DNA (cytosine-5)-methyltransferase 1 Research Articles

Grape seed extract prevents chlorpyrifos-induced toxicity in rat liver through the modulation of phase I detoxification pathway.

Chlorpyrifos (CPF) poisoning is a public health problem for which there is not currently any effective prophylaxis. In this study, we investigated the protective effect of grape seed extract (GSE) against CPF-induced hepatotoxicity. Rats were daily treated either with CPF (2mg/kg) or CPF and GSE (20mg/kg) for 1week, sacrificed, and their livers dissected for biochemical, molecular, and histopathological analyses. CPF generated liver dysfunction by altering carbohydrate, lipid, amino acid, ammonia and urea metabolism, and provoked mitochondrial impairment through disturbing tricarboxylic acid (TCA) cycle, oxidative phosphorylation (OXPHOS), and mitochondrial viability. CPF also induced cholinergic excitotoxicity along with oxidative stress and histopathological alterations. Interestingly, treatment with GSE prevented all the detrimental effects of CPF through the regulation of cytochrome P450 (CYP450) gene expression. Molecular docking analysis indicated that GSE-containing polyphenols acted as epigenetic modulators through inhibiting DNA (cytosine-5)-methyltransferase 1 (DNMT1), thus favoring the CYP2C6 detoxification pathway. Thereby, GSE might be a promising strategy in the protection of the liver against CPF toxicity.

A review on recent advances in assays for DNMT1: a promising diagnostic biomarker for multiple human cancers.

The abnormal expression of human DNA methyltransferases (DNMTs) is closely related with the occurrence and development of a wide range of human cancers. DNA (cytosine-5)-methyltransferase-1 (DNMT1) is the most abundant human DNA methyltransferase and is mainly responsible for genomic DNA methylation patterns. Abnormal expression of DNMT1 has been found in many kinds of tumors, and DNMT1 has become a valuable target for the diagnosis and drug therapy of diseases. Nowadays, DNMT1 has been found to be involved in multiple cancers such as pancreatic cancer, breast cancer, bladder cancer, lung cancer, gastric cancer and other cancers. In order to achieve early diagnosis and for scientific research, various analytical methods have been developed for qualitative or quantitative detection of low-abundance DNMT1 in biological samples and human tumor cells. Herein, we provide a brief explication of the research progress of DNMT1 involved in various cancer types. In addition, this review focuses on the types, principles, and applications of DNMT1 detection methods, and discusses the challenges and potential future directions of DNMT1 detection.

Prothymosin α accelerates dengue virus-induced thrombocytopenia

Thrombocytopenia is the hallmark finding in dengue virus (DENV) infection. Prothymosin α (ProT) has both intracellular and extracellular functions involved in cell cycle progression, cell differentiation, gene regulation, oxidative stress response, and immunomodulation. In this study, we found that ProT levels were elevated in dengue patient sera as well as DENV-infected megakaryoblasts and their culture supernatants. ProT transgenic mice had reduced platelet counts with prolonged bleeding times. Upon treatment with DENV plus anti-CD41 antibody, they exhibited severe skin hemorrhage. Furthermore, overexpression of ProT suppressed megakaryocyte differentiation. Infection with DENV inhibited miR-126 expression, upregulated DNA (cytosine-5)-methyltransferase 1 (DNMT1), downregulated GATA-1, and increased ProT expression. Upregulation of ProT led to Nrf2 activation and reduced reactive oxygen species production, thereby suppressing megakaryopoiesis. We report the pathophysiological role of ProT in DENV infection and propose an involvement of the miR-126-DNMT1-GATA-1-ProT-Nrf2 signaling axis in DENV-induced thrombocytopenia.

Epigenetic Modification of HepG2 Cells by Modulating DNA (cytosine-5)- methyltransferase 1 (DNMT1) and Ten-eleven Translocation Methylcytosine Dioxygenase 1 (TET1) Expression using Persian Shallot Extract

