Thrombomodulin Is Silenced in Malignant Mesothelioma by a Poly(ADP-ribose) Polymerase-1-mediated Epigenetic Mechanism

Malignant mesothelioma (MM) is often complicated by thromboembolic episodes, with thrombomodulin (TM) playing a critical role in the anticoagulant process. Heterogeneous expression of TM has been observed in cancer, and low or no TM expression in cancer cells is associated with poor prognosis. In this study, we analyzed TM expression in biopsies of MM patients and compared them with normal mesothelial tissue. The role of DNA methylation-associated gene silencing in TM expression was investigated. To evaluate poly(ADP-ribose) polymerase-1 (PARP1) as responsible for gene promoter epigenetic modifications, nonmalignant mesothelial cells (Met-5A) and MM cells (H28) were silenced for PARP1 and the DNA methylation/acetylation-associated TM expression evaluated. A correlation between low TM expression and high level of TM promoter methylation was found in MM biopsies. Low expression of TM was restored in MM cells by their treatment with 5-aza-2′-deoxycytidine and, to a lesser extent, with trichostatin, whereas the epigenetic agents did not affect TM expression in Met-5A cells. Silencing of PARP1 resulted in a strong down-regulation of TM expression in Met-5A cells, while restoring TM expression in H28 cells. PARP1 silencing induced TM promoter methylation in Met-5A cells and demethylation in MM cells, and this was paralleled by corresponding changes in the DNA methyltransferase activity. We propose that methylation of the TM promoter is responsible for silencing of TM expression in MM tissue, a process that is regulated by PARP1.

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Aldosterone reprograms promoter methylation to regulate αENaC transcription in the collecting cuct

AJP Renal Physiology ◽

10.1152/ajprenal.00407.2013 ◽

2013 ◽

Vol 305 (7) ◽

pp. F1006-F1013 ◽

Cited By ~ 16

Author(s):

Zhiyuan Yu ◽

Qun Kong ◽

Bruce C. Kone

Keyword(s):

Promoter Methylation ◽

Dna Methyltransferase ◽

Cpg Island ◽

Collecting Duct ◽

Epigenetic Mechanism ◽

Sequencing Analysis ◽

Control Of Gene Expression ◽

Bisulfite Treatment ◽

High Level ◽

Interfering Rna

Aldosterone increases tubular Na+ absorption largely by increasing α-epithelial Na+ channel (αENaC) transcription in collecting duct principal cells. How aldosterone reprograms basal αENaC transcription to high-level activity in the collecting duct is incompletely understood. Promoter methylation, a covalent but reversible epigenetic process, has been implicated in the control of gene expression in health and disease. We investigated the role of promoter methylation/demethylation in the epigenetic control of basal and aldosterone-stimulated αENaC transcription in mIMCD3 collecting duct cells. Bisulfite treatment and sequencing analysis after treatment of the cells with the DNA methyltransferase (DNMT) inhibitor 5-aza-2′-deoxycytidine (5-Aza-CdR) identified clusters of methylated cytosines in a CpG island near the transcription start site of the αENaC promoter. 5-Aza-CdR treatment or small interfering RNA-mediated knockdown of DNMT3b or methyl-CpG-binding domain protein (MBD)-4 derepressed basal αENaC transcription, indicating that promoter methylation suppresses basal αENaC transcription. Aldosterone triggered a time-dependent decrease in 5mC and DNMT3b and a concurrent enrichment in 5-hydroxymethylcytosine (5hmC) and ten-eleven translocation (Tet)2 at the αENaC promoter, consistent with active demethylation. 5-Aza-CdR mimicked aldosterone by enhancing Sp1 binding to the αENaC promoter. We conclude that DNMT3b- and MBD4-dependent methylation of the αENaC promoter limits basal αENaC transcription, in part by limiting Sp1 binding and trans-activation. Aldosterone stimulates the dispersal of DNMT3b and recruitment of Tet2 to demethylate the αENaC promoter to induce αENaC transcription. These results disclose a novel epigenetic mechanism for the control of basal and aldosterone-induced αENaC transcription that adds to previously described epigenetic controls exerted by histone modifications.

