Aberrant DNA Methylation Changes Research Articles

Significance of aberrant DNA methylation for cancer diagnostics and therapy.

Epigenetics is a scientific field that covers changes in gene expression that are not caused by the alteration of the nucleotide sequence in the DNA strand. Together with sequential changes, epigenetic reprogramming is a recognized cancer hallmark driving carcinogenesis. The underlying mechanisms of epigenetically-driven gene expression changes are diverse. However, one of the most extensively studied mechanisms is a change in DNA methylation. Under physiological conditions, DNA methylation ensures tissue-specific gene silencing and helps to maintain genome stability. With malignant transformation, genomic DNA undergoes global hypomethylation as well as locus-specific hypermethylation in promoters of tumor suppressor genes. In the last few decades, specific aberrant DNA methylation changes have emerged as both cancer-associated biomarkers and therapeutic targets and prompted ongoing efforts to enhance both diagnostic and therapeutic means in oncology. The main purpose of this review is to introduce both established and emerging DNA methylation-based biomarkers for cancer diagnostics with a focus on biomarkers that are either routinely used or have been developed as commercial tests with certification for their use within in vitro diagnostics. Furthermore, therapeutic options for targeting aberrant DNA methylation are described, including both approved compounds and newly developed agents undergoing clinical investigation.

Integrated epigenomic and network analysis identifies novel DNA methylation signature in atrial fibrillation

Abstract Background CD4+ T cells are associated with atrial fibrillation (AF) pathogenesis but mechanistic insights as well as epigenetic phenomena underlying the onset and progression of AF are not fully understood. Purpose To identify differential methylation signature in circulating CD4+ T lymphocytes isolated from AF patients and HS enrolled in the DIANA clinical trial through epigenome and network medicine approaches. Methods DNA was extracted from CD4+ T lymphocytes obtained from patients diagnosed with AF (n = 10) vs patients with no known arrhythmias (n = 11). DNA was then used for paired-end reduced representation bisulfite sequencing to quantify differentially methylated regions (DMRs). NetworkAnalyst tool was interrogated in order to build a specific protein-protein interaction network and identify hub differentially methylated genes (DMGs) in AF. We considered the centrality degree as main network topological measure and defined hubs those DMGs with a centrality degree > 10. Results The analysis provided 7,846 DMRs (P<0.05), associated with 5,373 annotated genes, among which 7,075 hyper-methylated and 771 hypo-methylated regions were uncovered (Figure 1A). Among the identified DMRs, 3,601 were located within gene promoters of which 3,513 were hypermethylated while 88 hypomethylated. Network analysis including the 50 top hyper- and hypomethylated DMGs, according to the differential methylation values, provided a heart specific subnetwork with 56 nodes and 611 edges and 51 seeds (Figure 1B). Although the DNA methylation trend showed a predominant hypermethylation pattern in CD4+ T cells from AF patients (Figure 1A), network analysis demonstrated that hypomethylation was prevalent in the top 15 hub DMGs. In particular, GSTK1, WWP1, PSMD6, TFRC, CDK5R1, HSPG2, WDFY3, WT1, USP49, ACOT7, LAMB2, ITGB7, SHARPIN, and PLXNB2 emerged as hypomethylated hub DMGs, while AIFM1, PFDN1, LMO3, CDK5RAP2, COL4A1, SEPT8, and CYTH4 resulted hypermethylated hub DMGs. Interestingly, several genes are involved in pathological mechanisms of AF in cardiac tissues such as inflammation, fibrosis and cardiac remodeling. Conclusions Our preliminary data represent the first global methylation analysis in circulating CD4+ T cells in AF. The validation of specific genomic regions and the detection of molecular pathways characterized by aberrant DNA methylation changes may help to clarify pathological mechanisms underlying AF, thus providing an important translational impact for novel diagnostic biomarker discovery and therapeutic target identification.Figure 1

Hypermethylation of DNA Methylation Markers in Non-Cirrhotic Hepatocellular Carcinoma.

