Epigenetic contribution to successful polyploidizations: variation in global cytosine methylation along an extensive ploidy series in Dianthus broteri (Caryophyllaceae).

Science.gov (United States)

Alonso, Conchita; Balao, Francisco; Bazaga, Pilar; Pérez, Ricardo

2016-11-01

Polyploidization is a significant evolutionary force in plants which involves major genomic and genetic changes, frequently regulated by epigenetic factors. We explored whether natural polyploidization in Dianthus broteri complex resulted in substantial changes in global DNA cytosine methylation associated to ploidy. Global cytosine methylation was estimated by high-performance liquid chromatography (HPLC) in 12 monocytotypic populations with different ploidies (2×, 4×, 6×, 12×) broadly distributed within D. broteri distribution range. The effects of ploidy level and local variation on methylation were assessed by generalized linear mixed models (GLMMs). Dianthus broteri exhibited a higher methylation percent (˜33%) than expected by its monoploid genome size and a large variation among study populations (range: 29.3-35.3%). Global methylation tended to increase with ploidy but did not significantly differ across levels due to increased variation within the highest-order polyploidy categories. Methylation varied more among hexaploid and dodecaploid populations, despite such cytotypes showing more restricted geographic location and increased genetic relatedness than diploids and tetraploids. In this study, we demonstrate the usefulness of an HPLC method in providing precise and genome reference-free global measure of DNA cytosine methylation, suitable to advance current knowledge of the roles of this epigenetic mechanism in polyploidization processes. © 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.

Genome-wide nucleosome map and cytosine methylation levels of an ancient human genome

DEFF Research Database (Denmark)

Pedersen, Jakob Skou; Valen, Eivind; Velazquez, Amhed Missael Vargas

2014-01-01

Epigenetic information is available from contemporary organisms, but is difficult to track back in evolutionary time. Here, we show that genome-wide epigenetic information can be gathered directly from next-generation sequence reads of DNA isolated from ancient remains. Using the genome sequence...... data generated from hair shafts of a 4000-yr-old Paleo-Eskimo belonging to the Saqqaq culture, we generate the first ancient nucleosome map coupled with a genome-wide survey of cytosine methylation levels. The validity of both nucleosome map and methylation levels were confirmed by the recovery...

Dynamic DNA cytosine methylation in the Populus trichocarpa genome: tissue-level variation and relationship to gene expression

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Vining Kelly J

2012-01-01

Full Text Available Abstract Background DNA cytosine methylation is an epigenetic modification that has been implicated in many biological processes. However, large-scale epigenomic studies have been applied to very few plant species, and variability in methylation among specialized tissues and its relationship to gene expression is poorly understood. Results We surveyed DNA methylation from seven distinct tissue types (vegetative bud, male inflorescence [catkin], female catkin, leaf, root, xylem, phloem in the reference tree species black cottonwood (Populus trichocarpa. Using 5-methyl-cytosine DNA immunoprecipitation followed by Illumina sequencing (MeDIP-seq, we mapped a total of 129,360,151 36- or 32-mer reads to the P. trichocarpa reference genome. We validated MeDIP-seq results by bisulfite sequencing, and compared methylation and gene expression using published microarray data. Qualitative DNA methylation differences among tissues were obvious on a chromosome scale. Methylated genes had lower expression than unmethylated genes, but genes with methylation in transcribed regions ("gene body methylation" had even lower expression than genes with promoter methylation. Promoter methylation was more frequent than gene body methylation in all tissues except male catkins. Male catkins differed in demethylation of particular transposable element categories, in level of gene body methylation, and in expression range of genes with methylated transcribed regions. Tissue-specific gene expression patterns were correlated with both gene body and promoter methylation. Conclusions We found striking differences among tissues in methylation, which were apparent at the chromosomal scale and when genes and transposable elements were examined. In contrast to other studies in plants, gene body methylation had a more repressive effect on transcription than promoter methylation.

Cytosine methylation dysregulation in neonates following intrauterine growth restriction.

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Francine Einstein

2010-01-01

Full Text Available Perturbations of the intrauterine environment can affect fetal development during critical periods of plasticity, and can increase susceptibility to a number of age-related diseases (e.g., type 2 diabetes mellitus; T2DM, manifesting as late as decades later. We hypothesized that this biological memory is mediated by permanent alterations of the epigenome in stem cell populations, and focused our studies specifically on DNA methylation in CD34+ hematopoietic stem and progenitor cells from cord blood from neonates with intrauterine growth restriction (IUGR and control subjects.Our epigenomic assays utilized a two-stage design involving genome-wide discovery followed by quantitative, single-locus validation. We found that changes in cytosine methylation occur in response to IUGR of moderate degree and involving a restricted number of loci. We also identify specific loci that are targeted for dysregulation of DNA methylation, in particular the hepatocyte nuclear factor 4alpha (HNF4A gene, a well-known diabetes candidate gene not previously associated with growth restriction in utero, and other loci encoding HNF4A-interacting proteins.Our results give insights into the potential contribution of epigenomic dysregulation in mediating the long-term consequences of IUGR, and demonstrate the value of this approach to studies of the fetal origin of adult disease.

Evidence Suggesting Absence of Mitochondrial DNA Methylation

DEFF Research Database (Denmark)

Mechta, Mie; Ingerslev, Lars R; Fabre, Odile

2017-01-01

, 16S, ND5 and CYTB, suggesting that mtDNA supercoiled structure blocks the access to bisulfite conversion. Here, we identified an artifact of mtDNA bisulfite sequencing that can lead to an overestimation of mtDNA methylation levels. Our study supports that cytosine methylation is virtually absent...

Cytosine Methylation Dysregulation in Neonates Following Intrauterine Growth Restriction

Science.gov (United States)

Bhagat, Tushar D.; Fazzari, Melissa J.; Verma, Amit; Barzilai, Nir; Greally, John M.

2010-01-01

Background Perturbations of the intrauterine environment can affect fetal development during critical periods of plasticity, and can increase susceptibility to a number of age-related diseases (e.g., type 2 diabetes mellitus; T2DM), manifesting as late as decades later. We hypothesized that this biological memory is mediated by permanent alterations of the epigenome in stem cell populations, and focused our studies specifically on DNA methylation in CD34+ hematopoietic stem and progenitor cells from cord blood from neonates with intrauterine growth restriction (IUGR) and control subjects. Methods and Findings Our epigenomic assays utilized a two-stage design involving genome-wide discovery followed by quantitative, single-locus validation. We found that changes in cytosine methylation occur in response to IUGR of moderate degree and involving a restricted number of loci. We also identify specific loci that are targeted for dysregulation of DNA methylation, in particular the hepatocyte nuclear factor 4α (HNF4A) gene, a well-known diabetes candidate gene not previously associated with growth restriction in utero, and other loci encoding HNF4A-interacting proteins. Conclusions Our results give insights into the potential contribution of epigenomic dysregulation in mediating the long-term consequences of IUGR, and demonstrate the value of this approach to studies of the fetal origin of adult disease. PMID:20126273

Epigenetic variation, inheritance, and parent-of-origin effects of cytosine methylation in maize (Zea mays).

Science.gov (United States)

Lauria, Massimiliano; Piccinini, Sara; Pirona, Raul; Lund, Gertrud; Viotti, Angelo; Motto, Mario

2014-03-01

Pure epigenetic variation, or epigenetic variation that is independent of genetic context, may provide a mechanism for phenotypic variation in the absence of DNA mutations. To estimate the extent of pure epigenetic variation within and across generations and to identify the DNA regions targeted, a group of eight plants derived from a highly inbred line of maize (Zea mays) was analyzed by the methylation-sensitive amplified polymorphism (MSAP) technique. We found that cytosine methylation (mC) differences among individuals accounted for up to 7.4% of CCGG sites investigated by MSAP. Of the differentially methylated fragments (DMFs) identified in the S0 generation, ∼12% were meiotically inherited for at least six generations. We show that meiotically heritable mC variation was consistently generated for an average of 0.5% CCGG sites per generation and that it largely occurred somatically. We provide evidence that mC variation can be established and inherited in a parent-of-origin manner, given that the paternal lineage is more prone to both forward and reverse mC changes. The molecular characterization of selected DMFs revealed that the variation was largely determined by CG methylation changes that map within gene regions. The expression analysis of genes overlapping with DMFs did not reveal an obvious correlation between mC variation and transcription, reinforcing the idea that the primary function of gene-body methylation is not to control gene expression. Because this study focuses on epigenetic variation in field-grown plants, the data presented herein pertain to spontaneous epigenetic changes of the maize genome in a natural context.

Evidence that steroid 5alpha-reductase isozyme genes are differentially methylated in human lymphocytes.

Science.gov (United States)

Rodríguez-Dorantes, M; Lizano-Soberón, M; Camacho-Arroyo, I; Calzada-León, R; Morimoto, S; Téllez-Ascencio, N; Cerbón, M A

2002-03-01

The synthesis of dihydrotestosterone (DHT) is catalyzed by steroid 5alpha-reductase isozymes 1 and 2, and this function determines the development of the male phenotype during embriogenesis and the growth of androgen sensitive tissues during puberty. The aim of this study was to determine the cytosine methylation status of 5alpha-reductase isozymes types 1 and 2 genes in normal and in 5alpha-reductase deficient men. Genomic DNA was obtained from lymphocytes of both normal subjects and patients with primary 5alpha-reductase deficiency due to point mutations in 5alpha-reductase 2 gene. Southern blot analysis of 5alpha-reductase types 1 and 2 genes from DNA samples digested with HpaII presented a different cytosine methylation pattern compared to that observed with its isoschizomer MspI, indicating that both genes are methylated in CCGG sequences. The analysis of 5alpha-reductase 1 gene from DNA samples digested with Sau3AI and its isoschizomer MboI which recognize methylation in GATC sequences showed an identical methylation pattern. In contrast, 5alpha-reductase 2 gene digested with Sau3AI presented a different methylation pattern to that of the samples digested with MboI, indicating that steroid 5alpha-reductase 2 gene possess methylated cytosines in GATC sequences. Analysis of exon 4 of 5alpha-reductase 2 gene after metabisulfite PCR showed that normal and deficient subjects present a different methylation pattern, being more methylated in patients with 5alpha-reductase 2 mutated gene. The overall results suggest that 5alpha-reductase genes 1 and 2 are differentially methylated in lymphocytes from normal and 5alpha-reductase deficient patients. Moreover, the extensive cytosine methylation pattern observed in exon 4 of 5alpha-reductase 2 gene in deficient patients, points out to an increased rate of mutations in this gene.

Isoschizomers and amplified fragment length polymorphism for the detection of specific cytosine methylation changes.

Science.gov (United States)

Ruiz-García, Leonor; Cabezas, Jose Antonio; de María, Nuria; Cervera, María-Teresa

2010-01-01

Different molecular techniques have been developed to study either the global level of methylated cytosines or methylation at specific gene sequences. One of them is a modification of the Amplified Fragment Length Polymorphism (AFLP) technique that has been used to study methylation of anonymous CCGG sequences in different fungi, plant and animal species. The main variation of this technique is based on the use of isoschizomers with different methylation sensitivity (such as HpaII and MspI) as a frequent cutter restriction enzyme. For each sample, AFLP analysis is performed using both EcoRI/HpaII and EcoRI/MspI digested samples. Comparative analysis between EcoRI/HpaII and EcoRI/MspI fragment patterns allows the identification of two types of polymorphisms: (1) "Methylation-insensitive polymorphisms" that show common EcoRI/HpaII and EcoRI/MspI patterns but are detected as polymorphic amplified fragments among samples; and (2) "Methylation-sensitive polymorphisms" that are associated with amplified fragments differing in their presence or absence or in their intensity between EcoRI/HpaII and EcoRI/MspI patterns. This chapter describes a detailed protocol of this technique and discusses modifications that can be applied to adjust the technology to different species of interest.

Pleiotropic phenotypes of the salt-tolerant and cytosine ...

Indian Academy of Sciences (India)

2SKA Institution for Research, Education and Development (SKAIRED), 4/11 Sarv Priya Vihar, ... wild-type parent cv Nirmal were characterized for overall cytosine methylation at .... (ii) altered in the expression of genes involved in the perfor-.

Identification of Differentially Methylated Sites with Weak Methylation Effects

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Hong Tran

2018-02-01

Full Text Available Deoxyribonucleic acid (DNA methylation is an epigenetic alteration crucial for regulating stress responses. Identifying large-scale DNA methylation at single nucleotide resolution is made possible by whole genome bisulfite sequencing. An essential task following the generation of bisulfite sequencing data is to detect differentially methylated cytosines (DMCs among treatments. Most statistical methods for DMC detection do not consider the dependency of methylation patterns across the genome, thus possibly inflating type I error. Furthermore, small sample sizes and weak methylation effects among different phenotype categories make it difficult for these statistical methods to accurately detect DMCs. To address these issues, the wavelet-based functional mixed model (WFMM was introduced to detect DMCs. To further examine the performance of WFMM in detecting weak differential methylation events, we used both simulated and empirical data and compare WFMM performance to a popular DMC detection tool methylKit. Analyses of simulated data that replicated the effects of the herbicide glyphosate on DNA methylation in Arabidopsis thaliana show that WFMM results in higher sensitivity and specificity in detecting DMCs compared to methylKit, especially when the methylation differences among phenotype groups are small. Moreover, the performance of WFMM is robust with respect to small sample sizes, making it particularly attractive considering the current high costs of bisulfite sequencing. Analysis of empirical Arabidopsis thaliana data under varying glyphosate dosages, and the analysis of monozygotic (MZ twins who have different pain sensitivities—both datasets have weak methylation effects of <1%—show that WFMM can identify more relevant DMCs related to the phenotype of interest than methylKit. Differentially methylated regions (DMRs are genomic regions with different DNA methylation status across biological samples. DMRs and DMCs are essentially the same

No Effect of Folic Acid Supplementation on Global DNA Methylation in Men and Women with Moderately Elevated Homocysteine

NARCIS (Netherlands)

Jung, A.Y.; Smulders, Y.; Verhoef, P.; Kok, F.J.; Blom, H.; Kok, R.M.; Kampman, E.; Durga, J.

