GuidingNet: revealing transcriptional cofactor and predicting binding for DNA methyltransferase by network regularization.

The DNA methyltransferases (DNMTs) (DNMT3A, DNMT3B and DNMT3L) are primarily responsible for the establishment of genomic locus-specific DNA methylation patterns, which play an important role in gene regulation and animal development. However, this important protein family's binding mechanism, i.e. how and where the DNMTs bind to genome, is still missing in most tissues and cell lines. This motivates us to explore DNMTs and TF's cooperation and develop a network regularized logistic regression model, GuidingNet, to predict DNMTs' genome-wide binding by integrating gene expression, chromatin accessibility, sequence and protein-protein interaction data. GuidingNet accurately predicted methylation experimental data validated DNMTs' binding, outperformed single data source based and sparsity regularized methods and performed well in within and across tissue prediction for several DNMTs in human and mouse. Importantly, GuidingNet can reveal transcription cofactors assisting DNMTs for methylation establishment. This provides biological understanding in the DNMTs' binding specificity in different tissues and demonstrate the advantage of network regularization. In addition to DNMTs, GuidingNet achieves good performance for other chromatin regulators' binding. GuidingNet is freely available at https://github.com/AMSSwanglab/GuidingNet.

Deconvolution of multiplexed transcriptional responses to wood smoke particles defines rapid aryl hydrocarbon receptor signaling dynamics.

The heterogeneity of respirable particulates and compounds complicates our understanding of transcriptional responses to air pollution. Here, we address this by applying precision nuclear run-on sequencing and the assay for transposase-accessible chromatin sequencing to measure nascent transcription and chromatin accessibility in airway epithelial cells after wood smoke particle (WSP) exposure. We used transcription factor enrichment analysis to identify temporally distinct roles for ternary response factor-serum response factor complexes, the aryl hydrocarbon receptor (AHR), and NFκB in regulating transcriptional changes induced by WSP. Transcription of canonical targets of the AHR, such as CYP1A1 and AHRR, was robustly increased after just 30 min of WSP exposure, and we discovered novel AHR-regulated pathways and targets including the DNA methyltransferase, DNMT3L. Transcription of these genes and associated enhancers rapidly returned to near baseline by 120 min after exposure. The kinetics of AHR- and NFκB-regulated responses to WSP were distinguishable based on the timing of both transcriptional responses and chromatin remodeling, with induction of several cytokines implicated in maintaining NFκB-mediated responses through 120 min of exposure. In aggregate, our data establish a direct and primary role for AHR in mediating airway epithelial responses to WSP and identify crosstalk between AHR and NFκB signaling in controlling proinflammatory gene expression. This work also defines an integrated genomics-based strategy for deconvoluting multiplexed transcriptional responses to heterogeneous environmental exposures.

Methylation-dependent transcriptional regulation of crescentin gene (creS) by GcrA in Caulobacter crescentus.

In Caulobacter crescentus the combined action of chromosome replication and the expression of DNA methyl-transferase CcrM at the end of S-phase maintains a cyclic alternation between a full- to hemi-methylated chromosome. This transition of the chromosomal methylation pattern affects the DNA-binding properties of the transcription factor GcrA that controls the several key cell cycle functions. However, the molecular mechanism by which GcrA and methylation are linked to transcription is not fully elucidated yet. Using a combination of cell biology, genetics, and in vitro analysis, we deciphered how GcrA integrates the methylation pattern of several S-phase expressed genes to their transcriptional output. We demonstrated in vitro that transcription of ctrA from the P1 promoter in its hemi-methylated state is activated by GcrA, while in its fully methylated state GcrA had no effect. Further, GcrA and methylation together influence a peculiar distribution of creS transcripts, encoding for crescentin, the protein responsible for the characteristic shape of Caulobacter cells. This gene is duplicated at the onset of chromosome replication and the two hemi-methylated copies are spatially segregated. Our results indicated that GcrA transcribed only the copy where coding strand is methylated. In vitro transcription assay further substantiated this finding. As several of the cell cycle-regulated genes are also under the influence of methylation and GcrA-dependent transcriptional regulation, this could be a mechanism responsible for maintaining the gene transcription dosage during the S-phase.