Aberrant DNA methylation changes have been reported to be associated with carcinogenesis in cirrhotic HCC, but DNA methylation patterns for these non-cirrhotic HCC cases were not examined. Therefore, we sought to investigate DNA methylation changes on non-cirrhotic HCC using reported promising DNA methylation markers (DMMs), including HOXA1, CLEC11A, AK055957, and TSPYL5, on 146 liver tissues using quantitative methylation-specific PCR and methylated DNA sequencing. We observed a high frequency of aberrant methylation changes in the four DMMs through both techniques in non-cirrhotic HCC compared to cirrhosis, hepatitis, and benign lesions (p < 0.05), suggesting that hypermethylation of these DMMs is specific to non-cirrhotic HCC development. Also, the combination of the four DMMs exhibited 78% sensitivity at 80% specificity with an AUC of 0.85 in discriminating non-cirrhotic HCC from hepatitis and benign lesions. In addition, HOXA1 showed a higher aberrant methylation percentage in non-cirrhotic HCC compared to cirrhotic HCC (43.3% versus 13.3%, p = 0.039), which was confirmed using multivariate linear regression (p < 0.05). In summary, we identified aberrant hypermethylation changes in HOXA1, CLEC11A, AK055957, and TSPYL5 in non-cirrhotic HCC tissues compared to cirrhosis, hepatitis, and benign lesions, providing information that could be used as potentially detectable biomarkers for these unusual HCC cases in clinical practice.

Abstract B016: Aberrant age-related methylation leads to tumor predisposition in colorectal organoids

Abstract Aging is one of the greatest risk factors for cancers, and epigenetic changes involving DNA methylation show close similarities during age-related cellular processes and tumor development. We have shown orthogonal relationships between the accrual of aberrant DNA methylation changes associated with cell division cycles during senescence and transformation. Consequently, we have hypothesized that while both senescent and proliferative aging cells coexist in the same tissues, the risk for cancer most likely comes from, and due to, epigenetic changes occurring in the latter, long-term, self-renewing cells. Using colon organoid models, we have studied the importance of genes undergoing DNA methylation mediated silencing during aging and their role in predisposing to tumorigenesis. Analyses of the in vivo and ex vivo aging-associated epigenetic/expression changes indicate the importance of age-related aberrant methylation in tumor promoting states. Citation Format: Daniel A. Petkovich, Rachael Powers, Yong Tao, Byunghak Kang Nibedita Patel, Kathy Gabrielson, Rafael de Cabo, Stephen B. Baylin, Hariharan P. Easwaran. Aberrant age-related methylation leads to tumor predisposition in colorectal organoids [abstract]. In: Proceedings of the AACR Special Conference: Aging and Cancer; 2022 Nov 17-20; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_1):Abstract nr B016.

Sex differences in saliva-based DNA methylation changes and environmental stressor in young African American adults.

Low socioeconomic status neighborhood exposure to stress and violence may be sources of negative stimuli that poses significant health risks for children, adolescents and throughout the life course of an individual. The study aims to investigate if aberrant epigenetic DNA methylation changes may be a potential mechanism for regulating neighborhood exposures and health outcomes. Exposure to environmental stressors identified in 98 young African American (AA) adults aged 18-25 years old from the Washington D.C., area were used in the study. We correlated the association between stress markers; cortisol, CRP, IgG, IGA, IgM, and self-reported exposure to violence and stress, with quantitative DNA methylation changes in a panel of gene-specific loci using saliva DNA. In all participants studied, the exposure to violence was significant and negatively correlated with DNA methylation of MST1R loci (p = 0.032; r = -0.971) and nominally significant with NR3C1 loci (p = 0.053; r = -0.948). In addition, we observed significant and negative correlation of DNA methylation changes of LINE1 (p = 0.044; r = -0.248); NR3C1 (p = 0.017; r = -0.186); MSTR1 (p = 0.022; r = -0.192); and DRD2 (p = 0.056; r = -0.184; albeit nominal significant correlation) with IgA expression. On the other hand, we observed a significant and position correlation of DNA methylation changes in DRD2 (p = 0.037; r = 0.184) with IgG expression. When participants were stratified by sex, we observed in AA young male adults, significant DNA methylation changes of MST1R (p< 0.05) and association with exposure to violence and IgG level. We also observed significant DNA methylation levels of DRD2 (p< 0.05) and association with IgA, IgG, and cortisol level. Furthermore, we observed significant DNA methylation changes of NR3C1 (p< 0.05) with stress, IgA, and IgG in the male participants only. On the other hand, we only observed significant and a positive association of IgG with DNA methylation levels of ESR1 (p = 0.041) in the young AA female participants. Our preliminary observation of significant DNA methylation changes in neuronal and immune genes in saliva samples supports our recently published genome-wide DNA methylations changes in blood samples from young AA male adults indicating that saliva offers a non-invasive means for DNA methylation prediction of exposure to environmental stressors in a gender-specific manner.