Background: Various forms of epigenetic modification, including DNA methylation, contribute to hepatocellular carcinoma (HCC) dissemination, invasion, and metastasis. The Persian shallot (Allium hirtifolium Bioss.) is reported to have antibacterial, antifungal, antioxidant, and anticarcinogenic properties. Aims: In the present study, we examined the expression of DNA (cytosine-5)-methyltransferase 1 (DNMT1) and ten-eleven translocation methylcytosine dioxygenase 1 (TET1) at the mRNA level in HepG2 cells following treatment with Persian shallot extract. Method: Ethanolic extracts of Persian shallot were prepared and dried at 80°C and 50°C for 20 and 30 minutes, respectively. Different concentrations of dried shallot extract over the range of 0-250 µg/ml were prepared. HepG2 cells were cultured and the cytotoxicity of each extract concentration was measured using an MTT assay. The gene expression in treated and untreated cells was assessed by Real-time polymerase chain reaction (RT-PCR). Results: The half maximal inhibitory concentration (IC50) was determined to be 149 µg/ml using an MTT assay. A concentration of 175 µg/ml was found to reduce the expression of DNMT1 in the treated group compared to the control group (P<0.001). Furthermore, the TET1 mRNA of HepG2 cells was down-regulated significantly after treatment with 100 and 1000 µg/ml of Persian shallot extract (P<0.05). These doses reduced the viability of the samples by 60% or higher. Conclusion: This study provides evidence for the potential use of Persian shallot extract as a supplementary herbal agent for the treatment of HCC. The concentrations of extract used in this study are near or above the level required for toxicity, and as such, further study is warranted.

Extremely low-frequency electromagnetic field (ELF-EMF) induces alterations in epigenetic regulation in the myometrium – An in vitro study

Previous research by the authors indicated that an extremely low-frequency electromagnetic field (ELF-EMF) evokes molecular alterations in the porcine myometrium. It was hypothesized that the ELF-EMF could induce alterations in the epigenetic regulation of gene expression in the myometrium. In the current study, slices of the porcine myometrium during the peri-implantation period (n = 4) were used for further in vitro exposition to ELF-EMF (50 Hz, 8 mT, 2 h treatment duration). The study tested whether the ELF-EMF may affect: 1/the expression of DNA (cytosine-5)-methyltransferase 1 (DNMT1) and DNA (cytosine-5)-methyltransferase 3a (DNMT3a), 2/the level of genomic DNA methylation, and 3/the level of amplification of methylated and unmethylated variants of promoter regions of selected genes with altered expression in response to ELF-EMF. It was found that ELF-EMF treatment increased DNMT1, decreased DNMT3a mRNA transcript and protein abundance, and increased the level of genomic DNA methylation. The direction of alterations in the level of amplification of methylated and unmethylated variants of the promoter region of selected genes with altered expression, i.e. prodynorphin (PDYN), interleukin 15 (IL15) signal transducer and activator of transcription 5A (STAT5A), tumor necrosis factor (TNF), and between down-regulated genes were early growth response 2 (EGR2), hyaluronan and proteoglycan link protein 1 (HAPLN1), and uteroferrin associated basic protein-2 (UABP2), mostly involving the direction of changes in their transcriptional activity, which was evaluated in a previous study by the authors. Thus, ELF-EMF radiation disturbs epigenetic mechanisms, which may underlay ELF-EMF-related transcriptomic alterations in the myometrium.

Isoflurane ameliorates oxygen-glucose deprivation-induced cardiomyocyte injury through SIRT6/DNMT1 pathway

The incidence of cardiovascular diseases is on the rise in the world, which poses a significant threat to human health. Myocardial ischemia can cause heart disease. Therefore, it is necessary to avoid myocardial hypoxia/reoxygenation (H/R) injury to attenuate the risk of heart disease. The present study focuses on the protective effect of isoflurane on H/R-induced cell injury through the Sirtuin 6 (SIRT6)/DNA (cytosine-5)-methyltransferase 1 (DNMT1) pathway. Quantitative reverse transcription PCR (RT‑qPCR) and Western blot analysis were used to measure protein levels and mRNA expression in H9c2 cells. Cell Counting Kit‑8 assays (CCK8 assay) was used to determine cell viability. The expression levels of pro-inflammatory molecule were assessed using commercial Enzyme-linked immunosorbent assay (ELISA) Kits. The ratio of cellular apoptosis was determined by flow cytometry. The contents of Lactate dehydrogenase (LDH), Cardiac Troponin I (cTnI), and Creatine Kinase MB (CK-MB) were detected using colorimetric assays. This study shows that Isoflurane reduces the expression of DNMT1 by activating SIRT6 in oxygen-glucose deprivation (OGD)-induced H/R injury. The damage of cardiomyocyte was decreased after Isoflurane treatment under OGD exposure condition. In addition, Isoflurane ameliorates OGD-induced inflammatory responses and cellular apoptosis in H9c2 cell via interaction with the SIRT6/DNMT1 pathway. Taken together, this study suggested the protective effect of Isoflurane on the process of OGD-induced damage and provided a new mechanism of action for Isoflurane in the treatment of H/R-induced cardiomyocyte injury.

