Binding Of DNA Methyltransferase Research Articles

Efficient Chemical Synthesis of Multi-Monoubiquitylated and Diubiquitylated Histones by the α-Halogen Ketone-Mediated Strategy.

The chemical synthesis of homogeneously ubiquitylated histones is a powerful approach to decipher histone ubiquitylation-dependent epigenetic regulation. Among the various methods, α-halogen ketone-mediated conjugation chemistry has recently been an attractive strategy to generate single-monoubiquitylated histones for biochemical and structural studies. Herein, we report the use of this strategy to prepare not only dual- and even triple-monoubiquitylated histones but also diubiquitin-modified histones. We were surprised to find that the synthetic efficiencies of multi-monoubiquitylated histones were comparable to those of single-monoubiquitylated ones, suggesting that this strategy is highly tolerant to the number of ubiquitin monomers installed onto histones. The facile generation of a series of single-, dual-, and triple-monoubiquitylated H3 proteins enabled us to evaluate the influence of ubiquitylation patterns on the binding of DNA methyltransferase 1 (DNMT1) to nucleosomes. Our study highlights the potential of site-specific conjugation chemistry to generate chemically defined histones for epigenetic studies.

Stroke Causes DNA Methylation at Ncx1 Heart Promoter in the Brain Via DNMT1/MeCP2/REST Epigenetic Complex.

REST (Repressor-Element 1 [RE1]-silencing transcription factor) inhibits Na+/Ca2+exchanger-1 (Ncx1) transcription in neurons through the binding of RE1 site on brain promoter (Br) after stroke. We identified a new putative RE1 site in Ncx1 heart promoter (Ht) sequence (Ht-RE1) that participates in neuronal Ncx1 transcription. Because REST recruits DNA-methyltransferase-1 (DNMT1) and MeCP2 (methyl-CpG binding protein 2) on different neuronal genes, we investigated the role of this complex in Ncx1 transcriptional regulation after stroke. Luciferase experiments performed in SH-SY5Y cells demonstrated that Br activity was selectively decreased by REST, whereas Ht activity was reduced by DNMT1, MeCP2, and REST. Notably, site-direct mutagenesis of Ht-RE1 prevented REST-dependent downregulation of Ncx1. Furthermore, in temporoparietal cortex of 8-week-old male wild-type mice (C57BL/6) subjected to transient middle cerebral artery occlusion, DNMT1, MeCP2, and REST binding to Ht promoter was increased, with a consequent DNA promoter hypermethylation. Intracerebroventricular injection of siREST prevented DNMT1/MeCP2 binding to Ht and Ncx1 downregulation, thus causing a reduction in stroke-induced damage. Consistently, in cortical neurons subjected to oxygen and glucose deprivation plus reoxygenation Ncx1 knockdown counteracted neuronal protection induced by the demethylating agent 5-azacytidine. For comparisons between 2 experimental groups, Student's ttest was used, whereas for more than 2 experimental groups, 1-way ANOVA was used, followed by Tukey or Newman Keuls. Statistical significance was set at P<0.05. If the results of this study are confirmed in humans, it could be asserted that DNMT1/MeCP2/REST complex disruption could be a new pharmacological strategy to reduce DNA methylation of Ht in the brain, ameliorating stroke damage.

DNMT1 mediates proliferation, migration and invasion of extravillous trophoblasts by regulating the methylation level of APLNR