Longitudinal Study of DNA Methylation and Epigenetic Clocks Prior to and Following Test-Confirmed COVID-19 and mRNA Vaccination.

The host epigenetic landscape rapidly changes during SARS-CoV-2 infection, and evidence suggest that severe COVID-19 is associated with durable scars to the epigenome. Specifically, aberrant DNA methylation changes in immune cells and alterations to epigenetic clocks in blood relate to severe COVID-19. However, a longitudinal assessment of DNA methylation states and epigenetic clocks in blood from healthy individuals prior to and following test-confirmed non-hospitalized COVID-19 has not been performed. Moreover, the impact of mRNA COVID-19 vaccines upon the host epigenome remains understudied. Here, we first examined DNA methylation states in the blood of 21 participants prior to and following test-confirmed COVID-19 diagnosis at a median time frame of 8.35 weeks; 756 CpGs were identified as differentially methylated following COVID-19 diagnosis in blood at an FDR adjusted p-value < 0.05. These CpGs were enriched in the gene body, and the northern and southern shelf regions of genes involved in metabolic pathways. Integrative analysis revealed overlap among genes identified in transcriptional SARS-CoV-2 infection datasets. Principal component-based epigenetic clock estimates of PhenoAge and GrimAge significantly increased in people over 50 following infection by an average of 2.1 and 0.84 years. In contrast, PCPhenoAge significantly decreased in people fewer than 50 following infection by an average of 2.06 years. This observed divergence in epigenetic clocks following COVID-19 was related to age and immune cell-type compositional changes in CD4+ T cells, B cells, granulocytes, plasmablasts, exhausted T cells, and naïve T cells. Complementary longitudinal epigenetic clock analyses of 36 participants prior to and following Pfizer and Moderna mRNA-based COVID-19 vaccination revealed that vaccination significantly reduced principal component-based Horvath epigenetic clock estimates in people over 50 by an average of 3.91 years for those who received Moderna. This reduction in epigenetic clock estimates was significantly related to chronological age and immune cell-type compositional changes in B cells and plasmablasts pre- and post-vaccination. These findings suggest the potential utility of epigenetic clocks as a biomarker of COVID-19 vaccine responses. Future research will need to unravel the significance and durability of short-term changes in epigenetic age related to COVID-19 exposure and mRNA vaccination.

27-Hydroxycholesterol, The Estrogen Receptor Modulator, Alters DNA Methylation in Breast Cancer.

27-hydroxycholesterol (27-HC) is the first known endogenous selective estrogen receptor modulator (SERM), and its elevation from normal levels is closely associated with breast cancer. A plethora of evidence suggests that aberrant epigenetic signatures in breast cancer cells can result in differential responses to various chemotherapeutics and often leads to the development of resistant cancer cells. Such aberrant epigenetic changes are mostly dictated by the microenvironment. The local concentration of oxygen and metabolites in the microenvironment of breast cancer are known to influence the development of breast cancer. Hence, we hypothesized that 27-HC, an oxysterol, which has been shown to induce breast cancer progression via estrogen receptor alpha (ERα) and liver X receptor (LXR) and by modulating immune cells, may also induce epigenetic changes. For deciphering the same, we treated the estrogen receptor-positive cells with 27-HC and identified DNA hypermethylation on a subset of genes by performing DNA bisulfite sequencing. The genes that showed significant DNA hypermethylation were phosphatidylserine synthase 2 (PTDSS2), MIR613, indoleamine 2,3-dioxygenase 1 (IDO1), thyroid hormone receptor alpha (THRA), dystrotelin (DTYN), and mesoderm induction early response 1, family member 3 (MIER). Furthermore, we found that 27-HC weakens the DNMT3B association with the ERα in MCF-7 cells. This study reports that 27-HC induces aberrant DNA methylation changes on the promoters of a subset of genes through modulation of ERα and DNMT3B complexes to induce the local DNA methylation changes, which may dictate drug responses and breast cancer development.

Olive Oil Improves While Trans Fatty Acids Further Aggravate the Hypomethylation of LINE-1 Retrotransposon DNA in an Environmental Carcinogen Model.

