischemia time. Global DNA methylation can be a useful tool to assess non-ideal kidneys and hence, could be used to expand the pool of kidneys donors.

Evaluation of the initial manifestations of the toxic process in conditions of chronic action of low subtoxic doses of dioxins polluting the environment

AIM: To study the state of the genome by indicators of retrotransposon activity the gene encoding DNA methyltransferase 1 (DNMT1); DNA damage in animals from the natural population of the bank vole (Clethrionomys glareolus) living in the vicinity of a preserved landfill contaminated with low concentrations of dioxins (landfill of production and consumption waste "Salariyevo", Moscow).&#x0D; MATERIAL AND METHODS: The activity of ERV-L, B1, and L1 retrotransposons and the transcription level of the DNMT1 gene were evaluated by real-time PCR. The stability of DNA in liver and bone marrow cells was characterized by the comet assay method. Afterward, the obtained characteristics of the state (stability, reactivity, and damage) of the genome in response to environmental stress factors were compared in groups of animals from the study and conditionally control samples.&#x0D; RESULTS: The effects of a decrease in the activity of retrotransposons of classes B1 and L1, and an increase in DNMT1 gene expression level were revealed in voles from the natural population living under the long-term chronic exposure to low doses of dioxins decrease in the activity of retrotransposons of classes B1 and L1, and an increase in the expression level of the DNMT1 gene were revealed. An increased level of DNA damage (on average up to 56% of the DNA in the tail of the comet) was detected in hepatocytes, with the addition of winter environmental factors to the chronic effect of small subtoxic doses of dioxins.&#x0D; CONCLUSION: Suppression of retrotransposon activity and increased expression of its epigenetic regulator (DNMT1) are regarded as adaptive responses to long-term chronic exposure to low doses of dioxins polluting the environment. The alteration in the reactivity and destabilization of the genome indicates the launch of the initial mechanisms of the toxic process formation. The created and tested methodological base for its study opens up prospects for establishing a threshold level, and as a result, substantiating indicators for screening assessment of local (territorial) risk to public health by biomonitoring.

Uncovering the role of DNA methyltransferases in grapevine – Plasmopara viticola interaction: From genome-wide characterization to global methylation patterns

Epigenetic regulation has recently gained prominence in the field of plant-pathogen interactions, providing a deeper understanding of the molecular mechanisms associated with plant infection. In grapevine interaction with pathogens, epigenetic regulation still remains a black box. In this work, we characterized grapevine DNA methyltransferase gene family and identified nine DNA methyltransferases genes across eight grapevine chromosomes coding for 17 proteins. We also assessed the modulation of global cytosine methylation and gene expression levels of these genes with the aim of establishing a connection between DNA methylation and grapevine resistance towards downy mildew. Our results revealed that, in the incompatible interaction, an early hypomethylation, coupled with downregulation of DNMT and CMT genes occurs very early after pathogen inoculation. Additionally, the compatible interaction is characterized by a hypermethylation at 6hpi. A temporal delay is evident between the shifts in DNA methyltransferases gene expression in both compatible and incompatible interactions which in turn may be reflected in the global methylation percentage. Overall, we present the first evidence of an epigenetic regulation role in grapevine defense against P. viticola.

Levels of epigenetic regulators TET2 and DNMT1 correlate with virus-stimulated pDC differentiation from IFN-α producing cells to antigen presenting cells

Abstract Plasmacytoid dendritic cells (pDC) are potent producers of IFN-α in response to DNA and RNA viruses and subsequently mature into antigen presenting cells. We investigated the role of DNA methyltransferase 1 (DNMT1) and Ten-eleven translocation methyl-cytosine dioxygenase 2 (TET2), which are generally known as gene silencers and activators, respectively, in pDC activation. PBMC from healthy donors were stimulated with Herpes Simplex Virus (HSV) or Influenza-A virus (IAV) for 6- and 24h. PBMC were surface-stained for pDC markers CD123, HLA-DR and CD11C and co-stimulatory markers CD80 and CD86, then intracellularly for DNMT1, TET2 and IFN-α, and acquired by flow cytometry. mRNA expression of DNMT1 in purified pDC was determined by qRT-PCR. At 6h, DNMT1 protein was expressed at equivalent levels in pDC with and without stimulation and in IFN-α+ and IFN-α-pDC, and in unstimulated controls. In contrast, TET2 proteins were elevated in IFN-α+ but not IFN-α-pDC and controls at 6h. At 24h, DNMT1 increased while TET2 was reduced in stimulated pDC, correlating with high IFN-α levels at 6h and low levels at 24h. There was no change in DNMT1 gene expression in virus-stimulated vs. unstimulated controls. CD80 and CD86 were elevated at 24h compared to 6h in stimulated pDC, confirming pDC differentiation from IFN-α producing to antigen presenting cells. At the point of pDC differentiation, TET2 levels were decreased and DNMT1 increased. These results suggest that TET2 is demethylating and causing activation of genes associated with pDC IFN-α production, while DNMT1 methylates and silences genes in this pathway. This results in an initial induction of TET2 and IFN-α production followed by upregulation of co-stimulatory markers, DNMT1 and loss of IFN-α. Supported by 1. AAI trainee fellowship Careers in Immunology Fellowship Program AAI EIN #: 52-2317193 2. R01Al106125

