Methylation Status Of Genomic DNA Research Articles

Abstract 6248: Multilayered molecular interactions underlying glioblastoma pathogenesis through multi-omics analysis

Abstract Glioblastoma, IDH-wild type (GBM) is the most common malignant and treatment-refractory brain tumor with a dismal prognosis. Despite the identification of various driver genes in previous large-scale genomic analyses, there has been limited progress in the development of novel treatments. Acquiring an extensive understanding of the molecular mechanisms underlying GBM pathogenesis is crucial to improve prognostic outcomes. We analyzed 289 whole-genome sequencing data (WGS) including 159 unpublished deep WGS (≥ ×120 coverage) along with RNA-seq, DNA methylation array, and assay for transposase-accessible chromatin with sequencing (ATAC-seq) to uncover the molecular mechanisms regulating development and progression of GBM. Our deep WGS enables us to delineate a fine view of clonal architecture, where mutational signature varies between clonal and subclonal mutations, suggesting that different mutational processes contribute to GBM pathogenesis depending on its developmental stage. The differentiation status of tumor cells detected by transcriptional deconvolution analysis is associated with multi-layer profiles including genomic alterations, expression subtypes, DNA methylation, and open chromatin status. Tumors predominantly comprised of differentiated cells display genetic and epigenetic profiles that align with the classical subtype, whereas tumors predominantly composed of stem-like cells exhibit profiles consistent with the proneural subtype. ATAC-seq unveils genome-wide chromatin accessibility features associated with gene expression subtypes. Motif enrichment analysis of differentially accessible sites among the subtypes identified specific transcription factors. The proneural subtype is enriched for the SOX10 binding motif, which is associated with regulating cell states, and exhibits higher expression of SOX10 compared to other subtypes. Conversely, the mesenchymal and classical subtypes are enriched for the CREB1 binding motif, which promotes cell proliferation and angiogenesis through TGFβ2 upregulation, wherein TGFβ2 expression is significantly elevated. These findings support a model in which the difference in chromatin structure also regulates the progression of GBM.Our analysis reveals the multi-layer molecular interactions underlying the progression of GBM enhancing our understanding of the pathogenesis. Citation Format: Takuma Nakashima, Yusuke Funakoshi, Ryo Yamamoto, Yuriko Sugihara, Shohei Nambu, Yoshiki Arakawa, Shota Tanaka, Joji Ishida, Ryuta Saito, Ryosuke Hanaya, Koji Yoshimoto, Yoshitaka Narita, Hiromichi Suzuki. Multilayered molecular interactions underlying glioblastoma pathogenesis through multi-omics analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6248.

Mitochondria-targeted Uncouplers Decrease Inflammatory Reactions in Endothelial Cells by Enhancing Methylation of the ICAM1 Gene Promoter.

The study aimed to investigate the effects of low concentrations of mitochondrial uncouplers in endothelial cells on the CpG dinucleotide methylation of the ICAM1 gene promoter. The excessive inflammatory response in the endothelium is responsible for the development of many cardiovascular diseases. Mitochondria are important regulators of endothelial cell functions. Mild uncoupling of oxidative phosphorylation and respiration in endothelial mitochondria exerts a long lasting anti-inflammatory effect. However, the detailed mechanism of the anti-inflammatory activity of mitochondrial uncouplers remains unclear.We hypothesized that mild mitochondrial uncoupling leads to epigenetic changes in genomic DNA contributing to the anti-inflammatory response. We studied the long-term effects of mitochondria-targeted compounds with the uncoupler's activities: the antioxidant plastoquinonyl-decyltriphenylphosphonium (SkQ1), dodecyl-triphenylphosphonium (C12TPP), and 2,4-dinitrophenol (DNP). The mRNA expression of the intercellular adhesion molecule 1 (ICAM1), a marker of inflammatory activation of endothelial cells, was measured by RT-qPCR. Cytosine methylation in the CpG sites of the ICAM1 gene promoter was estimated by bisulfite sequencing of individual clones. It was found that downregulation of ICAM1 expression caused by DNP and C12TPP was accompanied by an increase in the methylation of CpG sites in the ICAM1 gene promoter. None of the compounds affected intracellular or intramitochondrial ATP levels. Low concentrations of mitochondrial oxidative phosphorylation uncouplers are able to increase methylation of ICAM1 gene promoter, which corresponds to the observed decrease in the levels of mRNA of this gene. Thus, the change in methylation of the ICAM1 gene promoter may underlie the mechanism of decreased ICAM1 expression caused by mild mitochondrial depolarization. Mitochondrial uncouplers may be exploited as possible therapeutic candidates to treat excessive inflammation in endothelium, by changing the methylation status of genomic DNA.