IntroductionProliferation, migration and invasion of extravillous trophoblasts (EVTs) play an important role in the progression of preeclampsia (PE). The purpose of this study was to investigate the molecular mechanism by which DNA methylase regulates the transcription level of APLNR and affects the phenotypic function of EVTs. Materials and methodsPE mice model and H/R model in HTR8/Svneo cells were constructed. Clinical samples of normal pregnant women and PE patients were collected. Expression and methylation level of APLNR in vivo and in vitro were detected. ChIP-qPCR was used to detect the binding of DNA methyltransferase at the APLNR promoter. The expression of DNA methyltransferase 1 (DNMT1), NO and eNOS in vitro were detected. EVTs proliferation, migration and invasion in vitro were detected. ResultsIn placental tissues or HTR8/Svneo cells of the PE model group, the expression of APLNR was reduced and APLNR methylation level was up-regulated. There was no significant difference in the APLNR expression in placental tissues between normal pregnant women and PE patients. H/R conditions only promote the binding of DNMT1 at the APLNR promoter. DNMT1 interference decreased the enrichment degree of DNMT1 in APLNR promoter region and up-regulated the mRNA and protein levels of APLNR in vivo and in vitro. The activation of APLNR by Elabela (ELA) can promote eNOS transcription, thereby promoting cell proliferation and NO level, while eNOS inhibitor can reverse this effect. DNMT1 down-regulation inhibted APLNR methylation level, promoted eNOS transcription, and promoted EVTs proliferation, migration and invasion, which could be revised by the interference of APLNR. DiscussionDNMT1 promotes eNOS transcription by inhibting APLNR methylation level, and promotes EVTs proliferation, migration and invasion, thus providing a new and broad application prospect for PE treatment.

Metformin regulates the effects of IR and IGF-1R methylation on mast cell activation and airway reactivity in diabetic rats with asthma through miR-152-3p/DNMT1 axis.

Metformin is a drug for treating type 2 diabetes mellitus (T2DM). Recently, metformin has been shown to reduce the risks of asthma-associated outcomes and asthma deterioration, thereby holding promise as a superior medicine for diabetic patients with asthma. However, the mechanism by which metformin reduces diabetic asthma is yet to be clarified. This study aimed at ascertaining the downstream molecules underlying the effect of metformin on the activation of mast cells (MCs) and airway reactivity in a concomitant diabetic and asthmatic rat model. A T2DM model was induced utilizing ahigh-fat diet and streptozotocin. Then, 10% ovalbumin was utilized to stimulate asthma-like pathology in the T2DM rats. RBL-2H3 cells were induced by anti-dinitrophenyl-specific immunoglobulin E for constructing an in vitro model. Luciferase assay and RNA immunoprecipitation (IP) assay were conducted to identify the interaction between microRNA-152-3p (miR-152-3p) and DNA methyltransferase 1 (DNMT1), while chromatin IP to identify the binding of DNMT1 to insulin receptor (IR) and insulin-like growth factor 1 receptor (IGF-1R) promoters. The effects of metformin on both pathological changes in vivo and biological behaviors of cells were evaluated. Using gain- and loss-of-function approaches, we assessed the role of the two interactions in the metformin-induced effect. It was suggested that metformin could impede the MC activation and airway resistance in the concomitant diabetic and asthmatic rats. Additionally, metformin downregulated IR and IGF-1R through DNMT1-dependent methylation to repress MC activation and airway resistance. DNMT1 was testified to be a target gene of miR-152-3p. Furthermore, miR-152-3p-induced silencing of DNMT1 was blocked by metformin, hence restraining MC activation and airway resistance. The findings cumulatively demonstrate that metformin downregulates IR/IGF-1R to block MC activation and airway resistance via impairing the binding affinity between miR-152-3p and DNMT1.

TNF-α/NF-κB signaling epigenetically represses PSD4 transcription to promote alcohol-related hepatocellular carcinoma progression.

BackgroundChronic 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.MethodsR‐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.ResultsWe 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.ConclusionsPSD4 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.

Role of DNA methylation-related chromatin remodeling in aryl hydrocarbon receptor-dependent regulation of T-2 toxin highly inducible Cytochrome P450 1A4 gene.