Investigating the role of DNMT1 gene expression on myocardial ischemia reperfusion injury in rat and associated changes in mitochondria

Altered DNA methylation and mitochondrial dysfunction are the two key features of myocardial ischemia reperfusion injury (I/R), but their association with I/R remains unknown. In the present study, the relationship between DNA methyl transferase1 (DNMT1), the key methylation gene, and the mitochondrial quality control genes in rat heart during I/R was explored. We used the Langendorff rat heart model with 30 min of ischemia followed by 60 min of reperfusion and subsequent inhibition of DNMT1 with 5-azacytidine to evaluate the role of DNA methylation in I/R. Reperfusion significantly increased the expression of the DNMT1 gene, enzyme activity, and global DNA methylation levels, along with decreased mitochondrial copy, electron transport chain (ETC) activities, and ATP level. This was in agreement with the significant downregulation of 11 mitochondrial genes PGC-1α, TFAM, POLG, MFN1 and MFN2, FIS1, PARKIN, OPTN, ND1, ND4L, Cyt B and COX1 in I/R induced rat hearts. The expression pattern of the mitochondrial genes PGC-1α, TFAM, ND1 and Cyt B showed a significant negative correlation with DNMT1 expression. Rate pressure product, index of cardiac performance negatively correlated with DNMT1 expression (r = -0.8231, p = 0.0456). However, DNMT1 inhibited rat hearts via 5-azacytidine significantly improved the heart from I/R injury and reversed the I/R associated changes in the gene expression of TFAM, POLG, PGC-1α, ND1, COX1 and Cyt B, and improved the overall mtDNA copies, with a subsequent improvement in the ETC enzyme activity and ATP levels. To conclude, I/R augmented the DNMT1 activity with a subsequent increase in cardiac injury via downregulating the mitochondrial functional genes.

An Exploratory Pilot Study of Changes in Global DNA Methylation in Patients Undergoing Major Breast Surgery Under Opioid-Based General Anesthesia.

This study aimed to investigate DNA methylation levels in patients undergoing major breast surgery under opioid-based general anesthesia. Blood samples were collected from eleven enrolled patients, before, during and after anesthesia. PBMC were isolated and global DNA methylation levels as well as DNA methyltransferase (DNMT) and cytokine gene expression were assessed. DNA methylation levels significantly declined by 26%, reversing the direction after the end of surgery. Likewise, DNMT1a mRNA expression was significantly reduced at all time points, with lowest level of −68%. DNMT3a and DNMT3b decreased by 65 and 71%, respectively. Inflammatory cytokines IL6 and TNFα mRNA levels showed a trend for increased expression at early time-points to end with a significant decrease at 48 h after surgery. This exploratory study revealed for the first time intraoperative global DNA hypomethylation in patients undergoing major breast surgery under general anesthesia with fentanyl. The alterations of global DNA methylation here observed seem to be in agreement with DNMTs gene expression changes. Furthermore, based on perioperative variations of IL6 and TNFα gene expression, we hypothesize that DNA hypomethylation may occur as a response to surgical stress rather than to opiate exposure.

Mechanisms of DNA Methylation in Virus-Host Interaction in Hepatitis B Infection: Pathogenesis and Oncogenetic Properties.