G Protein-Coupled Receptor 37L1 Modulates Epigenetic Changes in Human Renal Proximal Tubule Cells.

Renal luminal sodium transport is essential for physiological blood pressure control, and abnormalities in this process are strongly implicated in the pathogenesis of essential hypertension. Renal G protein-coupled receptors (GPCRs) are critical for the regulation of the reabsorption of essential nutrients, ions, and water from the glomerular filtrate. Recently, we showed that GPCR 37L1 (GPR37L1) is expressed on the apical membrane of renal proximal tubules (RPT) and regulates luminal sodium transport and blood pressure by modulating the function of the sodium proton exchanger 3 (NHE3). However, little is known about GPR37L1 intracellular signaling. Here, we show that GPR37L1 is localized to the nuclear membrane, in addition to the plasma membrane in human RPT cells. Furthermore, GPR37L1 signals via the PI3K/AKT/mTOR pathway to decrease the expression of DNA (cytosine-5)-methyltransferase 1 (DNMT1) and enhance NHE3 transcription. Overall, we demonstrate the direct role of a nuclear membrane GPCR in the regulation of renal sodium through epigenetic gene regulation.

Long non-coding RNAs and small nucleolar RNA host gene 6 contribute to the depression-like behavior of hypothyroid mice by promoting methylation of the brain-derived neuropathic factor promoter that is mediated by DNA methyltransferase 1.

Long non-coding RNAs (lncRNAs) and small nucleolar RNA host gene 6 (SNHG6) have attracted extensive attention due to their involvement in various pathological processes. However, the functional role of lncRNA SNHG6 in depression-like behavior induced by hypothyroidism is still largely unknown. Our study was designed to explore the biological role of lncRNA SNHG6 in depression-like behavior induced by hypothyroidism and the underlying mechanisms. First, the depression-like behavior of hypothyroid mice was investigated after lncRNA SNHG6 knockdown. Subsequently, the regulation of the methylation levels of the brain-derived neurotrophic factor (BDNF) promoters by lncRNA SNHG6 was evaluated. To reveal the underlying mechanisms of lncRNA SNHG6 in the methylation of the BDNF promoters, RNA pull-down assays, RNA immunoprecipitation, and co-immunoprecipitation were performed. Further experiments were also conducted to investigate the roles of DNA (cytosine-5)-methyltransferase 1 (DNMT1) in the depression-like behavior induced by hypothyroidism. In this study, elevated levels of lncRNA SNHG6 were noted in the hippocampus in hypothyroid mice. Function assays proved that lncRNA SNHG6 knockdown alleviated the depression-like behavior induced by hypothyroidism. Furthermore, a mechanistic investigation validated that lncRNA SNHG6 stabilized DNMT1 by blocking UHRF1-mediated DNMT1 ubiquitination, which increased the methylation levels of the BDNF promoters. Moreover, DNMT1 was found to be involved in depression-like behavior in hypothyroid mice. Our findings revealed a novel mechanism by which lncRNA SNHG6 promotes the methylation levels of the BDNF promoters by stabilizing DNMT1 and sheds light on potential therapeutic strategies for depression-like behavior induced by hypothyroidism.

NOS2 and S-nitrosothiol signaling induces DNA hypomethylation and LINE-1 retrotransposon expression