Mycotoxins are toxic secondary metabolites produced by food-contaminating fungi, which lead to global epigenetic changes and cause toxicity to both farm animals and humans. However, whether mycotoxins induce gene-specific epigenetic alterations associated with inducible downstream gene expression is unclear as are the underlying regulatory mechanisms. Here, we found that T-2 toxin and its deacetylated metabolites but not deoxynivalenol (DON) or other representative mycotoxins highly induced the expression of cytochrome P450 1A4 (CYP1A4) in both Leghorn male hepatoma (LMH) cells and chicken primary hepatocytes, and this effect was related to the regulation of both aryl hydrocarbon receptor (AhR) and DNA methylation. We used methylation-sensitive restriction enzyme digestion-qPCR (MSRE-qPCR) and chromatin immunoprecipitation (ChIP) assays and found that the binding of DNA methyltransferase 1 (DNMT1) and histone deacetylase 2 (HDAC2) to highly methylated CpG island 3-2 at the enhancer of CYP1A4 was accompanied by the recruitment of the repressive histone modification marker H3K27me3, inducing a silent state. In turn, T-2 toxin stimulation enriched the binding of AhR to demethylated CpG island 3-2, which facilitated p300 and H3K9ac recruitment and ultimately generated an activated chromatin structure at the enhancer by increasing the active histone modification markers, including H3K4me3, H3K27ac, and H3K14ac. Interestingly, T-2 toxin-induced AhR activation also facilitated RNA polymerase II binding to CpG island 2, which may form a transcriptionally active chromatin structure at the promoter and ultimately transactivate CYP1A4. Our findings provide novel insights into the epigenetic regulation of T-2 toxin-induced gene expression.

Prenatal Stress Leads to the Altered Maturation of Corticostriatal Synaptic Plasticity and Related Behavioral Impairments Through Epigenetic Modifications of Dopamine D2 Receptor in Mice.

Prenatal stress (PRS) had a long-term adverse effect on motor behaviors. Corticostriatal synaptic plasticity, a cellular basis for motor controlling, has been proven to participate in the pathogenesis of many behavior disorders. Based on the reports about the involvement of epigenetic DNA alterations in PRS-induced long-term effects, this research investigated the influence of PRS on the development and maturation of corticostriatal synaptic plasticity and related behaviors and explored the underlying epigenetic mechanism. Subjects were male offspring of dams that were exposed to stress three times per day from the 10th day of pregnancy until delivery. The development and maturation of plasticity at corticostriatal synapses, dopamine signaling, behavioral habituation, and DNA methylation were examined and analyzed. Control mice expressed long-term potentiation (LTP) at corticostriatal synapses during postnatal days (PD) 12-14 and produced long-term depression (LTD) during PD 20-60. However, PRS mice exhibited sustained LTP during PD 12-60. The treatment with dopamine 2 receptor (D2R) agonist quinpirole recovered striatal LTD and improved the impaired behavioral habituation in PD 45 adult PRS mice. Additionally, adult PRS mice showed reduced D2R, excess DNA methyltransferase 1 (DNMT1), increased binding of DNMT1 to D2R promoter, and hypermethylation at D2R promoter in the striatum. The DNMT1 inhibitor 5-aza-deoxycytidine restored striatal synaptic plasticity and improved behavioral habituation in adult PRS mice via D2R-mediated dopamine signaling. DNMT1-associated D2R hypermethylation is responsible for altering the maturation of plasticity at corticostriatal synapses and impairing the behavioral habituation in PRS mice.

Hypermethylation of mitochondrial DNA in vascular smooth muscle cells impairs cell contractility

Vascular smooth muscle cell (SMC) from arterial stenotic-occlusive diseases is featured with deficiency in mitochondrial respiration and loss of cell contractility. However, the regulatory mechanism of mitochondrial genes and mitochondrial energy metabolism in SMC remains elusive. Here, we described that DNA methyltransferase 1 (DNMT1) translocated to the mitochondria and catalyzed D-loop methylation of mitochondrial DNA in vascular SMCs in response to platelet-derived growth factor-BB (PDGF-BB). Mitochondrial-specific expression of DNMT1 repressed mitochondrial gene expression, caused functional damage, and reduced SMC contractility. Hypermethylation of mitochondrial D-loop regions were detected in the intima-media layer of mouse carotid arteries subjected to either cessation of blood flow or mechanical endothelial injury, and also in vessel specimens from patients with carotid occlusive diseases. Likewise, the ligated mouse arteries exhibited an enhanced mitochondrial binding of DNMT1, repressed mitochondrial gene expression, defects in mitochondrial respiration, and impaired contractility. The impaired contractility of a ligated vessel could be restored by ex vivo transplantation of DNMT1-deleted mitochondria. In summary, we discovered the function of DNMT1-mediated mitochondrial D-loop methylation in the regulation of mitochondrial gene transcription. Methylation of mitochondrial D-loop in vascular SMCs contributes to impaired mitochondrial function and loss of contractile phenotype in vascular occlusive disease.