Down-regulation of lncRNA NEAT1 regulated by miR-194-5p/DNMT3A facilitates acute myeloid leukemia.

OBJECTIVE: miR-194-5p and NEAT1 have been reported to be associated with multiple malignancies, but their roles in acute myeloid leukemia (AML) remains not fully understood. METHODS: Bone marrow samples were collected for monocyte separation. qRT-PCR assay was performed to investigate the expression patterns of NEAT1 and miR-194-5p in AML. CCK-8, soft agar colony formation, flow cytometry and transwell assays were employed to explore the biological functions of NEAT1 or miR-194-5p. Methylation PCR was performed to monitor the methylation of NEAT1. Luciferase reporter assay was subjected to verify the relationship between miR-194-5p and DNMT3A. Immunofluorescence and western blotting were performed to detect the alterations of protein expression. RESULTS: NEAT1 and miR-194-5p were both down-regulated in AML. Overexpression of either NEAT1 or miR-194-5p repressed proliferation, induced apoptosis and restrained migration and invasion of AML cells. There was a negative correlation between NEAT1 and DNMT3A in AML. Knockdown of DNMT3A dramatically decreased the methylation of NEAT1. Moreover, DNMT3A was identified as a downstream target of miR-194-5p. Furthermore, down-regulation of DNMT3A rescued the impacts on the malignant phenotypes of NEAT1 inhibition by miR-194-5p inhibitor. CONCLUSION: Altogether, down-regulation of NEAT1 mediated by miR-194-5p/DNMT3A axis promotes AML progression, which might provide therapeutic targets in AML treatment.

The loss of global DNA methylation due to decreased DNMT expression in the postnatal mouse ovaries may associate with infertility emerging during ovarian aging.

Ovarian aging is one of the main causes of female infertility, and its molecular background is still largely unknown. As DNA methylation regulates many oogenesis/folliculogenesis-related genes, the expression levels and cellular localizations of DNA methyltransferases (DNMTs) playing key roles in this process is important in the ovaries from early to aged terms. In the present study, we aimed to evaluate the spatial and temporal expression of the Dnmt1, Dnmt3a, Dnmt3b, and Dnmt3l genes as well as global DNA methylation levels in the mouse ovaries during aging. For this purpose, the following groups were created: young (1- and 2-week old; n = 3 from each week), prepubertal (3- and 4-week-old; n = 3 from each week), pubertal (5- and 6-week-old; n = 3 from each week), postpubertal (16- and 18-week-old; n = 3 from each week), and aged (52-, 60- and 72-week-old; n = 3 from each week). We found here that Dnmt1, Dnmt3a, and Dnmt3l genes' expression at mRNA and protein levels as well as global DNA methylation profiles were gradually and significantly decreased in the postnatal ovaries from young to aged groups (P < 0.05). In contrast, there was a remarkable increase of Dnmt3b expression in the pubertal, postpubertal and aged groups (P < 0.05). Our findings suggest that the significantly altered DNMT expression and global DNA methylation levels during ovarian aging may contribute to female infertility development at the later terms of lifespan. Also, new researches are required to determine the molecular biological mechanism(s) that how altered DNMT expression and decreased DNA methylation lead to ovarian aging.

Dnmt3a2 in the Nucleus Accumbens Shell Is Required for Reinstatement of Cocaine Seeking.