Inducible nitric oxide synthase (NOS2) produces high local concentrations of nitric oxide (NO), and its expression is associated with inflammation, cellular stress signals, and cellular transformation. Additionally, NOS2 expression results in aggressive cancer cell phenotypes and is correlated with poor outcomes in patients with breast cancer. DNA hypomethylation, especially of noncoding repeat elements, is an early event in carcinogenesis and is a common feature of cancer cells. In addition to altered gene expression, DNA hypomethylation results in genomic instability via retrotransposon activation. Here, we show that NOS2 expression and associated NO signaling results in substantial DNA hypomethylation in human cell lines by inducing the degradation of DNA (cytosine-5)–methyltransferase 1 (DNMT1) protein. Similarly, NOS2 expression levels were correlated with decreased DNA methylation in human breast tumors. NOS2 expression and NO signaling also resulted in long interspersed noncoding element 1 (LINE-1) retrotransposon hypomethylation, expression, and DNA damage. DNMT1 degradation was mediated by an NO/p38-MAPK/lysine acetyltransferase 5–dependent mechanism. Furthermore, we show that this mechanism is required for NO-mediated epithelial transformation. Therefore, we conclude that NOS2 and NO signaling results in DNA damage and malignant cellular transformation via an epigenetic mechanism.

DNMT1-Mediated DNA Methylation Targets CDKN2B to Promote the Repair of Retinal Ganglion Cells in Streptozotocin-Induced Mongolian Gerbils during Diabetic Retinopathy.

Objective DNA methylation played a vital role in the progression of diabetic retinopathy. In this study, we aimed to explore the role of DNA cytosine-5-methyltransferase 1 (DNMT1) in the development of early diabetic retinopathy and its potential underlying mechanism. Methods Eight-week-old healthy Mongolian gerbils were used to establish type 1 diabetes using streptozotocin (STZ). Alteration of weight, fasting blood glucose, density of RGCs (Tuj1-labeled), and H&E-stained retinal cross sections were applied to evaluate the diabetic retinopathy mouse model. The global DNA methylation level of the retina at different time points after STZ injection was measured using the global methylation assay. Western blot was used to detect the protein expression of DNMT1, DNA methyltransferase 3A (DNMT3A), and 3B (DNMT3B). Quantitative reverse transcription-polymerase chain reactions (qRT-PCR) and western blot were used to determine the expression of CDKN2B. Cell proliferation and cell cycle were evaluated by the MTS assay and flow cytometry. Results STZ injection caused the increased global DNA methylation level, which reached a maximum at 6 weeks after injection. Moreover, STZ injection caused the damage of RGCs. At 6 weeks after STZ injection, the expression levels of DNMT1 and DNMT3B were significantly increased in the STZ group. DNMT1-induced DNA hypermethylation inhibited the expression of CDKN2B (a negative regulator of cell cycle). DNMT1-mediated DNA methylation facilitated RGC proliferation via regulating the expression of CDKN2B. Conclusion DNMT1-mediated DNA methylation played an important role in STZ-induced diabetic retinopathy via modulating CDKN2B expression.

DNA Methylation and Detoxification in the Earthworm Lumbricus terrestris Exposed to Cadmium and the DNA Demethylation Agent 5-aza-2'-deoxycytidine.

Earthworms are well-established model organisms for testing the effects of heavy metal pollution. How DNA methylation affects cadmium (Cd) detoxification processes such as the expression of metallothionein 2 (MT2), however, is largely unknown. We therefore exposed Lumbricus terrestris to 200 mg concentrations of Cd and 5-aza-2′-deoxycytidine (Aza), a demethylating agent, and sampled tissue and coelomocytes, cells of the innate immune system, for 48 h. MT2 transcription significantly increased in the Cd- and Cd-Aza-treated groups. In tissue samples, a significant decrease in MT2 in the Aza-treated group was detected, showing that Aza treatment inhibits basal MT2 gene activity but has no effect on Cd-induced MT2 levels. Although Cd repressed the gene expression of DNA-(cytosine-5)-methyltransferase-1 (DNMT1), which is responsible for maintaining DNA methylation, DNMT activity was unchanged, meaning that methylation maintenance was not affected in coelomocytes. The treatment did not influence DNMT3, which mediates de novo methylation, TET gene expression, which orchestrates demethylation, and global levels of hydroxymethylcytosine (5hmC), a product of the demethylation process. Taken together, this study indicates that Aza inhibits basal gene activity, in contrast to Cd-induced MT2 gene expression, but does not affect global DNA methylation. We therefore conclude that Cd detoxification based on the induction of MT2 does not relate to DNA methylation changes.

Quantitative proteomics revealed new functions of ALKBH4.