Longitudinal multi-omics alterations response to 8-week risperidone monotherapy: Evidence linking cortical thickness, transcriptomics and epigenetics

Antipsychotic treatment-related alterations of cortical thickness (CT) and clinical symptoms have been previously corroborated, but less is known about whether the changes are driven by gene expression and epigenetic modifications. Utilizing a prospective design, we recruited 42 treatment-naive first-episode schizophrenia patients (FESP) and 38 healthy controls. Patients were scanned by TI weighted imaging before and after 8-week risperidone monotherapy. CT estimation was automatically performed with the FreeSurfer software package. Participants' peripheral blood genomic DNA methylation (DNAm) status, quantified by using Infinium® Human Methylation 450K BeadChip, was examined in parallel with T1 scanning. In total, CT measures from 118 subjects and genomic DNAm status from 114 subjects were finally collected. Partial least squares (PLS) regression was used to detect the spatial associations between longitudinal CT variations after treatment and cortical transcriptomic data acquired from the Allen Human Brain Atlas. Canonical correlation analysis (CCA) was then performed to identify multivariate associations between DNAm of PLS1 genes and patients' clinical improvement. We detected the significant PLS1 component (2,098 genes) related to longitudinal alterations of CT, and the PLS1 genes were significantly enriched in neurobiological processes, and dopaminergic- and cancer-related pathways. Combining Laplacian score and CCA analysis, we further linked DNAm of 33 representative genes from the 2,098 PLS1 genes with patients' reduction rate of clinical symptoms. This study firstly revealed that changes of CT and clinical behaviors after treatment may be transcriptionally and epigenetically underlied. We define a "three-step" roadmap which represents a vital step toward the exploration of treatment- and treatment response-related biomarkers on the basis of multiple omics rather than a single omics type as a strategy for advancing precise care.

Interstrand crosslinking oligonucleotides elucidate the effect of metal ions on the methylation status of repetitive DNA elements.

DNA methylation plays an important physiological function in cells, and environmental changes result in fluctuations in DNA methylation levels. Metal ions have become both environmental and health concerns, as they have the potential to disrupt the genomic DNA methylation status, even on specific sequences. In the current research, the methylation status of two typical repetitive DNA elements, i.e., long-interspersed nuclear element-1 (LINE-1) and alpha satellite (α-sat), was imaged and assessed using methylation-specific fluorescence in situ hybridization (MeFISH). This technique elucidated the effect of several metal ions on the methylation levels of repetitive DNA sequences. The upregulation and downregulation of the methylation levels of repetitive DNA elements by various metal ions were confirmed and depended on their concentration. This is the first example to investigate the effects of metal ions on DNA methylation in a sequence-specific manner.

Global and Regional DNA methylation silencing of PPARγ Associated with Glioblastoma Multiforme Pathogenesis.

The relationship between peroxisome proliferator-activated receptor gamma (PPARγ) expression level and epigenetic modifications occurring in glioblastoma multiforme (GBM) pathogenesis is largely unknown. Herein, we examine the association of PPARγ expression with its promoter and genomic global DNA methylation status, as well as DNA methyltransferases (DNMTs) gene expression in GBM patients. We examined the patterns of promoter methylation and PPARγ expression in 26 GBM tissues and 13 adjacent non-tumor tissues by methylation-specific PCR (MSP), real-time PCR, and ELISA, respectively. Also, we examined the genomic global 5-methyl cytosine levels and DNMTs gene expression using ELISA and real-time PCR methods, respectively. We found that hypermethylation on a specific region of the PPARγ promoter is significantly associated with the downregulation of the PPARγ gene and protein level in GBM patients. Interestingly, the amount of 5-methyl cytosine level was significantly reduced in GBM patients and positively correlated with PPARγ protein expression. Furthermore, the expression level of DNMT1, DNMT3A, and 3B were upregulated in GBM patients and the average expression level of all three DNMTs was positively correlated with tumor area. Also, we found that tumors from cortical regions exhibited a higher global DNA hypomethylation and PPARγ hypermethylation was related to the increase in GBM risk. Our study demonstrated that global DNA methylation and PPARγ epigenetic silencing is associated with the GBM risk. Our data provide a novel molecular mechanistic insight into epigenetic silencing of PPARγ in GBM patients that may be relevant as a key tumor marker for GBM pathogenesis.