2011-01-01

A global loss of cytosine methylation in DNA has been implicated in a wide range of diseases. There is growing evidence that modifications in DNA methylation can be brought about by altering the intake of methyl donors such as folate. We examined whether long-term daily supplementation with 0.8 mg

No effect of folic acid supplementation on global DNA methylation in men and women with moderately elevated homocysteine

NARCIS (Netherlands)

Jung, A.Y.; Smulders, Y.; Verhoef, P.; Kok, F.J.; Blom, H.J.; Kok, R.M.; Kampman, E.; Durga, J.

2011-01-01

A global loss of cytosine methylation in DNA has been implicated in a wide range of diseases. There is growing evidence that modifications in DNA methylation can be brought about by altering the intake of methyl donors such as folate. We examined whether long-term daily supplementation with 0.8 mg

Methylation-Specific PCR Unraveled

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Sarah Derks

2004-01-01

Full Text Available Methylation‐specific PCR (MSP is a simple, quick and cost‐effective method to analyze the DNA methylation status of virtually any group of CpG sites within a CpG island. The technique comprises two parts: (1 sodium bisulfite conversion of unmethylated cytosine's to uracil under conditions whereby methylated cytosines remains unchanged and (2 detection of the bisulfite induced sequence differences by PCR using specific primer sets for both unmethylated and methylated DNA. This review discusses the critical parameters of MSP and presents an overview of the available MSP variants and the (clinical applications.

Genomic change, retrotransposon mobilization and extensive cytosine methylation alteration in Brassica napus introgressions from two intertribal hybridizations.

Science.gov (United States)

Zhang, Xueli; Ge, Xianhong; Shao, Yujiao; Sun, Genlou; Li, Zaiyun

2013-01-01

Hybridization and introgression represent important means for the transfer and/or de novo origination of traits and play an important role in facilitating speciation and plant breeding. Two sets of introgression lines in Brassica napus L. were previously established by its intertribal hybridizations with two wild species and long-term selection. In this study, the methods of amplified fragment length polymorphisms (AFLP), sequence-specific amplification polymorphism (SSAP) and methylation-sensitive amplified polymorphism (MSAP) were used to determine their genomic change, retrotransposon mobilization and cytosine methylation alteration in these lines. The genomic change revealed by the loss or gain of AFLP bands occurred for ∼10% of the total bands amplified in the two sets of introgressions, while no bands specific for wild species were detected. The new and absent SSAP bands appeared for 9 out of 11 retrotransposons analyzed, with low frequency of new bands and their total percentage of about 5% in both sets. MSAP analysis indicated that methylation changes were common in these lines (33.4-39.8%) and the hypermethylation was more frequent than hypomethylation. Our results suggested that certain extents of genetic and epigenetic alterations were induced by hybridization and alien DNA introgression. The cryptic mechanism of these changes and potential application of these lines in breeding were also discussed.

Disclosing bias in bisulfite assay: MethPrimers underestimate high DNA methylation.

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Andrea Fuso

Full Text Available Discordant results obtained in bisulfite assays using MethPrimers (PCR primers designed using MethPrimer software or assuming that non-CpGs cytosines are non methylated versus primers insensitive to cytosine methylation lead us to hypothesize a technical bias. We therefore used the two kinds of primers to study different experimental models and methylation statuses. We demonstrated that MethPrimers negatively select hypermethylated DNA sequences in the PCR step of the bisulfite assay, resulting in CpG methylation underestimation and non-CpG methylation masking, failing to evidence differential methylation statuses. We also describe the characteristics of "Methylation-Insensitive Primers" (MIPs, having degenerated bases (G/A to cope with the uncertain C/U conversion. As CpG and non-CpG DNA methylation patterns are largely variable depending on the species, developmental stage, tissue and cell type, a variable extent of the bias is expected. The more the methylome is methylated, the greater is the extent of the bias, with a prevalent effect of non-CpG methylation. These findings suggest a revision of several DNA methylation patterns so far documented and also point out the necessity of applying unbiased analyses to the increasing number of epigenomic studies.

Effects of TET2 mutations on DNA methylation in chronic myelomonocytic leukemia

Science.gov (United States)

TET2 enzymatically converts 5-methyl-cytosine to 5-hydroxymethyl-cytosine, possibly leading to loss of DNA methylation. TET2 mutations are common in myeloid leukemia and were proposed to contribute to leukemogenesis through DNA methylation. To expand on this concept, we studied chronic myelomonocyti...

Genomic change, retrotransposon mobilization and extensive cytosine methylation alteration in Brassica napus introgressions from two intertribal hybridizations.

Directory of Open Access Journals (Sweden)

Xueli Zhang

Full Text Available Hybridization and introgression represent important means for the transfer and/or de novo origination of traits and play an important role in facilitating speciation and plant breeding. Two sets of introgression lines in Brassica napus L. were previously established by its intertribal hybridizations with two wild species and long-term selection. In this study, the methods of amplified fragment length polymorphisms (AFLP, sequence-specific amplification polymorphism (SSAP and methylation-sensitive amplified polymorphism (MSAP were used to determine their genomic change, retrotransposon mobilization and cytosine methylation alteration in these lines. The genomic change revealed by the loss or gain of AFLP bands occurred for ∼10% of the total bands amplified in the two sets of introgressions, while no bands specific for wild species were detected. The new and absent SSAP bands appeared for 9 out of 11 retrotransposons analyzed, with low frequency of new bands and their total percentage of about 5% in both sets. MSAP analysis indicated that methylation changes were common in these lines (33.4-39.8% and the hypermethylation was more frequent than hypomethylation. Our results suggested that certain extents of genetic and epigenetic alterations were induced by hybridization and alien DNA introgression. The cryptic mechanism of these changes and potential application of these lines in breeding were also discussed.

DNA methyltransferase homologue TRDMT1 in Plasmodium falciparum specifically methylates endogenous aspartic acid tRNA.

Science.gov (United States)

Govindaraju, Gayathri; Jabeena, C A; Sethumadhavan, Devadathan Valiyamangalath; Rajaram, Nivethika; Rajavelu, Arumugam

2017-10-01

In eukaryotes, cytosine methylation regulates diverse biological processes such as gene expression, development and maintenance of genomic integrity. However, cytosine methylation and its functions in pathogenic apicomplexan protozoans remain enigmatic. To address this, here we investigated the presence of cytosine methylation in the nucleic acids of the protozoan Plasmodium falciparum. Interestingly, P. falciparum has TRDMT1, a conserved homologue of DNA methyltransferase DNMT2. However, we found that TRDMT1 did not methylate DNA, in vitro. We demonstrate that TRDMT1 methylates cytosine in the endogenous aspartic acid tRNA of P. falciparum. Through RNA bisulfite sequencing, we mapped the position of 5-methyl cytosine in aspartic acid tRNA and found methylation only at C38 position. P. falciparum proteome has significantly higher aspartic acid content and a higher proportion of proteins with poly aspartic acid repeats than other apicomplexan pathogenic protozoans. Proteins with such repeats are functionally important, with significant roles in host-pathogen interactions. Therefore, TRDMT1 mediated C38 methylation of aspartic acid tRNA might play a critical role by translational regulation of important proteins and modulate the pathogenicity of the malarial parasite. Copyright © 2017 Elsevier B.V. All rights reserved.

Identification and characterization of the cytosine-5 DNA methyltransferase gene family in Salvia miltiorrhiza

OpenAIRE

Jiang Li; Caili Li; Shanfa Lu

2018-01-01

Cytosine DNA methylation is highly conserved epigenetic modification involved in a wide range of biological processes in eukaryotes. It was established and maintained by cytosine-5 DNA methyltransferases (C5-MTases) in plants. Through genome-wide identification, eight putative SmC5-MTase genes were identified from the genome of Salvia miltiorrhiza, a well-known traditional Chinese medicine material and an emerging model medicinal plant. Based on conserved domains and phylogenetic analysis, ei...

DNA Methylation: A Frontier in Tooth Organogenesis and Developmental Dental Defects.

Science.gov (United States)

Wan, Mian; Li, Hongyu; Zhou, Yachuan; Du, Wei; Xu, Xin; Ye, Ling; Zhou, Xuedong; Zheng, Liwei

2018-01-01

Tooth development relies on interactions between epithelial and mesenchymal tissues, which are controlled by sophisticated networks of conserved signaling. The signaling networks regulating odontogenesis have been well characterized, but the epigenetic mechanisms underlying remain to be elucidated. In this review, we describe current researches regarding the control of various genes expression by DNA methylation during odontogenesis, summarize genomic mapping of DNA methylation in various stages of tooth formation and diverse dental tissues by high-throughput approaches, and highlight the roles of DNA methylation in odontogenesis. Researches on mammals have revealed that the genomic methylation, which occurs on cytosine residues, regulates certain genes transcription. Consequently, DNA methylation plays a crucial role in spatiotemporal organization of signaling pathways, and is essential for organogenesis. Recently, mounting evidence proves that methylation of genomes contributes to the spatiotemporal gene dynamics during odontogenesis. With emerging new technologies of mapping cytosine modifications in global genome, investigators are seeking an overall view of DNA methylome dynamics that characterize genetic information to manifest across incredibly varied tooth development stages, dental tissues, and developmental dental defects. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Epigenetic regulation during fetal femur development: DNA methylation matters.

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María C de Andrés

Full Text Available Epigenetic modifications are heritable changes in gene expression without changes in DNA sequence. DNA methylation has been implicated in the control of several cellular processes including differentiation, gene regulation, development, genomic imprinting and X-chromosome inactivation. Methylated cytosine residues at CpG dinucleotides are commonly associated with gene repression; conversely, strategic loss of methylation during development could lead to activation of lineage-specific genes. Evidence is emerging that bone development and growth are programmed; although, interestingly, bone is constantly remodelled throughout life. Using human embryonic stem cells, human fetal bone cells (HFBCs, adult chondrocytes and STRO-1(+ marrow stromal cells from human bone marrow, we have examined a spectrum of developmental stages of femur development and the role of DNA methylation therein. Using pyrosequencing methodology we analysed the status of methylation of genes implicated in bone biology; furthermore, we correlated these methylation levels with gene expression levels using qRT-PCR and protein distribution during fetal development evaluated using immunohistochemistry. We found that during fetal femur development DNA methylation inversely correlates with expression of genes including iNOS (NOS2 and COL9A1, but not catabolic genes including MMP13 and IL1B. Furthermore, significant demethylation was evident in the osteocalcin promoter between the fetal and adult developmental stages. Increased TET1 expression and decreased expression of DNA (cytosine-5--methyltransferase 1 (DNMT1 in adult chondrocytes compared to HFBCs could contribute to the loss of methylation observed during fetal development. HFBC multipotency confirms these cells to be an ideal developmental system for investigation of DNA methylation regulation. In conclusion, these findings demonstrate the role of epigenetic regulation, specifically DNA methylation, in bone development

Radiation effects on DNA methylation in mice

International Nuclear Information System (INIS)

Komura, J.; Kurishita, A.; Miyamura, Y.; Ono, T.; Tawa, R.; Sakurai, H.

1992-01-01

Effects of ionizing radiation on DNA methylation in liver, brain and spleen were examined by high performance liquid chromatography (HPLC). The total methylated cytosine level in the genome was reduced within 8 hours after 3.8 Gy of irradiation in liver of adult mice. But no appreciable effect was observed in brain and spleen. When mice were irradiated at newborn, liver DNA revealed no change in methylated cytosine level. Even though slight effects of radiation were detected in he methylation of the c-myc and c-fos genes, they were only temporary and no long-term effects were observed. These data suggest that the effect of radiation on DNA methylation in vivo is not prevailing a DNA damage, but rather influenced much through biological parameters. (author)

Determination of DNA methylation associated with Acer rubrum (red maple) adaptation to metals: analysis of global DNA modifications and methylation-sensitive amplified polymorphism.

Science.gov (United States)

Kim, Nam-Soo; Im, Min-Ji; Nkongolo, Kabwe

2016-08-01

Red maple (Acer rubum), a common deciduous tree species in Northern Ontario, has shown resistance to soil metal contamination. Previous reports have indicated that this plant does not accumulate metals in its tissue. However, low level of nickel and copper corresponding to the bioavailable levels in contaminated soils in Northern Ontario causes severe physiological damages. No differentiation between metal-contaminated and uncontaminated populations has been reported based on genetic analyses. The main objective of this study was to assess whether DNA methylation is involved in A. rubrum adaptation to soil metal contamination. Global cytosine and methylation-sensitive amplified polymorphism (MSAP) analyses were carried out in A. rubrum populations from metal-contaminated and uncontaminated sites. The global modified cytosine ratios in genomic DNA revealed a significant decrease in cytosine methylation in genotypes from a metal-contaminated site compared to uncontaminated populations. Other genotypes from a different metal-contaminated site within the same region appear to be recalcitrant to metal-induced DNA alterations even ≥30 years of tree life exposure to nickel and copper. MSAP analysis showed a high level of polymorphisms in both uncontaminated (77%) and metal-contaminated (72%) populations. Overall, 205 CCGG loci were identified in which 127 were methylated in either outer or inner cytosine. No differentiation among populations was established based on several genetic parameters tested. The variations for nonmethylated and methylated loci were compared by analysis of molecular variance (AMOVA). For methylated loci, molecular variance among and within populations was 1.5% and 13.2%, respectively. These values were low (0.6% for among populations and 5.8% for within populations) for unmethylated loci. Metal contamination is seen to affect methylation of cytosine residues in CCGG motifs in the A. rubrum populations that were analyzed.

Analysis of DNA methylation related to rice adult plant resistance to bacterial blight based on methylation-sensitive AFLP (MSAP) analysis.