ALKBH4 is a versatile demethylase capable of catalyzing the demethylation of monomethylated lysine-84 on actin and N6 -methyladenine in DNA. In this study, we conducted a quantitative proteomic experiment to reveal the altered expression of proteins in HEK293T cells upon genetic ablation of ALKBH4. Our results showed markedly diminished levels of GSTP1 and HSPB1 proteins in ALKBH4-depleted cells, which emanate from an augmented expression level of DNA (cytosine-5)-methyltransferase 1 (DNMT1) and the ensuing elevated cytosine methylation in the promoter regions of GSTP1 and HSPB1 genes. Together, our results revealed a role of ALKBH4 in modulating DNA cytosine methylation through regulating the expression level of DNMT1 protein.

Deubiquitination of FBP1 by USP7 blocks FBP1-DNMT1 interaction and decreases the sensitivity of pancreatic cancer cells to PARP inhibitors.

Poly[ADP‐ribose] polymerase (PARP) inhibitors can block DNA single‐strand damage repair and subsequently increase double‐stranded breaks (DSBs) by reducing the activity of the PARP1 protease and by preventing the PARP1 protein from dissociating from chromatin. Tumors with the BRCA mutation are particularly sensitive to PARP inhibitors. So far, PARP inhibitors (Olaparib) have been used to treat pancreatic cancer patients with BRCA mutation. However, these patients are prone to PARP inhibitor resistance. Our previous studies suggest that fructose‐1,6‐bisphosphatase 1 (FBP1) is responsible for the sensitivity to various anticancer agents, such as gemcitabine or mitogen‐activated protein kinase kinase (MEK) inhibitors. In this study, we demonstrate that FBP1 regulates the sensitivity to PARP inhibitors in pancreatic cancer. Then, we showed that nuclear FBP1 is responsible for this process by interacting with DNA (cytosine‐5)‐methyltransferase 1 (DNMT1) and trapping PARP1 in chromatin. Moreover, we revealed that ubiquitin carboxyl‐terminal hydrolase 7 (USP7) binds to and induces the deubiquitination of FBP1, which prevented FBP1 from translocating to the nucleus. Finally, we demonstrated that USP7 inhibitors enhanced the antitumor effect of PARP inhibitors in an FBP1‐dependent manner. Collectively, our results identify a novel USP7–FBP1–DNMT1 signaling axis in pancreatic cancer, which might indicate that USP7 inhibitors and PARP inhibitors might have more powerful antitumor effects than PARP inhibitors alone in pancreatic cancer patients.

Inhibition of DNMT-1 alleviates ferroptosis through NCOA4 mediated ferritinophagy during diabetes myocardial ischemia/reperfusion injury

The purpose of this study was to investigate whether inhibition of DNA (cytosine-5)-methyltransferase 1 (DNMT-1) alleviated ferroptosis through nuclear receptor coactivator 4 (NCOA4)-mediated ferritinophagy during diabetes myocardial (DM) ischemia/reperfusion (I/R) injury (IRI). Rat DM + sham (DS), I/R, and DM + I/R (DIR), H9c2 cell high glucose (HG), hypoxia reoxygenation (H/R), and high-glucose hypoxia reoxygenation (HH/R) models were established. DNMT-1 inhibitor 5-Aza-2’-deoxycytidine (5-aza-CdR) was administered to rat and cell models. The protein level of DNMT-1, NCOA4, FTH, GPX4, Beclin-1, and P62 was detected by western blotting. Compared with normal sham (NS) group, myocardial tissue was injured in DS and I/R models. The level of DNMT-1, NCOA4, and ferroptosis was increased. Moreover, the cell injury was more serious in rat DIR or HH/R model. 5-Aza-CdR could reduce NCOA4-mediated ferritinophagy and myocardial injury in DIR and HH/R models. Moreover, the siRNA for NCOA4 could also reduce the level of ferritinophagy and cell injury in HH/R model. 5-Aza-CdR enhanced the protective effect for NCOA4-siRNA in the process of cell injury. Inhibition of DNMT-1 could reduce ferroptosis during DIR, which the NCOA4-mediated ferritinophagy might be regulated.

Protective effect of plastrum testudinis extract on dopaminergic neurons in a Parkinson's disease model through DNMT1 nuclear translocation and SNCA's methylation.