Contribution of DNA methylation profiling to the reclassification of a variant of uncertain significance in the KDM5C gene

KDM5C encodes a demethylase of the histone H3 lysine 4 residue, involved in chromatin regulation and gene expression. Hemizygous KDM5C pathogenic variants cause X-linked intellectual disability of Claes-Jensen type. Because of its mode of inheritance and the low specificity of the clinical phenotype, interpretation of variants can be difficult, hence the need for functional studies and biomarkers specific to this disorder. We present the case of a male patient with intellectual disability, behavioral abnormalities and subtle dysmorphic features, in which genetic investigation identified a hemizygous novel missense KDM5C variant of uncertain significance (VUS), inherited from his asymptomatic mother and present in his paucisymptomatic sister. We assessed the global genomic DNA methylation status from a whole blood sample of the proband. Global DNA methylation profiling specifically identified the recently discovered epi-signature of Claes-Jensen syndrome. This result served as a biomarker which independently highlighted KDM5C as the cause of the disorder in this patient. Because of the X-linked mode of inheritance, variant reclassification had a high impact on genetic counseling in this family. This example highlights the value of global methylome profiling in situations of variants of uncertain significance in genes with a known specific epi-signature.

Genome-Wide Identification and Expression Analysis of Cytosine DNA Methyltransferase Genes Related to Somaclonal Variation in Pineapple (Ananas comosus L.)

DNA methylation plays an important role in somaclonal variation. Cytosine-5 DNA methyltransferases (C5-MTases) are crucial enzymes for maintaining the de novo genome DNA methylation status, and the gene family has been identified and characterized in several species. However, limited information is available about the role of C5-MTases in the somaclonal variation of pineapple. Herein, the characteristic of DNA methylation in the leaf variants of pineapple showed that the methylation rate of variants decreased and the demethylation was mainly. Six AcC5-MTases were identified from pineapple genome. Phylogenetic analysis indicated that the six members could be classified into four subgroups, namely, AcMET, AcCMT, AcDRM, and AcDNMT, demonstrating evolutionary conservation in the C5-MTases across pineapple and other plant species. Cis-elements were presented in AcC5-MTase promoters, including light-responsive, phytohormone-responsive, and plant growth and development elements. Furthermore, we investigated the expression profiles of AcC5-MTases in different tissues and their transcript abundance in the leaves of somaclonal variation. Interestingly, most AcC5-MTase genes were highly expressed in leaf and carpopodium. AcMET, AcCMT2, and AcDRM2 were upregulated in pineapple somaclonal variants, indicating that AcC5-MTases might play an important role in the somaclonal variation of pineapple. Our results will aid in understanding the complex roles of methylation in plants and provide a basis for analyzing the role of methylation modification in the somaclonal variation of pineapple.

Reprogramming of the Hevea brasiliensis Epigenome and Transcriptome in Response to Cold Stress.

Low temperature is a key factor limiting the rubber plantation extending to high latitude area. Previous work has shown that cold-induced DNA demethylation was coordinated with the expression of cold-responsive (COR) genes in Hevea brasiliensis. In this work, reduced representation bisulphite sequencing analysis of H. brasiliensis showed that cold treatment induced global genomic DNA demethylation and altered the sequence contexts of methylated cytosines, but the levels of mCG methylation in transposable elements were slightly enhanced by cold treatment. Integrated analysis of the DNA methylome and transcriptome revealed 400 genes whose expression correlated with altered DNA methylation. DNA demethylation in the upstream region of gene seems to correlate with higher gene expression, whereas demethylation in the gene body has less association. Our results suggest that cold treatment globally change the genomic DNA methylation status of the rubber tree, which might coordinate reprogramming of the transcriptome.

