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ÖzBu çalışmanın amacı, prenatal stresin (PS) dişi sıçanların serebral korteks ve hipokampüsünde DNA metiltransferazlar (DNMT) ve histon deasetilazların (HDAC) mRNA düzeylerine etkilerini araştırmaktır. PS sıçanlarda deksametazonla (Dex) indüklendi. Gebeliğin 14. gününden 21. gününe, gebe sıçanlara Dex (100 ug/kg/gün) veya salin enjekte edildi. Doğumdan sonra 3 aylıkken dişi sıçanlar (n=5) dekapite edildi. Dex'in epigenetik mekanizmalar üzerindeki etkileri gerçek zamanlı PCR ile DNMT1, DNMT3a, DNMT3b, HDAC1 ve HDAC2 mRNA düzeyleri aracılığıyla araştırıldı. İstatistiksel analizler tek yönlü varyans analiziyle yapıldı. Prenatal Dex maruziyeti, korteks ve hipokampüste DNMT3a, HDAC1 ve HDAC2 mRNA düzeylerinde anlamlı artışlara neden oldu. Ayrıca Dex grubunda DNMT3b mRNA düzeylerinin hipokampüste anlamlı bir şekilde artarken kortekste azaldığı bulundu. DNMT1 mRNA düzeylerinde anlamlı bir farklılık saptanmadı. PS'in dişi sıçanların korteks ve hipokampüsünde; DNMT3a, DNMT3b, HDAC1 ve HDAC2 genlerinin ekspresyon profillerindeki değişiklikler yoluyla epigenetik mekanizmaların düzensizliğini tetikleyebileceği sonucuna varılmıştır., The aim of this study was to investigate the effects of prenatal stress (PS) on mRNA levels of DNA methyltransferases (DNMTs) and histone deacetylases (HDACs) in cerebral cortex and hippocampus of female rats. PS was induced in rats with dexamethasone (Dex). From gestation day 14 to 21, pregnant rats were injected daily with Dex (100 μg/kg) or saline. After birth, at 3 months of age, female rats were decapitated (n=5). The effects of Dex on epigenetic mechanisms were investigated by real-time PCR through mRNA levels of DNMT1, DNMT3a, DNMT3b, HDAC1 and HDAC2. Statistical significant differences were determined with one-way analysis of variance. Prenatal Dex exposure caused significant increases in DNMT3a, HDAC1 and HDAC2 mRNA levels in cortex and hippocampus. We further found that DNMT3b mRNA levels significantly increased in hippocampus but decreased in cortex of Dex group. No significant differences were found in DNMT1 mRNA levels. It was concluded that PS may trigger dysregulation of epigenetic mechanisms in cortex and hippocampus of female rats through alterations in gene expression profiles of DNMT3a, DNMT3b, HDAC1 and HDAC2.

DNA methylation is associated with gene silencing, but its clinical role in cardiovascular diseases (CVDs) remains to be elucidated. We hypothesized that extracellular vesicles (EVs) may carry epigenetic changes, showing themselves as a potentially valuable non-invasive diagnostic liquid biopsy. We isolated and characterized circulating EVs of acute coronary syndrome (ACS) patients and assessed their role on DNA methylation in epigenetic modifications.EVs were recovered from plasma of 19 ACS patients and 50 healthy subjects (HS). Flow cytometry, qRT-PCR, and Western blot (WB) were performed to evaluate both intra-vesicular and intra-cellular signals. ShinyGO, PANTHER, and STRING tools were used to perform GO and PPI network analyses.ACS-derived EVs showed increased levels of DNA methyltransferases (DNMTs) (p0.001) and Ten-eleven translocation (TET) genes reduction. Specifically, de novo methylation transcripts, as DNMT3A and DNMT3B, were significantly increased in plasma ACS-EVs. DNA methylation analysis on PBMCs from healthy donors treated with HS- and ACS-derived EVs showed an important role of DNMTs carried by EVs. PPI network analysis evidenced that ACS-EVs induced changes in PBMC methylome. In the most enriched subnetwork, the hub gene SRC was connected to NOTCH1, FOXO3, CDC42, IKBKG, RXRA, DGKG, BAIAP2 genes that were showed to have many molecular effects on various cell types into onset of several CVDs. Modulation in gene expression after ACS-EVs treatment was confirmed for SRC, NOTCH1, FOXO3, RXRA, DGKG and BAIAP2 (p0.05).Our data showed an important role for ACS-derived EVs in gene expression modulation through de novo DNA methylation signals, and modulating signalling pathways in target cells.

Background: Low birth weight (LBW) is a risk factor associated with endometriosis. Our study aimed to analyze the risk of endometriosis in women with a LBW history and the relationships of progesterone receptor B (PR-B) gene promoter methylation, DNA methyltransferase-1 (DNMT1) expression, PR-B expression, and vascular endothelial growth factors (VEGF) with endometriosis. Methods: This study was conducted in two stages, a retrospective case-control design and a cross-sectional design, with 52 cases of endometriosis and 30 controls, which were further subdivided into LBW and non-LBW groups, at Hasan Sadikin General Hospital and its hospital networks from October 2017 to August 2021. Menstrual blood was taken from subjects and analyzed using pyrosequencing techniques to assess DNA methylation, while q-RT PCR was used to assess gene expression. Results: There were significant differences in PR-B methylation, DNMT1 expression, PR-B expression, and VEGF expression (p < 0.001) between the case and control groups. There was a significant negative correlation between PR-B methylation and PR-B expression (r = −0.558; p = 0.047). Based on a multiple logistic analysis, the most dominant factor affecting endometriosis incidence is PR-B (OR 10.40, 95% CI 3.24–33.4, R2 = 45.8). We found that patients with a low birth weight history had a 1.41-times-higher risk of developing endometriosis (95% CI 0.57–3.49, p = 0.113), although the relationship was not statistically significant. Conclusion: Endometriosis is associated with PR-B gene promoter hypermethylation, decreased PR-B expression, and increased DNMT1 and VEGF expression. The methylation of PR-B is the most dominant factor affecting endometriosis incidence.

Wilms tumour (WT), an embryonal kidney cancer, has been extensively characterised for genetic and epigenetic alterations, but a proportion of WTs still lack identifiable abnormalities. To uncover DNA methylation changes critical for WT pathogenesis, we compared the epigenome of foetal kidney with two WT cell lines, filtering our results to remove common cancer‐associated epigenetic changes and to enrich for genes involved in early kidney development. This identified four hypermethylated genes, of which ESRP2 (epithelial splicing regulatory protein 2) was the most promising for further study. ESRP2 was commonly repressed by DNA methylation in WT, and this occurred early in WT development (in nephrogenic rests). ESRP2 expression was reactivated by DNA methyltransferase inhibition in WT cell lines. When ESRP2 was overexpressed in WT cell lines, it inhibited cellular proliferation in vitro, and in vivo it suppressed tumour growth of orthotopic xenografts in nude mice. RNA‐seq of the ESRP2‐expressing WT cell lines identified several novel splicing targets. We propose a model in which epigenetic inactivation of ESRP2 disrupts the mesenchymal to epithelial transition in early kidney development to generate WT., ESRP2 regulates alternative splicing events that are critical for the mesenchymal to epithelial transition (MET) that occurs during kidney development. We show that in Wilms tumour, ESRP2 is commonly inactivated by DNA methylation at an early stage of carcinogenesis. We propose that epigenetic inactivation of ESRP2 disrupts the regulation of alternative splicing during MET. Disrupted MET leads in turn to persistence of undifferentiated foetal kidney cells that may progress to a tumour.

Group rearing is a common housing condition, but group-housed older mice show increased adrenal hypertrophy, a marker of stress. However, the ingestion of theanine, an amino acid unique to tea leaves, suppressed stress. We aimed to elucidate the mechanism of theanine’s stress-reducing effects using group-reared older mice. The expression of repressor element 1 silencing transcription factor (REST), which represses excitability-related genes, was increased in the hippocampus of group-reared older mice, whereas the expression of neuronal PAS domain protein 4 (Npas4), which is involved in the regulation of excitation and inhibition in the brain, was lower in the hippocampus of older group-reared mice than in same-aged two-to-a-house mice. That is, the expression patterns of REST and Npas4 were found to be just inversely correlated. On the other hand, the expression levels of the glucocorticoid receptor and DNA methyltransferase, which suppress Npas4 transcription, were higher in the older group-housed mice. In mice fed theanine, the stress response was reduced and Npas4 expression tended to be increased. These results suggest that Npas4 expression was suppressed by the increased expression of REST and Npas4 downregulators in the group-fed older mice, but that theanine avoids the decrease in Npas4 expression by suppressing the expression of Npas4 transcriptional repressors.

