Association of intronic DNA methylation and hydroxymethylation alterations in the epigenetic etiology of dilated cardiomyopathy.

In this study, we investigated the role of DNA methylation [5-methylcytosine (5mC)] and 5-hydroxymethylcytosine (5hmC), epigenetic modifications that regulate gene activity, in dilated cardiomyopathy (DCM). A MYBPC3 mutant mouse model of DCM was compared with wild type and used to profile genomic 5mC and 5hmC changes by Chip-seq, and gene expression levels were analyzed by RNA-seq. Both 5mC-altered genes (957) and 5hmC-altered genes (2,022) were identified in DCM hearts. Diverse gene ontology and KEGG pathways were enriched for DCM phenotypes, such as inflammation, tissue fibrosis, cell death, cardiac remodeling, cardiomyocyte growth, and differentiation, as well as sarcomere structure. Hierarchical clustering of mapped genes affected by 5mC and 5hmC clearly differentiated DCM from wild-type phenotype. Based on these data, we propose that genomewide 5mC and 5hmC contents may play a major role in DCM pathogenesis. NEW & NOTEWORTHY Our data demonstrate that development of dilated cardiomyopathy in mice is associated with significant epigenetic changes, specifically in intronic regions, which, when combined with gene expression profiling data, highlight key signaling pathways involved in pathological cardiac remodeling and heart contractile dysfunction.

Changes in DNA methylation induced by multi-walled carbon nanotube exposure in the workplace.

This study was designed to assess the epigenetic alterations in blood cells, induced by occupational exposure to multi-wall carbon nanotubes (MWCNT). The study population comprised of MWCNT-exposed workers (n=24) and unexposed controls (n=43) from the same workplace. We measured global DNA methylation/hydroxymethylation levels on the 5th cytosine residues using a validated liquid chromatography tandem-mass spectrometry (LC-MS/MS) method. Sequence-specific methylation of LINE1 retrotransposable element 1 (L1RE1) elements, and promoter regions of functionally important genes associated with epigenetic regulation [DNA methyltransferase-1 (DNMT1) and histone deacetylase 4 (HDAC4)], DNA damage/repair and cell cycle pathways [nuclear protein, coactivator of histone transcription/ATM serine/threonine kinase (NPAT/ATM)], and a potential transforming growth factor beta (TGF-ß) repressor [SKI proto-oncogene (SKI)] were studied using bisulfite pyrosequencing. Analysis of global DNA methylation levels and hydroxymethylation did not reveal significant difference between the MWCNT-exposed and control groups. No significant changes in Cytosine-phosphate-Guanine (CpG) site methylation were observed for the LINE1 (L1RE1) elements. Further analysis of gene-specific DNA methylation showed a significant change in methylation for DNMT1, ATM, SKI, and HDAC4 promoter CpGs in MWCNT-exposed workers. Since DNA methylation plays an important role in silencing/regulation of the genes, and many of these genes have been associated with occupational and smoking-induced diseases and cancer (risk), aberrant methylation of these genes might have a potential effect in MWCNT-exposed workers.

Genetic epidemiology of titin-truncating variants in the etiology of dilated cardiomyopathy.

Heart failure (HF) is a complex clinical syndrome defined by the inability of the heart to pump enough blood to meet the body's metabolic demands. Major causes of HF are cardiomyopathies (diseases of the myocardium associated with mechanical and/or electrical dysfunction), among which the most common form is dilated cardiomyopathy (DCM). DCM is defined by ventricular chamber enlargement and systolic dysfunction with normal left ventricular wall thickness, which leads to progressive HF. Over 60 genes are linked to the etiology of DCM. Titin (TTN) is the largest known protein in biology, spanning half the cardiac sarcomere and, as such, is a basic structural and functional unit of striated muscles. It is essential for heart development as well as mechanical and regulatory functions of the sarcomere. Next-generation sequencing (NGS) in clinical DCM cohorts implicated truncating variants in titin (TTNtv) as major disease alleles, accounting for more than 25% of familial DCM cases, but these variants have also been identified in 2-3% of the general population, where these TTNtv blur diagnostic and clinical utility. Taking into account the published TTNtv and their association to DCM, it becomes clear that TTNtv harm the heart with position-dependent occurrence, being more harmful when present in the A-band TTN, presumably with dominant negative/gain-of-function mechanisms. However, these insights are challenged by the depiction of position-independent toxicity of TTNtv acting via haploinsufficient alleles, which are sufficient to induce cardiac pathology upon stress. In the current review, we provide an overview of TTN and discuss studies investigating various TTN mutations. We also present an overview of different mechanisms postulated or experimentally validated in the pathogenicity of TTNtv. DCM-causing genes are also discussed with respect to non-truncating mutations in the etiology of DCM. One way of understanding pathogenic variants is probably to understand the context in which they may or may not affect protein-protein interactions, changes in cell signaling, and substrate specificity. In this regard, we also provide a brief overview of TTN interactions in situ. Quantitative models in the risk assessment of TTNtv are also discussed. In summary, we highlight the importance of gene-environment interactions in the etiology of DCM and further mechanistic studies used to delineate the pathways which could be targeted in the management of DCM.

Changes in DNA Methylation in Mouse Lungs after a Single Intra-Tracheal Administration of Nanomaterials.