Epigenetic mechanisms have gained increasing attention as regulators of synaptic plasticity and responsiveness to drugs of abuse. In particular, it has been shown that the activity of the DNA methyltransferase 3a (Dnmt3a) mediates certain long-lasting effects of cocaine. Here we examined the role of the Dnmt isoforms, Dnmt3a1 and Dnmt3a2, within the nucleus accumbens (NAc) on transcriptional activity of immediate early genes (IEGs) and acute and long-lasting responsiveness to cocaine and cocaine conditioned cues. Using primary striatal cultures, we show that transcription of Dnmt3a2, but not that of Dnmt3a1, is activated by dopamine D1 receptor signaling and that knockdown of Dnmt3a2 using viral vector-mediated expression of Dnmt3a2-specific shRNAs impairs induction of the IEGs, Arc, FosB, and Egr2 Acute cocaine administration increases expression of Dnmt3a2 but not that of Dnmt3a1 in the NAc shell. In contrast, in the NAc core, expression of Dnmt3a1 and Dnmt3a2 was unaffected by cocaine administration. shRNA-mediated knockdown of Dnmt3a2 in vivo impairs the induction of IEGs, including Egr2 and FosB indicating that Dnmt3a2 regulates cocaine-dependent expression of plasticity genes in the rat NAc shell. Cocaine self-administration experiments in rats revealed that Dnmt3a2 regulates drug cue memories that drive reinstatement of cocaine seeking as well as incubation of this phenomenon within the NAc shell. Dnmt3a2 does not influence the primary reinforcing effects of cocaine. Thus, Dnmt3a2 mediates long-lasting cocaine cue memories within the NAc shell. Targeting Dnmt3a2 expression or function may interfere with cocaine craving and relapse.SIGNIFICANCE STATEMENT In humans, drug craving can occur in response to conditioned cues, even after extended periods of abstinence. In rats, cue-induced cocaine seeking has been shown to increase progressively during the first 2 months of abstinence from drug self-administration. This phenomenon, referred to as incubation of cocaine seeking, is consistent with the hypothesis that in humans craving increases over time and remains high following prolonged abstinence. Those long-lasting behavioral changes are likely to be mediated by epigenetic effects and neuroplastic changes within the mesolimbic brain reward system. Here we show that a specific isoform of DNA-methyltransferases in the NAc shell regulates drug cue memories that drive reinstatement of cocaine seeking after both early abstinence and incubation of cocaine craving.

DNMT1 mutations found in HSANIE patients affect interaction with UHRF1 and neuronal differentiation.

DNMT1 is recruited to substrate sites by PCNA and UHRF1 to maintain DNA methylation after replication. The cell cycle dependent recruitment of DNMT1 is mediated by the PCNA-binding domain (PBD) and the targeting sequence (TS) within the N-terminal regulatory domain. The TS domain was found to be mutated in patients suffering from hereditary sensory and autonomic neuropathies with dementia and hearing loss (HSANIE) and autosomal dominant cerebellar ataxia deafness and narcolepsy (ADCA-DN) and is associated with global hypomethylation and site specific hypermethylation. With functional complementation assays in mouse embryonic stem cells, we showed that DNMT1 mutations P496Y and Y500C identified in HSANIE patients not only impair DNMT1 heterochromatin association, but also UHRF1 interaction resulting in hypomethylation. Similar DNA methylation defects were observed when DNMT1 interacting domains in UHRF1, the UBL and the SRA domain, were deleted. With cell-based assays, we could show that HSANIE associated mutations perturb DNMT1 heterochromatin association and catalytic complex formation at methylation sites and decrease protein stability in late S and G2 phase. To investigate the neuronal phenotype of HSANIE mutations, we performed DNMT1 rescue assays and could show that cells expressing mutated DNMT1 were prone to apoptosis and failed to differentiate into neuronal lineage. Our results provide insights into the molecular basis of DNMT1 dysfunction in HSANIE patients and emphasize the importance of the TS domain in the regulation of DNA methylation in pluripotent and differentiating cells.

MIR29B mediates epigenetic mechanisms of HBG gene activation.