Science.gov (United States)

Sha, A H; Lin, X H; Huang, J B; Zhang, D P

2005-07-01

DNA methylation is known to play an important role in the regulation of gene expression in eukaryotes. The rice cultivar Wase Aikoku 3 becomes resistant to the blight pathogen Xanthomonas oryzae pv. oryzae at the adult stage. Using methylation-sensitive amplified polymorphism (MSAP) analysis, we compared the patterns of cytosine methylation in seedlings and adult plants of the rice cultivar Wase Aikoku 3 that had been inoculated with the pathogen Xanthomonas oryzae pv. oryzae, subjected to mock inoculation or left untreated. In all, 2000 DNA fragments, each representing a recognition site cleaved by either or both of two isoschizomers, were amplified using 60 pairs of selective primers. A total of 380 sites were found to be methylated. Of these, 45 showed differential cytosine methylation among the seedlings and adult plants subjected to different treatments, and overall levels of methylation were higher in adult plants than in seedlings. All polymorphic fragments were sequenced, and six showed homology to genes that code for products of known function. Northern analysis of three fragments indicated that their expression varied with methylation pattern, with hypermethylation being correlated with repression of transcription, as expected. The results suggest that significant differences in cytosine methylation exist between seedlings and adult plants, and that hypermethylation or hypomethylation of specific genes may be involved in the development of adult plant resistance (APR) in rice plants.

Microhydration of cytosine and its radical anion: Cytosine.(H2O)n (n=1-5)

Science.gov (United States)

Kim, Sunghwan; Schaefer, Henry F.

2007-02-01

Microhydration effects on cytosine and its radical anion have been investigated theoretically, by explicitly considering various structures of cytosine complexes with up to five water molecules. Each successive water molecule (through n =5) is bound by 7-10kcalmol-1 to the relevant cytosine complex. The hydration energies are uniformly higher for the analogous anion systems. While the predicted vertical detachment energy (VDE) of the isolated cytosine is only 0.48eV, it is predicted to increase to 1.27eV for the lowest-lying pentahydrate of cytosine. The adiabatic electron affinity (AEA) of cytosine was also found to increase from 0.03to0.61eV for the pentahydrate, implying that the cytosine anion, while questionable in the gas phase, is bound in aqueous solution. Both the VDE and AEA values for cytosine are smaller than those of uracil and thymine for a given hydration number. These results are in qualitative agreement with available experimental results from photodetachment-photoelectron spectroscopy studies of Schiedt et al. [Chem. Phys. 239, 511 (1998)].

Kismeth: Analyzer of plant methylation states through bisulfite sequencing

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Martienssen Robert A

2008-09-01

Full Text Available Abstract Background There is great interest in probing the temporal and spatial patterns of cytosine methylation states in genomes of a variety of organisms. It is hoped that this will shed light on the biological roles of DNA methylation in the epigenetic control of gene expression. Bisulfite sequencing refers to the treatment of isolated DNA with sodium bisulfite to convert unmethylated cytosine to uracil, with PCR converting the uracil to thymidine followed by sequencing of the resultant DNA to detect DNA methylation. For the study of DNA methylation, plants provide an excellent model system, since they can tolerate major changes in their DNA methylation patterns and have long been studied for the effects of DNA methylation on transposons and epimutations. However, in contrast to the situation in animals, there aren't many tools that analyze bisulfite data in plants, which can exhibit methylation of cytosines in a variety of sequence contexts (CG, CHG, and CHH. Results Kismeth http://katahdin.mssm.edu/kismeth is a web-based tool for bisulfite sequencing analysis. Kismeth was designed to be used with plants, since it considers potential cytosine methylation in any sequence context (CG, CHG, and CHH. It provides a tool for the design of bisulfite primers as well as several tools for the analysis of the bisulfite sequencing results. Kismeth is not limited to data from plants, as it can be used with data from any species. Conclusion Kismeth simplifies bisulfite sequencing analysis. It is the only publicly available tool for the design of bisulfite primers for plants, and one of the few tools for the analysis of methylation patterns in plants. It facilitates analysis at both global and local scales, demonstrated in the examples cited in the text, allowing dissection of the genetic pathways involved in DNA methylation. Kismeth can also be used to study methylation states in different tissues and disease cells compared to a reference sequence.

miRNAting control of DNA methylation

Indian Academy of Sciences (India)

DNA methylation is a type of epigenetic modification where a methyl group is added to the cytosine or adenine residue of a given DNA sequence. It has been observed that DNA methylation is achieved by some collaborative agglomeration of certain proteins and non-coding RNAs. The assembly of IDN2 and its ...

Effects of cytosine methylation on transcription factor binding sites

KAUST Repository

Medvedeva, Yulia A; Khamis, Abdullah M.; Kulakovskiy, Ivan V; Ba Alawi, Wail; Bhuyan, Md Shariful I; Kawaji, Hideya; Lassmann, Timo; Harbers, Matthias; Forrest, Alistair RR; Bajic, Vladimir B.

2014-01-01

Background: DNA methylation in promoters is closely linked to downstream gene repression. However, whether DNA methylation is a cause or a consequence of gene repression remains an open question. If it is a cause, then DNA methylation may affect

Methylation effect on the ohmic resistance of a poly-GC DNA-like chain

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Moura, F.A.B.F. de, E-mail: fidelis@fis.ufal.br [Instituto de Física, Universidade Federal de Alagoas, Maceió AL 57072-970 (Brazil); Lyra, M.L. [Instituto de Física, Universidade Federal de Alagoas, Maceió AL 57072-970 (Brazil); Almeida, M.L. de; Ourique, G.S.; Fulco, U.L.; Albuquerque, E.L. [Departamento de Biofísica e Farmacologia, Universidade Federal do Rio Grande do Norte, 59072-970, Natal-RN (Brazil)

2016-10-14

We determine, by using a tight-binding model Hamiltonian, the characteristic current–voltage (IxV) curves of a 5-methylated cytosine single strand poly-GC DNA-like finite segment, considering the methyl groups attached laterally to a random fraction of the cytosine basis. Striking, we found that the methylation significantly impacts the ohmic resistance (R) of the DNA-like segments, indicating that measurements of R can be used as a biosensor tool to probe the presence of anomalous methylation. - Highlights: • Ohmic resistance of finite segments of poly-CG DNA-like segments. • Possibility for the development of biosensor devices. • Methylation effect and electronic transport in DNA-like segments.

DNA methylation inhibits expression and transposition of the Neurospora Tad retrotransposon.

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Zhou, Y; Cambareri, E B; Kinsey, J A

2001-06-01

Tad is a LINE-like retrotransposon of the filamentous fungus Neurospora crassa. We have analyzed both expression and transposition of this element using strains with a single copy of Tad located in the 5' noncoding sequences of the am (glutamate dehydrogenase) gene. Tad in this position has been shown to carry a de novo cytosine methylation signal which causes reversible methylation of both Tad and am upstream sequences. Here we find that methylation of the Tad sequences inhibits both Tad expression and transposition. This inhibition can be relieved by the use of 5-azacytidine, a drug which reduces cytosine methylation, or by placing the Tad/am sequences in a dim-2 genetic background.

Holocaust Exposure Induced Intergenerational Effects on FKBP5 Methylation.

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Yehuda, Rachel; Daskalakis, Nikolaos P; Bierer, Linda M; Bader, Heather N; Klengel, Torsten; Holsboer, Florian; Binder, Elisabeth B

2016-09-01

The involvement of epigenetic mechanisms in intergenerational transmission of stress effects has been demonstrated in animals but not in humans. Cytosine methylation within the gene encoding for FK506 binding protein 5 (FKBP5) was measured in Holocaust survivors (n = 32), their adult offspring (n = 22), and demographically comparable parent (n = 8) and offspring (n = 9) control subjects, respectively. Cytosine-phosphate-guanine sites for analysis were chosen based on their spatial proximity to the intron 7 glucocorticoid response elements. Holocaust exposure had an effect on FKBP5 methylation that was observed in exposed parents as well in their offspring. These effects were observed at bin 3/site 6. Interestingly, in Holocaust survivors, methylation at this site was higher in comparison with control subjects, whereas in Holocaust offspring, methylation was lower. Methylation levels for exposed parents and their offspring were significantly correlated. In contrast to the findings at bin 3/site 6, offspring methylation at bin 2/sites 3 to 5 was associated with childhood physical and sexual abuse in interaction with an FKBP5 risk allele previously associated with vulnerability to psychological consequences of childhood adversity. The findings suggest the possibility of site specificity to environmental influences, as sites in bins 3 and 2 were differentially associated with parental trauma and the offspring's own childhood trauma, respectively. FKBP5 methylation averaged across the three bins examined was associated with wake-up cortisol levels, indicating functional relevance of the methylation measures. This is the first demonstration of an association of preconception parental trauma with epigenetic alterations that is evident in both exposed parent and offspring, providing potential insight into how severe psychophysiological trauma can have intergenerational effects. Published by Elsevier Inc.

Adenine N6-methylation in diverse fungi

NARCIS (Netherlands)

Seidl, Michael F.

2017-01-01

A DNA modification - methylation of cytosines and adenines - has important roles in diverse processes such as regulation of gene expression and genome stability, yet until recently adenine methylation had been considered to be only a hallmark of prokaryotes. A new study identifies abundant

Methods for detection of methyl-CpG dinucleotides

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Dunn, John J.

2012-09-11

The invention provides methods for enriching methyl-CpG sequences from a DNA sample. The method makes use of conversion of cytosine residues to uracil under conditions in which methyl-cytosine residues are preserved. Additional methods of the invention enable to preservation of the context of me-CpG dinucleotides. The invention also provides a recombinant, full length and substantially pure McrA protein (rMcrA) for binding and isolation of DNA fragments containing the sequence 5'-C.sup.MeCpGG-3'. Methods for making and using the rMcrA protein, and derivatives thereof are provided.

Comparison of methods for quantification of global DNA methylation in human cells and tissues.

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Sofia Lisanti

Full Text Available DNA methylation is a key epigenetic modification which, in mammals, occurs mainly at CpG dinucleotides. Most of the CpG methylation in the genome is found in repetitive regions, rich in dormant transposons and endogenous retroviruses. Global DNA hypomethylation, which is a common feature of several conditions such as ageing and cancer, can cause the undesirable activation of dormant repeat elements and lead to altered expression of associated genes. DNA hypomethylation can cause genomic instability and may contribute to mutations and chromosomal recombinations. Various approaches for quantification of global DNA methylation are widely used. Several of these approaches measure a surrogate for total genomic methyl cytosine and there is uncertainty about the comparability of these methods. Here we have applied 3 different approaches (luminometric methylation assay, pyrosequencing of the methylation status of the Alu repeat element and of the LINE1 repeat element for estimating global DNA methylation in the same human cell and tissue samples and have compared these estimates with the "gold standard" of methyl cytosine quantification by HPLC. Next to HPLC, the LINE1 approach shows the smallest variation between samples, followed by Alu. Pearson correlations and Bland-Altman analyses confirmed that global DNA methylation estimates obtained via the LINE1 approach corresponded best with HPLC-based measurements. Although, we did not find compelling evidence that the gold standard measurement by HPLC could be substituted with confidence by any of the surrogate assays for detecting global DNA methylation investigated here, the LINE1 assay seems likely to be an acceptable surrogate in many cases.

Direct evidence for sequence-dependent attraction between double-stranded DNA controlled by methylation.

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Yoo, Jejoong; Kim, Hajin; Aksimentiev, Aleksei; Ha, Taekjip

2016-03-22

Although proteins mediate highly ordered DNA organization in vivo, theoretical studies suggest that homologous DNA duplexes can preferentially associate with one another even in the absence of proteins. Here we combine molecular dynamics simulations with single-molecule fluorescence resonance energy transfer experiments to examine the interactions between duplex DNA in the presence of spermine, a biological polycation. We find that AT-rich DNA duplexes associate more strongly than GC-rich duplexes, regardless of the sequence homology. Methyl groups of thymine acts as a steric block, relocating spermine from major grooves to interhelical regions, thereby increasing DNA-DNA attraction. Indeed, methylation of cytosines makes attraction between GC-rich DNA as strong as that between AT-rich DNA. Recent genome-wide chromosome organization studies showed that remote contact frequencies are higher for AT-rich and methylated DNA, suggesting that direct DNA-DNA interactions that we report here may play a role in the chromosome organization and gene regulation.

Differential DNA Methylation Patterns Are Related to Phellogen Origin and Quality of Quercus suber Cork.

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Inácio, Vera; Barros, Pedro M; Costa, Augusta; Roussado, Cristóvão; Gonçalves, Elsa; Costa, Rita; Graça, José; Oliveira, M Margarida; Morais-Cecílio, Leonor

2017-01-01

DNA methylation is thought to influence Quercus suber cork quality, which is the main constraint for its economic valorisation. However, a deep knowledge of the cytosine methylation patterns disclosing the epigenetic variability of trees with different cork quality types is totally missing. This study investigates the hypothesis that variations in DNA methylation contribute to differences in cork cellular characteristics directly related to original or traumatic phellogen activity. We used MSAPs (Methylation Sensitive Amplified Polymorphism) to assess DNA methylation patterns of cork and leaf tissues of Q. suber adult trees growing in three cork oak stands. The relationship between the detected polymorphisms and the diversity of cork quality traits was explored by a marker-trait analysis focusing on the most relevant quality characteristics. Populations differed widely in cork quality, but only slightly in degree of epigenetic differentiation. Four MSAP markers (1.3% of the total) were significantly associated with the most noteworthy quality traits: wood inclusions (nails) and porosity. This evidence supports the potential role of cytosine methylation in the modulation of differential phellogen activity either involved in localized cell death or in pore production, resulting in different cork qualities. Although, the underlying basis of the methylation polymorphism of loci affecting cork quality traits remain unclear, the disclosure of markers statistically associated with cork quality strengthens the potential role of DNA methylation in the regulation of these traits, namely at the phellogen level.

Promoter methylation and age-related downregulation of Klotho in rhesus monkey.

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King, Gwendalyn D; Rosene, Douglas L; Abraham, Carmela R

2012-12-01

While overall DNA methylation decreases with age, CpG-rich areas of the genome can become hypermethylated. Hypermethylation near transcription start sites typically decreases gene expression. Klotho (KL) is important in numerous age-associated pathways including insulin/IGF1 and Wnt signaling and naturally decreases with age in brain, heart, and liver across species. Brain tissues from young and old rhesus monkeys were used to determine whether epigenetic modification of the KL promoter underlies age-related decreases in mRNA and protein levels of KL. The KL promoter in genomic DNA from brain white matter did not show evidence of oxidation in vivo but did exhibit an increase in methylation with age. Further analysis identified individual CpG motifs across the region of interest with increased methylation in old animals. In vitro methyl modification of these individual cytosine residues confirmed that methylation of the promoter can decrease gene transcription. These results provide evidence that changes in KL gene expression with age may, at least in part, be the result of epigenetic changes to the 5' regulatory region.