DNA methylation is an epigenetic mechanism that is crucial for mammalian development and genomic stability. Aberrant DNA methylation changes have been detected not only in malignant tumor tissues; the decrease of global DNA methylation levels is also characteristic for aging. The consumption of extra virgin olive oil (EVOO) as part of a balanced diet shows preventive effects against age-related diseases and cancer. On the other hand, consuming trans fatty acids (TFA) increases the risk of cardiovascular diseases as well as cancer. The aim of the study was to investigate the LINE-1 retrotransposon (L1-RTP) DNA methylation pattern in liver, kidney, and spleen of mice as a marker of genetic instability. For that, mice were fed with EVOO or TFA and were pretreated with environmental carcinogen 7,12-dimethylbenz[a]anthracene (DMBA)—a harmful substance known to cause L1-RTP DNA hypomethylation. Our results show that DMBA and its combination with TFA caused significant L1-RTP DNA hypomethylation compared to the control group via inhibition of DNA methyltransferase (DNMT) enzymes. EVOO had the opposite effect by significantly decreasing DMBA and DMBA + TFA-induced hypomethylation, thereby counteracting their effects.

Genetic Studies on Mammalian DNA Methyltransferases.

Cytosine methylation at the C5-position-generating 5-methylcytosine (5mC)-is a DNA modification found in many eukaryotic organisms, including fungi, plants, invertebrates, and vertebrates, albeit its levels vary greatly in different organisms. In mammals, cytosine methylation occurs predominantly in the context of CpG dinucleotides, with the majority (60-80%) of CpG sites in their genomes being methylated. DNA methylation plays crucial roles in the regulation of chromatin structure and gene expression and is essential for mammalian development. Aberrant changes in DNA methylation and genetic alterations in enzymes and regulators involved in DNA methylation are associated with various human diseases, including cancer and developmental disorders. In mammals, DNA methylation is mediated by two families of DNA methyltransferases (Dnmts), namely Dnmt1 and Dnmt3 proteins. Over the last three decades, genetic manipulations of these enzymes, as well as their regulators, in mice have greatly contributed to our understanding of the biological functions of DNA methylation in mammals. In this chapter, we discuss genetic studies on mammalian Dnmts, focusing on their roles in embryogenesis, cellular differentiation, genomic imprinting, and human diseases.

Editing of DNA Methylation Patterns Using CRISPR-Based Tools.

DNA methylation is an epigenetic modification with an established role in both normal cellular function and mammalian disease. Despite well-characterized associations between aberrant DNA methylation changes and gene expression, evidence for a causal relationship in this context has been difficult to obtain. Early techniques for interrogating the role of DNA methylation in the regulation of gene transcription lack specificity and, where more specific techniques such and ZNFs and TALEs have been developed, they are limited by their extensive cost and labor requirements. However, the recent advent of CRISPR-based technologies has revolutionized our potential for site-specific epigenomic editing. Here, we provide a detailed protocol for the design, construction, and utilization of a transient, CRISPR-based DNA methylation-editing system in mammalian cells.

The Role of DNA Methylation and DNA Methyltransferases in Cancer.

The malignant transformation of normal cells is driven by both genetic and epigenetic changes. With the advent of next-generation sequencing and large-scale international consortia, it is now possible to profile the genomes and epigenomes of thousands of primary tumors from nearly every cancer type. These studies clearly demonstrate that the dynamic regulation of DNA methylation is a critical epigenetic mechanism of cancer initiation, maintenance, and progression. Proper control of DNA methylation is not only crucial for regulating gene transcription and tissue-specific cellular functions, but its broader consequences include maintaining the integrity of the genome and modulating the immune response. Here, we describe the aberrant DNA methylation changes in human cancers and how they contribute to the disease phenotypes. Aside from CpG island promoter DNA hypermethylation-based gene silencing, human cancers also display gene body DNA hypomethylation that is also associated with downregulated gene expression. In addition, the implementation of whole genome bisulfite sequencing (WGBS) has unveiled DNA hypomethylation of large blocks of the genome, known as partially methylated domains (PMDs), as well as cancer-specific DNA methylation aberrancies at enhancers and super-enhancers. Integrating WGBS and DNA methylation array data with mutation, copy number, and gene expression data has allowed for the identification of novel tumor suppressor genes and candidate driver genes of the disease state. Finally, we highlight potential clinical implications of these changes in the context of prognostic and diagnostic biomarkers, as well as therapeutic targets. Mounting evidence shows that DNA methylation data are effective and highly-sensitive disease classifiers, not only from analyses of the primary tumor but also from tumor-derived, cell free DNA (cfDNA) in blood of cancer patients. These findings highlight the power of DNA methylation aberrancies in providing efficacious biomarkers for clinical utility in improving patient diagnostics and their reversal using DNA methylation inhibitors in cancer treatment may be key in surveillance, treatment, and quality of life for cancer patients.