BACKGROUND Albeit a relatively stable epigenetic modification, DNA methylation in plants can be repatterned and play important roles in response to biotic and abiotic stresses. However, whether DNA methylation dynamics may contribute to cope with mercury (Hg) stress in plants remains to be fully investigated. OBJECTIVE To probe the potential roles of DNA methylation dynamics in coping with Hg stress in rice. METHODS Whole-genome bisulfite sequencing was used to profile the DNA methylation patterns of a rice Hg-resistant line (RHg) selected from a heterozygous mutant of the DNA methyltransferase 1 gene (OsMET1+/-), together with its Hg-sensitive wild-type plants of cv. Nipponbare (Nip) under both normal and Hg stress conditions. RESULTS Genome-wide locus-specific differential methylation regions (DMRs) were detected between RHg and Nip under normal condition, the predominant DMR patterns were CG hypo-DMRs, CHG hypo-DMRs and CHH hyper-DMRs. In both lines, more hyper- than hypo-DMRs were detected at all three sequence contexts (CG, CHG and CHH) under Hg stress relative to normal condition. Comparison of DNA methylation changes in the two lines under Hg stress indicates that RHg had a more dynamic methylome than the control (Nip). Original DMRs in RHg trended to transform to opposite status (from hyper- to hypo- or vice versa) under Hg stress condition. Gene ontology analysis revealed that Hg-resistance-related DMGs were enriched in diverse biological processes. CONCLUSIONS Our results suggest genome-wide locus-specific DNA methylation repatterning can facilitate rapid acquisition of Hg resistance in rice.

Brown adipocytes share the same developmental origin with skeletal muscle. Here we find that a brown adipocyte-to-myocyte remodeling also exists in mature brown adipocytes, and is induced by prolonged high fat diet (HFD) feeding, leading to brown fat dysfunction. This process is regulated by the interaction of epigenetic pathways involving histone and DNA methylation. In mature brown adipocytes, the histone demethylase UTX maintains persistent demethylation of the repressive mark H3K27me3 at Prdm16 promoter, leading to high Prdm16 expression. PRDM16 then recruits DNA methyltransferase DNMT1 to Myod1 promoter, causing Myod1 promoter hypermethylation and suppressing its expression. The interaction between PRDM16 and DNMT1 coordinately serves to maintain brown adipocyte identity while repressing myogenic remodeling in mature brown adipocytes, thus promoting their active brown adipocyte thermogenic function. Suppressing this interaction by HFD feeding induces brown adipocyte-to-myocyte remodeling, which limits brown adipocyte thermogenic capacity and compromises diet-induced thermogenesis, leading to the development of obesity., Brown adipocytes contribute to energy balance, and adipocyte development and brown adipocyte thermogenesis are in part regulated by epigenetic modifications. Here the authors report that the histone demethylase Utx maintains brown adipocyte identity via demethylation of PRDM16, which in turn represses myogenic remodelling via DNMT1-mediated Myod1 promoter hypermethylation in mice.

DNA methylation is a chemical modification that defines cell type and lineage through the control of gene expression and genome stability. Disruption of DNA methylation control mechanisms causes a variety of diseases, including cancer. Cancer cells are characterized by aberrant DNA methylation (i.e., genome-wide hypomethylation and site-specific hypermethylation), mainly targeting CpG islands in gene expression regulatory elements. In particular, the early findings that a variety of tumor suppressor genes (TSGs) are targets of DNA hypermethylation in cancer led to the proposal of a model in which aberrant DNA methylation promotes cellular oncogenesis through TSGs silencing. However, recent genome-wide analyses have revealed that this classical model needs to be reconsidered. In this review, we will discuss the molecular mechanisms of DNA methylation abnormalities in cancer as well as their therapeutic potential.

Background Gallbladder cancer (GBC) is known for its high malignancy and multidrug resistance. Previously, we uncovered that impaired integrity and stability of the elongator complex leads to GBC chemotherapy resistance, but whether its restoration can be an efficient therapeutic strategy for GBC remains unknown. Methods RT-qPCR, MS-qPCR and ChIP-qPCR were used to evaluate the direct association between ELP5 transcription and DNA methylation in tumour and non-tumour tissues of GBC. EMSA, chromatin accessibility assays, and luciferase assays were utilized to analysis the DNA methylation in interfering PAX5-DNA interactions. The functional experiments in vitro and in vivo were performed to investigate the effects of DNA demethylating agent decitabine (DAC) on the transcription activation of elongator complex and the enhanced sensitivity of gemcitabine in GBC cells. Tissue microarray contains GBC tumour tissues was used to evaluate the association between the expression of ELP5, DNMT3A and PAX5. Results We demonstrated that transcriptional repression of ELP5 in GBC was highly correlated with hypermethylation of the promoter. Mechanistically, epigenetic analysis revealed that DNA methyltransferase DNMT3A-catalysed hypermethylation blocked transcription factor PAX5 activation of ELP5 by disrupting PAX5-DNA interaction, resulting in repressed ELP5 transcription. Pharmacologically, the DNA demethylating agent DAC eliminated the hypermethylated CpG dinucleotides in the ELP5 promoter and then facilitated PAX5 binding and reactivated ELP5 transcription, leading to the enhanced function of the elongator complex. To target this mechanism, we employed a sequential combination therapy of DAC and gemcitabine to sensitize GBC cells to gemcitabine-therapy through epigenetic activation of the elongator complex. Conclusions Our findings suggest that ELP5 expression in GBC is controlled by DNA methylation-sensitive induction of PAX5. The sequential combination therapy of DAC and gemcitabine could be an efficient therapeutic strategy to overcome chemotherapy resistance in GBC.

Background Metastasis occurs in the majority of pancreatic ductal adenocarcinoma (PDAC) patients at diagnosis or following resection. Patients with liver metastasis and those with lung metastasis have significantly different prognosis. Here, we sought to understand how cancer-associated fibroblasts (CAFs) play roles in the development of organ-specific metastasis. Methods PDAC tumor cell lines established from the primary tumors with liver and lung metastasis potentials, respectively, in Kras/p53 mutation conditional knock-in (KPC) mice were co-cultured with matched CAFs or mouse mesenchymal stem cells. CAFs were isolated from metastases and subjected to DNA methylation and whole transcriptomic RNA sequencing analysis. Results The ability of mouse PDAC tumor cell lines in developing liver or lung-specific metastases was demonstrated in orthotopic models. Tumor cells associated with liver metastasis potential, but not those associated with lung metastasis potential, induced the methylation of metabolism genes including NQO1 and ALDH1a3 and subsequent downregulated mRNA expression of a broader group of metabolism genes in CAFs. DNA methylation and downregulation of metabolism genes in CAFs in liver metastasis, but not those in lung metastasis, appeared to be regulated by DNA methyltransferase. Tumor cells associated with liver metastasis potential, but not those associated with lung metastasis potential, induce inflammatory CAF (iCAF) signatures. CAFs from liver metastasis demonstrated a more homogenous iCAF phenotype, whereas CAFs from lung metastasis maintained the heterogeneity. Conclusions PDAC with organ-specific metastatic potentials has different capacities in inducing methylation of metabolism genes in CAFs, modulating CAF phenotypes, and resulting in different levels of heterogeneity of CAFs in different metastatic niches.