The Aorta-Gonad-Mesonephros Organ Culture Recapitulates 5hmC Reorganization and Replication-Dependent and Independent Loss of DNA Methylation in the Germline.

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Calvopina, Joseph Hargan; Cook, Helene; Vincent, John J; Nee, Kevin; Clark, Amander T

2015-07-01

Removal of cytosine methylation from the genome is critical for reprogramming and transdifferentiation and plays a central role in our understanding of the fundamental principles of embryo lineage development. One of the major models for studying cytosine demethylation is the mammalian germ line during the primordial germ cell (PGC) stage of embryo development. It is now understood that oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) is required to remove cytosine methylation in a locus-specific manner in PGCs; however, the mechanisms downstream of 5hmC are controversial and hypothesized to involve either active demethylation or replication-coupled loss. In the current study, we used the aorta-gonad-mesonephros (AGM) organ culture model to show that this model recapitulates germ line reprogramming, including 5hmC reorganization and loss of cytosine methylation from Snrpn and H19 imprinting control centers (ICCs). To directly address the hypothesis that cell proliferation is required for cytosine demethylation, we blocked PI3-kinase-dependent PGC proliferation and show that this leads to a G1 and G2/M cell cycle arrest in PGCs, together with retained levels of cytosine methylation at the Snrpn ICC, but not at the H19 ICC. Taken together, the AGM organ culture model is an important tool to evaluate mechanisms of locus-specific demethylation and the role of PI3-kinase-dependent PGC proliferation in the locus-specific removal of cytosine methylation from the genome.

Infraspecific DNA methylation polymorphism in cotton (Gossypium hirsutum L.).

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Keyte, Anna L; Percifield, Ryan; Liu, Bao; Wendel, Jonathan F

2006-01-01

Cytosine methylation is important in the epigenetic regulation of gene expression and development in plants and has been implicated in silencing duplicate genes after polyploid formation in several plant groups. Relatively little information exists, however, on levels and patterns of methylation polymorphism (MP) at homologous loci within species. Here we explored the levels and patterns of methylation-polymorphism diversity at CCGG sites within allotetraploid cotton, Gossypium hirsutum, using a methylation-sensitive amplified fragment length polymorphism screen and a selected set of 20 G. hirsutum accessions for which we have information on genetic polymorphism levels and relationships. Methylation and MP exist at high levels within G. hirsutum: of 150 HpaII/MspI sites surveyed, 48 were methylated at the inner cytosine (32%) and 32 of these were polymorphic (67%). Both these values are higher than comparable measures of genetic diversity using restriction fragment length polymorphisms. The high percentage of methylation-polymorphic sites and potential relationship to gene expression underscore the potential significance of MP within and among populations. We speculate that biased correlation of methylation-polymorphic sites and genes in cotton may be a consequence of polyploidy and the attendant doubling of all genes.

Quantification of 5-methyl-2'-deoxycytidine in the DNA.

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Giel-Pietraszuk, Małgorzata; Insińska-Rak, Małgorzata; Golczak, Anna; Sikorski, Marek; Barciszewska, Mirosława; Barciszewski, Jan

2015-01-01

Methylation at position 5 of cytosine (Cyt) at the CpG sequences leading to formation of 5-methyl-cytosine (m(5)Cyt) is an important element of epigenetic regulation of gene expression. Modification of the normal methylation pattern, unique to each organism, leads to the development of pathological processes and diseases, including cancer. Therefore, quantification of the DNA methylation and analysis of changes in the methylation pattern is very important from a practical point of view and can be used for diagnostic purposes, as well as monitoring of the treatment progress. In this paper we present a new method for quantification of 5-methyl-2'deoxycytidine (m(5)C) in the DNA. The technique is based on conversion of m(5)C into fluorescent 3,N(4)-etheno-5-methyl-2'deoxycytidine (εm(5)C) and its identification by reversed-phase high-performance liquid chromatography (RP-HPLC). The assay was used to evaluate m(5)C concentration in DNA of calf thymus and peripheral blood of cows bred under different conditions. This approach can be applied for measuring of 5-methylcytosine in cellular DNA from different cells and tissues.

Genome-wide identification and comparative analysis of cytosine-5 DNA methyltransferases and demethylase families in wild and cultivated peanut

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Pengfei eWang

2016-02-01

Full Text Available AbstractDNA methylation plays important roles in genome protection, regulation of gene expression and was associated with plants development. Plant DNA methylation pattern was mediated by cytosine-5 DNA methyltransferases and demethylase. Although the genomes of AA and BB wild peanuts have been fully sequence, these two gene families have not been studied. In this study we report the identification and analysis of putative cytosine-5 DNA methyltransferases (C5-MTases and demethylase in AA and BB wild peanuts. Cytosine-5 DNA methyltransferases in AA and BB wild peanuts could be classified in known MET, CMT and DRM2 groups based on their domain organization. This result was supported by the gene and protein structural characteristics and phylogenetic analysis. We found that some wild peanut DRM2 numbers didn’t contain UBA domain which was different from other plants such as Arabidopsis, maize, soybean. Five DNA demethylase were found in AA genome and five in BB genome. The selective pressure analysis showed that wild peanut C5-MTases gene mainly underwent purifying selection but many positive selection sites can be detected. Conversely, DNA demethylase genes mainly underwent positive selection during evolution. Additionally, the expression dynamic of cytosine-5 DNA methyltransferases and demethylase genes in different cultivated peanut tissues were analyzed. Expression result showed that cold, heat or drought stress could influence the expression level of C5-MTases and DNA demethylase genes in cultivated peanut. These results are useful for better understanding the complexity of these two gene families, and will facilitate epigenetic studies in peanut.

AID/APOBEC cytosine deaminase induces genome-wide kataegis

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Lada Artem G

2012-12-01

Full Text Available Abstract Clusters of localized hypermutation in human breast cancer genomes, named “kataegis” (from the Greek for thunderstorm, are hypothesized to result from multiple cytosine deaminations catalyzed by AID/APOBEC proteins. However, a direct link between APOBECs and kataegis is still lacking. We have sequenced the genomes of yeast mutants induced in diploids by expression of the gene for PmCDA1, a hypermutagenic deaminase from sea lamprey. Analysis of the distribution of 5,138 induced mutations revealed localized clusters very similar to those found in tumors. Our data provide evidence that unleashed cytosine deaminase activity is an evolutionary conserved, prominent source of genome-wide kataegis events. Reviewers This article was reviewed by: Professor Sandor Pongor, Professor Shamil R. Sunyaev, and Dr Vladimir Kuznetsov.

DNA cytosine methylation in the bovine leukemia virus promoter is associated with latency in a lymphoma-derived B-cell line: potential involvement of direct inhibition of cAMP-responsive element (CRE)-binding protein/CRE modulator/activation transcription factor binding.

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Pierard, Valérie; Guiguen, Allan; Colin, Laurence; Wijmeersch, Gaëlle; Vanhulle, Caroline; Van Driessche, Benoît; Dekoninck, Ann; Blazkova, Jana; Cardona, Christelle; Merimi, Makram; Vierendeel, Valérie; Calomme, Claire; Nguyên, Thi Liên-Anh; Nuttinck, Michèle; Twizere, Jean-Claude; Kettmann, Richard; Portetelle, Daniel; Burny, Arsène; Hirsch, Ivan; Rohr, Olivier; Van Lint, Carine

2010-06-18

Bovine leukemia virus (BLV) proviral latency represents a viral strategy to escape the host immune system and allow tumor development. Besides the previously demonstrated role of histone deacetylation in the epigenetic repression of BLV expression, we showed here that BLV promoter activity was induced by several DNA methylation inhibitors (such as 5-aza-2'-deoxycytidine) and that overexpressed DNMT1 and DNMT3A, but not DNMT3B, down-regulated BLV promoter activity. Importantly, cytosine hypermethylation in the 5'-long terminal repeat (LTR) U3 and R regions was associated with true latency in the lymphoma-derived B-cell line L267 but not with defective latency in YR2 cells. Moreover, the virus-encoded transactivator Tax(BLV) decreased DNA methyltransferase expression levels, which could explain the lower level of cytosine methylation observed in the L267(LTaxSN) 5'-LTR compared with the L267 5'-LTR. Interestingly, DNA methylation inhibitors and Tax(BLV) synergistically activated BLV promoter transcriptional activity in a cAMP-responsive element (CRE)-dependent manner. Mechanistically, methylation at the -154 or -129 CpG position (relative to the transcription start site) impaired in vitro binding of CRE-binding protein (CREB) transcription factors to their respective CRE sites. Methylation at -129 CpG alone was sufficient to decrease BLV promoter-driven reporter gene expression by 2-fold. We demonstrated in vivo the recruitment of CREB/CRE modulator (CREM) and to a lesser extent activating transcription factor-1 (ATF-1) to the hypomethylated CRE region of the YR2 5'-LTR, whereas we detected no CREB/CREM/ATF recruitment to the hypermethylated corresponding region in the L267 cells. Altogether, these findings suggest that site-specific DNA methylation of the BLV promoter represses viral transcription by directly inhibiting transcription factor binding, thereby contributing to true proviral latency.

Shotgun Bisulfite Sequencing of the Betula platyphylla Genome Reveals the Tree’s DNA Methylation Patterning

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Chang Su

2014-12-01

Full Text Available DNA methylation plays a critical role in the regulation of gene expression. Most studies of DNA methylation have been performed in herbaceous plants, and little is known about the methylation patterns in tree genomes. In the present study, we generated a map of methylated cytosines at single base pair resolution for Betula platyphylla (white birch by bisulfite sequencing combined with transcriptomics to analyze DNA methylation and its effects on gene expression. We obtained a detailed view of the function of DNA methylation sequence composition and distribution in the genome of B. platyphylla. There are 34,460 genes in the whole genome of birch, and 31,297 genes are methylated. Conservatively, we estimated that 14.29% of genomic cytosines are methylcytosines in birch. Among the methylation sites, the CHH context accounts for 48.86%, and is the largest proportion. Combined transcriptome and methylation analysis showed that the genes with moderate methylation levels had higher expression levels than genes with high and low methylation. In addition, methylated genes are highly enriched for the GO subcategories of binding activities, catalytic activities, cellular processes, response to stimulus and cell death, suggesting that methylation mediates these pathways in birch trees.

No effect of folic acid supplementation on global DNA methylation in men and women with moderately elevated homocysteine.

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Audrey Y Jung

Full Text Available A global loss of cytosine methylation in DNA has been implicated in a wide range of diseases. There is growing evidence that modifications in DNA methylation can be brought about by altering the intake of methyl donors such as folate. We examined whether long-term daily supplementation with 0.8 mg of folic acid would increase global DNA methylation compared with placebo in individuals with elevated plasma homocysteine. We also investigated if these effects were modified by MTHFR C677T genotype. Two hundred sixteen participants out of 818 subjects who had participated in a randomized double-blind placebo-controlled trial were selected, pre-stratified on MTHFR C677T genotype and matched on age and smoking status. They were allocated to receive either folic acid (0.8 mg/d; n = 105 or placebo treatment (n = 111 for three years. Peripheral blood leukocyte DNA methylation and serum and erythrocyte folate were assessed. Global DNA methylation was measured using liquid chromatography-tandem mass spectrometry and expressed as a percentage of 5-methylcytosines versus the total number of cytosine. There was no difference in global DNA methylation between those randomized to folic acid and those in the placebo group (difference = 0.008, 95%CI = -0.05,0.07, P = 0.79. There was also no difference between treatment groups when we stratified for MTHFR C677T genotype (CC, n = 76; CT, n = 70; TT, n = 70, baseline erythrocyte folate status or baseline DNA methylation levels. In moderately hyperhomocysteinemic men and women, long-term folic acid supplementation does not increase global DNA methylation in peripheral blood leukocytes.ClinicalTrials.gov NCT00110604.

No effect of folic acid supplementation on global DNA methylation in men and women with moderately elevated homocysteine.

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Jung, Audrey Y; Smulders, Yvo; Verhoef, Petra; Kok, Frans J; Blom, Henk; Kok, Robert M; Kampman, Ellen; Durga, Jane

2011-01-01

A global loss of cytosine methylation in DNA has been implicated in a wide range of diseases. There is growing evidence that modifications in DNA methylation can be brought about by altering the intake of methyl donors such as folate. We examined whether long-term daily supplementation with 0.8 mg of folic acid would increase global DNA methylation compared with placebo in individuals with elevated plasma homocysteine. We also investigated if these effects were modified by MTHFR C677T genotype. Two hundred sixteen participants out of 818 subjects who had participated in a randomized double-blind placebo-controlled trial were selected, pre-stratified on MTHFR C677T genotype and matched on age and smoking status. They were allocated to receive either folic acid (0.8 mg/d; n = 105) or placebo treatment (n = 111) for three years. Peripheral blood leukocyte DNA methylation and serum and erythrocyte folate were assessed. Global DNA methylation was measured using liquid chromatography-tandem mass spectrometry and expressed as a percentage of 5-methylcytosines versus the total number of cytosine. There was no difference in global DNA methylation between those randomized to folic acid and those in the placebo group (difference = 0.008, 95%CI = -0.05,0.07, P = 0.79). There was also no difference between treatment groups when we stratified for MTHFR C677T genotype (CC, n = 76; CT, n = 70; TT, n = 70), baseline erythrocyte folate status or baseline DNA methylation levels. In moderately hyperhomocysteinemic men and women, long-term folic acid supplementation does not increase global DNA methylation in peripheral blood leukocytes.ClinicalTrials.gov NCT00110604.

Cytosine deamination and the precipitous decline of spontaneous mutation during Earth's history.