DNA Methylation Signatures and the Contribution of Age-Associated Methylomic Drift to Carcinogenesis in Early-Onset Colorectal Cancer.

Simple SummaryThe role of DNA methylation in the carcinogenesis of colorectal cancer (CRC) diagnosed <50 years of age (early-onset CRC or EOCRC) is currently unknown. In this study, we investigated the genome-wide DNA methylation of 97 tumour and 54 normal colonic mucosa samples from people with EOCRC with the aim of identifying unique DNA methylation signatures and determining the role of ageing-related DNA methylation drift and age-acceleration in EOCRC aetiology. We found extensive DNA methylation alterations associated with EOCRC carcinogenesis, including a unique signature comprising 234 loci compared with CRCs from people >50 years of age. CpGs that undergo ageing-related methylation drift were significantly altered in EOCRC, and accelerated ageing was also evident in normal mucosa from people with EOCRC. Our study is the first study to identify unique DNA methylation changes in EOCRC, contributing novel information that may aid future efforts towards EOCRC prevention.We investigated aberrant DNA methylation (DNAm) changes and the contribution of ageing-associated methylomic drift and age acceleration to early-onset colorectal cancer (EOCRC) carcinogenesis. Genome-wide DNAm profiling using the Infinium HM450K on 97 EOCRC tumour and 54 normal colonic mucosa samples was compared with: (1) intermediate-onset CRC (IOCRC; diagnosed between 50–70 years; 343 tumour and 35 normal); and (2) late-onset CRC (LOCRC; >70 years; 318 tumour and 40 normal). CpGs associated with age-related methylation drift were identified using a public dataset of 231 normal mucosa samples from people without CRC. DNAm-age was estimated using epiTOC2. Common to all three age-of-onset groups, 88,385 (20% of all CpGs) CpGs were differentially methylated between tumour and normal mucosa. We identified 234 differentially methylated genes that were unique to the EOCRC group; 13 of these DMRs/genes were replicated in EOCRC compared with LOCRCs from TCGA. In normal mucosa from people without CRC, we identified 28,154 CpGs that undergo ageing-related DNAm drift, and of those, 65% were aberrantly methylated in EOCRC tumours. Based on the mitotic-based DNAm clock epiTOC2, we identified age acceleration in normal mucosa of people with EOCRC compared with normal mucosa from the IOCRC, LOCRC groups (p = 3.7 × 10−16) and young people without CRC (p = 5.8 × 10−6). EOCRC acquires unique DNAm alterations at 234 loci. CpGs associated with ageing-associated drift were widely affected in EOCRC without needing the decades-long accrual of DNAm drift as commonly seen in intermediate- and late-onset CRCs. Accelerated ageing in normal mucosa from people with EOCRC potentially underlies the earlier age of diagnosis in CRC carcinogenesis.

A long intergenic non-coding RNA regulates nuclear localization of DNA methyl transferase-1

SummaryDNA methyl transferase-1 or DNMT1 maintains DNA methylation in the genome and is important for regulating gene expression in cells. Aberrant changes in DNMT1 activity and DNA methylation are commonly observed in cancers and many other diseases. Recently, a number of long intergenic non-protein-coding RNAs or lincRNAs have been shown to play a role in regulating DNMT1 activity. CCDC26 is a nuclear lincRNA that is frequently mutated in cancers and is a hotbed for disease-associated single nucleotide changes. However, the functional mechanism of CCDC26 is not understood. Here, we show that this lincRNA is concentrated on the nuclear periphery. Strikingly, in the absence of CCDC26 lincRNA, DNMT1 is mis-located in the cytoplasm, and the genomic DNA is significantly hypomethylated. This is accompanied by double-stranded DNA breaks and increased cell death. These results point to a previously unrecognized mechanism of lincRNA-mediated subcellular localization of DNMT1 and regulation of DNA methylation.

Could high serum folate be associated with adverse effects?