LncRNA MIR210HG promotes proliferation and invasion of non-small cell lung cancer by upregulating methylation of CACNA2D2 promoter via binding to DNMT1.

Background: In recent years, a large number of studies have shown that differentially expressed lncRNAs are capable of promoting the occurrence and development of tumors by regulating cell proliferation and differentiation. However, the biological effects of lncRNAs in non-small cell lung cancer (NSCLC) are still needed to be further investigated.Methods: The differentially expressed lncRNAs in NSCLC tissues in the downloaded profiles from GEO database were analyzed and further verified in 100 pairs of NSCLC samples collected in our hospital. After identification of the target gene MIR210HG, the relationship between MIR210HG expression and clinical data of NSCLC patients was analyzed. Regulatory effects of MIR210HG on proliferation, migration, and invasion of NSCLC cells were detected by CCK-8, colony formation, and transwell assay, respectively. The binding condition of MIR210HG and DNA methyltransferase 1 (DNMT1) was detected by RNA binding protein immunoprecipitation. Subsequently, chromatin immunoprecipitation assay assessed the promoter binding of DNMT1 to CACNA2D2. Rescue experiments were conducted to assess whether CACNA2D2 can reverse the function of MIR210HG.Results: MIR210HG was highly expressed in NSCLC tissues not only in GSE30219 dataset but also in our collected NSCLC tissues. MIR210HG expression was correlated to tumor stage and lymph node metastasis of NSCLC patients. Besides, lower disease-free survival (DFS) and overall survival (OS) were found in NSCLC patients with high-level MIR210HG compared with those with low-level MIR210HG. Regression analysis indicated that MIR210HG was the independent risk factor for DFS and OS of NSCLC patients. In vitro experiments demonstrated that MIR210HG knockdown remarkably inhibited proliferation and migration of NSCLC cells. MIR210HG could recruit DNMT1, thereafter promoting methylation of CACNA2D2 promoter region. CACNA2D2 overexpression remarkably inhibited cell proliferation. Moreover, inhibited proliferation induced by MIR210HG knockdown was reversed by CACNA2D2 knockdown.Conclusion: MIR210HG can promote the tumorigenesis of NSCLC by inhibiting the expression of CACNA2D2. Our findings provide new therapeutic strategies for the future treatment of NSCLC.

Hypermethylation of endoplasmic reticulum disulfide oxidase 1α leads to trophoblast cell apoptosis through endoplasmic reticulum stress in preeclampsia.

Abnormal trophoblast cell apoptosis is implicated in the pathogenesis of pregnancy-related disorders including preeclampsia (PE), and endoplasmic reticulum (ER) stress has been considered as a novel pathway in the regulation of cell apoptosis. In this study, we observed that both apoptosis and ER stress are triggered in trophoblast cells under hypoxia as well as in the placenta of PE rats. Quantitative polymerase chain reaction and Western blot analysis showed that the expression of endoplasmic reticulum disulfide oxidase 1α (ERO1α) is suppressed in trophoblast cells under hypoxia due to the hypermethylation of the ERO1α promoter region, and the inhibition of ERO1α expression plays an important role in ER stress and trophoblast cell apoptosis. Furthermore, we found that DNA methyltransferase 1 (DNMT1) is a key methyltransferase for DNA methylation in the regulation of ERO1α expression, and the binding level of DNMT1 to the ERO1α promoter is markedly elevated under hypoxia although DNMT1 expression is inhibited by hypoxia, suggesting that the binding level of DNMT1 to the ERO1α promoter region rather than the DNMT1 expression level contributes to the hypermethylation of ERO1α. Taken together, these results demonstrate that the hypermethylation of ERO1α mediated by increased binding of DNMT1 to the ERO1α promoter leads to trophoblast cell apoptosis through ER stress in the placenta of PE rats, which shed insight into the etiology of PE and might present a validated therapeutic target for the treatment of PE.

Doxorubicin-induced loss of DNA topoisomerase II and DNMT1- dependent suppression of MiR-125b induces chemoresistance in ALK-positive cells.