Sickle cell disease (SCD) affects over 2 million people worldwide with high morbidity and mortality in underdeveloped countries. Therapeutic interventions aimed at reactivating fetal haemoglobin (HbF) is an effective approach for improving survival and ameliorating the clinical severity of SCD. A class of agents that inhibit DNA methyltransferase (DNMT) activity show promise as HbF inducers because off-target effects are not observed at low concentrations. However, these compounds are rapidly degraded by cytidine deaminase when taken by oral administration, creating a critical barrier to clinical development for SCD. We previously demonstrated that microRNA29B (MIR29B) inhibits de novo DNMT synthesis, therefore, the goal of our study was to determine if MIR29 mediates HbF induction. Overexpression of MIR29B in human KU812 cells and primary erythroid progenitors significantly increased the percentage of HbF positive cells, while decreasing the expression of DNMT3A and the HBG repressor MYB. Furthermore, HBG promoter methylation levels decreased significantly following MIR29B overexpression in human erythroid progenitors. We subsequently, observed higher MIR29B expression in SCD patients with higher HbF levels compared to those with low HbF. Our findings provide evidence for the ability of MIR29B to induce HbF and supports further investigation to expand treatment options for SCD.

Ectopic expression of DNA methyltransferases DNMT3A2 and DNMT3L leads to aberrant hypermethylation and postnatal lethality in mice.

DNA methylation is generally known to inactivate gene expression. The DNA methyltransferases (DNMTs), DNMT3A and DNMT3B, catalyze somatic cell lineage-specific DNA methylation, while DNMT3A and DNMT3L catalyze germ cell lineage-specific DNA methylation. How such lineage- and gene-specific DNA methylation patterns are created remains to be elucidated. To better understand the regulatory mechanisms underlying DNA methylation, we generated transgenic mice that constitutively expressed DNMT3A and DNMT3L, and analyzed DNA methylation, gene expression, and their subsequent impact on ontogeny. All transgenic mice were born normally but died within 20 weeks accompanied with cardiac hypertrophy. Several genes were repressed in the hearts of transgenic mice compared with those in wild-type mice. CpG islands of these downregulated genes were highly methylated in the transgenic mice. This abnormal methylation occurred in the perinatal stage. Conversely, monoallelic DNA methylation at imprinted loci was faithfully maintained in all transgenic mice, except H19. Thus, the loci preferred by DNMT3A and DNMT3L differ between somatic and germ cell lineages.

Epigenetic Modification as a Regulatory Mechanism for Spatiotemporal Dynamics of ANO1 Expression in Salivary Glands.

Anoctamin1 (ANO1), a calcium activated chloride channel, is known to play a critical role in salivary secretion. In the salivary gland, ANO1 is expressed exclusively in the acinar cells, with no expression in the ductal cells. However, the mechanisms that determine this distinctive cell type-dependent expression pattern of ANO1 remain unknown. In this study, we discovered that the cell-dependent expression of ANO1 during salivary gland organogenesis is regulated by DNA methylation of ANO1 CpG islands. ANO1 CpG islands in e12 embryonic submandibular glands (eSMG) are highly methylated, but those in e14 eSMG or adult SMG are significantly unmethylated. The differential expression pattern of ANO1 in duct and acini is defined at e14. Artificial demethylation by treatment with the demethylating agent 5-aza-2'-deoxycytidine (5-Aza-CdR), induced the expression of ANO1 in both the ductal cell line Human Submandibular Gland (HSG) and in the duct cells of adult mouse SMG. During the trans-differentiation in Matrigel of duct-origin HSG cells into acinar-like phenotype, significant demethylation of ANO1 CpG islands is observed. This may be due to the reduced expression of DNA methyltransferase (DNMT) 3a and 3b. These results suggest that the differential expression of ANO1 in salivary glands during organogenesis and differentiation is mainly regulated by epigenetic demethylation of the ANO1 gene.

The role of DNA methylation in directing the functional organization of the cancer epigenome.