The pathological characteristics of Parkinson's disease (PD) include dopaminergic neuron damage, specifically disorders caused by dopamine synthesis, in vivo. Plastrum testudinis extract (PTE) and its bioactive ingredient ethyl stearate (PubChem CID: 8122) were reported to be correlated with tyrosine hydroxylase (TH), which is a biomarker of dopaminergic neurons. This suggests that PTE and its small-molecule active ingredient ethyl stearate have potential for development as a therapeutic drug for PD. In this study, we treated 6-hydroxydopamine (6-OHDA)-induced model rats and PC12 cells with PTE. The mechanism of action of PTE and ethyl stearate was investigated by western blotting, bisulfite sequencing PCR (BSP), real-time PCR, immunofluorescence and siRNA transfection. PTE effectively upregulated the TH expression and downregulated the alpha-synuclein expression in both the substantia nigra and the striatum of the midbrain in a PD model rat. The PC12 cell model showed that both PTE and its active monomer ethyl stearate significantly promoted TH expression and blocked alpha-synuclein, agreeing with the in vivo results. BSP showed that PTE and ethyl stearate increased the methylation level of the Snca intron 1 region. These findings suggest that some of the protective effects of PTE on dopaminergic neurons are mediated by ethyl stearate. The mechanism of ethyl stearate may involve disrupting the abnormal aggregation of DNA (cytosine-5)-methyltransferase 1 (DNMT1) with alpha-synuclein by releasing DNMT1, upregulating Snca intron 1 CpG island methylation, and ultimately, reducing the expression of alpha-synuclein.

Detection of the level of DNMT1 based on self-assembled probe signal amplification technique in plasma

DNA (cytosine-5)-methyltransferase1 (DNMT1) is the most abundant DNA methyltransferase in somatic cells, and it plays an important role in the initiation, occurrence, and rehabilitation of tumors. Herein, we developed a novel strategy for the detection of the level of DNMT1 in human plasma using the self-assembled nucleic acid probe signal amplification technology. In this method, the DNMT1 monoclonal antibody (McAbDNMT1) was immobilized on carboxyl magnetic beads to form immunomagnetic beads and then captured DNMT1 specifically. After that, DNMT1 polyclonal antibody (PcAbDNMT1) and biotinylated sheep anti-rabbit IgG (sheep anti rabbit IgG-Biotin) were sequentially added into the system to react with DNMT1 and form biotinylated double antibody sandwich immunomagnetic beads. In the presence of the bridging medium streptavidin, the biotinylated double antibody sandwich immunomagnetic beads would form a complex with biotinylated poly-fluorescein (Biotin-poly FAM), and the fluorescence intensity of the complex was proportional to the concentration of DNMT1. Immunomagnetic beads can capture the target DNMT1 in the sample, and Biotin-poly FAM can realize signal amplification. Using these strategies, we got a linear range of the system for DNMT1 level detection was from 2 nmol/L to 200 nmol/L, and the limit of detection (LOD) was 0.05 nmol/L. The method was successfully applied for the determination of DNMT1 in human plasma with the recovery of 101.3–106.0%. Therefore, this method has the potential for the detection of DNMT1 level in clinical diagnosis.

DNA Methyltransferase 1 (DNMT1) Shapes Neuronal Activity of Human iPSC-Derived Glutamatergic Cortical Neurons.

Epigenetic mechanisms are emerging key players for the regulation of brain function, synaptic activity, and the formation of neuronal engrams in health and disease. As one important epigenetic mechanism of transcriptional control, DNA methylation was reported to distinctively modulate synaptic activity in excitatory and inhibitory cortical neurons in mice. Since DNA methylation signatures are responsive to neuronal activity, DNA methylation seems to contribute to the neuron’s capacity to adapt to and integrate changing activity patterns, being crucial for the plasticity and functionality of neuronal circuits. Since most studies addressing the role of DNA methylation in the regulation of synaptic function were conducted in mice or murine neurons, we here asked whether this functional implication applies to human neurons as well. To this end, we performed calcium imaging in human induced pluripotent stem cell (iPSC)-derived excitatory cortical neurons forming synaptic contacts and neuronal networks in vitro. Treatment with DNMT1 siRNA that diminishs the expression of the DNA (cytosine-5)-methyltransferase 1 (DNMT1) was conducted to investigate the functional relevance of DNMT1 as one of the main enzymes executing DNA methylations in the context of neuronal activity modulation. We observed a lowered proportion of actively firing neurons upon DNMT1-knockdown in these iPSC-derived excitatory neurons, pointing to a correlation of DNMT1-activity and synaptic transmission. Thus, our experiments suggest that DNMT1 decreases synaptic activity of human glutamatergic neurons and underline the relevance of epigenetic regulation of synaptic function also in human excitatory neurons.