Systemic anaplastic large-cell lymphoma (ALCL) is a childhood T cell neoplasm defined by the presence or absence of translocations that lead to the ectopic expression of anaplastic lymphoma kinase (ALK), with nucleophosmin-ALK (NPM-ALK) fusions being the most common. Polychemotherapy involving doxorubicin is the standard first-line treatment but for the 25 to 35% of patients who relapse and develop resistance the prognosis remains poor. We studied the potential role of the microRNA miR-125b in the development of resistance to doxorubicin in NPM-ALK(+) ALCL. Our results show that miR-125b expression is repressed in NPM-ALK(+) cell lines and patient samples through hypermethylation of its promoter. NPM-ALK activity, in cooperation with DNA topoisomerase II (Topo II) and DNA methyltransferase 1 (DNMT1), is responsible for miR-125b repression through DNA hypermethylation. MiR-125b repression was reversed by the inhibition of DNMTs with decitabine or the inhibition of DNA topoisomerase II with either doxorubicin or etoposide. In NPM-ALK(+) cell lines, doxorubicin treatment led to an increase in miR-125b levels by inhibiting the binding of DNMT1 to the MIR125B1 promoter and downregulating the pro-apoptotic miR-125b target BAK1. Reversal of miR-125b silencing, increased miR-125b levels and reduced BAK1 expression also led to a lower efficacy of doxorubicin, suggestive of a pharmacoresistance mechanism. In line with this, miR-125b repression and increased BAK1 expression correlated with early relapse in human NPM-ALK(+) ALCL primary biopsies. Collectively our findings suggest that miR-125b could be used to predict therapeutic outcome in NPM-ALK(+) ALCL.

Synthetic lethality and beyond.

DNA damage–induced binding of DNA methyltransferases maximizes the efficacy of poly(ADP-ribose) polymerase inhibitors.

Long non-coding RNA DBCCR1-003 regulate the expression of DBCCR1 via DNMT1 in bladder cancer.

BackgroundMany long non coding RNAs have been identified as key modulators in cancer development. A lncRNA, DBCCR1-003, derived from the locus of tumor suppressor gene DBCCR1 (deleted in bladder cancer chromosome region 1), has unknown function. In the present study, we explored function and molecular mechanism of DBCCR1-003 in bladder cancer (BC) development.MethodsWe evaluated the expression levels of DBCCR1-003 in tissues and cells with western blot and quantitative real-time polymerase chain reaction. Multiple approaches including chromatin immunoprecipitation assay and RNA immunoprecipitation were used to confirm the direct binding of DBCCR1-003 to DNMT1. The recombinant vector overexpressing DBCCR1-003 was constructed. Cell proliferation assay, colony formation assay and flow cytometric analysis were employed to measure the role of DBCCR1-003 in regulation of cell proliferation, cycle and apoptosis.ResultsFirstly we detected the expression of DBCCR1-003, DBCCR1, DNMT1 (DNA methyltransferase 1) and DNA methylation in the promoter of DBCCR1. We found low expression of DBCCR1-003, same as DBCCR1, while high expression of DNMT1 and hypermethylation of DBCCR1 gene promoter in BC tissues and T24 cells line. Further studies revealed that treatment of DNMT inhibitor, 5-aza-2-deoxycytidine(DAC), or overexpression of DBCCR1-003 led to increased DBCCR1 expression by reversion of promoter hypermethylation and DNMT1 binding to DBCCR1 promoter in T24 cells. Importantly, RNA immunoprecipitation (RIP) showed that DBCCR1-003 physically associates with DNMT1. The binding of them was increased with the inhibition of DBCCR1 promoter methylation, indicating that DBCCR1-003 may bind to DNMT1 and prevent DNMT1-mediated the methylation of DBCCR1. Furthermore, overexpression of DBCCR1-003 resulted in significant inhibition of T24 cells growth through the inducing G0/G1 arrest and apoptosis.ConclusionsTaken together, these findings demonstrated that a novel tumor suppressor DBCCR1-003 regulates the expression of DBCCR1 via binding to DNMT1 and preventing DNMT1-mediated the methylation of DBCCR1 in BC. LncRNA DBCCR1-003 may serve as a novel biomarker and therapeutic target for BC in future cancer clinic.Electronic supplementary materialThe online version of this article (doi:10.1186/s12935-016-0356-8) contains supplementary material, which is available to authorized users.