The holistic role of DNA methylation in the organization of the cancer epigenome is not well understood. Here we perform a comprehensive, high-resolution analysis of chromatin structure to compare the landscapes of HCT116 colon cancer cells and a DNA methylation-deficient derivative. The NOMe-seq accessibility assay unexpectedly revealed symmetrical and transcription-independent nucleosomal phasing across active, poised, and inactive genomic elements. DNA methylation abolished this phasing primarily at enhancers and CpG island (CGI) promoters, with little effect on insulators and non-CGI promoters. Abolishment of DNA methylation led to the context-specific reestablishment of the poised and active states of normal colon cells, which were marked in methylation-deficient cells by distinct H3K27 modifications and the presence of either well-phased nucleosomes or nucleosome-depleted regions, respectively. At higher-order genomic scales, we found that long, H3K9me3-marked domains had lower accessibility, consistent with a more compact chromatin structure. Taken together, our results demonstrate the nuanced and context-dependent role of DNA methylation in the functional, multiscale organization of cancer epigenomes.

Biochemical characterization of ParI, an orphan C5-DNA methyltransferase from Psychrobacter arcticus 273-4.

Cytosine-specific DNA methyltransferases are important enzymes in most living organisms. In prokaryotes, most DNA methyltransferases are members of the type II restriction-modification system where they methylate host DNA, thereby protecting it from digestion by the accompanying restriction endonucleases. DNA methyltransferases can also act as solitary enzymes having important roles in controlling gene expression, DNA replication, cell cycle and DNA post-replicative mismatch repair. They have potential applications in biotechnology, such as in labeling of biopolymers, DNA mapping or epigenetic analysis, as well as for general DNA-protein interaction studies. The parI gene from the psychrophilic bacterium Psychrobacter arcticus 273-4 encodes a cytosine-specific DNA methyltransferase. In this work, recombinant ParI was expressed and purified in fusion to either an N-terminal hexahistidine affinity tag, or a maltose binding protein following the hexahistidine affinity tag, for solubility improvement. After removal of the fusion partners, recombinant ParI was found to be monomeric by size exclusion chromatography, with its molecular mass estimated to be 54 kDa. The apparent melting temperature of the protein was 53 °C with no detectable secondary structures above 65 °C. Both recombinant and native ParI showed methyltransferase activity in vivo. In addition, MBP- and His-tagged ParI also demonstrated in vitro activity. Although the overall structure of ParI exhibits high thermal stability, the loss of in vitro activity upon removal of solubility tags or purification from the cellular milieu indicates that the catalytically active form is more labile. Horizontal gene transfer may explain the acquisition of a protein-encoding gene that does not display common cold-adapted features.

Overexpression of DNA (Cytosine-5)-Methyltransferase 1 (DNMT1) And DNA (Cytosine-5)-Methyltransferase 3A (DNMT3A) Is Associated with Aggressive Behavior and Hypermethylation of Tumor Suppressor Genes in Human Pituitary Adenomas.

BACKGROUND Alteration of DNA methylation of tumor suppressor genes (TSGs) is one of the most consistent epigenetic changes in human cancers. DNMTs play several important roles in DNA methylation and development of cancers. Regarding DNMTs protein expressions, little is known about the clinical significance and correlation with promoter methylation status of TSGs in human pituitary adenomas. MATERIAL AND METHODS We analyzed the protein expression of 3 DNMTs using immunohistochemistry and assessed DNA hypermethylation of RASSF1A, CDH13, CDH1, and CDKN2A (p16) in 63 pituitary adenomas. We examined associations between DNMTs expression and clinicopathological features or promoter methylation status of TSGs. RESULTS Overexpression of DNMTs was detected in pituitary adenomas. Frequencies of DNMT1 overexpression were significantly higher in macroadenomas, invasive tumors, and grade III and IV tumors. DNMT3A was frequently detected in invasive tumors and grade IV tumors. In addition, DNMT1 and DNMT3A were frequently detected in high-methylation tumors. Furthermore, in multivariate logistic regression, the significant association between DNMT1 or DNMT3A and high-methylation status persisted after adjusting for clinicopathological features. CONCLUSIONS Our findings suggested that tumor overexpression of DNMT1 and DNMT3A is associated with tumor aggressive behavior and high-methylation status in pituitary adenomas. Our data support a possible role of DNMT1 and DNMT3A in TSG promoter methylation leading to pituitary adenoma invasion and suggest that inhibition of DNMTs has the potential to become a new therapeutic approach for invasive pituitary adenoma.