Dissertation, RWTH Aachen University, 2023; Aachen : RWTH Aachen University 1 Online-Ressource : Illustrationen, Diagramme (2023). = Dissertation, RWTH Aachen University, 2023, Mammalian DNA methyltransferases (DNA MTases) methylate the genomic DNA within the short 5´-CG-3´(CpG) DNA sequence leading to the epigenetic base 5-methyl¬cytosine (5mC). DNA methylation is important for regulating gene expression and changes in promotor regions are known markers for cancer diagnosis, making DNA methylation detection significant for early stage cancer diagnostics. DNA MTases naturally transfer the methyl group from S adenosyl L methionine (SAM) onto their target base within specific double-stranded DNA sequences and can be repurposed for DNA labeling using synthetic cofactors analogues. The bacterial DNA MTase M.MpeI forms 5mC within CpG sequences and, therefore, could be used as a tool for indirect mammalian DNA methylation detection by labeling only unmethylated cytosine residues in CpG sequences using fluorescent reporter groups. Fluorescent DNA can be analyzed in nanochannels or on array chips to make a fast DNA methylation detection and quantification possible. In the course of this work the labeling reaction was investigated and optimized for CpG specific MTases combined with customized cofactor analogues. Two classes of analogues, aziridine-based and double-activated cofactor analogues, were synthesized. For both types of cofactor analogues fluorescent groups could be directly transferred to DNA or coupled with the target base. The labeling reactions were analyzed by protection against fragmentation by restriction endonucleases using agarose gel electrophoresis or by rp-HPLC of the complete fragmentation of the DNA into nucleosides. In the case of double-activated analogues, a two-step approach using click chemistry to introduce the fluorescent group after enzymatic transfer of a suitable functional group was used for fluorescent labeling as well. Protein engineering based on sequence comparisons of homologues enzymes, computational docking and molecular dynamic simulations were used to expand cofactor specificity. Assays investigating the binding affinity of the wildtype and mutants were performed and based on the results the labeling reaction was optimized. The highest yields for the investigated cofactor classes and types of labeling reactions were 4 % using fluorescent aziridine-based cofactor analogues and the wildtype of M.MpeI, 79 % for the two-step approach using double-activated cofactor analogues followed by click-chemistry with a fluorescent label and a mutant of M.MpeI and 30 % for the one-step labeling using fluorescent double-activated cofactor analogues and an advanced mutant of M.MpeI., Published by RWTH Aachen University, Aachen

Impairments of the genes that encode enzymes that are involved in one-carbon metabolism because of the presence of gene polymorphisms can affect the methylation pattern. The altered methylation profiles of the genes involved in cardiogenesis may result in congenital heart defects (CHDs). The aim of this study was to investigate the association between the MTHFR rs1801133, MTHFR rs1801131, MTRR rs1801394, DNMT1 rs2228611, DNMT3A rs1550117, DNMT3B rs1569686, and DNMT3B rs2424913 gene polymorphisms and congenital heart defects in Down syndrome (DS) individuals. The study was conducted on 350 participants, including 134 DS individuals with CHDs (DSCHD+), 124 DS individuals without CHDs (DSCHD−), and 92 individuals with non-syndromic CHD. The genotyping was performed using the PCR–RFLP method. A statistically significant higher frequency of the DNMT3B rs2424913 TT in the DSCHD+ individuals was observed. The DNMT3B rs2424913 TT genotype, as well as the T allele, had significantly higher frequencies in the individuals with DS and atrial septal defects (ASDs) in comparison with the individuals with DS and other CHDs. Furthermore, our results indicate a statistically significant effect of the DNMT3B rs1569686 TT genotype in individuals with non-syndromic CHDs. The results of the study suggest that the DNMT3B rs2424913 TT genotypes may be a possible predisposing factor for CHDs in DS individuals, and especially those with ASDs.

Inteins, often referred to as protein introns, are highly mobile genetic elements that invade conserved genes throughout the tree of life. Inteins have been found to invade a wide variety of key genes within actinophages. While in the process of conducting a survey of these inteins in actinophages, we discovered that one protein family of methylases contained a putative intein, and two other unique insertion elements. These methylases are known to occur commonly in phages as orphan methylases (possibly as a form of resistance to restriction–modification systems). We found that the methylase family is not conserved within phage clusters and has a disparate distribution across divergent phage groups. We determined that two of the three insertion elements have a patchy distribution within the methylase protein family. Additionally, we found that the third insertion element is likely a second homing endonuclease, and that all three elements (the intein, the homing endonuclease, and what we refer to as the ShiLan domain) have different insertion sites that are conserved in the methylase gene family. Furthermore, we find strong evidence that both the intein and ShiLan domain are partaking in long-distance horizontal gene transfer events between divergent methylases in disparate phage hosts within the already dispersed methylase distribution. The reticulate evolutionary history of methylases and their insertion elements reveals high rates of gene transfer and within-gene recombination in actinophages.

Hypothesis Squamous cell carcinomas (SCC) of the external auditory canal (EAC) may harbor unique genomic alterations that may explain aggressive behavior and differentiate these tumors from cutaneous SCCs of other subsites. Background EAC SCCs arise in a non-ultraviolet-exposed region of the head and neck, are often locally aggressive and may metastasize to lymph nodes or distant sites. The genomic alterations underlying cutaneous SCC of other sites are well-documented; however, mutational profiles of EAC SCC are less well characterized and may contribute to the unique anatomic site, high rates of recurrence and tumor spread. We performed targeted sequencing of a cohort of primary EAC SCCs to identify recurring and potentially targetable genomic alterations. Methods Genomic DNA was extracted from formalin-fixed paraffin-embedded specimens of 7 EAC SCCs and subjected to targeted DNA sequencing using a 227-gene panel. Somatic alterations and gene copy number alterations were annotated using our validated, in-house bioinformatics pipelines. Results In our EAC SCCs, we found recurrent alterations in TP53 and genes of receptor tyrosine kinase (eg, EGFR, FGFR) and PI3K pathways (eg, PIK3CA), similar to cutaneous SCCs of other head and neck sites. We also observed a high frequency of telomerase reverse transcriptase amplification and DNA methyltransferase 1 alterations, both of which are rarely observed in cutaneous SCCs of other sites. Conclusion These data represent the first step toward precise molecular characterization of EAC SCCs that may lead to an enhanced understanding of tumor biology and modernized precision medicine approaches for unique tumors.Level of Evidence: NA.

Blind mole rats (BMRs) are small rodents, characterized by an exceptionally long lifespan (>21 years) and resistance to both spontaneous and induced tumorigenesis. Here we report that cancer resistance in the BMR is mediated by retrotransposable elements (RTEs). Cells and tissues of BMRs express very low levels of DNA methyltransferase 1. Following cell hyperplasia, the BMR genome DNA loses methylation, resulting in the activation of RTEs. Upregulated RTEs form cytoplasmic RNA-DNA hybrids, which activate the cGAS-STING pathway to induce cell death. Although this mechanism is enhanced in the BMR, we show that it functions in mice and humans. We propose that RTEs were co-opted to serve as tumor suppressors that monitor cell proliferation and are activated in premalignant cells to trigger cell death via activation of the innate immune response. Activation of RTEs is a double-edged sword, serving as a tumor suppressor but contributing to aging in late life via the induction of sterile inflammation.

Background: Glioblastoma is a malignant and very aggressive brain tumor with a poor prognosis. Despite having chemotherapy concomitant with surgery and/or radiation therapy, the median survival of ...

Background : It is well established that obesity is a major health risk in diabetes and associated diseases. Epigenetic changes, specially DNA methylation, play an important role in regulation of adipokines. The objective of the present study was to evaluate the DNA methylation status at the promoter region of the leptin gene in obese individuals and its association with metabolic risk factors. Methods : The study included obese (n=100) and non-obese (n=75) individuals aged 25-45 years, and measured their physical, biochemical parameters (glucose, insulin, and lipid profiles) and leptin, DNA methyltransferase 1 (DNMT1), and DNA methyltransferase 3 beta (DNMT3b) mRNA expressions with real-time reverse transcription-polymerase chain reaction (qRT-PCR). DNA methylation of the leptin gene at the promoter region was analyzed by methyl-specific qPCR . Results : The study found that the DNA methylation level at the promoter area of the leptin gene was negatively associated with weight in obese subjects. Furthermore, study findings showed that the DNA methylation level was negatively associated with fasting insulin, glucose, homeostatic model assessment for insulin resistance, and total cholesterol. There was also a higher expression of DNMT1 and DNMT-3b in obese subjects as compared with non-obese subjects. Conclusion : The leptin epigenetic profile may be associated with obesity and its associated metabolic risk factors.