Folate is an important water-soluble vitamin that is presented naturally in foods in particular vegetables, fruits, and whole grains. To which extent is this vitamin needed in our daily regimen is not fully known. Several studies have indicated that many complications, such as megaloblastic anemia, cardiovascular disease, neural tube defects, and numerous cancers, occur in humans when the body becomes deficient in folic acid. On the other hand, a few studies have shown thier concerns regarding the supplementation of folic acid, resulting in the development of existing tumors and alteration of normal patterns of DNA methylation. Although there is no clear evidence of aberrant DNA methylation and gene expression changes in response to “high” levels of folate or folic acid intake, there are still some concerns. Therefore, its adverse effects especially on fetus and later stages of life should be carefully investigated.

Environmental Epigenetics and Genome Flexibility: Focus on 5-Hydroxymethylcytosine.

Convincing evidence accumulated over the last decades demonstrates the crucial role of epigenetic modifications for mammalian genome regulation and its flexibility. DNA methylation and demethylation is a key mechanism of genome programming and reprogramming. During ontogenesis, the DNA methylome undergoes both programmed changes and those induced by environmental and endogenous factors. The former enable accurate activation of developmental programs; the latter drive epigenetic responses to factors that directly or indirectly affect epigenetic biochemistry leading to alterations in genome regulation and mediating organism response to environmental transformations. Adverse environmental exposure can induce aberrant DNA methylation changes conducive to genetic dysfunction and, eventually, various pathologies. In recent years, evidence was derived that apart from 5-methylcytosine, the DNA methylation/demethylation cycle includes three other oxidative derivatives of cytosine—5-hydroxymethylcytosine (5hmC), 5-formylcytosine, and 5-carboxylcytosine. 5hmC is a predominantly stable form and serves as both an intermediate product of active DNA demethylation and an essential hallmark of epigenetic gene regulation. This makes 5hmC a potential contributor to epigenetically mediated responses to environmental factors. In this state-of-the-art review, we consolidate the latest findings on environmentally induced adverse effects on 5hmC patterns in mammalian genomes. Types of environmental exposure under consideration include hypnotic drugs and medicines (i.e., phenobarbital, diethylstilbestrol, cocaine, methamphetamine, ethanol, dimethyl sulfoxide), as well as anthropogenic pollutants (i.e., heavy metals, particulate air pollution, bisphenol A, hydroquinone, and pentachlorophenol metabolites). We put a special focus on the discussion of molecular mechanisms underlying environmentally induced alterations in DNA hydroxymethylation patterns and their impact on genetic dysfunction. We conclude that DNA hydroxymethylation is a sensitive biosensor for many harmful environmental factors each of which specifically targets 5hmC in different organs, cell types, and DNA sequences and induces its changes through a specific metabolic pathway. The associated transcriptional changes suggest that environmentally induced 5hmC alterations play a role in epigenetically mediated genome flexibility. We believe that knowledge accumulated in this review together with further studies will provide a solid basis for new approaches to epigenetic therapy and chemoprevention of environmentally induced epigenetic toxicity involving 5hmC patterns.

PM2.5-induced extensive DNA methylation changes in the heart of zebrafish embryos and the protective effect of folic acid

We previously found that folic acid (FA) attenuated cardiac defects in zebrafish embryos exposed to extractable organic matter (EOM) from PM2.5, but the underlining mechanisms remain to be elucidated. Since DNA methylation is crucial to cardiac development, we hypothesized that EOM-induced aberrant DNA methylation changes could be diminished by FA supplementation. In this study, zebrafish embryos were exposed to EOM in the absence or presence of FA. Genomic-wide DNA methylation analysis identified both DNA hypo- and hyper-methylation changes in CCGG sites in zebrafish embryos exposed to EOM, which were attenuated by FA supplementation. We identified a total of 316 genes with extensive DNA methylation changes in EOM samples but little or no DNA methylation changes in EOM plus FA samples. The genes were involved in critical cellular processes and signaling pathways important for embryo development. In addition, the EOM-decreased SAM/SAH ratio was counteracted by FA supplementation. Furthermore, FA attenuated the EOM-induced changes in the expression of genes involved in the regulation of DNA methylation and in folate biosynthesis. In conclusion, our data suggest that FA supplementation protected zebrafish embryos from the cardiac developmental toxicity of PM2.5 by alleviating EOM-induced DNA methylation changes.