Binding of 14-3-3 reader proteins to phosphorylated DNMT1 facilitates aberrant DNA methylation and gene expression.

Mammalian DNA (cytosine-5) methyltransferase 1 (DNMT1) is essential for maintenance methylation. Phosphorylation of Ser143 (pSer143) stabilizes DNMT1 during DNA replication. Here, we show 14-3-3 is a reader protein of DNMT1pSer143. In mammalian cells 14-3-3 colocalizes and binds DNMT1pSer143 post-DNA replication. The level of DNMT1pSer143 increased with overexpression of 14-3-3 and decreased by its depletion. Binding of 14-3-3 proteins with DNMT1pSer143 resulted in inhibition of DNA methylation activity in vitro. In addition, overexpression of 14-3-3 in NIH3T3 cells led to decrease in DNMT1 specific activity resulting in hypomethylation of the genome that was rescued by transfection of DNMT1. Genes representing cell migration, mobility, proliferation and focal adhesion pathway were hypomethylated and overexpressed. Furthermore, overexpression of 14-3-3 also resulted in enhanced cell invasion. Analysis of TCGA breast cancer patient data showed significant correlation for DNA hypomethylation and reduced patient survival with increased 14-3-3 expressions. Therefore, we suggest that 14-3-3 is a crucial reader of DNMT1pSer143 that regulates DNA methylation and altered gene expression that contributes to cell invasion.

Oregonin from Alnus incana bark affects DNA methyltransferases expression and mitochondrial DNA copies in mouse embryonic fibroblasts.

Oregonin is an open-chain diarylheptanoid isolated from Alnus incana bark that possesses remarkable antioxidant and anti-inflammatory properties, inhibits adipogenesis, and can be used in the prevention of obesity and related metabolic disorders. Here, we aimed to investigate the effects of oregonin on the epigenetic regulation in cells as well as its ability to modulate DNA methylating enzymes expression and mitochondrial DNA (mtDNA) copies. Our results show that oregonin altered the expression of DNA methyltransferases and mtDNA copy numbers in dependency on concentration and specificity of cells genotype. A close correlation between mtDNA copy numbers and mRNA expression of the mtDnmt1 and Dnmt3b was established. Moreover, molecular modeling suggested that oregonin fits the catalytic site of DNMT1 and partially overlaps with binding of the cofactor. These findings further extend the knowledge on oregonin, and elucidate for the first time its potential to affect the key players of the DNA methylation process, namely DNMTs transcripts and mtDNA.

Dnmt3a in the Medial Prefrontal Cortex Regulates Anxiety-Like Behavior in Adult Mice.