The DNA methyltransferase family contains a conserved set of DNA-modifying enzymatic proteins. They are responsible for epigenetic gene modulation, such as transcriptional silencing, transcription activation, and post-transcriptional modulation. Recent research has revealed that the canonical DNA methyltransferases (DNMTs) biological roles go beyond their traditional functions of establishing and maintaining DNA methylation patterns. Although a complete DNA methylation toolkit is absent in most insect orders, recent evidence indicates the de novo DNA methylation and maintenance function remain conserved. Studies using various molecular approaches provided evidence that DNMTs are multi-functional proteins. However, still in-depth studies on their biological role lack due to the least studied area in insects. Here, we review the DNA methylation toolkit of insects, focusing on recent research on various insect orders, which exhibit DNA methylation at different levels, and for which DNMTs functional studies have become available in recent years. We survey research on the potential roles of DNMTs in the regulation of gene transcription in insect species. DNMTs participate in different physiological processes by interacting with other epigenetic factors. Future studies on insect's DNMTs will benefit to understand developmental processes, responses to various stimuli, and adaptability of insects to different environmental conditions.

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.

Predatory mites, that are important natural enemies of small sap-feeding pests, often tolerate various fluctuating environmental stresses. DNA methylation has been demonstrated to play critical roles in regulating transcriptional changes in response to environmental stress. To investigate whether the thermal tolerance of a high temperature acclimated strain (HTAS) of the predatory mite Neoseiulus barkeri Hughes is associated with DNA methylation mechanisms, we cloned and sequenced its DNA methyltransferase 1-associated protein1 (DMAP1). Quantitative real-time PCR analysis of transcript abundances revealed that NbDMAP1 was expressed ubiquitously at all developmental stages of N. barkeri and was also significantly upregulated after exposure to 38 °C and 42 °C. The expression of NbDMAP1 in the HTAS N. barkeri showed a significant increase compared to the conventional strain N. barkeri after exposure to 42 °C. Feeding on double-stranded RNA specific for NbDMAP1, however, significantly decreased the thermal tolerance of HTAS and also increased its mortality rate. This study suggests DNA methylation played a potential role in thermal tolerance of N. barkeri, which is an important mechanism for its successful colonization under thermal environments and commercialization for pest control.

Intragenic DNA methylation, also called gene body methylation, is an evolutionarily-conserved epigenetic mechanism in animals and plants. In social insects, gene body methylation is thought to contribute to behavioral plasticity, for example between foragers and nurse workers, by modulating gene expression. However, recent studies have suggested that the majority of DNA methylation is sequence-specific, and therefore cannot act as a flexible mediator between environmental cues and gene expression. To address this paradox, we examined whole-genome methylation patterns in the brains and ovaries of young honey bee workers that had been subjected to divergent social contexts: the presence or absence of the queen. Although these social contexts are known to bring about extreme changes in behavioral and reproductive traits through differential gene expression, we found no significant differences between the methylomes of workers from queenright and queenless colonies. In contrast, thousands of regions were differentially methylated between colonies, and these differences were not associated with differential gene expression in a subset of genes examined. Methylation patterns were highly similar between brain and ovary tissues and only differed in nine regions. These results strongly indicate that DNA methylation is not a driver of differential gene expression between tissues or behavioral morphs. Finally, despite the lack of difference in methylation patterns, queen presence affected the expression of all four DNA methyltransferase genes, suggesting that these enzymes have roles beyond DNA methylation. Therefore, the functional role of DNA methylation in social insect genomes remains an open question.

The mandarin fish (Siniperca chuatsi) DNA methyltransferase gene 1 (dnmt1) was highly expressed in the mesonephros, head kidney and gonad, whereas dnmt2 was expressed in most tissues. dnmt3a was highly expressed in the brain and spleen, but dnmt3b was mainly expressed in the brain and head kidney. The genes dnmt1 and dnmt2 were highly expressed in the early stages of embryonic development, and dnmt3a and dnmt3b were expressed later. These genes also showed certain changes after artificial diet acclimation, salinity adaptation and immune stress.

DNA methyltransferase (DNMT) and histone deacetylase (HDAC) are well recognized epigenetic targets for discovery of antitumor agents. In this study, we designed and synthesized a series of nucleoside base hydroxamic acid derivatives as DNMT and HDAC dual inhibitors. MTT assays and enzymatic inhibitory activity tests indicated that compound 204 exhibited potent DNMT1 and HDAC1/6 inhibitory potency simultaneously in enzymatic levels and at cellular levels, inducing hypomethylation of p16 and hyperacetylation of histones H3K9 and H4K8. Besides, 204 remarkably inhibited proliferation against cancer cells U937 by prompting G0/G1 cell cycle arrest. Molecular docking models explained the functional mechanism of 204 inhibiting DNMT1 and HDAC. Preliminary studies on metabolic profiles revealed that 204 showed desirable stability in liver microsomes. Our study suggested that 204 inhibiting DNMT and HDAC concurrently can be a potential lead compound for epigenetic cancer therapy.

We previously found that DNA methyltransferase 3a (DNMT3a) plays an important role in regulating embryonic cardiomyocyte gene expression, morphology, and function. In this study, we investigated the role of the most abundant DNMT in mammalian cells, DNMT1, in these processes. It is known that DNMT1 is essential for embryonic development, during which it is involved in regulating cardiomyocyte DNA methylation and gene expression. We used siRNA to knock down DNMT1 expression in primary cultures of mouse embryonic cardiomyocytes. Immunofluorescence staining and multielectrode array were, respectively, utilized to evaluate cardiomyocyte growth and electrophysiology. RNA sequencing (RNA‐Seq) and multiplex bisulfite sequencing were, respectively, performed to examine gene expression and promoter methylation. At 72 h post‐transfection, reduction of DNMT1 expression decreased the number and increased the size of embryonic cardiomyocytes. Beat frequency and the amplitude of field action potentials were decreased by DNMT1 siRNA. RNA‐Seq analysis identified 801 up‐regulated genes and 494 down‐regulated genes in the DNMT1 knockdown cells when compared to controls. Pathway analysis of the differentially expressed genes revealed pathways that were associated with cell death and survival, cell morphology, cardiac function, and cardiac disease. Alternative splicing analysis identified 929 differentially expressed exons, including 583 up‐regulated exons and 308 down‐regulated exons. Moreover, decreased methylation levels were found in the promoters of cardiac genes Myh6, Myh7, Myh7b, Tnnc1, Tnni3, Tnnt2, Nppa, Nppb, mef2c, mef2d, Camta2, Cdkn1A, and Cdkn1C. Of these 13 genes, 6 (Myh6, Tnnc1, Tnni3, Tnnt2, Nppa, Nppb) and 1 (Cdkn1C) had increased or decreased gene expression, respectively. Altogether, these data show that DNMT1 is important in embryonic cardiomyocytes by regulating DNA methylation, gene expression, gene splicing, and cell function., Knockdown of DNMT1 expression in mouse embryonic cardiomyocytes decreased promoter DNA methylation levels, altered activities of transcription factors, changed gene expression and splicing, and decreased cell viability, beating frequency, and amplitude of field action potential.

Cancer is a genetic disease resulting from genomic changes; however, epigenetic alterations act synergistically with these changes during tumorigenesis and cancer progression. Epigenetic variations are gaining more attention as an important regulator in tumor progression, metastasis and therapy resistance. Aberrant DNA methylation at CpG islands is a central event in epigeneticmediated gene silencing of various tumor suppressor genes. DNA methyltransferase 1 (DNMT1) predominately methylates at CpG islands on hemimethylated DNA substrates in proliferation of cells. DNMT1 has been shown to be overexpressed in various cancer types and exhibits tumor-promoting potential. The major drawbacks to DNMT1-targeted cancer therapy are the adverse effects arising from nucleoside and non-nucleoside based DNMT1 inhibitors. This paper focuses on the regulation of DNMT1 by various microRNAs (miRNAs), which may be assigned as future DNMT1 modulators, and highlights how DNMT1 regulates various miRNAs involved in tumor suppression. Importantly, the role of reciprocal inhibition between DNMT1 and certain miRNAs in tumorigenic potential is approached in this review. Hence, this review seeks to project an efficient and strategic approach using certain miRNAs in conjunction with conventional DNMT1 inhibitors as a novel cancer therapy. It has also been pinpointed to select miRNA candidates associated with DNMT1 regulation that may not only serve as potential biomarkers for cancer diagnosis and prognosis, but may also predict the existence of aberrant methylation activity in cancer cells.