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Lewis, Charles A; Crayle, Jesse; Zhou, Shuntai; Swanstrom, Ronald; Wolfenden, Richard

2016-07-19

The hydrolytic deamination of cytosine and 5-methylcytosine residues in DNA appears to contribute significantly to the appearance of spontaneous mutations in microorganisms and in human disease. In the present work, we examined the mechanism of cytosine deamination and the response of the uncatalyzed reaction to changing temperature. The positively charged 1,3-dimethylcytosinium ion was hydrolyzed at a rate similar to the rate of acid-catalyzed hydrolysis of 1-methylcytosine, for which it furnishes a satisfactory kinetic model and a probable mechanism. In agreement with earlier reports, uncatalyzed deamination was found to proceed at very similar rates for cytosine, 1-methylcytosine, cytidine, and cytidine 5'-phosphate, and also for cytosine residues in single-stranded DNA generated from a phagemid, in which we sequenced an insert representing the gene of the HIV-1 protease. Arrhenius plots for the uncatalyzed deamination of cytosine were linear over the temperature range from 90 °C to 200 °C and indicated a heat of activation (ΔH(‡)) of 23.4 ± 0.5 kcal/mol at pH 7. Recent evidence indicates that the surface of the earth has been cool enough to support life for more than 4 billion years and that life has been present for almost as long. If the temperature at Earth's surface is assumed to have followed Newton's law of cooling, declining exponentially from 100 °C to 25 °C during that period, then half of the cytosine-deaminating events per unit biomass would have taken place during the first 0.2 billion years, and <99.4% would have occurred during the first 2 billion years.

Defining Driver DNA Methylation Changes in Human Cancer

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Gerd P. Pfeifer

2018-04-01

Full Text Available Human malignant tumors are characterized by pervasive changes in the patterns of DNA methylation. These changes include a globally hypomethylated tumor cell genome and the focal hypermethylation of numerous 5′-cytosine-phosphate-guanine-3′ (CpG islands, many of them associated with gene promoters. It has been challenging to link specific DNA methylation changes with tumorigenesis in a cause-and-effect relationship. Some evidence suggests that cancer-associated DNA hypomethylation may increase genomic instability. Promoter hypermethylation events can lead to silencing of genes functioning in pathways reflecting hallmarks of cancer, including DNA repair, cell cycle regulation, promotion of apoptosis or control of key tumor-relevant signaling networks. A convincing argument for a tumor-driving role of DNA methylation can be made when the same genes are also frequently mutated in cancer. Many of the most commonly hypermethylated genes encode developmental transcription factors, the methylation of which may lead to permanent gene silencing. Inactivation of such genes will deprive the cells in which the tumor may initiate from the option of undergoing or maintaining lineage differentiation and will lock them into a perpetuated stem cell-like state thus providing an additional window for cell transformation.

Detection of DNA methylation changes in micropropagated banana plants using methylation-sensitive amplification polymorphism (MSAP).

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Peraza-Echeverria, S; Herrera-Valencia, V A.; Kay, A -J.

2001-07-01

The extent of DNA methylation polymorphisms was evaluated in micropropagated banana (Musa AAA cv. 'Grand Naine') derived from either the vegetative apex of the sucker or the floral apex of the male inflorescence using the methylation-sensitive amplification polymorphism (MSAP) technique. In all, 465 fragments, each representing a recognition site cleaved by either or both of the isoschizomers were amplified using eight combinations of primers. A total of 107 sites (23%) were found to be methylated at cytosine in the genome of micropropagated banana plants. In plants micropropagated from the male inflorescence explant 14 (3%) DNA methylation events were polymorphic, while plants micropropagated from the sucker explant produced 8 (1.7%) polymorphisms. No DNA methylation polymorphisms were detected in conventionally propagated banana plants. These results demonstrated the usefulness of MSAP to detect DNA methylation events in micropropagated banana plants and indicate that DNA methylation polymorphisms are associated with micropropagation.

[Analysis of genomic DNA methylation level in radish under cadmium stress by methylation-sensitive amplified polymorphism technique].

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Yang, Jin-Lan; Liu, Li-Wang; Gong, Yi-Qin; Huang, Dan-Qiong; Wang, Feng; He, Ling-Li

2007-06-01

The level of cytosine methylation induced by cadmium in radish (Raphanus sativus L.) genome was analysed using the technique of methylation-sensitive amplified polymorphism (MSAP). The MSAP ratios in radish seedling exposed to cadmium chloride at the concentration of 50, 250 and 500 mg/L were 37%, 43% and 51%, respectively, and the control was 34%; the full methylation levels (C(m)CGG in double strands) were at 23%, 25% and 27%, respectively, while the control was 22%. The level of increase in MSAP and full methylation indicated that de novo methylation occurred in some 5'-CCGG sites under Cd stress. There was significant positive correlation between increase of total DNA methylation level and CdCl(2) concentration. Four types of MSAP patterns: de novo methylation, de-methylation, atypical pattern and no changes of methylation pattern were identified among CdCl(2) treatments and the control. DNA methylation alteration in plants treated with CdCl(2) was mainly through de novo methylation.

Methylation-sensitive amplified polymorphism (MSAP) marker to investigate drought-stress response in Montepulciano and Sangiovese grape cultivars.

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Albertini, Emidio; Marconi, Gianpiero

2014-01-01

Methylation-sensitive amplified polymorphism (MSAP) is a technique developed for assessing the extent and pattern of cytosine methylation and has been applied to genomes of several species (Arabidopsis, grape, maize, tomato, and pepper). The technique relies on the use of isoschizomers that differ in their sensitivity to methylation.

Analysis of DNA methylation in various swine tissues.

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Chun Yang

Full Text Available DNA methylation is known to play an important role in regulating gene expression during biological development and tissue differentiation in eukaryotes. In this study, we used the fluorescence-labeled methylation-sensitive amplified polymorphism (F-MSAP method to assess the extent and pattern of cytosine methylation in muscle, heart, liver, spleen, lung, kidney and stomach from the swine strain Laiwu, and we also examined specific methylation patterns in the seven tissues. In total, 96,371 fragments, each representing a recognition site cleaved by either or both EcoRI + HpaII and EcoRI + MspI, the HpaII and MspI are isoschizomeric enzymes, were amplified using 16 pairs of selective primers. A total of 50,094 sites were found to be methylated at cytosines in seven tissues. The incidence of DNA methylation was approximately 53.99% in muscle, 51.24% in the heart, 50.18% in the liver, 53.31% in the spleen, 51.97% in the lung, 51.15% in the kidney and 53.39% in the stomach, as revealed by the incidence of differential digestion. Additionally, differences in DNA methylation levels imply that such variations may be related to specific gene expression during tissue differentiation, growth and development. Three types of bands were generated in the F-MSAP profile, the total numbers of these three types of bands in the seven tissues were 46,277, 24,801 and 25,293, respectively.In addition, different methylation patterns were observed in seven tissues from pig, and almost all of the methylation patterns detected by F-MSAP could be confirmed by Southern analysis using the isolated amplified fragments as probes. The results clearly demonstrated that the F-MSAP technique can be adapted for use in large-scale DNA methylation detection in the pig genome.

Forensic DNA methylation profiling from evidence material for investigative leads

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Lee, Hwan Young; Lee, Soong Deok; Shin, Kyoung-Jin

2016-01-01

DNA methylation is emerging as an attractive marker providing investigative leads to solve crimes in forensic genetics. The identification of body fluids that utilizes tissue-specific DNA methylation can contribute to solving crimes by predicting activity related to the evidence material. The age estimation based on DNA methylation is expected to reduce the number of potential suspects, when the DNA profile from the evidence does not match with any known person, including those stored in the forensic database. Moreover, the variation in DNA implicates environmental exposure, such as cigarette smoking and alcohol consumption, thereby suggesting the possibility to be used as a marker for predicting the lifestyle of potential suspect. In this review, we describe recent advances in our understanding of DNA methylation variations and the utility of DNA methylation as a forensic marker for advanced investigative leads from evidence materials. [BMB Reports 2016; 49(7): 359-369] PMID:27099236

Analysis of DNA methylation in Arabidopsis thaliana based on methylation-sensitive AFLP markers.

Science.gov (United States)

Cervera, M T; Ruiz-García, L; Martínez-Zapater, J M

2002-12-01

AFLP analysis using restriction enzyme isoschizomers that differ in their sensitivity to methylation of their recognition sites has been used to analyse the methylation state of anonymous CCGG sequences in Arabidopsis thaliana. The technique was modified to improve the quality of fingerprints and to visualise larger numbers of scorable fragments. Sequencing of amplified fragments indicated that detection was generally associated with non-methylation of the cytosine to which the isoschizomer is sensitive. Comparison of EcoRI/ HpaII and EcoRI/ MspI patterns in different ecotypes revealed that 35-43% of CCGG sites were differentially digested by the isoschizomers. Interestingly, the pattern of digestion among different plants belonging to the same ecotype is highly conserved, with the rate of intra-ecotype methylation-sensitive polymorphisms being less than 1%. However, pairwise comparisons of methylation patterns between samples belonging to different ecotypes revealed differences in up to 34% of the methylation-sensitive polymorphisms. The lack of correlation between inter-ecotype similarity matrices based on methylation-insensitive or methylation-sensitive polymorphisms suggests that whatever the mechanisms regulating methylation may be, they are not related to nucleotide sequence variation.

Photochemistry of DNA containing iodinated cytosine

Energy Technology Data Exchange (ETDEWEB)

Rahn, R O; Stafford, R S [Oak Ridge National Lab., TN (USA)

1979-10-01

Irradiation at 313 nm of compounds containing iodinated cytosine moieties results in the photolysis of iodine. Photolysis occurs with a quantum yield of 0.022-0.024 for 5-iododeoxycytidine and 5-iododeoxycytidine monophosphate, and 0.004-0.008 for iodinated DNA as well as for iodinated polycytidylate. Photodegradation of the cytosine moiety occurs when air is present during irradiation, presumably due to the reaction of oxygen with the cytosyl radical formed when iodine is lost. This oxygen promoted photodegradation destroys the cytosine chromophore and is complete in the monomers but occurs to only a limited extent in the polymers. In the absence of oxygen or in the presence of ethanol, photodegradation is prevented and the loss of iodine leads exclusively to the formation of the cytosine chromophore. In DNA, the loss of iodine is accompanied by the formation of sugar damage and/or chain breaks. As measured by sedimentation in alkaline sucrose gradients, approximately one break is made for every six iodines lost in denatured DNA. The frequency of chain breakage per iodine photolyzed is reduced 2-fold in renatured DNA. Analysis in neutral gradients suggests that half of the breaks observed in alkali are alkali-labile bonds. Both ethanol and cysteamine reduce the number of chain breaks in alkali by approximately 3-fold.

Hydroxyl radical induced cross-linking of cytosine and tyrosine in nucleohistone

International Nuclear Information System (INIS)

Gajewski, E.; Dizdaroglu, M.

1990-01-01

Hydroxyl radical induced formation of a DNA-protein cross-link involving cytosine and tyrosine in nucleohistone in buffered aqueous solution is reported. The technique of gas chromatography-mass spectrometry was used for this investigation. A γ-irradiated aqueous mixture of cytosine and tyrosine was first investigated in order to obtain gas chromatographic-mass spectrometric properties of possible cytosine-tyrosine cross-links. One cross-link was observed, and its structure was identified as the product from the formation of a covalent bond between carbon 6 of cytosine and carbon 3 of tyrosine. With the use of gas chromatography-mass spectrometry with selected-ion monitoring, this cytosine-tyrosine cross-link was identified in acidic hydrolysates of calf thymus nucleohistone γ-irradiated in N 2 O-saturated aqueous solution. The yield of this DNA-protein cross-link in nucleohistone was found to be a linear function of the radiation dose in the range of 100-500 Gy (J·kg -1 ). This yield amounted to 0.05 nmol·J -1 . Mechanisms underlying the formation of the cytosine-tyrosine cross-link in nucleohistone were proposed to involve radical-radical and/or radical addition reactions of hydroxyl adduct radicals of cytosine and tyrosine moieties, forming a covalent bond between carbon 6 of cytosine and carbon 3 of tyrosine. When oxygen was present in irradiated solutions, no cytosine-tyrosine cross-links were observed

DNA methylation analysis of allotetraploid hybrids of red crucian carp (Carassius auratus red var. and common carp (Cyprinus carpio L..

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Jun Xiao

Full Text Available Hybridization and polyploidization may lead to divergence in adaptation and boost speciation in angiosperms and some lower animals. Epigenetic change plays a significant role in the formation and adaptation of polyploidy. Studies of the effects of methylation on genomic recombination and gene expression in allopolyploid plants have achieved good progress. However, relevant advances in polyploid animals have been relatively slower. In the present study, we used the bisexual, fertile, genetically stable allotetraploid generated by hybridization of Carassius auratus red var. and Cyprinus carpio L. to investigate cytosine methylation level using methylation-sensitive amplification polymorphism (MSAP analysis. We observed 38.31% of the methylation changes in the allotetraploid compared with the parents at 355 randomly selected CCGG sites. In terms of methylation status, these results indicate that the level of methylation modification in the allotetraploid may have increased relative to that in the parents. We also found that the major methylation changes were hypermethylation on some genomic fragments and genes related to metabolism or cell cycle regulation. These results provide circumstantial evidence that DNA methylation might be related to the gene expression and phenotype variation in allotetraploid hybrids. Our study partly fulfils the need for epigenetic research in polyploid animals, and provides evidence for the epigenetic regulation of allopolyploids.

The Role of DNA Methylation in Xylogenesis in Different Tissues of Poplar

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Qingshi Wang

2016-07-01

Full Text Available In trees, xylem tissues play a key role in the formation of woody tissues, which have important uses for pulp and timber production; also DNA methylation plays an important part in gene regulation during xylogenesis in trees. In our study, methylation-sensitive amplified polymorphism (MSAP analysis was used to analyze the role cytosine methylation plays in wood formation in the commercially important tree species Populus tomentosa. This analysis compared the methylation patterns between xylem tissues (developing xylem and mature xylem and non-xylem tissues (cambium, shoot apex, young leaf, mature leaf, phloem, root, male catkin, and female catkin and found 10,316 polymorphic methylation sites. MSAP identified 132 candidate genes with the same methylation patterns in xylem tissues, including seven wood-related genes. The expression of these genes differed significantly between xylem and non-xylem tissue types (P<0.01. This indicated that the difference of expression of specific genes with unique methylation patterns, rather than relative methylation levels between the two tissue types plays a critical role in wood biosynthesis. However, 46.2% of candidate genes with the same methylation pattern in vascular tissues (cambium, phloem, and developing xylem did not have distinct expression patterns in xylem and non-xylem tissue. Also, bisulfite sequencing and transcriptome sequencing of MYB, NAC and FASCICLIN-LIKE AGP 13 revealed that the location of cytosine methylation in the gene might affect the expression of different transcripts from the corresponding gene. The expression of different transcripts that produce distinct proteins from a single gene might play an important role in the regulation of xylogenesis.