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Domain-Specific Response of Imprinted Genes to Reduced DNMT1

Molecular and Cellular Biology ◽

10.1128/mcb.01278-09 ◽

2010 ◽

Vol 30 (16) ◽

pp. 3916-3928 ◽

Cited By ~ 32

Author(s):

Jamie R. Weaver ◽

Garnik Sarkisian ◽

Christopher Krapp ◽

Jesse Mager ◽

Mellissa R. W. Mann ◽

...

Keyword(s):

Dna Methylation ◽

Dna Methyltransferase ◽

Critical Role ◽

Imprinted Genes ◽

Specific Response ◽

Loss Of Function ◽

Partial Loss ◽

Domain Specific ◽

Parent Of Origin

ABSTRACT Imprinted genes are expressed in a monoallelic, parent-of-origin-specific manner. Clusters of imprinted genes are regulated by imprinting control regions (ICRs) characterized by DNA methylation of one allele. This methylation is critical for imprinting; a reduction in the DNA methyltransferase DNMT1 causes a widespread loss of imprinting. To better understand the role of DNA methylation in the regulation of imprinting, we characterized the effects of Dnmt1 mutations on the expression of a panel of imprinted genes in the embryo and placenta. We found striking differences among imprinted domains. The Igf2 and Peg3 domains showed imprinting perturbations with both null and partial loss-of-function mutations, and both domains had pairs of coordinately regulated genes with opposite responses to loss of DNMT1 function, suggesting these domains employ similar regulatory mechanisms. Genes in the Kcnq1 domain were less sensitive to the absence of DNMT1. Cdkn1c exhibited imprinting perturbations only in null mutants, while Kcnq1 and Ascl2 were largely unaffected by a loss of DNMT1 function. These results emphasize the critical role for DNA methylation in imprinting and reveal the different ways it controls gene expression.

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Abstract 636: Regulation of Galectin-3 Expression in Pulmonary Vascular Smooth Muscle by Dna Methylation

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.37.suppl_1.636 ◽

2017 ◽

Vol 37 (suppl_1) ◽

Author(s):

Xueyi Li ◽

Feng Chen ◽

Stephen Haigh ◽

Yusi Wang ◽

Zsuzsana Bordan ◽

...

Keyword(s):

Dna Methylation ◽

Cell Proliferation ◽

Dna Methyltransferase ◽

Significant Loss ◽

Epigenetic Mechanism ◽

Proximal Promoter ◽

Vascular Cell ◽

Galectin 3 ◽

The Media

Pulmonary Arterial Hypertension (PAH) is characterized by excessive vascular cell proliferation, inward remodeling and increased stiffness and inflammation of the pulmonary blood vessels. We found that galectin-3 (Gal-3) is upregulated in PA from multiple models of PAH including monocrotaline (MCT), MCT + pneumonectomy, and SUGEN/hypoxia rats as well as in human PAH and correlated with severity of disease. Gal-3 is a β-galactoside binding lectin implicated in signaling pathways regulating cell proliferation, inflammation and fibrosis, but its role in PAH is poorly defined. Confocal analysis revealed the majority of Gal-3 expression in the media of PA of both rodent models and humans. Selective inhibitors of Gal-3 attenuated PAH in MCT-treated rats and reduced indices of proliferation, fibrosis and increased apoptosis in PA. Overexpression of Gal-3 in PASMC increased proliferation, migration and expression of profibrotic molecules and protected from apoptosis. Acute exposure of cultured HPASMC with various mitogens and factors important in the development of PAH, failed to increase Gal-3 expression. In contrast, PASMC isolated from rats with PAH exhibited an enduring capacity for increased proliferation and expressed higher levels of Gal-3 suggesting an epigenetic mechanism regulating Gal-3 expression. We found that treatment of PASMC with inhibitors of DNA methylation robustly increased Gal-3 expression in control human and rat PASMC but not in MCT-derived PASMC. Methylation analysis of DNA isolated from PA using MeDIP-qPCR and pyrosequencing revealed hypomethylation of Gal-3 proximal promoter. Analysis of DNA methyltransferase expression in PA revealed a significant loss of only Dnmt3A expression in hypertensive PA. To assess the role of local methylation in the regulation of Gal-3 expression we used CRISPR-dCas9. Targeted Gal-3 promoter methylation using multiple RNA guides and dCas9-Dnmt3A-Dnmt3L effectively reduced Gal-3 expression in SMC isolated from MCT rat PA and reversed the excessive proliferation. These results advance an important role of methylation-dependent mechanisms in Gal-3 signaling and provide a mechanism for the enduring changes in vascular cell behavior observed in PAH.