Comparative methylomics and chromatin accessibility analysis in Osmanthus fragrans uncovers regulation of genic transcription and mechanisms of key floral scent production.

Linalool and ionone are two important aromatic components in sweet osmanthus petals, and the regulatory mechanisms that produce these two components remain unclear. In this study, we employed whole-genome methylation sequencing and ATAC-seq technology to analyze the genomic DNA methylation status and chromatin accessibility of the sweet osmanthus cultivars ‘Zaohuang’ and ‘Chenghong Dangui’. Results showed that the promoter region of TPS2, a key gene in the linalool synthesis pathway, was less methylated in ‘Chenghong Dangui’ than in ‘Zaohuang’. The chromatin was more accessible in ‘Chenghong Dangui’ than in ‘Zaohuang’, which resulted in a much stronger expression of this gene in ‘Chenghong Dangui’ than in ‘Zaohuang’. This eventually led to a high quantity of linalool and its oxides in the petals of ‘Chenghong Dangui’, but there were lower levels present in the petals of ‘Zaohuang’. These results suggest that DNA methylation and chromatin accessibility play major roles in linalool synthesis in sweet osmanthus. The methylation level of the promoter region of CCD4, a key gene for ionone synthesis, was higher in ‘Zaohuang’ than in ‘Chenghong Dangui’. The chromatin accessibility was lower in ‘Zaohuang’ than in ‘Chenghong Dangui’, although the expression of this gene was significantly higher in ‘Zaohuang’ than in ‘Chenghong Dangui’. ChIP-seq analysis and a series of experiments showed that the differential expression of CCD4 and CCD1 in the two cultivars may predominantly be the result of regulation by ERF2 and other transcription factors. However, a 183-bp deletion involving the CCD4 promoter region in ‘Chenghong Dangui’ may be the main reason for the low expression of this gene in its petals. This study provides an important theoretical basis for improving selective breeding of key floral fragrance components in sweet osmanthus.

Nicotine attenuates global genomic DNA methylation by influencing DNMTs gene expression in human endometrial stromal cells

BackgroundThere is increasing evidence indicating an incidence of infertility and also the risk of endometrial cancers among smokers. However, the mechanism underlying nicotine adverse effect on female reproduction remains unclear. Growing evidence has suggested that environmental exposures such as nicotine could modulate the epigenome. No study has yet been published to evaluate the direct effect of nicotine on the epigenome profiling of human endometrial stromal cells (HESC). Herein, we decided to examine the direct effects of nicotine on global genomic DNA methylation status and DNA methyl- transferases (DNMTs) gene expression in HESC. HESC were treated with different doses of nicotine (0 or control, 10− 11, 10− 8 and 10− 6) M for 24 h and their genomic global DNA methylation and gene expression of DNMTs (DNMT1, DNMT3A, and DNMT3B) were investigated using ELISA and real-time PCR, respectively.ResultsNicotine treatments reduced the average level of DNMTs gene expression by 90, 79, and 73.4% in 10− 11, 10− 8 and 10− 6 M of nicotine treated cells as compared to control cells, respectively (p < 0.05). Also, 10− 8 and 10− 6 M of nicotine concentrations effectively reduced the amounts of 5-methylated cytosine (5-mC) by 1.09 and 1.87% compared to control cells, respectively (p < 0.05). The 5-mC percentages were positively correlated with the relative cellular DNMTs expression in HESC as verified by the Pearson correlation test.ConclusionAn interesting possibility raised by the current study is that the reduced genomic global DNA methylation level in HESC may be partly due to the suppression of DNMTs gene expression caused by nicotine in these cells.Graphical abstract

Systematic Analysis of the DNA Methylase and Demethylase Gene Families in Rapeseed (Brassica napus L.) and Their Expression Variations After Salt and Heat stresses.