Tobacco exposure is well known to induce genetic and epigenetic changes that contribute to the pathogenesis of lung cancer. 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a significant tobacco-specific carcinogen, but the oncogenic mechanisms of NNK have not been thoroughly elucidated. In this study we found that DNA methyltransferase 1 (DNMT1) was overexpressed in malignantly transformed human bronchial epithelial Beas-2B cells induced by NNK (2B-NNK cells), by treatment with NNK (400 μg/mL) for 7 days. An Arraystar Human noncoding RNA Promoter Microarray was used to detect the DNA methylation status of the promoter region of long noncoding RNAs (lncRNAs). The result showed that 1010 differentially methylated fragments were present in the lncRNA promoter region. QRT-PCR revealed that the expression of lncRNA AC007255.8 was remarkably downregulated in 2B-NNK cells and lung cancer tissues. Furthermore, Methylation-specific PCR showed that the methylation of the lncRNA AC007255.8 promoter was increased in 2B-NNK cells and lung cancer tissues. The reduced expression of lncRNA AC007255.8 was significantly associated with hypermethylation of lncRNA AC007255.8 promoter region. LncRNA AC007255.8 overexpression could result in decreased cell proliferation and increased cell apoptosis in 2B-NNK cells. In conclusion, NNK induced lncRNA AC007255.8 promoter hypermethylation via upregulation of DNMT1 in Beas-2B cells, leading to downregulation of lncRNA AC007255.8, and ultimately the enhancement of cell proliferation and the inhibition of apoptosis. This research affords novel insights into the epigenetic mechanisms of lung cancer, and will stimulate further research into the involvement of aberrant DNA methylation of non-coding regions of the genome in the pathogenesis of lung cancer.

DNA methylation is an epigenetic mark associated with gene repression. It is now well established that tumor development involves alterations in DNA methylation patterns, which include both gains (hypermethylation) and losses (hypomethylation) of methylation marks in different genomic regions. The mechanisms underlying these two opposite, yet co-existing, alterations in tumors remain unclear. While studying the human MAGEA6/GABRA3 gene locus, we observed that DNA hypomethylation in tumor cells can lead to the activation of a long transcript (CT-GABRA3) that overlaps downstream promoters (GABRQ and GABRA3) and triggers their hypermethylation. Overlapped promoters displayed increases in H3K36me3, a histone mark known to be deposited during progression of the transcription machinery and to stimulate de novo DNA methylation. Consistent with such a processive mechanism, increases in H3K36me3 and DNA methylation were observed over the entire region covered by the CT-GABRA3 overlapping transcript. Importantly, experimental induction of CT-GABRA3 by depletion of DNMT1 DNA methyltransferase, resulted in a similar pattern of increased DNA methylation in the MAGEA6/GABRA3 locus. Bioinformatics analyses in lung cancer datasets identified other genomic loci displaying this process of coupled DNA hypo- and hypermethylation. In several of these loci, DNA hypermethylation affected tumor suppressor genes, e.g. RERG and PTPRO. Together, our work reveals that focal DNA hypomethylation in tumors can indirectly contribute to hypermethylation of nearby promoters through activation of overlapping transcription, and establishes therefore an unsuspected connection between these two opposite epigenetic alterations.

Renal fibrosis, the final pathway of chronic kidney disease, is caused by genetic and epigenetic mechanisms. Although DNA methylation has drawn attention as a developing mechanism of renal fibrosis, its contribution to renal fibrosis has not been clarified. To address this issue, the effect of zebularine, a DNA methyltransferase inhibitor, on renal inflammation and fibrosis in the murine unilateral ureteral obstruction (UUO) model was analyzed. Zebularine significantly attenuated renal tubulointerstitial fibrosis and inflammation. Zebularine decreased trichrome, α-smooth muscle actin, collagen IV, and transforming growth factor-β1 staining by 56.2%. 21.3%, 30.3%, and 29.9%, respectively, at 3 days, and by 54.6%, 41.9%, 45.9%, and 61.7%, respectively, at 7 days after UUO. Zebularine downregulated mRNA expression levels of matrix metalloproteinase (MMP)-2, MMP-9, fibronectin, and Snail1 by 48.6%. 71.4%, 31.8%, and 42.4%, respectively, at 7 days after UUO. Zebularine also suppressed the activation of nuclear factor-κB (NF-κB) and the expression of pro-inflammatory cytokines, including tumor necrosis factor-α, interleukin (IL)-1β, and IL-6, by 69.8%, 74.9%, and 69.6%, respectively, in obstructed kidneys. Furthermore, inhibiting DNA methyltransferase buttressed the nuclear expression of nuclear factor (erythroid-derived 2)-like factor 2, which upregulated downstream effectors such as catalase (1.838-fold increase at 7 days, p < 0.01), superoxide dismutase 1 (1.494-fold increase at 7 days, p < 0.05), and NAD(P)H: quinone oxidoreduate-1 (1.376-fold increase at 7 days, p < 0.05) in obstructed kidneys. Collectively, these findings suggest that inhibiting DNA methylation restores the disrupted balance between pro-inflammatory and anti-inflammatory pathways to alleviate renal inflammation and fibrosis. Therefore, these results highlight the possibility of DNA methyltransferases as therapeutic targets for treating renal inflammation and fibrosis.

Piwi-interacting RNAs (piRNAs/piRs) are small non-coding RNAs that play important roles in stablizing genome through silencing transposable genetic elements. The piR-651 was reported to be dysregulated in several human solid cancer tissues, such as gastric and lung cancers. However, the role of piRNA-651 in carcinogenesis of breast cancer has not been defined. We found that piR-651 was highly expressed in breast cancer tissues and cell lines. Overexpression of piR-651 facilitated cell proliferation and invasion, restrained cell apoptosis and the percentage of arrested cells in G0/G1 phase, accompanied by upregulated expression of oncogenes (MDM2, CDK4 and Cyclin D1), whereas piR-651 downregulation showed the opposite effects. Additionally, piR-651 could promote phosphatase and tensin homolog (PTEN) methylation and its downregulated expression by recruiting DNA methyltransferase 1 (DNMT1) to the PTEN promoter region through complex formation with PIWIL2. PTEN overexpression reversed the effects of upregulated piR-651 on cell functions. This study reveals that piR-651 promotes proliferation and migration and induces apoptosis of breast cancer cells by facilitating DNMT1-mediated PTEN promoter methylation, which may provide a potential therapeutic mechanism for breast cancer.

Background: O6-methylguanine-DNA methyltransferase (MGMT) is a DNA-repair enzyme that correlates with tumor resistance mechanism to chemotherapy. Methylation of the MGMT promoter inhibits the cells from producing MGMT and is useful to predict chemotherapy's effectiveness with alkylating agents. This study aims to evaluate the MGMT promoter methylation of low-grade and high-grade glioma in the Neurosurgery Department of Cipto Mangunkusumo National General HospitalMethods: We evaluated MGMT promoter methylation status using methylation-specific polymerase chain reaction in low and high-grade glioma patients who underwent surgical resection in the Neurosurgery Department of Cipto Mangunkusomo Hospital Jakarta. The result then correlated with age, sex, Karnofsky Performance Scale (KPS), and glioma grading. Data were analyzed using SPSS version 20 for Windows.Results: MGMT promoter methylation was observed more often in patients diagnosed with age more than 40 years old than in patients less than 40 years old (85.7% vs. 50.0%), also more in men than women (77.7% vs. 50.0%). In patients with KPS more than 70 and KPS 70 or less, methylation of MGMT promoter was observed in 70.0% and 57.1%, respectively. Based on tumor grading, MGMT promoter methylation was observed more often in low-grade gliomas (WHO grade II) than high-grade gliomas (WHO grade II and IV) (85.7% vs. 50.0%). There was no significant relationship between gender, age, KPS, malignancy degree, and Overall Survival (OS) to the MGMT promoter methylation (p>0.05).Conclusion: MGMT promoter methylation was observed less in the higher grade of tumors (grade IV), lower KPS, younger age at the time of diagnosis, and female patients, although the differences were not statistically significant. MGMT promoter methylation was observed more often in gliomas with oligodendroglioma components.