AIMS: This study aimed to investigate the effects of nanomaterial (NM) exposure on DNA methylation. METHODS AND RESULTS: Intra-tracheal administration of NM: gold nanoparticles (AuNPs) of 5-, 60- and 250-nm diameter; single-walled carbon nanotubes (SWCNTs) and multi-walled carbon nanotubes (MWCNTs) at high dose of 2.5 mg/kg and low dose of 0.25 mg/kg for 48 h to BALB/c mice. Study showed deregulations in immune pathways in NM-induced toxicity in vivo. NM administration had the following DNA methylation effects: AuNP 60 nm induced CpG hypermethylation in Atm, Cdk and Gsr genes and hypomethylation in Gpx; Gsr and Trp53 showed changes in methylation between low- and high-dose AuNP, 60 and 250 nm respectively, and AuNP had size effects on methylation for Trp53. CONCLUSION: Epigenetics may be implicated in NM-induced disease pathways.

Assessment of Changes in Global DNA Methylation Levels by Pyrosequencing® of Repetitive Elements.

Transposable elements (TE) comprise half of the human genome. LINE-1 and ALU are the most common TE, and they have been used to assess changes in the DNA methylation of repetitive elements in response to intrinsic and extrinsic cellular events. Pyrosequencing(®) is a real-time sequencing technology that enables quantitative assessment of TE methylation at single-base resolution. In Pyrosequencing, a region of interest is first amplified from bisulfite-converted DNA by polymerase chain reaction (PCR), before PCR amplicons are rendered single stranded and annealed with the Pyrosequencing primer prior to sequencing. In this chapter, we provide an overview of the analysis of repetitive element DNA methylation by bisulfite Pyrosequencing, and we describe a protocol that can be used for such purposes.

Epigenetic changes in lymphocytes of solvent-exposed individuals.

AIM: We investigated global DNA methylation alterations in lymphocytes of solvent workers and chronic toxic encephalopathy (CTE) patients and explored potential gene-environment interactions for GST. POPULATION & METHODS: A cross-sectional study was set up in 41 referents, 128 solvent workers and 23 CTE patients. RESULTS: We found a global DNA hypermethylation in the solvent-exposed population compared with the referents (p = 0.001, r = -0.544). Global DNA methylation was negatively associated with exposure. Furthermore, GSTP1 genotypic polymorphism was found to be significantly associated (p = 0.033) with global DNA hypomethylation, which indicates a potential role for gene-environment interaction in the etiology of solvent-induced neurobehavioral disorders. CONCLUSION: This study indicates that solvent-induced DNA methylation alterations have an impact on neurotoxicity and development of CTE.

Effect of chemical mutagens and carcinogens on gene expression profiles in human TK6 cells.

Characterization of toxicogenomic signatures of carcinogen exposure holds significant promise for mechanistic and predictive toxicology. In vitro transcriptomic studies allow the comparison of the response to chemicals with diverse mode of actions under controlled experimental conditions. We conducted an in vitro study in TK6 cells to characterize gene expression signatures of exposure to 15 genotoxic carcinogens frequently used in European industries. We also examined the dose-responsive changes in gene expression, and perturbation of biochemical pathways in response to these carcinogens. TK6 cells were exposed at 3 dose levels for 24 h with and without S9 human metabolic mix. Since S9 had an impact on gene expression (885 genes), we analyzed the gene expression data from cells cultures incubated with S9 and without S9 independently. The ribosome pathway was affected by all chemical-dose combinations. However in general, no similar gene expression was observed among carcinogens. Further, pathways, i.e. cell cycle, DNA repair mechanisms, RNA degradation, that were common within sets of chemical-dose combination were suggested by clustergram. Linear trends in dose-response of gene expression were observed for Trichloroethylene, Benz[a]anthracene, Epichlorohydrin, Benzene, and Hydroquinone. The significantly altered genes were involved in the regulation of (anti-) apoptosis, maintenance of cell survival, tumor necrosis factor-related pathways and immune response, in agreement with several other studies. Similarly in S9+ cultures, Benz[a]pyrene, Styrene and Trichloroethylene each modified over 1000 genes at high concentrations. Our findings expand our understanding of the transcriptomic response to genotoxic carcinogens, revealing the alteration of diverse sets of genes and pathways involved in cellular homeostasis and cell cycle control.

Epigenetic factors in cancer risk: effect of chemical carcinogens on global DNA methylation pattern in human TK6 cells.

In the current study, we assessed the global DNA methylation changes in human lymphoblastoid (TK6) cells in vitro in response to 5 direct and 10 indirect-acting genotoxic agents. TK6 cells were exposed to the selected agents for 24 h in the presence and/or absence of S9 metabolic mix. Liquid chromatography-mass spectrometry was used for quantitative profiling of 5-methyl-2'-deoxycytidine. The effect of exposure on 5-methyl-2'-deoxycytidine between control and exposed cultures was assessed by applying the marginal model with correlated residuals on % global DNA methylation data. We reported the induction of global DNA hypomethylation in TK6 cells in response to S9 metabolic mix, under the current experimental settings. Benzene, hydroquinone, styrene, carbon tetrachloride and trichloroethylene induced global DNA hypomethylation in TK6 cells. Furthermore, we showed that dose did not have an effect on global DNA methylation in TK6 cells. In conclusion we report changes in global DNA methylation as an early event in response to agents traditionally considered as genotoxic.