DNA methylation is a process through which methyl groups are added to the DNA molecule, thereby modifying the activity of a DNA segment without changing the sequence. Increasing evidence has shown that DNA methylation is involved in various aspects of plant growth and development via a number of key processes including genomic imprinting and repression of transposable elements. DNA methylase and demethylase are two crucial enzymes that play significant roles in dynamically maintaining genome DNA methylation status in plants. In this work, 22 DNA methylase genes and six DNA demethylase genes were identified in rapeseed (Brassica napus L.) genome. These DNA methylase and DNA demethylase genes can be classified into four (BnaCMTs, BnaMET1s, BnaDRMs and BnaDNMT2s) and three (BnaDMEs, BnaDML3s and BnaROS1s) subfamilies, respectively. Further analysis of gene structure and conserved domains showed that each sub-class is highly conserved between rapeseed and Arabidopsis. Expression analysis conducted by RNA-seq as well as qRT-PCR suggested that these DNA methylation/demethylation-related genes may be involved in the heat/salt stress responses in rapeseed. Taken together, our findings may provide valuable information for future functional characterization of these two types of epigenetic regulatory enzymes in polyploid species such as rapeseed, as well as for analyzing their evolutionary relationships within the plant kingdom.

Relative Efficiency of Recognition of 5-Methylcytosine and 5-Hydroxymethylcytosine by Methyl-Dependent DNA Endonuclease GlaI

Only a limited number of tools are available to study cytosine methylation in DNA. One of the representatives of the recently discovered methyl-dependent DNA endonucleases is an enzyme GlaI. It is of great interest for determining the methylation status of eukaryotic genomic DNA due to its ability to cleave only methylated DNA. However, the ability of the GlaI endonuclease to recognize oxidized derivatives of 5-methylcytosine (mC), in particular another epigenetic base, 5-hydroxymethylcytosine (hmC), has not yet been characterized. It is not possible to fully use the potential of GlaI in the analysis of methylation due to the notable occurrence of the latter in the DNA of mammals. In this study, the efficiency of cleavage of DNA substrates with various combinations of mC and hmC by methyl-dependent DNA-endonuclease GlaI was compared; the kinetic parameters of cleavage reactions for fully methylated and fully hydroxymethylated recognition site were determined. It was shown that in most cases GlaI recognized substrates containing mC better than substrates containing hmC in the same positions. The most effective hydrolysis of substrates containing modifications in the sequence 5'-GCGC-3'/3'-CGCG-5' required the presence of hmC not only in the central but also in the edge positions in both DNA chains as in the case of mC.

DNA methyltransferase expression and DNA hypomethylation status in human hepatocytes following trichloroacetic acid exposure

Trichloroacetic acid (TCA) is one of the major metabolites of trichloroethylene (TCE) as the significant factor of environmental and occupational pollution. TCA has been shown to induce a series of epigenetic mutation in mouse liver. However, the epigenetic cytotoxicity of TCA is still in infancy. In this study, we explored the cellular biological characteristics, the genome DNA methylation status and the expression profile of DNA methyltransferases in human hepatic L-02 cells treated with TCA with certain time and dose effects. The cell cycle measured by flow cytometry revealed an increasing S + G2 (M) phase of TCA (0.9 mM 24 h, 48 h and 72 h) treated cells after a recovery day, and sub-G1 phase was not appeared. The levels of 5 -mC were decreased in TCA (0.9 mM 24 h and 72 h) treated cells by 5-mC immunolocalization process and HPCE (decreased from 27.2% to 50.1% respectively). Meanwhile, the mCpG% in normal L-02 cells and TCA (0.9 mM 48 h) treated cells was 79.6% ± 6.5% and 50.8% ± 3.8%, respectively (P < 0.05). It also revealed that treatment of L-02 cells with TCA induced decreased in DNMT1 and DNMT3a mRNA and protein levels with a time-dependent manner and a dose-response relationship, while DNMT3b had no obvious change. These results establish a link between DNA methyltransferases and Genome DNA hypomethylation, which is associated with TCA exposure.