Histone Methyltransferase Enhancer of Zeste Homolog 2-Mediated ABCA1 Promoter DNA Methylation Contributes to the Progression of Atherosclerosis.

ATP-binding cassette transporter A1 (ABCA1) plays a critical role in maintaining cellular cholesterol homeostasis. The purpose of this study is to identify the molecular mechanism(s) underlying ABCA1 epigenetic modification and determine its potential impact on ABCA1 expression in macrophage-derived foam cell formation and atherosclerosis development. DNA methylation induced foam cell formation from macrophages and promoted atherosclerosis in apolipoprotein E-deficient (apoE−/−) mice. Bioinformatics analyses revealed a large CpG island (CGI) located in the promoter region of ABCA1. Histone methyltransferase enhancer of zeste homolog 2 (EZH2) downregulated ABCA1 mRNA and protein expression in THP-1 and RAW264.7 macrophage-derived foam cells. Pharmacological inhibition of DNA methyltransferase 1 (DNMT1) with 5-Aza-dC or knockdown of DNMT1 prevented the downregulation of macrophage ABCA1 expression, suggesting a role of DNA methylation in ABCA1 expression. Polycomb protein EZH2 induced DNMT1 expression and methyl-CpG-binding protein-2 (MeCP2) recruitment, and stimulated the binding of DNMT1 and MeCP2 to ABCA1 promoter, thereby promoting ABCA1 gene DNA methylation and atherosclerosis. Knockdown of DNMT1 inhibited EZH2-induced downregulation of ABCA1 in macrophages. Conversely, EZH2 overexpression stimulated DNMT1-induced ABCA1 gene promoter methylation and atherosclerosis. EZH2-induced downregulation of ABCA1 gene expression promotes foam cell formation and the development of atherosclerosis by DNA methylation of ABCA1 gene promoter.

The replication foci targeting sequence (RFTS) of DNMT1 functions as a potent histone H3 binding domain regulated by autoinhibition

DNA methyltransferase 1 (DNMT1) plays an essential role in propagation of the DNA methylation pattern to daughter cells. The replication foci targeting sequence (RFTS) of DNMT1 is required for the recruitment of DNMT1 to DNA methylation sites through direct binding to ubiquitylated histone H3 mediated by UHRF1 (Ubiquitin-like containing PHD and RING finger domains 1). Recently, it has been reported that the RFTS plugs the catalytic pocket of DNMT1 in an intermediated manner and inhibits its DNA methyltransferase activity. However, it is unclear whether this binding affects RFTS function in terms of recruitment to DNA methylation sites. Using Xenopus egg extracts, we demonstrate here that abrogation of the interaction between the RFTS and the catalytic center of DNMT1, by deletion of the C-terminal portion or disruption of the hydrogen bond, results in non-ubiquitylated histone H3 binding and abnormal accumulation of DNMT1 on the chromatin. Interestingly, DNMT1 mutants identified in patients with a neurodegenerative disease, ADCA-DN, bound to non-ubiquitylated histone H3 and accumulated on chromatin during S phase in Xenopus egg extracts. These results suggest that the interaction between the RFTS and the catalytic center of DNMT1 serves as an autoinhibitory mechanism for suppressing the histone H3 binding of DNMT1 and ensuring the accurate recruitment of DNMT1 to sites of DNA methylation. The autoinhibitory mechanism may play an important role in the regulation of gene expression in neurogenesis.

Structure and dynamics of H. pylori 98-10 C5-cytosine specific DNA methyltransferase in complex with S-adenosyl-l-methionine and DNA.