Hepatitis B virus (HBV), the well-studied oncovirus that contributes to the majority of hepatocellular carcinomas (HCC) worldwide, can cause a severe inflammatory microenvironment leading to genetic and epigenetic changes in hepatocyte clones. HBV replication contributes to the regulation of DNA methyltransferase gene expression, particularly by X protein (HBx), and subsequent methylation changes may lead to abnormal transcription activation of adjacent genes and genomic instability. Undoubtedly, the altered expression of these genes has been known to cause diverse aspects of infected hepatocytes, including apoptosis, proliferation, reactive oxygen species (ROS) accumulation, and immune responses. Additionally, pollutant-induced DNA methylation changes and aberrant methylation of imprinted genes in hepatocytes also complicate the process of tumorigenesis. Meanwhile, hepatocytes also contribute to epigenetic modification of the viral genome to affect HBV replication or viral protein production. Meanwhile, methylation levels of HBV integrants and surrounding host regions also play crucial roles in their ability to produce viral proteins in affected hepatocytes. Both host and viral changes can provide novel insights into tumorigenesis, individualized responses to therapeutic intervention, disease progress, and early diagnosis. As such, DNA methylation-mediated epigenetic silencing of cancer-related genes and viral replication is a compelling therapeutic goal to reduce morbidity and mortality from liver cancer caused by chronic HBV infection. In this review, we summarize the most recent research on aberrant DNA methylation associated with HBV infection, which is involved in HCC development, and provide an outlook on the future direction of the research.

Effects of Berberine on the Chondrogenic Differentiation of Embryonic Limb Skeletal Progenitors.

IntroductionBerberine (BBR) is an isoquinoline plant alkaloid with demonstrated anti-inflammatory, anti-tumor and immunosuppressive pharmacological properties that functions via multiple signaling pathways and epigenetic modulators. Numerous studies have proposed BBR as a promising therapeutic agent for joint cartilage degeneration, and other connective tissue diseases.Purpose and MethodsThis work aimed to evaluate the effects of BBR on the growth and differentiation of embryonic skeletal progenitors using the limb mesoderm micromass culture assay.ResultsOur findings show that at difference of its apoptotic influence on a variety of tumor tissues, cell death was not induced in skeletal progenitors by the addition of 12 or 25 µM BBR concentration to the culture medium. Morphological and transcriptional analysis revealed dual and opposite effects of BBR treatments on chondrogenesis depending on the stage of differentiation of the cultured progenitors. At early stage of culture, BBR was a potent chondrogenic inhibitor, while chondrogenesis was intensified in treatments at advanced stages of culture. The chondrogenic promoting effect was accompanied by a moderate upregulation of gene markers of prehypertrophic cartilage, including ColXa1, alkaline phosphatase Alpl, Runx2, and Indian Hedgehog Ihh. We further observed a positive transcriptional influence of BBR in the expression of DNA methyltransferase genes, Dnmt1, Dnmt3a and Dnmt3b, suggesting a potential involvement of epigenetic factors in its effects.ConclusionOur study uncovers a new pharmacological influence of BBR in cartilage differentiation that must be taken into account in designing clinical protocols for its employment in the treatment of cartilage degenerative diseases.

Low-dose cadmium exposure facilitates cell proliferation by promoter hypermethylation of RASSF1A and DAPK1 genes.

Cadmium (Cd) at low concentrations has a potential to promote cell proliferation. However, the molecular mechanisms of Cd-induced proliferation are not well understood. Here, we reported that Cd (0-500 nM) significantly promoted the proliferation of HepG2 cells as demonstrated by elevated cell viability, more EdU-positive cells and increased gene expression of KI-67 and COX-2. Meanwhile, the gene expression of DNA methyltransferases was found to be elevated while that of tumor suppressor genes DAPK1 and RASSF1A were decreased under Cd exposure. Correspondingly, the methylation level of promoters in DAPK1 and RASSF1A were increased. Specifically, the CpG sites at -461 (Chr3:50, 374, 481) of RASSF1A promoter, and that at -260 (Chr9:90, 113, 207), -239 (Chr9:90, 113, 228), and -68 (Chr9:90, 113, 399) of DAPK1 promoter, were significantly hypermethylated. Moreover, 5-azacytidine (an inhibitor of DNA methyltransferase) partly impaired Cd-induced promoter hypermethylation of RASSF1A and DAPK1 genes, increased their expressions and slowed down Cd-induced cell proliferation, suggesting that DNA methylation play an essential part in Cd-boosted proliferation. The study showed that Cd caused promoter hypermethylation of RASSF1A and DAPK1, decreasing their expression and leading to higher level of cell proliferation. Furthermore, Cd at low concentrations could influence DNA methylation, which may serve as the proliferative mechanism of Cd.