Developmental Dioxin Exposure Alters the Methylome of Adult Male Zebrafish Gonads.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a persistent environmental toxicant and endocrine disrupting compound with reproductive and developmental effects in humans and model organisms, including zebrafish. Our previous microarray and histological studies found defects in spermatogenesis and fertility of zebrafish in response to acute developmental TCDD exposure. These effects are apparent following exposure during reproductive development, modeling fetal basis of adult-onset disease. Some outcomes of these previous studies (reduced fertility, changes in sex ratio, transcriptomic alterations) are also transgenerational – persisting to unexposed generations – through the male germline. We hypothesized that DNA methylation could be a possible mechanism for these reproductive effects and performed whole genome bisulfite sequencing (WGBS), which identifies whole genome DNA methylation status at the base pair level, on testes of adult zebrafish exposed to TCDD (two separate hour-long exposures to 50 pg/mL TCDD at 3 and 7 weeks post fertilization). In response to TCDD exposure, multiple genes were differentially methylated; many of which are involved in reproductive processes or epigenetic modifications, suggesting a role of DNA methylation in later-life health outcomes. Additionally, several differentially methylated genes corresponded with gene expression changes identified in TCDD-exposed zebrafish testes, indicating a potential link between DNA methylation and gene expression. Ingenuity pathway analysis of WGBS and microarray data revealed genes involved in reproductive processes and development, RNA regulation, the cell cycle, and cellular morphology and development. We conclude that site-specific changes in DNA methylation of adult zebrafish testes occur in response to acute developmental TCDD exposure.

Cucurbitacin B Induces Hypermethylation of Oncogenes in Breast Cancer Cells.

Breast cancer is a complex disease driven by multiple factors including both genetic and epigenetic alterations. Recent studies revealed that abnormal gene expression induced by epigenetic changes including aberrant promoter methylation plays a critical role in human breast carcinogenesis. Cucurbitacin B has antiproliferative activity against various human breast cancer cells, but the molecular mechanism is not completely understood. In this study, we explore the influence of cucurbitacin B from Trichosanthes cucumerina on the methylation status at the promoter of oncogenes c-Myc, cyclin D1, and survivin in breast cancer cell lines. Growth inhibitory effect of cucurbitacin B on breast cancer cells was assessed by MTT assay and colony formation assay. Methylation status of genomic DNA was determined by methylation-specific PCR. Gene and protein expression levels of all genes studied were analyzed by real-time RT-PCR and western blot. The results indicated that cucurbitacin B could inhibit cell growth in breast cancer cells. The oncogene promoters are usually hypomethylated in cancer cells. Upon cucurbitacin B treatment, upregulation of DNMT1 and obvious heavy methylation in the promoters of c-Myc, cyclin D1, and survivin, which consequently downregulated the expression of all these oncogenes, were observed. Hence, cucurbitacin B proved to be a potential cancer therapeutic agent, in part by inducing hypermethylation and silences the oncogenic activation.

DNA methylation affects the lifespan of honey bee (Apis mellifera L.) workers – Evidence for a regulatory module that involves vitellogenin expression but is independent of juvenile hormone function

The canonic regulatory module for lifespan of honey bee (Apis mellifera) workers involves a mutual repressor relationship between juvenile hormone (JH) and vitellogenin (Vg). Compared to vertebrates, however, little is known about a possible role of epigenetic factors. The full genomic repertoire of DNA methyltransferases (DNMTs) makes the honey bee an attractive emergent model for studying the role of epigenetics in the aging process of invertebrates, and especially so in social insects. We first quantified the transcript levels of the four DNMTs encoding genes in the head thorax and abdomens of workers of different age, showing that dnmt1a and dnmt3 expression is up-regulated in abdomens of old workers, whereas dnmt1b and dnmt2 are down-regulated in heads of old workers. Pharmacological genome demethylation by RG108 treatment caused an increase in worker lifespan. Next, we showed that the genomic DNA methylation status indirectly affects vitellogenin gene expression both in vitro and in vivo in young workers, and that this occurs independent of caloric restriction or JH levels, suggesting that a non-canonical circuitry may be acting in parallel with the JH/Vg module to regulate the adult life cycle of honey bee workers. Our data provide evidence that epigenetic factors play a role in regulatory networks associated with complex life history traits of a social insect.

DNA methylation dysregulations in valvular atrial fibrillation.