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Dietary-phytochemical mediated reversion of cancer-specific splicing inhibits Warburg effect in head and neck cancer

BMC Cancer ◽

10.1186/s12885-019-6257-1 ◽

2019 ◽

Vol 19 (1) ◽

Cited By ~ 5

Author(s):

Sandhya Yadav ◽

Somnath D. Bhagat ◽

Amit Gupta ◽

Atul Samaiya ◽

Aasheesh Srivastava ◽

...

Keyword(s):

Dna Methylation ◽

Head And Neck Cancer ◽

Alternative Splicing ◽

Growth Inhibition ◽

Cancer Cells ◽

Warburg Effect ◽

Dna Methyltransferase ◽

Epigenetic Mechanism ◽

Exon 10

Abstract Background The deregulated alternative splicing of key glycolytic enzyme, Pyruvate Kinase muscle isoenzyme (PKM) is implicated in metabolic adaptation of cancer cells. The splicing switch from normal PKM1 to cancer-specific PKM2 isoform allows the cancer cells to meet their energy and biosynthetic demands, thereby facilitating the cancer cells growth. We have investigated the largely unexplored epigenetic mechanism of PKM splicing switch in head and neck cancer (HNC) cells. Considering the reversible nature of epigenetic marks, we have also examined the utility of dietary-phytochemical in reverting the splicing switch from PKM2 to PKM1 isoform and thereby inhibition of HNC tumorigenesis. Methods We present HNC-patients samples, showing the splicing-switch from PKM1-isoform to PKM2-isoform analyzed via immunoblotting and qRT-PCR. We performed methylated-DNA-immunoprecipitation to examine the DNA methylation level and chromatin-immunoprecipitation to assess the BORIS (Brother of Regulator of Imprinted Sites) recruitment and polII enrichment. The effect of dietary-phytochemical on the activity of denovo-DNA-methyltransferase-3b (DNMT3B) was detected by DNA-methyltransferase-activity assay. We also analyzed the Warburg effect and growth inhibition using lactate, glucose uptake assay, invasion assay, cell proliferation, and apoptosis assay. The global change in transcriptome upon dietary-phytochemical treatment was assayed using Human Transcriptome Array 2.0 (HTA2.0). Results Here, we report the role of DNA-methylation mediated recruitment of the BORIS at exon-10 of PKM-gene regulating the alternative-splicing to generate the PKM2-splice-isoform in HNC. Notably, the reversal of Warburg effect was achieved by employing a dietary-phytochemical, which inhibits the DNMT3B, resulting in the reduced DNA-methylation at exon-10 and hence, PKM-splicing switch from cancer-specific PKM2 to normal PKM1. Global-transcriptome-analysis of dietary-phytochemical-treated cells revealed its effect on alternative splicing of various genes involved in HNC. Conclusion This study identifies the epigenetic mechanism of PKM-splicing switch in HNC and reports the role of dietary-phytochemical in reverting the splicing switch from cancer-specific PKM2 to normal PKM1-isoform and hence the reduced Warburg effect and growth inhibition of HNC. We envisage that this approach can provide an effective way to modulate cancer-specific-splicing and thereby aid in the treatment of HNC.