A unique regulatory phase of DNA methylation in the early mammalian embryo

OpenAIRE

Smith, Zachary D.; Chan, Michelle M.; Mikkelsen, Tarjei S.; Gu, Hongcang; Gnirke, Andreas; Regev, Aviv; Meissner, Alexander

2012-01-01

Summary DNA methylation is highly dynamic during mammalian embryogenesis. It is broadly accepted that the paternal genome is actively depleted of 5-methyl cytosine at fertilization, followed by passive loss that reaches a minimum at the blastocyst stage. However, this model is based on limited data, and to date no base-resolution maps exist to support and refine it. Here, we generated genome-scale DNA methylation maps in mouse gametes and through post-implantation embryogenesis. We find that ...

Use of MSAP markers to analyse the effects of salt stress on DNA methylation in rapeseed (Brassica napus var. oleifera.

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Gianpiero Marconi

Full Text Available Excessive soil salinity is a major ecological and agronomical problem, the adverse effects of which are becoming a serious issue in regions where saline water is used for irrigation. Plants can employ regulatory strategies, such as DNA methylation, to enable relatively rapid adaptation to new conditions. In this regard, cytosine methylation might play an integral role in the regulation of gene expression at both the transcriptional and post-transcriptional levels. Rapeseed, which is the most important oilseed crop in Europe, is classified as being tolerant of salinity, although cultivars can vary substantially in their levels of tolerance. In this study, the Methylation Sensitive Amplified Polymorphism (MSAP approach was used to assess the extent of cytosine methylation under salinity stress in salinity-tolerant (Exagone and salinity-sensitive (Toccata rapeseed cultivars. Our data show that salinity affected the level of DNA methylation. In particular methylation decreased in Exagone and increased in Toccata. Nineteen DNA fragments showing polymorphisms related to differences in methylation were sequenced. In particular, two of these were highly similar to genes involved in stress responses (Lacerata and trehalose-6-phosphatase synthase S4 and were chosen to further characterization. Bisulfite sequencing and quantitative RT-PCR analysis of selected MSAP loci showed that cytosine methylation changes under salinity as well as gene expression varied. In particular, our data show that salinity stress influences the expression of the two stress-related genes. Moreover, we quantified the level of trehalose in Exagone shoots and found that it was correlated to TPS4 expression and, therefore, to DNA methylation. In conclusion, we found that salinity could induce genome-wide changes in DNA methylation status, and that these changes, when averaged across different genotypes and developmental stages, accounted for 16.8% of the total site

Social Crowding during Development Causes Changes in GnRH1 DNA Methylation.

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Alvarado, Sebastian G; Lenkov, Kapa; Williams, Blake; Fernald, Russell D

2015-01-01

Gestational and developmental cues have important consequences for long-term health, behavior and adaptation to the environment. In addition, social stressors cause plastic molecular changes in the brain that underlie unique behavioral phenotypes that also modulate fitness. In the adult African cichlid, Astatotilapia burtoni, growth and social status of males are both directly regulated by social interactions in a dynamic social environment, which causes a suite of plastic changes in circuits, cells and gene transcription in the brain. We hypothesized that a possible mechanism underlying some molecular changes might be DNA methylation, a reversible modification made to cytosine nucleotides that is known to regulate gene function. Here we asked whether changes in DNA methylation of the GnRH1 gene, the central regulator of the reproductive axis, were altered during development of A. burtoni. We measured changes in methylation state of the GnRH1 gene during normal development and following the gestational and developmental stress of social crowding. We found differential DNA methylation within developing juveniles between 14-, 28- and 42-day-old. Following gestational crowding of mouth brooding mothers, we saw differential methylation and transcription of GnRH1 in their offspring. Taken together, our data provides evidence for social control of GnRH1 developmental responses to gestational cues through DNA methylation.

[Novel Approaches in DNA Methylation Studies - MS-HRM Analysis and Electrochemistry].

Science.gov (United States)

Bartošík, M; Ondroušková, E

Cytosine methylation in DNA is an epigenetic mechanism regulating gene expression and plays a vital role in cell differentiation or proliferation. Tumor cells often exhibit aberrant DNA methylation, e.g. hypermethylation of tumor suppressor gene promoters. New methods, capable of determining methylation status of specific DNA sequences, are thus being developed. Among them, MS-HRM (methylation-specific high resolution melting) and electrochemistry offer relatively inexpensive instrumentation, fast assay times and possibility of screening multiple samples/DNA regions simultaneously. MS-HRM is due to its sensitivity and simplicity an interesting alternative to already established techniques, including methylation-specific PCR or bisulfite sequencing. Electrochemistry, when combined with suitable electroactive labels and electrode surfaces, has been applied in several unique strategies for discrimination of cytosines and methylcytosines. Both techniques were successfully tested in analysis of DNA methylation within promoters of important tumor suppressor genes and could thus help in achieving more precise diagnostics and prognostics of cancer. Aberrant methylation of promoters has already been described in hundreds of genes associated with tumorigenesis and could serve as important biomarker if new methods applicable into clinical practice are sufficiently advanced.Key words: DNA methylation - 5-methylcytosine - HRM analysis - melting temperature - DNA duplex - electrochemistry - nucleic acid hybridizationThis work was supported by MEYS - NPS I - LO1413.The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study.The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers.Submitted: 6. 5. 2016Accepted: 16. 5. 2016.

Evidence for methyl group transfer between the methyl-accepting chemotaxis proteins in Bacillus subtilis

International Nuclear Information System (INIS)

Bedale, W.A.; Nettleton, D.O.; Sopata, C.S.; Thoelke, M.S.; Ordal, G.W.

1988-01-01

The authors present evidence for methyl (as methyl or methoxy) transfer from the methyl-accepting chemotaxis proteins H1 and possibly H3 of Bacillus subtilis to the methyl-accepting chemotaxis protein H2. This methyl transfer, which has been observed in vitro was strongly stimulated by the chemoattractant aspartate and thus may plan an important role in the sensory processing system of this organism. Although radiolabeling of H1 and H3 began at once after the addition of [ 3 H] methionine, radiolabeling of H2 showed a lag. Furthermore, the addition of excess nonradioactive methionine caused immediate exponential delabeling of H1 and H3 while labeling of H2 continued to increase. Methylation of H2 required the chemotactic methyltransferase, probably to first methylate H1 and H3. Aspartate caused increased labeling of H2 and strongly decreased labeling of H1 and H3 after the addition of nonradioactive methionine. Without the addition of nonradioactive methionine, aspartate caused demethylation of H1 and to a lesser extent H3, with an approximately equal increase of methylation of H2

Syntheses of [5-2H]-uracil, [5-2H]-cytosine, [6-2H]-uracil and [6-2H]-cytosine

International Nuclear Information System (INIS)

Kiritani, Reiko; Asano, Takeyoshi; Fujita, Shin-ichi; Dohmaru, Takaaki; Kawanishi, Tetsuro

1986-01-01

Syntheses of [5- 2 H]-, [6- 2 H]-uracil and [5- 2 H]-, [6- 2 H]-cytosine were investigated. The catalytic reaction of uracil or cytosine with 2 H 2 gas in alkaline media gave rise to [6- 2 H]-compounds almost exclusively. On the other hand, the reaction of 5-bromouracil or 5-bromocytosine with 2 H 2 gas gave rise to a mixture of [5- 2 H]-, [6- 2 H]- and [5- 2 H, 6- 2 H]-compounds depending on the experimental conditions. By controlling the temperature, the pressure of 2 H 2 gas and the amount of catalyst, [5- 2 H]-uracil and [5- 2 H]-cytosine were obtained. The isotopic distribution in each product was measured by 1 H NMR spectroscopy combined with an HPLC method. (author)

The role of DNA methylation on Octopus vulgaris development and their perspectives

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Eva eDíaz-Freije

2014-02-01

Full Text Available DNA methylation is a common regulator of gene expression and development in mammalian and other vertebrate genomes. DNA methylation has been studied so far in a few bivalve mollusk species, finding a wide spectrum of levels. We focused our study in the common octopus, Octopus vulgaris, an important organism for neuroscience, physiology and ethology research as well as for human consumption. We aim to confirm the existence of DNA methylation in O. vulgaris and ultimately, if methylation plays a role in gene regulation during octopus development. We used a genome-wide approach, methylation-sensitive amplified polymorphism (MSAP, firstly in four different tissues from the same specimens from adult benthonic individuals to test whether gene expression is regulated by methylation. Secondly, we tested the hypothesis that methylation underlies development by assessing MSAP patters from paralarvae to adult developmental stages. Our data indicate that octopus genome is widely methylated since clear differences can be observed, and the methylation pattern change with the development. The statistical analyses showed significant differences in methylation pattern between paralarvae, where higher internal cytosine methylation is observed, and the three other post-hatching stages. This suggests an important role of cytosine methylation during the first step of development, when major morphological changes take place. However, methylation seems to have little effect on gene expression during the benthonic phase, since any significant effect was revealed in the AMOVA performed. Our observations highlight the importance of epigenetic mechanism in the first developmental steps of the common octopus and open new perspectives to overcome high mortality rate during paralarvae growth. Thus, better understanding the molecular regulation patterns could lead to new approaches that increase the efficiency of husbandry of this emergent species for aquaculture.

Methylation by a unique α-class N4-cytosine methyltransferase is required for DNA transformation of Caldicellulosiruptor bescii DSM6725.

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Daehwan Chung

Full Text Available Thermophilic microorganisms capable of using complex substrates offer special advantages for the conversion of lignocellulosic biomass to biofuels and bioproducts. Members of the gram-positive bacterial genus Caldicellulosiruptor are anaerobic thermophiles with optimum growth temperatures between 65°C and 78°C and are the most thermophilic cellulolytic organisms known. In fact, they efficiently use biomass non-pretreated as their sole carbon source and in successive rounds of application digest 70% of total switchgrass substrate. The ability to genetically manipulate these organisms is a prerequisite to engineering them for use in conversion of these complex substrates to products of interest as well as identifying gene products critical for their ability to utilize non-pretreated biomass. Here, we report the first example of DNA transformation of a member of this genus, C. bescii. We show that restriction of DNA is a major barrier to transformation (in this case apparently absolute and that methylation with an endogenous unique α-class N4-Cytosine methyltransferase is required for transformation of DNA isolated from E. coli. The use of modified DNA leads to the development of an efficient and reproducible method for DNA transformation and the combined frequencies of transformation and recombination allow marker replacement between non-replicating plasmids and chromosomal genes providing the basis for rapid and efficient methods of genetic manipulation.

DNA (Cytosine-C5) Methyltransferase Inhibition by Oligodeoxyribonucleotides Containing 2-(1H)-Pyrimidinone (Zebularine Aglycon) at the Enzymatic Target Site

OpenAIRE

van Bemmel, Dana M.; Brank, Adam S.; Eritja, Ramon; Marquez, Victor E.; Christman, Judith K.

2009-01-01

Aberrant cytosine methylation in promoter regions leads to gene silencing associated with cancer progression. A number of DNA methyltransferase inhibitors are known to reactivate silenced genes; including 5-azacytidine and 2-(1H)-pyrimidinone riboside (zebularine). Zebularine is a more stable, less cytotoxic inhibitor compared to 5-azacytidine. To determine the mechanistic basis for this difference, we carried out a detailed comparisons of the interaction between purified DNA methyltransferas...

Methylation changes of H19 gene in sperms of X-irradiated mouse and maintenance in offspring

International Nuclear Information System (INIS)

Zhu Bin; Huang Xinghua; Chen Jindong; Lu Yachao; Chen Ying; Zhao Jingyong

2006-01-01

The nature of imprinting is just differential methylation of imprinted genes. Unlike the non-imprinted genes, the methylation pattern of imprinted genes established during the period of gametogenesis remains unchangeable after fertilization and during embryo development. It implies that gametogenesis is the key stage for methylation pattern of imprinted genes. The imprinting interfered by exogenous factors during this stage could be inherited to offspring and cause genetic effect. Now many studies have proved that ionizing irradiation could disturb DNA methylation. Here we choose BALB/c mice as a research model and X-ray as interfering source to further clarify it. We discovered that the whole-body irradiation of X-ray to male BALB/c mice could influence the methylation pattern of H 19 gene in sperms, which resulted in some cytosines of partial CpG islands in the imprinting control region could not transform to methylated cytosines. Furthermore, by copulating the interfered male mice with normal female, we analyzed the promoter methylation pattern of H 19 in offspring fetal liver and compared the same to the pattern of male parent in sperms. We found that the majority of methylation changes in offspring liver were related to the ones in their parent sperms. Our data proved that the changes of the H 19 gene methylation pattern interfered by X-ray irradiation could be transmitted and maintained in First-generation offspring

DNA Methylation Alterations at 5'-CCGG Sites in the Interspecific and Intraspecific Hybridizations Derived from Brassica rapa and B. napus.

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Wanshan Xiong

Full Text Available DNA methylation is an important regulatory mechanism for gene expression that involved in the biological processes of development and differentiation in plants. To investigate the association of DNA methylation with heterosis in Brassica, a set of intraspecific hybrids in Brassica rapa and B. napus and interspecific hybrids between B. rapa and B. napus, together with parental lines, were used to monitor alterations in cytosine methylation at 5'-CCGG sites in seedlings and buds by methylation-sensitive amplification polymorphism analysis. The methylation status of approximately a quarter of the methylation sites changed between seedlings and buds. These alterations were related closely to the genomic structure and heterozygous status among accessions. The methylation status in the majority of DNA methylation sites detected in hybrids was the same as that in at least one of the parental lines in both seedlings and buds. However, the association between patterns of cytosine methylation and heterosis varied among different traits and between tissues in hybrids of Brassica, although a few methylation loci were associated with heterosis. Our data suggest that changes in DNA methylation at 5'-CCGG sites are not associated simply with heterosis in the interspecific and intraspecific hybridizations derived from B. rapa and B. napus.