Recently, it has been suggested that alterations in DNA methylation mediate the molecular changes and psychopathologies that can occur following trauma. Despite the abundance of DNA methyltransferases (Dnmts) in the brain, which are responsible for catalyzing DNA methylation, their roles in behavioral regulation and in response to stressful challenges remain poorly understood. Here, we demonstrate that adult mice which underwent chronic social defeat stress (CSDS) displayed elevated anxiety-like behavior that was accompanied by a reduction in medial prefrontal cortex (mPFC)-DNA methyltransferase 3a (Dnmt3a) mRNA levels and a subsequent decrease in mPFC-global DNA methylation. To explore the role of mPFC-Dnmt3a in mediating the behavioral responses to stressful challenges we established lentiviral-based mouse models that express lower (knockdown) or higher (overexpression) levels of Dnmt3a specifically within the mPFC. Nonstressed mice injected with knockdown Dnmt3a lentiviruses specifically into the mPFC displayed the same anxiogenic phenotype as the CSDS mice, whereas overexpression of Dnmt3a induced an opposite, anxiolytic, effect in wild-type mice. In addition, overexpression of Dnmt3a in the mPFC of CSDS mice attenuated stress-induced anxiety. Our results indicate a central role for mPFC-Dnmt3a as a mediator of stress-induced anxiety. Significance statement: DNA methylation is suggested to mediate the molecular mechanisms linking environmental challenges, such as chronic stress or trauma, to increased susceptibility to psychopathologies. Here, we show that chronic stress-induced increase in anxiety-like behavior is accompanied by a reduction in DNA methyltransferase 3a (Dnmt3a) mRNA levels and global DNA methylation in the medial prefrontal cortex (mPFC). Overexpression or knockdown of mPFC-Dnmt3a levels induces decrease or increase in anxiety-like behavior, respectively. In addition, overexpression of Dnmt3a in the mPFC of chronic stressed mice attenuated stress-induced anxiety. We suggest that mPFC-Dnmt3a levels mediates anxiety-like behavior, which may be a primary molecular link between chronic stress and the development of anxiety disorders, including post-traumatic stress disorder.

miRNA-342 Regulates CEACAM1-induced Lumen Formation in a Three-dimensional Model of Mammary Gland Morphogenesis.

Lumen formation of breast epithelium is rapidly lost during tumorigenesis along with expression of cell adhesion molecule CEACAM1. CEACAM1 induces lumena in a three-dimensional culture of MCF7/CEACAM1 cells that otherwise fail to form lumena. We hypothesized miRNAs may be involved because >400 genes were up- or down-regulated in MCF7/CEACAM1 cells and miRNAs may modify global expression patterns. Comparative analysis of miRNA expression in MCF7 versus MCF7/CEACAM1 cells revealed two miRNAs significantly down-regulated (hsa-miR-30a-3p by 6.73-fold and hsa-miR-342-5p by 5.68-fold). Location of miR-342 within an intron of the EVL gene, hypermethylated and involved in tumorigenesis, suggested that miR-342 overexpression may block lumen formation. In fact, overexpression of miR-342 in MCF7/CEACAM1 cells significantly blocked lumen formation (p < 0.001). ID4, a dominant-negative inhibitor of basic helix-loop-helix transcription factors, up-regulated in MCF7/CEACAM1 cells, down-regulated in breast cancer, and containing a miR-342 binding site, was tested as a potential target of miR-342. The ratio of ID4 to miR-342 increased from 1:2 in MCF7 cells to 30:1 in MCF7/CEACAM1 cells and a miR-342 inhibitor was able to induce 3'-UTR ID4 reporter activity in MCF7 cells. Because 5-methylcytosine methyltransferase DNMT1 is also a potential target of miR-342, we inhibited miR-342 in MCF7 cells and found DNMT1 was up-regulated with no change in EVL expression, suggesting that miR-342 regulates DNMT1 expression but DNMT1 does not affect the EVL expression in these cells. We conclude that the regulation of lumen formation by miR-342 involves at least two of its known targets, namely ID4 and DNMT1.

Transforming Growth Factor-ß1 Increases DNA Methyltransferase 1 and 3a Expression through Distinct Post-transcriptional Mechanisms in Lung Fibroblasts.