Chronic kidney disease (CKD) is a common and complex disease in kidneys which has been associated with an increased risk of renal cell carcinoma. Elevated homocysteine (Hcy) levels are known to influence the development and progression of CKD by regulating podocyte injury and apoptosis. To investigate the molecular mechanisms triggered in podocytes by Hcy, we used cbs+/− mice and observed that higher Hcy levels increased the apoptosis rate of podocytes with accompanying glomerular damage. Hcy‐induced podocyte injury and apoptosis in cbs+/− mice was regulated by inhibition of microRNA (miR)‐1929‐5p expression. Overexpression of miR‐1929‐5p in podocytes inhibited apoptosis by upregulating Bcl‐2. Furthermore, the expression of miR‐1929‐5p was regulated by epigenetic modifications of its promoter. Hcy upregulated DNA methyltransferase 1 (DNMT1) and enhancer of zeste homolog 2 (EZH2) levels, resulting in increased DNA methylation and H3K27me3 levels on the miR‐1929‐5p promoter. Additionally, we observed that c‐Myc recruited DNMT1 and EZH2 to the miR‐1929‐5p promoter and suppressed the expression of miR‐1929‐5p. In summary, we demonstrated that Hcy promotes podocyte apoptosis through the regulation of the epigenetic modifiers DNMT1 and EZH2, which are recruited by c‐Myc to the promoter of miR‐1929‐5p to silence miR‐1929‐5p expression., Chronic kidney disease is a common and complex disease of the kidneys, associated with an increased risk of renal cell carcinoma. In the cbs+/− mouse model, we observed that homocysteine (Hcy) induced podocyte apoptosis by regulating miR‐1929‐5p expression. Hcy‐mediated upregulation of DNMT1 and EZH2 led to epigenetic miR‐1929‐5p silencing, following c‐Myc‐dependent recruitment of DNMT1 and EZH2 to the miR‐1929‐5p promoter. Inhibition of miR‐1929‐5p expression induced apoptosis in podocytes and led to kidney injury.

Glioma‐initiating cells (GICs), a major source of glioblastoma recurrence, are characterized by the expression of neural stem cell markers and the ability to grow by forming nonadherent spheres under serum‐free conditions. Bone morphogenetic proteins (BMPs), members of the transforming growth factor‐β family, induce differentiation of GICs and suppress their tumorigenicity. However, the mechanisms underlying the BMP‐induced loss of GIC stemness have not been fully elucidated. Here, we show that paired related homeobox 1 (PRRX1) induced by BMPs decreases the CD133‐positive GIC population and inhibits tumorigenic activity of GICs in vivo. Of the two splice isoforms of PRRX1, the longer isoform, pmx‐1b, but not the shorter isoform, pmx‐1a, induces GIC differentiation. Upon BMP stimulation, pmx‐1b interacts with the DNA methyltransferase DNMT3A and induces promoter methylation of the PROM1 gene encoding CD133. Silencing DNMT3A maintains PROM1 expression and increases the CD133‐positive GIC population. Thus, pmx‐1b promotes loss of stem cell‐like properties of GICs through region‐specific epigenetic regulation of CD133 expression by recruiting DNMT3A, which is associated with decreased tumorigenicity of GICs., BMPs induce differentiation and apoptosis of glioma‐initiating cells (GICs). Here, we identified PRRX1 as a target of BMPs in GICs. Of the two splice isoforms, only pmx‐1b induced GIC differentiation. Through interaction with DNMT3A, pmx‐1b induced the methylation of the PROM1 gene promoter and suppressed the CD133+ GIC population. Thus, site‐specific epigenetic regulation plays a critical role in the differentiation of GICs.

Pendahuluan: Kanker paru masih menjadi penyebab utama kematian akibat kanker. Metode skrining terkini masih memiliki banyak kekurangan dalam mendiagnosis kanker paru, sehingga dibutuhkan metode skrining yang lebih cermat dan aplikatif dalam mendiagnosis individu dengan kanker paru. Salah satu metode skrining yang berpotensi membantu penegakkan diagnosis kanker paru adalah metilasi DNA yang diduga memiliki keunggulan sebagai biomarka pada kanker paru. Metode: Pencarian dan seleksi jurnal sesuai kriteria inklusi melalui database PubMed, ScienceDirect, dan Cochrane Library. Ditemukan sejumlah 13 studi yang memenuhi kriteria inklusi. Pembahasan: Metilasi DNA terjadi pada fase awal perkembangan kanker dan bersifat spesifik pada jenis tumor yang berbeda. Pada kanker paru, regulasi DNA metiltransferase (DNMT) dan enzim ten-eleven translocation (TET) mengalami disrupsi sehingga terjadi inaktivasi tumor suppressor gene dan perkembangan kanker. Secara keseluruhan, metilasi DNA pada gen HOXD10 / PAX9 / PTPRN2 / STAG3, SHOX2 / PTGER4 / FOXL2, SOX17 / TAC1 / HOXA7 / CDO1 / HOXA9 / ZFP42, dan CDO1 / SOX17 / HOXA7 memiliki sensitivitas hingga >90%. Beberapa panel metilasi DNA lainnya seperti DCC, TMEM196, SHOX2 / PTGER4, dan CDO1 / SOX17 / HOXA7 memiliki spesifisitas yang tinggi hingga 90-100%. Selain itu, nilai AUC menunjukan angka diatas 0,80 pada mayoritas studi. Metilasi DNA sebagai penunjang metode skrining dapat meningkatkan efektivitas deteksi dini dan dapat menurunkan tingkat false positive. Simpulan: Biomarka metilasi DNA efektif sebagai metode deteksi dini kanker paru.

It was found that short exogenous peptides GlyGly and GlyAsp and the amino acid Gly can have a selective effect on the expression of genes for DNA-methyltransferases and SNF2 proteins from Nicotiana tabacum regenerants. It was noted that Gly and GlyGly decrease the expression of DNA methyltransferase genes (except for CMT3) and increase the expression of CMT3 by almost 30%. It was established that the presence of the acidic dipeptide GlyAsp in the nutrient medium increases the expression of genes not only of the CMT3 DNA methyltransferase but also of the supporting MET1 DNA methyltransferase as compared to the control variant. It was determined that all dipeptides and free glycine reduce (by two to four times) the expression of the DRM2 methyltransferase gene, which is responsible for DNA methylation de novo. It was shown that Gly, GlyGly, and GlyAsp modulate the expression of genes encoding the SNF2 family of proteins, decreasing the expression of almost all genes of the SNF2 family, only GlyGly increasing the expression of genes encoding an actin-dependent protein and CHR12. It was found that a decrease in the expression of the SNF2 genes leads to a decrease in the availability of free DNA for methylation, especially for de novo methylation.

The thirteen-lined ground squirrel, Ictidomys tridecemlineatus, is a mammal capable of lowering its Tb to almost 0 °C while undergoing deep torpor bouts over the winter. To decrease its metabolic rate to such a drastic extent, the squirrel must undergo multiple physiological, biological, and molecular alterations including downregulation of almost all nonessential processes. Epigenetic regulation allows for a dynamic range of transient phenotypes, allowing the squirrel to downregulate energy-expensive and nonessential pathways during torpor. DNA methylation is a prominent form of epigenetic regulation; therefore, the DNA methyltransferase (DNMT) family of enzymes were studied by measuring expression and activity levels of the five major proteins during torpor bouts. Additionally, specific cytosine marks on genomic DNA were quantified to further elucidate DNA methylation during hibernation. A tissue-specific response was observed that highlighted variant degrees of DNA methylation and DNMT expression/activity, demonstrating that DNA methylation is a highly complex form of epigenetic regulation and likely one of many regulatory mechanisms that enables metabolic rate depression in response to torpor.