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Exosomal miR-214-5p Released from Glioblastoma Cells Modulates Inflammatory Response of Microglia after Lipopolysaccharide Stimulation through Targeting CXCR5

CNS & Neurological Disorders - Drug Targets ◽

10.2174/1871527317666181105112009 ◽

2019 ◽

Vol 18 (1) ◽

pp. 78-87 ◽

Cited By ~ 7

Author(s):

Jian-kai Yang ◽

Hong-jiang Liu ◽

Yuanyu Wang ◽

Chen Li ◽

Ji-peng Yang ◽

...

Keyword(s):

Inflammatory Response ◽

Critical Role ◽

Luciferase Reporter ◽

Clinical Prognosis ◽

Direct Target ◽

And Migration ◽

High Level ◽

Cxcr5 Expression ◽

Proliferation And Migration

Background and Objective: Exosomes communicate inter-cellularly and miRNAs play critical roles in this scenario. MiR-214-5p was implicated in multiple tumors with diverse functions uncovered. However, whether miR-214-5p is mechanistically involved in glioblastoma, especially via exosomal pathway, is still elusive. Here we sought to comprehensively address the critical role of exosomal miR-214-5p in glioblastoma (GBM) microenvironment.Methods:The relative expression of miR-214-5p was determined by real-time PCR. Cell viability and migration were measured by MTT and transwell chamber assays, respectively. The secretory cytokines were measured with ELISA kits. The regulatory effect of miR-214-5p on CXCR5 expression was interrogated by luciferase reporter assay. Protein level was analyzed by Western blot.Results:We demonstrated that miR-214-5p was aberrantly overexpressed in GBM and associated with poorer clinical prognosis. High level of miR-214-5p significantly contributed to cell proliferation and migration. GBM-derived exosomal miR-214-5p promoted inflammatory response in primary microglia upon lipopolysaccharide challenge. We further identified CXCR5 as the direct target of miR-214- 5p in this setting.Conclusion:Overexpression of miR-214-5p in GBM modulated the inflammatory response in microglia via exosomal transfer.

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Lowering DNA binding affinity of SssI DNA methyltransferase does not enhance the specificity of targeted DNA methylation in E. coli

Scientific Reports ◽

10.1038/s41598-021-94528-3 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Krystyna Ślaska-Kiss ◽

Nikolett Zsibrita ◽

Mihály Koncz ◽

Pál Albert ◽

Ákos Csábrádi ◽

...

Keyword(s):

Dna Methylation ◽

Dna Binding ◽

Binding Affinity ◽

Dna Methyltransferase ◽

Restriction Enzymes ◽

Dna Binding Protein ◽

E Coli ◽

Dna Binding Affinity ◽

Higher Eukaryotes

AbstractTargeted DNA methylation is a technique that aims to methylate cytosines in selected genomic loci. In the most widely used approach a CG-specific DNA methyltransferase (MTase) is fused to a sequence specific DNA binding protein, which binds in the vicinity of the targeted CG site(s). Although the technique has high potential for studying the role of DNA methylation in higher eukaryotes, its usefulness is hampered by insufficient methylation specificity. One of the approaches proposed to suppress methylation at unwanted sites is to use MTase variants with reduced DNA binding affinity. In this work we investigated how methylation specificity of chimeric MTases containing variants of the CG-specific prokaryotic MTase M.SssI fused to zinc finger or dCas9 targeting domains is influenced by mutations affecting catalytic activity and/or DNA binding affinity of the MTase domain. Specificity of targeted DNA methylation was assayed in E. coli harboring a plasmid with the target site. Digestions of the isolated plasmids with methylation sensitive restriction enzymes revealed that specificity of targeted DNA methylation was dependent on the activity but not on the DNA binding affinity of the MTase. These results have implications for the design of strategies of targeted DNA methylation.

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Genome-wide analysis identifies critical DNA methylations within NTRKs genes in colorectal cancer

Journal of Translational Medicine ◽

10.1186/s12967-021-02740-6 ◽

2021 ◽

Vol 19 (1) ◽

Author(s):

Zijian Chen ◽

Zenghong Huang ◽

Yanxin Luo ◽

Qi Zou ◽

Liangliang Bai ◽

...