Helicobacter pylori is a Gram-negative bacterium that inhabits the human gastrointestinal tract, and some strains of this bacterium cause gastric ulcers and cancer. DNA methyltransferases (MTases) are promising drug targets for the treatment of cancer and other diseases that are also caused by epigenetic alternations of the genome. The C5-cytosine specific DNA methyltransferase from H. pylori (M. Hpy C5mC) catalyzes the transfer of the methyl group from the cofactor S-adenosyl-l-methionine (AdoMet) to the flipped cytosine of the substrate DNA. Herein we report the sequence analyses, 3-D structure modeling and molecular dynamics simulations of M. Hpy C5mC, when complexed with AdoMet as well as DNA. We analyzed the protein-DNA interactions prominently established by the flipped cytosine and the interactions between the protein and cofactor in the active site. We propose that the contacts made by cytosine O2 with Arg155 and Arg157, and the water-mediated interactions with cytosine N3 may be essential for the activity of methyl transfer as well as the deprotonation at the C5 position in our C5mC model. Specific recognition of DNA was mediated mainly by residues from Ser221-Arg229 and Ser243-Gln246 of the target recognition domain (TRD) and some residues of the loop Ser75-Lys83 from the large domain. These findings are further supported by alanine scanning mutagenesis studies. The results reported here explain the sequence, structure and binding features necessary for the recognition between the cofactor and the substrate by the key epigenetic enzyme, M. Hpy C5mC.

Behavioral and molecular neuroepigenetic alterations in prenatally stressed mice: relevance for the study of chromatin remodeling properties of antipsychotic drugs.

We have recently reported that mice born from dams stressed during pregnancy (PRS mice), in adulthood, have behavioral deficits reminiscent of behaviors observed in schizophrenia (SZ) and bipolar (BP) disorder patients. Furthermore, we have shown that the frontal cortex (FC) and hippocampus of adult PRS mice, like that of postmortem chronic SZ patients, are characterized by increases in DNA-methyltransferase 1 (DNMT1), ten-eleven methylcytosine dioxygenase 1 (TET1) and exhibit an enrichment of 5-methylcytosine (5MC) and 5-hydroxymethylcytosine (5HMC) at neocortical GABAergic and glutamatergic gene promoters. Here, we show that the behavioral deficits and the increased 5MC and 5HMC at glutamic acid decarboxylase 67 (Gad1), reelin (Reln) and brain-derived neurotrophic factor (Bdnf) promoters and the reduced expression of the messenger RNAs (mRNAs) and proteins corresponding to these genes in FC of adult PRS mice is reversed by treatment with clozapine (5 mg kg−1 twice a day for 5 days) but not by haloperidol (1 mg kg−1 twice a day for 5 days). Interestingly, clozapine had no effect on either the behavior, promoter methylation or the expression of these mRNAs and proteins when administered to offspring of nonstressed pregnant mice. Clozapine, but not haloperidol, reduced the elevated levels of DNMT1 and TET1, as well as the elevated levels of DNMT1 binding to Gad1, Reln and Bdnf promoters in PRS mice suggesting that clozapine, unlike haloperidol, may limit DNA methylation by interfering with DNA methylation dynamics. We conclude that the PRS mouse model may be useful preclinically in screening for the potential efficacy of antipsychotic drugs acting on altered epigenetic mechanisms. Furthermore, PRS mice may be invaluable for understanding the etiopathogenesis of SZ and BP disorder and for predicting treatment responses at early stages of the illness allowing for early detection and remedial intervention.

The DAXX co-repressor is directly recruited to active regulatory elements genome-wide to regulate autophagy programs in a model of human prostate cancer.

While carcinoma of the prostate is the second most common cause of cancer death in the US, current methods and markers used to predict prostate cancer (PCa) outcome are inadequate. This study was aimed at understanding the genome-wide binding and regulatory role of the DAXX transcriptional repressor, recently implicated in PCa. ChIP-Seq analysis of genome-wide distribution of DAXX in PC3 cells revealed over 59,000 DAXX binding sites, found at regulatory enhancers and promoters. ChIP-Seq analysis of DNA methyltransferase 1 (DNMT1), which is a key epigenetic partner for DAXX repression, revealed that DNMT1 binding was restricted to a small number of DAXX sites. DNMT1 and DAXX bound close to transcriptional activator motifs. DNMT1 sites were found to be dependent on DAXX for recruitment by analyzing DNMT1 ChIP-Seq following DAXX knockdown (K/D), corroborating previous findings that DAXX recruits DNMT1 to repress its target genes. Massively parallel RNA sequencing (RNA-Seq) was used to compare the transcriptomes of WT and DAXX K/D PC3 cells. Genes induced by DAXX K/D included those involved in autophagy, and DAXX ChIP-Seq peaks were found close to the transcription start sites (TSS) of autophagy genes, implying they are more likely to be regulated by DAXX. In conclusion, DAXX binds active regulatory elements and co-localizes with DNMT1 in the prostate cancer genome. Given DAXX's putative regulatory role in autophagy, future studies may consider DAXX as a candidate marker and therapeutic target for prostate cancer.