The epigenetic changes underlying the development of atrial fibrillation (AF) remain incompletely understood. Limited evidence suggests that abnormal DNA methylation might be involved in the pathogenesis of AF. In the present study, we evaluated the methylation status of genomic DNA from myocardial tissue in AF patients and sinus rhythm (SR) patients systematically. DNA methylation dysregulations will be associated with valvular AF. Right atrial myocardial tissue was obtained from rheumatic valvular patients who had undergone valve replacement surgery (SR group, n = 10; AF group, n = 10). The global DNA methylation level, the promoter methylation level of the natriuretic peptide receptor-A gene (NPRA), and its correlation with the mRNA expression level of DNA methyltransferase genes were detected. The global DNA methylation level was significantly higher in the AF group than in the SR group (P < 0.05). The NPRA mRNA expression was decreased and the NPRA gene was hypermethylated in the AF group (P < 0.05). Meanwhile, the NPRA mRNA expression level has a negative correlation with the mean methylation level in the promoter region of the NPRA gene. DNA methylation dysregulations may be relevant in the pathogenesis of AF. DNA methyltransferase 3B likely plays an essential role in the DNA methylation dysregulations in AF.

Molecular cloning of chicken TET family genes and role of chicken TET1 in erythropoiesis

Ten-eleven translocation (TET) methylcytosine dioxygenase has potential as an active eraser to regulate the genomic DNA methylation status. We herein cloned chicken TET (cTET) family genes, and confirmed their functions. Quantitative reverse-transcription PCR showed that cTET1 was strongly expressed in erythrocytes throughout development. This cTET1 expression pattern, together with the results of methylated or hydroxymethylated DNA immunoprecipitation, suggests that cTET1 contributes to demethylation around the promoter region of the definitive-type β-globin gene βΑ in erythroid cells. The knockdown of cTET1 in T2ECs chicken erythroid progenitor cells suppressed the induction of βΑ expression under differentiation conditions. These results suggest that cTET1 plays an important role in erythroid cell differentiation.

Skin score correlates with global DNA methylation and GSTO1 A140D polymorphism in arsenic-affected population of Eastern India

Arsenic is a potent environmental toxicant causing serious public health concerns in India, Bangladesh and other parts of the world. Gene- and promoter-specific hypermethylation has been reported in different arsenic-exposed cell lines, whereas whole genome DNA methylation study suggested genomic hypo- and hypermethylation after arsenic exposure in in vitro and in vivo studies. Along with other characteristic biomarkers, arsenic toxicity leads to typical skin lesions. The present study demonstrates significant correlation between severities of skin manifestations with their whole genome DNA methylation status as well as with a particular polymorphism (Ala 140 Asp) status in arsenic metabolizing enzyme Glutathione S-transferase Omega-1 (GSTO1) in arsenic-exposed population of the district of Nadia, West Bengal, India.

Genome-wide DNA methylation profiles of maternal peripheral blood and placentas: potential risk factors for preeclampsia and validation of GRK5

Hypoxic placentation has been considered as a key step for the development of preeclampsia (PE); however, the underlying epigenetic mechanisms are still not fully understood. The purpose of this study is to investigate the whole genome DNA methylation status of PE. A microarray analysis using the Infinium HumanMethylation450 BeadChip assay in the placentas and maternal peripheral blood (PB) from PE patients and normal controls was performed. For validation, a quantitative RT-PCR analysis was used. Maternal PB showed 71 differentially methylated CpG loci (44 hypermethylated and 27 hypomethylated), while placenta revealed 365 loci (37 hypermethylated and 328 hypomethylated) at the statistical significance level of |Δβ| ≥ 0.17 and P ≤ 0.01. Notably, among the candidates showing significant signals, GRK5 (a member of G protein-coupled receptor kinase family that has previously been known to be associated with PE) showed a significantly hypomethylated level in the placentas of PE patients (Δβ = −0.176, P = 2.8 × 10−5). In the validation for the potential effect of GRK5 methylation on the gene regulation, GRK5 expression was significantly increased in the placentas from PE patients compared to those from controls (P = 0.027). In further GO analysis, genes of MHC class II protein complex showed the most significant differential methylation in the maternal PB of PE patients, while genes of palate development were differentially methylated in the placenta. Although further replication and functional studies are required, our preliminary results suggest that PE has distinct DNA methylation profiles in the maternal PB and placentas, which may provide insight into future research.