Keyword(s):

Colorectal Cancer ◽

Dna Methylation ◽

Promoter Methylation ◽

Expression Profiles ◽

Methylation Status ◽

Gene Promoter ◽

Normal Mucosa ◽

Suppressor Gene ◽

Survival Outcome ◽

Prognostic Models

Abstract Background Neurotrophic tropomyosin receptor kinases (NTRKs) are a gene family function as oncogene or tumor suppressor gene in distinct cancers. We aimed to investigate the methylation and expression profiles and prognostic value of NTRKs gene in colorectal cancer (CRC). Methods An analysis of DNA methylation and expression profiles in CRC patients was performed to explore the critical methylations within NTRKs genes. The methylation marker was validated in a retrospectively collected cohort of 229 CRC patients and tested in other tumor types from TCGA. DNA methylation status was determined by quantitative methylation-specific PCR (QMSP). Results The profiles in six CRC cohorts showed that NTRKs gene promoter was more frequently methylated in CRC compared to normal mucosa, which was associated with suppressed gene expression. We identified a specific methylated region within NTRK3 promoter targeted by cg27034819 and cg11525479 that best predicted survival outcome in CRC. NTRK3 promoter methylation showed independently predictive value for survival outcome in the validation cohort (P = 0.004, HR 2.688, 95% CI [1.355, 5.333]). Based on this, a nomogram predicting survival outcome was developed with a C-index of 0.705. Furthermore, the addition of NTRK3 promoter methylation improved the performance of currently-used prognostic model (AIC: 516.49 vs 513.91; LR: 39.06 vs 43.64, P = 0.032). Finally, NTRK3 promoter methylation also predicted survival in other tumors, including pancreatic cancer, glioblastoma and stomach adenocarcinoma. Conclusions This study highlights the essential value of NTRK3 methylation in prognostic evaluation and the potential to improve current prognostic models in CRC and other tumors.

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DNA Methyltransferase 3A Is Involved in the Sustained Effects of Chronic Stress on Synaptic Functions and Behaviors

Cerebral Cortex ◽

10.1093/cercor/bhaa337 ◽

2020 ◽

Author(s):

Jing Wei ◽

Jia Cheng ◽

Nicholas J Waddell ◽

Zi-Jun Wang ◽

Xiaodong Pang ◽

...

Keyword(s):

Dna Methylation ◽

Dna Methyltransferase ◽

Epigenetic Modification ◽

Substantial Reduction ◽

Dna Methyltransferases ◽

Male Rats ◽

Neural Pathways ◽

Dnmt Inhibitors ◽

Synaptic Functions

Abstract Emerging evidence suggests that epigenetic mechanisms regulate aberrant gene transcription in stress-associated mental disorders. However, it remains to be elucidated about the role of DNA methylation and its catalyzing enzymes, DNA methyltransferases (DNMTs), in this process. Here, we found that male rats exposed to chronic (2-week) unpredictable stress exhibited a substantial reduction of Dnmt3a after stress cessation in the prefrontal cortex (PFC), a key target region of stress. Treatment of unstressed control rats with DNMT inhibitors recapitulated the effect of chronic unpredictable stress on decreased AMPAR expression and function in PFC. In contrast, overexpression of Dnmt3a in PFC of stressed animals prevented the loss of glutamatergic responses. Moreover, the stress-induced behavioral abnormalities, including the impaired recognition memory, heightened aggression, and hyperlocomotion, were partially attenuated by Dnmt3a expression in PFC of stressed animals. Finally, we found that there were genome-wide DNA methylation changes and transcriptome alterations in PFC of stressed rats, both of which were enriched at several neural pathways, including glutamatergic synapse and microtubule-associated protein kinase signaling. These results have therefore recognized the potential role of DNA epigenetic modification in stress-induced disturbance of synaptic functions and cognitive and emotional processes.