RNA-mediated epigenetic heredity requires the cytosine methyltransferase Dnmt2.

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Jafar Kiani

2013-05-01

Full Text Available RNA-mediated transmission of phenotypes is an important way to explain non-Mendelian heredity. We have previously shown that small non-coding RNAs can induce hereditary epigenetic variations in mice and act as the transgenerational signalling molecules. Two prominent examples for these paramutations include the epigenetic modulation of the Kit gene, resulting in altered fur coloration, and the modulation of the Sox9 gene, resulting in an overgrowth phenotype. We now report that expression of the Dnmt2 RNA methyltransferase is required for the establishment and hereditary maintenance of both paramutations. Our data show that the Kit paramutant phenotype was not transmitted to the progeny of Dnmt2(-/- mice and that the Sox9 paramutation was also not established in Dnmt2(-/- embryos. Similarly, RNA from Dnmt2-negative Kit heterozygotes did not induce the paramutant phenotype when microinjected into Dnmt2-deficient fertilized eggs and microinjection of the miR-124 microRNA failed to induce the characteristic giant phenotype. In agreement with an RNA-mediated mechanism of inheritance, no change was observed in the DNA methylation profiles of the Kit locus between the wild-type and paramutant mice. RNA bisulfite sequencing confirmed Dnmt2-dependent tRNA methylation in mouse sperm and also indicated Dnmt2-dependent cytosine methylation in Kit RNA in paramutant embryos. Together, these findings uncover a novel function of Dnmt2 in RNA-mediated epigenetic heredity.

The Variation Analysis of DNA Methylation in Wheat Carrying Gametocidal Chromosome 3C from Aegilops triuncialis.

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Wang, Dan; Zhao, Jieyu; Bai, Yan; Ao, You; Guo, Changhong

2017-08-10

Gametocidal (Gc) chromosomes can ensure their preferential transmission by killing the gametes without themselves through causing chromosome breakage and therefore have been exploited as an effective tool for genetic breeding. However, to date very little is known about the molecular mechanism of Gc action. In this study, we used methylation-sensitive amplified polymorphism (MSAP) technique to assess the extent and pattern of cytosine methylation alterations at the whole genome level between two lines of wheat Gc addition line and their common wheat parent. The results indicated that the overall levels of cytosine methylation of two studied Gc addition lines (CS-3C and CS-3C3C, 48.68% and 48.65%, respectively) were significantly increased when compared to common wheat CS (41.31%) and no matter fully methylated or hemimethylated rates enhanced in Gc addition lines. A set of 30 isolated fragments that showed different DNA methylation or demethylation patterns between the three lines were sequenced and the results indicated that 8 fragments showed significant homology to known sequences, of which three were homologous to MITE transposon (Miniature inverted-repeat transposable elements), LTR-retrotransposon WIS-1p and retrotransposon Gypsy , respectively. Overall, our results showed that DNA methylation could play a role in the Gc action.

The Variation Analysis of DNA Methylation in Wheat Carrying Gametocidal Chromosome 3C from Aegilops triuncialis

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Dan Wang

2017-08-01

Full Text Available Gametocidal (Gc chromosomes can ensure their preferential transmission by killing the gametes without themselves through causing chromosome breakage and therefore have been exploited as an effective tool for genetic breeding. However, to date very little is known about the molecular mechanism of Gc action. In this study, we used methylation-sensitive amplified polymorphism (MSAP technique to assess the extent and pattern of cytosine methylation alterations at the whole genome level between two lines of wheat Gc addition line and their common wheat parent. The results indicated that the overall levels of cytosine methylation of two studied Gc addition lines (CS–3C and CS–3C3C, 48.68% and 48.65%, respectively were significantly increased when compared to common wheat CS (41.31% and no matter fully methylated or hemimethylated rates enhanced in Gc addition lines. A set of 30 isolated fragments that showed different DNA methylation or demethylation patterns between the three lines were sequenced and the results indicated that 8 fragments showed significant homology to known sequences, of which three were homologous to MITE transposon (Miniature inverted–repeat transposable elements, LTR-retrotransposon WIS-1p and retrotransposon Gypsy, respectively. Overall, our results showed that DNA methylation could play a role in the Gc action.

HPLC-UV, MALDI-TOF-MS and ESI-MS/MS analysis of the mechlorethamine DNA crosslink at a cytosine-cytosine mismatch pair.

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Pornchai Rojsitthisak

Full Text Available Mechlorethamine [ClCH(2CH(2N(CH(3CH(2CH(2Cl], a nitrogen mustard alkylating agent, has been proven to form a DNA interstrand crosslink at a cytosine-cytosine (C-C mismatch pair using gel electrophoresis. However, the atomic connectivity of this unusual crosslink is unknown.HPLC-UV, MALDI-TOF-MS, and ESI-MS/MS were used to determine the atomic connectivity of the DNA C-C crosslink formed by mechlorethamine, MALDI-TOF-MS of the HPLC-purified reaction product of mechlorethamine with the DNA duplex d[CTCACACCGTGGTTC]•d[GAACCACCGTGTGAG] (underlined bases are a C-C mismatch pair indicated formation of an interstrand crosslink at m/z 9222.088 [M-2H+Na](+. Following enzymatic digestion of the crosslinked duplex by snake venom phosphodiesterase and calf intestinal phosphatase, ESI-MS/MS indicated the presence of dC-mech-dC [mech = CH(2CH(2N(CH(3CH(2CH(2] at m/z 269.2 [M](2+ (expected m/z 269.6, exact mass 539.27 and its hydrolytic product dC-mech-OH at m/z 329.6 [M](+ (expected m/z 329.2. Fragmentation of dC-mech-dC gave product ions at m/z 294.3 and 236.9 [M](+, which are both due to loss of the 4-amino group of cytosine (as ammonia, in addition to dC and dC+HN(CH(3CH = CH(2, respectively. The presence of m/z 269.2 [M](2+ and loss of ammonia exclude crosslink formation at cytosine N(4 or O(2 and indicate crosslinking through cytosine N(3 with formation of two quaternary ammonium ions.Our results provide an important addition to the literature, as the first example of the use of HPLC and MS for analysis of a DNA adduct at the N(3 position of cytosine.

The Dnmt3L ADD Domain Controls Cytosine Methylation Establishment during Spermatogenesis

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Georgios Vlachogiannis

2015-02-01

Full Text Available A critical aspect of mammalian gametogenesis is the reprogramming of genomic DNA methylation. The catalytically inactive adaptor Dnmt3L is essential to ensuring this occurs correctly, but the mechanism by which it functions is unclear. Using gene targeting to engineer a single-amino-acid mutation, we show that the Dnmt3L histone H3 binding domain (ADD is necessary for spermatogenesis. Genome-wide single-base-resolution DNA methylome analysis of mutant germ cells revealed overall reductions in CG methylation at repetitive sequences and non-promoter CpG islands. Strikingly, we also observe an even more severe loss of non-CG methylation, suggesting an unexpected role for the ADD in this process. These epigenetic deficiencies were coupled with defects in spermatogonia, with mutant cells displaying marked changes in gene expression and reactivation of retrotransposons. Our results demonstrate that the Dnmt3L ADD is necessary for Dnmt3L function and full reproductive fitness.

Heritable alteration of DNA methylation induced by whole-chromosome aneuploidy in wheat.

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Gao, Lihong; Diarso, Moussa; Zhang, Ai; Zhang, Huakun; Dong, Yuzhu; Liu, Lixia; Lv, Zhenling; Liu, Bao

2016-01-01

Aneuploidy causes changes in gene expression and phenotypes in all organisms studied. A previous study in the model plant Arabidopsis thaliana showed that aneuploidy-generated phenotypic changes can be inherited to euploid progenies and implicated an epigenetic underpinning of the heritable variations. Based on an analysis by amplified fragment length polymorphism and methylation-sensitive amplified fragment length polymorphism markers, we found that although genetic changes at the nucleotide sequence level were negligible, extensive changes in cytosine DNA methylation patterns occurred in all studied homeologous group 1 whole-chromosome aneuploid lines of common wheat (Triticum aestivum), with monosomic 1A showing the greatest amount of methylation changes. The changed methylation patterns were inherited by euploid progenies derived from the aneuploid parents. The aneuploidy-induced DNA methylation alterations and their heritability were verified at selected loci by bisulfite sequencing. Our data have provided empirical evidence supporting earlier suggestions that heritability of aneuploidy-generated, but aneuploidy-independent, phenotypic variations may have an epigenetic basis. That at least one type of aneuploidy - monosomic 1A - was able to cause significant epigenetic divergence of the aneuploid plants and their euploid progenies also lends support to recent suggestions that aneuploidy may have played an important and protracted role in polyploid genome evolution. © 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.

Exploring the Link between Nucleosome Occupancy and DNA Methylation

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Cecilia Lövkvist

2018-01-01

Full Text Available Near promoters, both nucleosomes and CpG sites form characteristic spatial patterns. Previously, nucleosome depleted regions were observed upstream of transcription start sites and nucleosome occupancy was reported to correlate both with CpG density and the level of CpG methylation. Several studies imply a causal link where CpG methylation might induce nucleosome formation, whereas others argue the opposite, i.e., that nucleosome occupancy might influence CpG methylation. Correlations are indeed evident between nucleosomes, CpG density and CpG methylation—at least near promoter sites. It is however less established whether there is an immediate causal relation between nucleosome occupancy and the presence of CpG sites—or if nucleosome occupancy could be influenced by other factors. In this work, we test for such causality in human genomes by analyzing the three quantities both near and away from promoter sites. For data from the human genome we compare promoter regions with given CpG densities with genomic regions without promoters but of similar CpG densities. We find the observed correlation between nucleosome occupancy and CpG density, respectively CpG methylation, to be specific to promoter regions. In other regions along the genome nucleosome occupancy is statistically independent of the positioning of CpGs or their methylation levels. Anti-correlation between CpG density and methylation level is however similarly strong in both regions. On promoters, nucleosome occupancy is more strongly affected by the level of gene expression than CpG density or CpG methylation—calling into question any direct causal relation between nucleosome occupancy and CpG organization. Rather, our results suggest that for organisms with cytosine methylation nucleosome occupancy might be primarily linked to gene expression, with no strong impact on methylation.

Surface study of gallium- and aluminum- doped graphenes upon adsorption of cytosine: DFT calculations

International Nuclear Information System (INIS)

Shokuhi Rad, Ali; Zareyee, Daryoush; Peyravi, Majid; Jahanshahi, Mohsen

2016-01-01

Highlights: • P1 and P4 are the most stable adsorption configurations for cytosine. • NBO analysis show n-type semiconductor property for both Al- and Ga-doped graphenes. • Important changes in the HOMO and LUMO of doped graphene upon adsorption of cytosine. • Increase in the conductivity of system when cytosine is adsorbed on doped graphenes. - Abstract: The adsorption of cytosine molecule on Al- and Ga- doped graphenes is studied using first-principles density functional theory (DFT) calculations. The energetically most stable geometries of cytosine on both Al- and Ga- doped graphenes are determined and the adsorption energies are calculated. The net charge of transfer as well as local charge of doped atoms upon adsorption of cytosine are studied by natural bond orbitals (NBO) analysis. Orbital hybridizing of complexes was searched by frontier molecular orbital theory (FMO), and density of states (DOS). Depending on the side of cytosine, there are four possible sites for its adsorption on doped graphene; denoted as P1, P2, P3, and P4, respectively. The order of binding energy in the case of Al-doped graphene is found as P1 > P4 > P3 > P2. Interestingly, the order in the case of Ga-doped graphene changes to: P4 ∼ P1 > P3 > P2. Both surfaces show superior adsorbent property, resulting chemisorption of cytosine, especially at P1 and P4 position configurations. The NBO charge analysis reveals that the charge transfers from Al- and Ga- doped graphene sheets to cytosine. The electronic properties of both surfaces undertake important changes after cytosine adsorption, which indicates notable change in its electrical conductivity.

Surface study of gallium- and aluminum- doped graphenes upon adsorption of cytosine: DFT calculations

Energy Technology Data Exchange (ETDEWEB)

Shokuhi Rad, Ali, E-mail: a.shokuhi@gmail.com [Department of Chemical Engineering, Qaemshahr Branch, Islamic Azad University, Qaemshahr (Iran, Islamic Republic of); Zareyee, Daryoush [Department of Chemistry, Qaemshahr Branch, Islamic Azad University, Qaemshahr (Iran, Islamic Republic of); Peyravi, Majid; Jahanshahi, Mohsen [Faculty of Chemical Engineering, Babol University of Technology, Babol (Iran, Islamic Republic of)

2016-12-30

Highlights: • P1 and P4 are the most stable adsorption configurations for cytosine. • NBO analysis show n-type semiconductor property for both Al- and Ga-doped graphenes. • Important changes in the HOMO and LUMO of doped graphene upon adsorption of cytosine. • Increase in the conductivity of system when cytosine is adsorbed on doped graphenes. - Abstract: The adsorption of cytosine molecule on Al- and Ga- doped graphenes is studied using first-principles density functional theory (DFT) calculations. The energetically most stable geometries of cytosine on both Al- and Ga- doped graphenes are determined and the adsorption energies are calculated. The net charge of transfer as well as local charge of doped atoms upon adsorption of cytosine are studied by natural bond orbitals (NBO) analysis. Orbital hybridizing of complexes was searched by frontier molecular orbital theory (FMO), and density of states (DOS). Depending on the side of cytosine, there are four possible sites for its adsorption on doped graphene; denoted as P1, P2, P3, and P4, respectively. The order of binding energy in the case of Al-doped graphene is found as P1 > P4 > P3 > P2. Interestingly, the order in the case of Ga-doped graphene changes to: P4 ∼ P1 > P3 > P2. Both surfaces show superior adsorbent property, resulting chemisorption of cytosine, especially at P1 and P4 position configurations. The NBO charge analysis reveals that the charge transfers from Al- and Ga- doped graphene sheets to cytosine. The electronic properties of both surfaces undertake important changes after cytosine adsorption, which indicates notable change in its electrical conductivity.

Correlating Gene-specific DNA Methylation Changes with Expression and Transcriptional Activity of Astrocytic KCNJ10 (Kir4.1).