Bioinformatics analysis of DNMT1 expression and its role in head and neck squamous cell carcinoma prognosis

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common type of malignancy in the world. DNA cytosine-5-methyltransferase 1 (DNMT1) play key roles in carcinogenesis and regulation of the immune micro-environment, but the gene expression and the role of DNMT1 in HNSCC is unknown. In this study, we utilized online tools and databases for pan-cancer and HNSCC analysis of DNMT1 expression and its association with clinical cancer characteristics. We also identified genes that positively and negatively correlated with DNMT1 expression and identified eight hub genes based on protein–protein interaction (PPI) network analysis. Enrichment analyses were performed to explore the biological functions related with of DNMT1. The Tumor Immune Estimation Resource (TIMER) database was performed to explore the relationship between DNMT1 expression and immune-cell infiltration. We demonstrated that DNMT1 gene expression was upregulated in HNSCC and associated with poor prognosis. Based on analysis of the eight hub genes, we determined that DNMT1 may be involved in cell cycle, proliferation and metabolic related pathways. We also found that significant difference of B cells infiltration based on TP 53 mutation. These findings suggest that DNMT1 related epigenetic alterations have close relationship with HNSCC progression, and DNMT1 could be a novel diagnostic biomarker and a promising therapeutic target for HNSCC.

Notch3 and its CADASIL mutants differentially regulate cellular phenotypes.

Notch3 is a member of the Notch family and its mutations are known to cause a hereditary human disorder called cerebral autosomal-dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL). However, the specific function and signaling cascade initiated by CADASIL mutants remain unknown. To gain further insight into mechanism of action of CADASIL mutants, the present study conducted several experiments on the effects of Notch3 mutants in multiple cell lines. The protein levels of Notch3, fibronectin, collagen, inducible nitric oxide synthase and DNA (cytosine-5)-methyltransferase 1 (DNMT1) were determined by western blotting. The mRNA levels of IL-1β and TNF-α were measured by reverse transcription semi-quantitative PCR and DNMT1 mRNA levels were determined by quantitative PCR. Trypan blue staining was used for proliferation analysis and wound healing assays were performed to determine cell migration capability. The present study reported that R90C and R169C Notch3 mutants, and wild-type Notch3 had different effects on several cell lines. In T/GHA-VSMC cells, following the transfection of the two mutants, collagen and fibronectin expression increased, whereas expression decreased in IMR-90 cells. In BV2 cells, the two mutants resulted in decreased nitric oxide and iNOS production. In HeLa cells, proliferation and migration increased significantly following the transfection of the two mutants, whereas in the MCF-7 and HCC1937 cell lines, cell proliferation and migration decreased. In addition, the two mutants suppressed the expression of DNMT1 in HeLa and IMR-90 cells. Overall, the present study provided novel insights that further explored the underlying mechanisms of CADASIL.

Characterization of Recombinant Maintenance DNA Methyltransferase (Dnmt1) and Application to DNA Methylation Analysis

A change doesn't occur to base sequence of DNA in the process of the human development. However, one fertilized egg differentiates into cells with sixty trillion of abundant individuality. The reason is DNA is methylated. The DNA methylated correctly is necessary for correct differentiation. Aberrant methylation induce various diseases including cancer, mental disorder and lifestyle disease.Elucidating essential of diseases needs analyzing methylation of DNA.Various DNA methylation analysis methods have been developed, among which bisulfite sequencing (BS-seq) is common.BS-seq has problems such as low sensitivity, which can not analyze cell types with insufficient sample amount, and may miss specific methylation of individual cells because it is an averaged data of cell population.Conventional BS-seq, hydroxymethylation (hmC), which is an intermediate of demethylation, is detected as methylation (mC). Since hmC is not recognized as mC in vivo, conventional BS-seq can not perform accurate DNA methylation analysis.New analytical methods are needed to assess pure DNA methylation even from small numbers of cells, such as single cells. In this study, we aim to develop a novel methylation analysis method that enables accurate methylation to be performed even with a small amount of DNA by repeating amplification and methylation of DNA using PCR and DNA (cytosine-5)-methyltransferase 1 (DNMT1).