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Role of Methylation in the Induced and Spontaneous Expression of the Avian Endogenous Virusev-1: DNA Structure and Gene Products

Molecular and Cellular Biology ◽

10.1128/mcb.2.6.638 ◽

1982 ◽

Vol 2 (6) ◽

pp. 638-652 ◽

Cited By ~ 37

Author(s):

Kathleen F. Conklin ◽

John M. Coffin ◽

Harriet L. Robinson ◽

Mark Groudine ◽

Robert Eisenman

Keyword(s):

Dna Methylation ◽

Strong Support ◽

Reverse Transcriptase Activity ◽

Structural Defects ◽

Long Terminal Repeats ◽

Control Of Gene Expression ◽

Rna Molecules ◽

Hypersensitive Sites ◽

High Level

The endogenous avian provirusev-1 is widespread in white leghorn chickens. Although it has no major structural defects,ev-1 has not been associated with any phenotype and is ordinarily expressed at a very low level. In this report, we describe a chicken embryo (Number 1836) cell culture containing bothev-1 andev-6 which spontaneously expressed theev-1 provirus. This culture released a high level of noninfectious virions containing a full complement of virion structural (gag) proteins but devoid of reverse transcriptase activity or antigen. These virions contained 70S RNA closely related to the genome of Rous-associated virus type 0, but identifiable as theev-1 genome by oligonucleotide mapping. A fraction of the RNA molecules in the 70S complex were unusual in that they were polyadenylated 100 to 200 nucleotides downstream of the usual polyadenylation site. Eight sibling embryo cultures did not share this unusual phenotype with 1836, indicating that it was not inherited. However, an identical phenotype was inducible in the sibling cultures by treatment with 5-azacytidine, an inhibitor of DNA methylation, and the induced expression was stable for more than 10 generations. Analysis of chromatin structure and DNA methylation of theev-1 provirus in 1836 cells revealed the presence (in a fraction of the proviruses) of both DNase I hypersensitive sites in the long terminal repeats and ingagand a pattern of cleavage sites for methyl-sensitive restriction endonuclease not found in a nonexpressing sibling. These results lend strong support to the role of DNA methylation in the control of gene expression. Additionally, they explain the lack of phenotype associated withev-1 as due to a combination of its low expression and defectiveness inpolandenv.

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Potential Role of Epigenetic Mechanism in Manganese Induced Neurotoxicity

BioMed Research International ◽

10.1155/2016/2548792 ◽

2016 ◽

Vol 2016 ◽

pp. 1-18 ◽

Cited By ~ 19

Author(s):

Prashant Tarale ◽

Tapan Chakrabarti ◽

Saravanadevi Sivanesan ◽

Pravin Naoghare ◽

Amit Bafana ◽

...

Keyword(s):

Oxidative Stress ◽

Parkinson’S Disease ◽

Parkinson's Disease ◽

Dna Methyltransferase ◽

Apoptotic Cell ◽

Genetic Difference ◽

Epigenetic Mechanism ◽

Dna Hypomethylation ◽

Manganese Neurotoxicity

Manganese is a vital nutrient and is maintained at an optimal level (2.5–5 mg/day) in human body. Chronic exposure to manganese is associated with neurotoxicity and correlated with the development of various neurological disorders such as Parkinson’s disease. Oxidative stress mediated apoptotic cell death has been well established mechanism in manganese induced toxicity. Oxidative stress has a potential to alter the epigenetic mechanism of gene regulation. Epigenetic insight of manganese neurotoxicity in context of its correlation with the development of parkinsonism is poorly understood. Parkinson’s disease is characterized by theα-synuclein aggregation in the form of Lewy bodies in neuronal cells. Recent findings illustrate that manganese can cause overexpression ofα-synuclein.α-Synuclein acts epigenetically via interaction with histone proteins in regulating apoptosis.α-Synuclein also causes global DNA hypomethylation through sequestration of DNA methyltransferase in cytoplasm. An individual genetic difference may also have an influence on epigenetic susceptibility to manganese neurotoxicity and the development of Parkinson’s disease. This review presents the current state of findings in relation to role of epigenetic mechanism in manganese induced neurotoxicity, with a special emphasis on the development of Parkinson’s disease.

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