Aberrant DNA methylation of M1-macrophage genes in coronary artery disease

M1 and M2 macrophage balance in atherosclerosis has attracted much interest. Though, it remains unknown how macrophage heterogeneity is regulated. Moreover, the regulation of macrophage polarization and activation also involve DNA methylation. However, it remains ambiguous which genes are under direct regulation by DNA methylation. Our aim was to evaluate the gene-specific promoter DNA methylation status of M1/M2 polarization markers in PBMCs of CAD patients. A case-control study was performed with 25 CAD patients and 25 controls to study the promoter DNA methylation status of STAT1, STAT6, MHC2, IL12b, iNOS, JAK1, JAK2 and SOCS5 using MS-HRM analysis. Our data indicates that there was a clear-cut difference in the pattern of gene-specific promoter DNA methylation of CAD patients in comparison to controls. A significant difference was observed between the percentage methylation of STAT1, IL12b, MHC2, iNOS, JAK1 and JAK2 in CAD patients and control subjects. In conclusion, our data show that MS-HRM assay is a rapid and inexpensive method for qualitatively identifying aberrant gene-specific promoter DNA methylation changes in CAD. Furthermore, we propose that gene-specific promoter DNA methylation based on monocyte/macrophage might aid as diagnostic marker for clinical application or DNA methylation-related drug interventions may offer novel possibilities for atherosclerotic disease management.

The effects of methionine on TCE-induced DNA methylation and mRNA expression changes in mouse liver

Trichloroethylene (TCE) had been shown to induce aberrant DNA methylation changes in mouse liver, which could be critical in the development of hepatocellular carcinoma. To understand the effect of methionine, a methyl donor, on TCE-induced mouse liver cancer, we exposed B6C3F1 mice to TCE in the presence or absence of methionine. We found that supplementation with methionine attenuated the TCE-induced hypo- and hyper-methylation status of Cdkn1a and Ihh in mouse liver, respectively. The TCE-induced mRNA expression changes of Cdkn1a, Ihh and Jun were also alleviated by methionine. However, methionine supplementation accelerated the TCE-induced mRNA over-expression of Mki67, and enhanced the TCE-induced mRNA down-regulation of Dnmt3a and Tet2. In summary, our results showed that methionine could attenuate at least part of the TCE-induced DNA methylation changes, but the additional gene expression changes induced by supplementary of methionine does not support the assumption that methionine could prevent TCE-induced liver carcinogenesis.

DNA methylation as mechanism of apoptotic resistance development in endometrial cancer patients.

DNA methylation is a significant epigenetic modification which plays a key role in regulation of gene expression and influences functional changes in endometrial tissue. Aberrant DNA methylation changes result in deregulation of important apoptotic proteins during endometrial carcinogenesis and apoptosis resistance development. Evading apoptosis is still a major problem in the successful treatment of endometrial cancer patients. The aim of our study was to examine the promoter DNA methylation changes in 22 apoptosis-associated genes in endometrioid endometrial cancer patients, precancerous lesions and healthy tissue from various normal menstrual cycle phases using a unique pre-designed methylation platform. We observed as the first a significant difference in promoter DNA methylation status in genes: BCL2L11 (p < 0.001), CIDEB (p < 0.03) and GADD45A (p < 0.05) during endometrial carcinogenesis and BIK gene (p < 0.03) in different phases of normal menstrual cycle. The results of our study indicate that deregulation of mitochondrial apoptotic pathway can considerably contributes to the apoptosis resistance development and may be helpful in identifying of new potent biomarkers in endometrial cancer.

New insights on the role of DNA methylation from a global view.

In mammals, DNA methylation is a crucial epigenetic modification with key functions during development. Cellular processes that are regulated by DNA methylation comprise X chromosome inactivation, gene imprinting, genomic stability and transcriptional regulation. Generally, the methylation status of the majority of target sites is reliably propagated during mitosis. However, advances in genome-wide DNA methylation analysis at base-resolution have discovered a substantial amount of differential DNA methylation between normal cells of different tissue-origin. Moreover, aberrant DNA methylation changes are linked with a significant number of human diseases, particularly with cancer. Sites of differential and aberrant DNA methylation include regulatory DNA sequences, such as CpG islands in promoters and distal cis-regulatory elements, like enhancers. In this review, we will discuss novel aspects of DNA methylation dynamics, during normal development and in association with diseases.