DNA methylation is a fundamental epigenetic mark that plays a critical role in differentiation and is mediated by the actions of DNA methyltransferases (DNMTs). TGF-ß1 is one of the most potent inducers of fibroblast differentiation, and although many of its actions on fibroblasts are well described, the ability of TGF-ß1 to modulate DNA methylation in mesenchymal cells is less clear. Here, we examine the ability of TGF-ß1 to modulate the expression of various DNMTs in primary lung fibroblasts (CCL210). TGF-ß1 increased the protein expression, but not RNA levels, of both DNMT1 and DNMT3a. The increases in DNMT1 and DNMT3a were dependent on TGF-ß1 activation of focal adhesion kinase and PI3K/Akt. Activation of mammalian target of rapamycin complex 1 by Akt resulted in increased protein translation of DNMT3a. In contrast, the increase in DNMT1 by TGF-ß1 was not dependent on new protein synthesis and instead was due to decreased protein degradation. TGF-ß1 treatment led to the phosphorylation and inactivation of glycogen synthase kinase-3ß, which resulted in inhibition of DNMT1 ubiquitination and proteosomal degradation. The phosphorylation and inactivation of glycogen synthase kinase-3ß was dependent on mammalian target of rapamycin complex 1. These results demonstrate that TGF-ß1 increases expression of DNMT1 and DNMT3a through different post-transcriptional mechanisms. Because DNA methylation is critical to many processes including development and differentiation, for which TGF-ß1 is known to be crucial, the ability of TGF-ß1 to increase expression of both DNMT1 and DNMT3a demonstrates a novel means by which TGF-ß1 may regulate DNA methylation in these cells.

Kaempferol Modulates DNA Methylation and Downregulates DNMT3B in Bladder Cancer.

BACKGROUND: Genomic DNA methylation plays an important role in both the occurrence and development of bladder cancer. Kaempferol (Kae), a natural flavonoid that is present in many fruits and vegetables, exhibits potent anti-cancer effects in bladder cancer. Similar to other flavonoids, Kae possesses a flavan nucleus in its structure. This structure was reported to inhibit DNA methylation by suppressing DNA methyltransferases (DNMTs). However, whether Kae can inhibit DNA methylation remains unclear. METHODS: Nude mice bearing bladder cancer were treated with Kae for 31 days. The genomic DNA was extracted from xenografts and the methylation changes was determined using an Illumina Infinium HumanMethylation 450 BeadChip Array. The ubiquitination was detected using immuno-precipitation assay. RESULTS: Our data indicated that Kae modulated DNA methylation in bladder cancer, inducing 103 differential DNA methylation positions (dDMPs) associated with genes (50 hyper-methylated and 53 hypo-methylated). DNA methylation is mostly relied on the levels of DNMTs. We observed that Kae specifically inhibited the protein levels of DNMT3B without altering the expression of DNMT1 or DNMT3A. However, Kae did not downregulate the transcription of DNMT3B. Interestingly, we observed that Kae induced a premature degradation of DNMT3B by inhibiting protein synthesis with cycloheximide (CHX). By blocking proteasome with MG132, we observed that Kae induced an increased ubiquitination of DNMT3B. These results suggested that Kae could induce the degradation of DNMT3B through ubiquitin-proteasome pathway. CONCLUSION: Our data indicated that Kae is a novel DNMT3B inhibitor, which may promote the degradation of DNMT3B in bladder cancer.

MiR-143 inhibits cell proliferation and invasion by targeting DNMT3A in gastric cancer.

Increasing evidence has suggested that MircroRNAs (miRNAs) dysregulated in pathogenesis and tumorigenicity in human cancers including gastric cancer (GC). MiR-143 had been reported to function as tumor suppressor in GC progression, however, the underlying function of miR-143 in GC still need to be well known. In the study, we revealed that miR-143 was significantly down-regulated in GC cell lines. Upregulation of miR-143 inhibited cell proliferation, invasion, S phase cell proportion and cell cycle related protein levels of Cyclin D1, CDK4 and CDK6 in GC. Furthermore, luciferase reporter assays demonstrated that DNMT3A was a direct target of miR-143 and Upregulation of miR-143 inhibited the DNMT3A mRNA and protein expression levels in GC cells. Moreover, we demonstrated that DNMT3A knockdown rescued the promoting effect of miR-143 inhibitor on cell proliferation in GC. Thus, these results demonstrated that miR-143 targeted DNMT3A in GC cells and inhibit GC tumorigenesis and progression, which may provide a novel therapeutic target of GC.