Endometrial cancer (EC) is the most common gynecological malignancy worldwide. However, the molecular mechanisms underlying EC progression are still largely unknown, and chemotherapeutic options for EC patients are currently very limited. In this study, we found that histone methyltransferase EZH2 and DNA methyltransferase DNMT3B were upregulated in EC samples from patients, and promoted EC cell proliferation as evidenced by assays of cell viability, cell cycle, colony formation. Mechanistically, we found that EZH2 promoted EC cell proliferation by epigenetically repressing TCF3, a direct transcriptional activator of CCKN1A (p21WAF1/Cip1), in vitro and in vivo. In addition, we found that DNMT3B specifically methylated the TCF3 promoter, repressing TCF3 expression and accelerating EC cell proliferation independently of EZH2. Importantly, elevated expression of EZH2 or DNMT3B in EC patients inversely correlated with expression of TCF3 and p21, and was associated with shorter overall survival. We show that combined treatment with GSK126 and 5-Aza-2d treatment wit synergistically inhibited methyltransferase activity of EZH2 and DNMT3B, resulting in a profound block of EC cell proliferation as well as EC tumor progression in cell line-derived xenograft (CDX) and patient-derived xenograft (PDX) mouse models. These findings reveal that TCF3 functions as a tumor suppressor epigenetically silenced by EZH2 and DNMT3B in EC, and support the notion that targeting the EZH2/DNMT3B/TCF3/p21 axis may be a novel and effective therapeutic strategy for treatment of EC.

Background and aims Cholangiocarcinoma (CCA) is a devastating disease often detected at advanced stages when surgery cannot be performed. Conventional and targeted systemic therapies perform poorly, and therefore effective drugs are urgently needed. Different epigenetic modifications occur in CCA and contribute to malignancy. Targeting epigenetic mechanisms may thus open therapeutic opportunities. However, modifications such as DNA and histone methylation often coexist and cooperate in carcinogenesis. We tested the therapeutic efficacy and mechanism of action of a class of dual G9a histone-methyltransferase and DNA-methyltransferase 1 (DNMT1) inhibitors. Approach and results Expression of G9a, DNMT1, and their molecular adaptor, ubiquitin-like with PHD and RING finger domains-1 (UHRF1), was determined in human CCA. We evaluated the effect of individual and combined pharmacological inhibition of G9a and DNMT1 on CCA cell growth. Our lead G9a/DNMT1 inhibitor, CM272, was tested in human CCA cells, patient-derived tumoroids and xenograft, and a mouse model of cholangiocarcinogenesis with hepatocellular deletion of c-Jun-N-terminal-kinase (Jnk)-1/2 and diethyl-nitrosamine (DEN) plus CCl4 treatment (JnkΔhepa + DEN + CCl4 mice). We found an increased and correlative expression of G9a, DNMT1, and UHRF1 in CCAs. Cotreatment with independent pharmacological inhibitors G9a and DNMT1 synergistically inhibited CCA cell growth. CM272 markedly reduced CCA cell proliferation and synergized with Cisplatin and the ERBB-targeted inhibitor, Lapatinib. CM272 inhibited CCA tumoroids and xenograft growth and significantly antagonized CCA progression in JnkΔhepa + DEN + CCl4 mice without apparent toxicity. Mechanistically, CM272 reprogrammed the tumoral metabolic transcriptome and phenotype toward a differentiated and quiescent status. Conclusions Dual targeting of G9a and DNMT1 with epigenetic small molecule inhibitors such as CM272 is a potential strategy to treat CCA and/or enhance the efficacy of other systemic therapies.

Precise deposition of CpG methylation is critical for mammalian development and tissue homeostasis and is often dysregulated in human diseases. The localization of de novo DNA methyltransferase DNMT3A is facilitated by its PWWP domain recognizing histone H3 lysine 36 (H3K36) methylation(1,2) and is normally depleted at CpG islands (CGIs)(3). However, methylation of CGIs regulated by Polycomb repressive complexes (PRCs) has also been observed(4–8). Here, we report that DNMT3A PWWP domain mutations identified in paragangliomas(9) and microcephalic dwarfism(10) promote aberrant localization of DNMT3A to CGIs in a PRC1-dependent manner. DNMT3A PWWP mutants accumulate at regions containing PRC1-mediated formation of monoubiquitylated histone H2A lysine 119 (H2AK119ub), irrespective of the amounts of PRC2-catalyzed formation of trimethylated histone H3 lysine 27 (H3K27me3). DNMT3A interacts with H2AK119ub-modified nucleosomes through a putative amino-terminal ubiquitin-dependent recruitment region, providing an alternative form of DNMT3A genomic targeting that is augmented by the loss of PWWP reader function. Ablation of PRC1 abrogates localization of DNMT3A PWWP mutants to CGIs and prevents aberrant DNA hypermethylation. Our study implies that a balance between DNMT3A recruitment by distinct reader domains guides de novo CpG methylation and may underlie the abnormal DNA methylation landscapes observed in select human cancer subtypes and developmental disorders.

It is known that autophagy-deficient cells are prone to DNA damage, but the specific role of autophagy in DNA damage repair is not fully known. Here, we show that autophagy-deficient liver cancer cells exhibit increased DNA damage caused by the chemotherapeutic agent epirubicin. Autophagy deficiency promotes downregulation of the DNA repair enzyme O6methylguanine-DNA methyltransferase (MGMT) in liver cancer cells. However, autophagy induction with epirubicin had no impact on MGMT gene or protein expression in liver cancer cells. In the absence of autophagy, the chemosensitivity of liver cancer cells was increased, but this was reversed by MGMT overexpression, indicating that autophagy mediates resistance to chemotherapy in liver cancer cells via MGMT. These findings demonstrate a direct link between autophagy, MGMT, and DNA damage repair in liver cancer cells, and show that MGMT not only regulates chemosensitivity to alkylating agents, but may also be involved in other DNA damage repair processes in autophagy-deficient cells.

Background Epidemiological data show that traffic-related air pollution contributes to the increasing prevalence and severity of asthma. DNA methylation (DNAm) changes may elucidate adverse health effects of environmental exposures. Objectives We sought to assess the effects of allergen and diesel exhaust (DE) exposures on global DNAm and its regulation enzymes in human airway epithelium. Methods A total of 11 participants, including 7 with and 4 without airway hyperresponsiveness, were recruited for a randomized, double-blind crossover study. Each participant had 3 exposures: filtered air + saline, filtered air + allergen, and DE + allergen. Forty-eight hours postexposure, endobronchial biopsies and bronchoalveolar lavages were collected. Levels of DNA methyltransferases (DNMTs) and ten-eleven translocation (TET) enzymes, 5-methylcytosine, and 5-hydroxymethylcytosine were determined by immunohistochemistry. Cytokines and chemokines in bronchoalveolar lavages were measured by electrochemiluminescence multiplex assays. Results Predominant DNMT (the most abundant among DNMT1, DNMT3A, and DNMT3B) and predominant TET (the most abundant among TET1, TET2, and TET3) were participant-dependent. 5-Methylcytosine and its regulation enzymes differed between participants with and without airway hyperresponsiveness at baseline (filtered air + saline) and in response to allergen challenge (regardless of DE exposure). Predominant DNMT and predominant TET correlated with lung function. Allergen challenge effect on IL-8 in bronchoalveolar lavages was modified by TET2 baseline levels in the epithelium. Conclusions Response to allergen challenge is associated with key DNAm regulation enzymes. This relationship is generally unaltered by DE coexposure but is rather dependent on airway hyperresponsiveness status. These enzymes therefore warranted further inquiry regarding their potential in diagnosis, prognosis, and treatment of asthma.

O6-methylguanine DNA methyltransferase (MGMT) is one of the most premutagenic, precarcinogenic and precytotoxic DNA lesions formed by alkylating agents. Repairing DNA damage is achieved by the protein MGMT which transfers the alkyl groups from the O6-position of guanine to a cysteine residue in the active center of MGMT. The most common assays of MGMT activity are time-consuming and have to employ radioactivity. In order to avoid these shortcomings, in this study we have developed a novel biosensing strategy for measuring the activity of MGMT based on fluorescence of DNA-silver nanoclusters. The results showed that a wide range from 0.01 to 1.0 µg/mL was achieved for MGMT assay and limit of detection of 8 ng/mL. The method has high sensitivity, good reproducibility, simplicity, also it is label free, rapid and cheap. Therefore, we can conclude that the developed strategy holds a great potential for quantitative measurement of MGMT activity with desirable sensitivity and reproducibility.