Effects of Flaxseed‐supplementation on DNA Methyltransferase and Obesity‐associated Gene Expression in Adipose Tissue from Obese Mice

Diet‐induced obesity (DIO) has been shown to increase DNA methyltransferase (DNMT) expression and DNMT binding at obesity‐associated genes. Natural compounds, such as flaxseed, have the potential to reverse obesity‐associated gene expression via the regulation of DNA methylation. The objective of this study was to determine the effect of the bioactive compounds of flaxseed on DNMT and obesity‐associated gene expression. Sixty C57BL/6J male mice were randomly assigned into one of the following diet groups and fed for eight weeks: 1) 45% Kcal fat; 2) 45% Kcal fat, 10% whole flaxseed; 3) 45% Kcal fat, 6% defatted flaxseed; 4) 45% Kcal fat, 4% flaxseed oil; and 5) 16% Kcal fat. DNMT1, DNMT3a, DNMT3b, leptin, and peroxisome proliferator‐activated receptor (PPAR)‐α expression levels in mouse adipose tissues were determined by real‐time PCR. DNMT1, DNMT3a, or leptin expression was significantly decreased in whole flaxseed, defatted flaxseed, flaxseed oil, or low fat diet groups compared to the high fat diet group. DNMT1 or DNMT3a expression was positively correlated with PPAR‐α and leptin expression in the defatted flaxseed, flaxseed oil, or low fat diet groups. The results indicate that bioactive compounds of flaxseed affect selected gene expression; however, further research is needed to identify the specific mechanisms regulating leptin or PPAR‐α expression during DIO development.

Effects of Melatonin on Prenatal Dexamethasone-Induced Epigenetic Alterations in Hippocampal Morphology and Reelin and Glutamic Acid Decarboxylase 67 Levels

Prenatal glucocorticoid exposure causes brain damage in adult offspring; however, the underlying mechanisms remain unclear. Melatonin has been shown to have beneficial effects in compromised pregnancies. Pregnant Sprague-Dawley rats were administered vehicle (VEH) or dexamethasone between gestation days 14 and 21. The programming effects of prenatal dexamethasone exposure on the brain were assessed at postnatal days (PND) 7, 42, and ∼120. Melatonin was administered from PND21 to the rats exposed to dexamethasone, and the outcome was assessed at ∼PND120. In total, there were four groups: VEH, vehicle plus melatonin (VEHM), prenatal dexamethasone-exposure (DEX), and prenatal dexamethasone exposure plus melatonin (DEXM). Spatial memory, gross hippocampal morphology, and hippocampal biochemistry were examined. Spatial memory assessed by the Morris water maze showed no significant differences among the four groups. Brain magnetic resonance imaging showed that all rats with prenatal dexamethasone exposure (DEX + DEXM) exhibited increased T2-weighted signals in the hippocampus. There were no significant differences in the levels of mRNA expression of hippocampal reln, which encodes reelin, and GAD1, which encodes glutamic acid decarboxylase 67, at PND7. At both PND42 and ∼PND120, reln and GAD1 mRNA expression levels were decreased. At ∼PND120, melatonin restored the reduced levels of hippocampal reln and GAD1 mRNA expression in the DEXM group. In addition, melatonin restored the reln mRNA expression levels by (1) reducing DNA methyltransferase 1 (DNMT1) mRNA expression and (2) reducing the binding of DNMT1 and the methyl-CpG binding protein 2 (MeCP2) to the reln promoter. The present study showed that prenatal dexamethasone exposure induced gross alterations in hippocampal morphology and reduced the levels of hippocampal mRNA expression of reln and GAD1. Spatial memory was unimpaired. Thus, melatonin had a beneficial effect in restoring hippocampal reln mRNA expression by reducing DNMT1 and MeCP2 binding to the reln promoter.