Background Chronic alcohol consumption is more frequently associated with advanced, aggressive hepatocellular carcinoma (HCC) tumors. Alcohol adversely impacts ER/Golgi membrane trafficking and Golgi protein N‐glycosylation in hepatocytes; these effects have been attributed (in part) to dysregulated adenosine diphosphate‐ribosylation factor (ARF) GTPase signaling. Here, we investigated the role of the ARF GTPase guanine exchange factor PSD4 in HCC progression. Methods R‐based bioinformatics analysis was performed on publicly available array data. Modulating gene expression was accomplished via lentiviral vectors. Gene expression was analyzed using quantitative real‐time PCR and immunoblotting. PSD4 promoter methylation was assessed using quantitative methylation‐specific PCR. Phospho‐p65(S276)/DNMT1 binding to the PSD4 promoter was analyzed via chromatin immunoprecipitation. We constructed ethanol/DEN‐induced and DEN only‐induced transgenic murine models of HCC. Results We identified PSD4 as a hypermethylated, suppressed gene in alcohol‐related HCC tumors; however, PSD4 was not dysregulated in all‐cause HCC tumors. Certain HCC cell lines also displayed varying degrees of PSD4 downregulation. PSD4 overexpression or knockdown decreased and increased cell migration and invasiveness, respectively. Mechanistically, PSD4 transcription was repressed by TNF‐α‐induced phospho‐p65(S276)’s recruitment of DNA methyltransferase 1 (DNMT1), resulting in PSD4 promoter methylation. PSD4 inhibited pro‐EMT CDC42 activity, resulting in downregulation of E‐cadherin and upregulation of N‐cadherin and vimentin. Hepatocyte‐specific PSD4 overexpression reduced ethanol/DEN‐induced HCC tumor progression and EMT marker expression in vivo. Conclusions PSD4 is a hypermethylated, suppressed gene in alcohol‐related HCC tumors that negatively modulated pro‐EMT CDC42 activity. Furthermore, we present a novel phospho‐NF‐κB p65(S276)/DNMT1‐mediated promoter methylation mechanism by which TNF‐α/NF‐κB signaling represses PSD4 transcription in HCC cells., PSD4 is a hypermethylated, suppressed gene in alcohol‐related HCC tumors. PSD4 decreases cell migration and invasiveness in HCC cells. PSD4 is epigenetically repressed by TNF‐α‐induced phospho‐p65(S276)’s recruitment of DNMT1.

The tea plant (Camellia sinensis) is a thermophilic cash crop and contains a highly duplicated and repeat-rich genome. It is still unclear how DNA methylation regulates the evolution of duplicated genes and chilling stress in tea plants. We therefore generated a single-base-resolution DNA methylation map of tea plants under chilling stress. We found that, compared with other plants, the tea plant genome is highly methylated in all three sequence contexts, including CG, CHG and CHH (where H = A, T, or C), which is further proven to be correlated with its repeat content and genome size. We show that DNA methylation in the gene body negatively regulates the gene expression of tea plants, whereas non-CG methylation in the flanking region enables a positive regulation of gene expression. We demonstrate that transposable element-mediated methylation dynamics significantly drives the expression divergence of duplicated genes in tea plants. The DNA methylation and expression divergence of duplicated genes in the tea plant increases with evolutionary age and selective pressure. Moreover, we detect thousands of differentially methylated genes, some of which are functionally associated with chilling stress. We also experimentally reveal that DNA methyltransferase genes of tea plants are significantly downregulated, whereas demethylase genes are upregulated at the initial stage of chilling stress, which is in line with the significant loss of DNA methylation of three well-known cold-responsive genes at their promoter and gene body regions. Overall, our findings underscore the importance of DNA methylation regulation and offer new insights into duplicated gene evolution and chilling tolerance in tea plants.

DNA methylation is an important epigenetic regulator of gene expression. Application of 5-azacytidine (a methylation inhibitor) significantly promoted bud sprouting rate and the elongation of branches and leaves in "Luhehong" and "Fengdanbai." In total, 11,166 and 11,443 fragments were obtained by methylation-sensitive amplified polymorphism (MSAP) analysis during chilling-induced dormancy release in the two varieties, respectively. Total methylation levels were high in dormant buds, mainly for hemimethylation, which were slowly increased by short-term chilling (7 days) and decreased by long-term chilling. Compared with 0 day, the ratio of the methylation downregulated group increased during dormancy release, whereas that of the upregulated group declined gradually. These variations were consistent with the dynamic expressions of DNA methyltransferase/demethylase genes and their enzyme activity changes. In total, 13 polymorphic MSAP fragments were similar to known proteins (E-value

DNA methylation, and consequent down-regulation, of tumour suppressor genes occurs in response to epigenetic stimuli during cancer development. Similarly, human oncoviruses, including human papillomavirus (HPV), up-regulate and augment DNA methyltransferase (DNMT) and histone deacetylase (HDAC) activities, thereby decreasing tumour suppressor genes (TSGs) expression. Ubiquitin-like containing PHD and RING finger domain 1 (UHRF1), an epigenetic regulator of DNA methylation, is overexpressed in HPV-induced cervical cancers. Here, we investigated the role of UHRF1 in cervical cancer by knocking down its expression in HeLa cells using lentiviral-encoded short hairpin (sh)RNA and performing cDNA microarrays. We detected significantly elevated expression of thioredoxin-interacting protein (TXNIP), a known TSG, in UHRF1-knockdown cells, and this gene is hypermethylated in cervical cancer tissue and cell lines, as indicated by whole-genome methylation analysis. Up-regulation of UHRF1 and decreased TXNIP were further detected in cervical cancer by western blot and immunohistochemistry and confirmed by Oncomine database analysis. Using chromatin immunoprecipitation, we identified the inverted CCAAT domain-containing UHRF1-binding site in the TXNIP promoter and demonstrated UHRF1 knockdown decreases UHRF1 promoter binding and enhances TXNIP expression through demethylation of this region. TXNIP promoter CpG methylation was further confirmed in cervical cancer tissue by pyrosequencing and methylation-specific polymerase chain reaction. Critically, down-regulation of UHRF1 by siRNA or UHRF1 antagonist (thymoquinone) induces cell cycle arrest and apoptosis, and ubiquitin-specific protease 7 (USP7), which stabilises and promotes UHRF1 function, is increased by HPV viral protein E6/E7 overexpression. These results indicate HPV might induce carcinogenesis through UHRF1-mediated TXNIP promoter methylation, thus suggesting a possible link between CpG methylation and cervical cancer.

DNA methyltransferase 1 (DNMT1) is a major epigenetic regulator of the formation of large macromolecular complexes that repress human γ-globin expression by maintaining DNA methylation. However, very little is known about the association of DNMT1 variants with β-thalassemia phenotypes. We systematically investigated associations between variants in DNMT1 and phenotypes in 1142 β-thalassemia subjects and identified a novel missense mutation (c.2633G>A, S878F) in the DNMT1 bromo-adjacent homology-1 (BAH1) domain. We functionally characterized this mutation in CD34+ cells from patients and engineered HuDEP-2 mutant cells. Our results demonstrate that DNMT1 phosphorylation is abrogated by substituting serine with phenylalanine at position 878, resulting in lower stability and catalytic activity loss. S878F mutation also attenuated DNMT1 interactions with BCL11A, GATA1, and HDAC1/2, and reduced recruitment of DNMT1 to the γ-globin (HBG) promoters, leading to epigenetic derepression of γ-globin expression. By analyzing the F-cell pattern, we demonstrated that the effect of DNMT1 mutation on increased fetal hemoglobin (HbF) is heterocellular. Furthermore, introduction of S878F mutation into erythroid cells by clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated protein 9 (Cas9) recapitulated γ-globin reactivation. Thus, the natural S878F DNMT1 mutation is a novel modulator of HbF synthesis and represents a potential new therapeutic target for β-hemoglobinopathies.

Concomitant inhibition of key epigenetic pathways involved in silencing tumor suppressor genes has been recognized as a promising strategy for cancer therapy. Herein, we report a first-in-class series of quinoline-based analogues that simultaneously inhibit histone deacetylases (from a low nanomolar range) and DNA methyltransferase-1 (from a mid-nanomolar range, IC50 1 log unit), and a suitable pharmacokinetic profile. In vivo, 12a achieved significant antitumor efficacy in a xenograft mouse model of human multiple myeloma.