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Nwaobi, Sinifunanya E; Olsen, Michelle L

2015-09-26

DNA methylation serves to regulate gene expression through the covalent attachment of a methyl group onto the C5 position of a cytosine in a cytosine-guanine dinucleotide. While DNA methylation provides long-lasting and stable changes in gene expression, patterns and levels of DNA methylation are also subject to change based on a variety of signals and stimuli. As such, DNA methylation functions as a powerful and dynamic regulator of gene expression. The study of neuroepigenetics has revealed a variety of physiological and pathological states that are associated with both global and gene-specific changes in DNA methylation. Specifically, striking correlations between changes in gene expression and DNA methylation exist in neuropsychiatric and neurodegenerative disorders, during synaptic plasticity, and following CNS injury. However, as the field of neuroepigenetics continues to expand its understanding of the role of DNA methylation in CNS physiology, delineating causal relationships in regards to changes in gene expression and DNA methylation are essential. Moreover, in regards to the larger field of neuroscience, the presence of vast region and cell-specific differences requires techniques that address these variances when studying the transcriptome, proteome, and epigenome. Here we describe FACS sorting of cortical astrocytes that allows for subsequent examination of a both RNA transcription and DNA methylation. Furthermore, we detail a technique to examine DNA methylation, methylation sensitive high resolution melt analysis (MS-HRMA) as well as a luciferase promoter assay. Through the use of these combined techniques one is able to not only explore correlative changes between DNA methylation and gene expression, but also directly assess if changes in the DNA methylation status of a given gene region are sufficient to affect transcriptional activity.

msap: a tool for the statistical analysis of methylation-sensitive amplified polymorphism data.

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Pérez-Figueroa, A

2013-05-01

In this study msap, an R package which analyses methylation-sensitive amplified polymorphism (MSAP or MS-AFLP) data is presented. The program provides a deep analysis of epigenetic variation starting from a binary data matrix indicating the banding pattern between the isoesquizomeric endonucleases HpaII and MspI, with differential sensitivity to cytosine methylation. After comparing the restriction fragments, the program determines if each fragment is susceptible to methylation (representative of epigenetic variation) or if there is no evidence of methylation (representative of genetic variation). The package provides, in a user-friendly command line interface, a pipeline of different analyses of the variation (genetic and epigenetic) among user-defined groups of samples, as well as the classification of the methylation occurrences in those groups. Statistical testing provides support to the analyses. A comprehensive report of the analyses and several useful plots could help researchers to assess the epigenetic and genetic variation in their MSAP experiments. msap is downloadable from CRAN (http://cran.r-project.org/) and its own webpage (http://msap.r-forge.R-project.org/). The package is intended to be easy to use even for those people unfamiliar with the R command line environment. Advanced users may take advantage of the available source code to adapt msap to more complex analyses. © 2013 Blackwell Publishing Ltd.

Analysis of DNA Methylation of Gracilariopsis lemaneiformis Under Temperature Stress Using the Methylation Sensitive Amplification Polymorphism (MSAP) Technique

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Peng, Chong; Sui, Zhenghong; Zhou, Wei; Hu, Yiyi; Mi, Ping; Jiang, Minjie; Li, Xiaodong; Ruan, Xudong

2018-06-01

Gracilariopsis lemaneiformis is an economically important agarophyte, which contains high quality gel and shows a high growth rate. Wild population of G. lemaneiformis displayed resident divergence, though with a low genetic diversity as was revealed by amplified fragment length polymorphism (AFLP) and simple sequence repeat (SSR) analyses. In addition, different strains of G. lemaneiformis are diverse in morphology. The highly inconsistence between genetic background and physiological characteristics recommends strongly to the regulation at epigenetic level. In this study, the DNA methylation change in G. lemaneiformis among different generation branches and under different temperature stresses was assessed using methylation sensitive amplified polymorphism (MSAP) technique. It was shown that DNA methylation level among different generation branches was diverse. The full and total methylated DNA level was the lowest in the second generation branch and the highest in the third generation. The total methylation level was 61.11%, 60.88% and 64.12% at 15°C, 22°C and 26°C, respectively. Compared with the control group (22°C), the fully methylated and totally methylated ratios were increased in both experiment groups (15°C and 26°C). All of the cytosine methylation/demethylation transform (CMDT) was further analyzed. High temperature treatment could induce more CMDT than low temperature treatment did.

Statistical method evaluation for differentially methylated CpGs in base resolution next-generation DNA sequencing data.

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Zhang, Yun; Baheti, Saurabh; Sun, Zhifu

2018-05-01

High-throughput bisulfite methylation sequencing such as reduced representation bisulfite sequencing (RRBS), Agilent SureSelect Human Methyl-Seq (Methyl-seq) or whole-genome bisulfite sequencing is commonly used for base resolution methylome research. These data are represented either by the ratio of methylated cytosine versus total coverage at a CpG site or numbers of methylated and unmethylated cytosines. Multiple statistical methods can be used to detect differentially methylated CpGs (DMCs) between conditions, and these methods are often the base for the next step of differentially methylated region identification. The ratio data have a flexibility of fitting to many linear models, but the raw count data take consideration of coverage information. There is an array of options in each datatype for DMC detection; however, it is not clear which is an optimal statistical method. In this study, we systematically evaluated four statistic methods on methylation ratio data and four methods on count-based data and compared their performances with regard to type I error control, sensitivity and specificity of DMC detection and computational resource demands using real RRBS data along with simulation. Our results show that the ratio-based tests are generally more conservative (less sensitive) than the count-based tests. However, some count-based methods have high false-positive rates and should be avoided. The beta-binomial model gives a good balance between sensitivity and specificity and is preferred method. Selection of methods in different settings, signal versus noise and sample size estimation are also discussed.

Understanding the connection between epigenetic DNA methylation and nucleosome positioning from computer simulations.

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Guillem Portella

Full Text Available Cytosine methylation is one of the most important epigenetic marks that regulate the process of gene expression. Here, we have examined the effect of epigenetic DNA methylation on nucleosomal stability using molecular dynamics simulations and elastic deformation models. We found that methylation of CpG steps destabilizes nucleosomes, especially when these are placed in sites where the DNA minor groove faces the histone core. The larger stiffness of methylated CpG steps is a crucial factor behind the decrease in nucleosome stability. Methylation changes the positioning and phasing of the nucleosomal DNA, altering the accessibility of DNA to regulatory proteins, and accordingly gene functionality. Our theoretical calculations highlight a simple physical-based explanation on the foundations of epigenetic signaling.

Characterization of in vitro haploid and doubled haploid Chrysanthemum morifolium plants via unfertilized ovule culture for phenotypical traits and DNA methylation pattern.

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Haibin eWang

2014-12-01

Full Text Available Chrysanthemum is one of important ornamental species in the world. Its highly heterozygous state complicates molecular analysis, so it is of interest to derive haploid forms. A total of 2,579 non-fertilized chrysanthemum ovules pollinated by Argyranthemum frutescens were cultured in vitro to isolate haploid progeny. One single regenerant emerged from each of three of the 105 calli produced. Chromosome counts and microsatellite fingerprinting showed that only one of the regenerants was a true haploid. Nine doubled haploid derivatives were subsequently generated by colchicine treatment of 80 in vitro cultured haploid nodal segments. Morphological screening showed that the haploid plant was shorter than the doubled haploids, and developed smaller leaves, flowers and stomata. An in vitro pollen germination test showed that few of the haploid's pollen were able to germinate and those which did so were abnormal. Both the haploid and the doubled haploids produced yellow flowers, whereas those of the maternal parental cultivar were mauve. Methylation-sensitive amplification polymorphism (MSAP profiling was further used to detect alterations in cytosine methylation caused by the haploidization and/or the chromosome doubling processes. While 52.2% of the resulting amplified fragments were cytosine methylated in the maternal parent's genome, the corresponding proportions for the haploid's and doubled haploids' genomes were, respectively, 47.0% and 51.7%, demonstrating a reduction in global cytosine methylation caused by haploidization and a partial recovery following chromosome doubling.

DNA methyltransferase 1 mutations and mitochondrial pathology: is mtDNA methylated?

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Alessandra eMaresca

2015-03-01

Full Text Available Autosomal dominant cerebellar ataxia, deafness and narcolepsy (ADCA-DN and Hereditary sensory neuropathy with dementia and hearing loss (HSN1E are two rare, overlapping neurodegenerative syndromes that have been recently linked to allelic dominant pathogenic mutations in the DNMT1 gene, coding for DNA (cytosine-5-methyltransferase 1. DNMT1 is the enzyme responsible for maintaining the nuclear genome methylation patterns during the DNA replication and repair, thus regulating gene expression. The mutations responsible for ADCA-DN and HSN1E affect the replication foci targeting sequence domain, which regulates DNMT1 binding to chromatin. DNMT1 dysfunction is anticipated to lead to a global alteration of the DNA methylation pattern with predictable downstream consequences on gene expression. Interestingly, ADCA-DN and HSN1E phenotypes share some clinical features typical of mitochondrial diseases, such as optic atrophy, peripheral neuropathy and deafness, and some biochemical evidence of mitochondrial dysfunction. The recent discovery of a mitochondrial isoform of DNMT1 and its proposed role in methylating mitochondrial DNA (mtDNA suggests that DNMT1 mutations may directly affect mtDNA and mitochondrial physiology. On the basis of this latter finding the link between DNMT1 abnormal activity and mitochondrial dysfunction in ADCA-DN and HSN1E appears intuitive, however mtDNA methylation remains highly debated. In the last years several groups demonstrated the presence of 5-methylcytosine in mtDNA by different approaches, but, on the other end, the opposite evidence that mtDNA is not methylated has also been published. Since over 1500 mitochondrial proteins are encoded by the nuclear genome, the altered methylation of these genes may well have a critical role in leading to the mitochondrial impairment observed in ADCA-DN and HSN1E. Thus, many open questions still remain unanswered, such as why mtDNA should be methylated, and how this process is

Cytosine modifications after gamma irradiation in aerated aqueous solution of Escherichia coli DNA

International Nuclear Information System (INIS)

Polverelli, M.

1983-04-01

After gamma irradiation of cytosine in aerated aqueous solution and utilization of various spectrometric methods (mass spectrometry, proton nuclear magnetic resonance and infrared spectrometry) about ten new radiolysis products were identified. The formation of N-glycolylbiuret in H 2 18 O aqueous solution of irradiated cytosine at pH 4,5 indicated that the preferred 18 OH hydroxyl radical attack was at C-5. The formation of trans 1-carbamoyl-4,5 dihydroxyimidazolidin-2 oxo which is the major product after cytosine pyrimidine ring rearrangement took place preferentially at neutral pH, while N-glycolylbiuret predominated at pH 4,5. The deamination pathway was predominant when cytosine was irradiated at acidic pH values (pH 2 ) or in copper complexes. The development of a new acid hydrolysis method using fluorhydric acid stabilized in pyridine made easier the evaluation of cytosine modifications after gamma irradiation in aerated aqueous solution of E. Coli DNA- 14 C-2 cytosine. This hydrolytic agent removed the modified bases from the polynucleotidic chain. A difference was found between the proportion of radiolytic products removed by acid hydrolysis and by irradiation of the free base in solution [fr

Intrauterine Exposure to Maternal Stress Alters Bdnf IV DNA Methylation and Telomere Length in the Brain of Adult Rat Offspring

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Blaze, Jennifer; Asok, Arun; Borrelli, Kristyn; Tulbert, Christine; Bollinger, Justin; Ronca Finco, April E.; Roth, Tania L.

2017-01-01

DNA methylation (addition of methyl groups to cytosines which normally represses gene transcription) and changes in telomere length (TTAGGG repeats on the ends of chromosomes) are two molecular modifications that result from stress and could contribute to the long-term effects of intrauterine exposure to maternal stress on offspring behavioral outcomes. Here, we measured methylation of Brain-derived neurotrophic factor (Bdnf), a gene important in development and plasticity, and telomere length in the brains of adult rat male and female offspring whose mothers were exposed to unpredictable and variable stressors throughout gestation. Males exposed to prenatal stress had greater methylation (Bdnf IV) in the medial prefrontal cortex (mPFC) compared to non-stressed controls. Further, prenatally-stressed males had shorter telomeres than controls in the mPFC. This study provides the first evidence in a rodent model of an association between prenatal stress exposure and subsequent shorter brain telomere length. Together findings indicate a long-term impact of prenatal stress on DNA methylation and telomere biology with relevance for behavioral and health outcomes, and contribute to a growing literature linking stress to intergenerational epigenetic alterations and changes in telomere length.

Genetic analysis of the cardiac methylome at single nucleotide resolution in a model of human cardiovascular disease.

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Michelle D Johnson

2014-12-01

Full Text Available Epigenetic marks such as cytosine methylation are important determinants of cellular and whole-body phenotypes. However, the extent of, and reasons for inter-individual differences in cytosine methylation, and their association with phenotypic variation are poorly characterised. Here we present the first genome-wide study of cytosine methylation at single-nucleotide resolution in an animal model of human disease. We used whole-genome bisulfite sequencing in the spontaneously hypertensive rat (SHR, a model of cardiovascular disease, and the Brown Norway (BN control strain, to define the genetic architecture of cytosine methylation in the mammalian heart and to test for association between methylation and pathophysiological phenotypes. Analysis of 10.6 million CpG dinucleotides identified 77,088 CpGs that were differentially methylated between the strains. In F1 hybrids we found 38,152 CpGs showing allele-specific methylation and 145 regions with parent-of-origin effects on methylation. Cis-linkage explained almost 60% of inter-strain variation in methylation at a subset of loci tested for linkage in a panel of recombinant inbred (RI strains. Methylation analysis in isolated cardiomyocytes showed that in the majority of cases methylation differences in cardiomyocytes and non-cardiomyocytes were strain-dependent, confirming a strong genetic component for cytosine methylation. We observed preferential nucleotide usage associated with increased and decreased methylation that is remarkably conserved across species, suggesting a common mechanism for germline control of inter-individual variation in CpG methylation. In the RI strain panel, we found significant correlation of CpG methylation and levels of serum chromogranin B (CgB, a proposed biomarker of heart failure, which is evidence for a link between germline DNA sequence variation, CpG methylation differences and pathophysiological phenotypes in the SHR strain. Together, these results will

Search for methylation-sensitive amplification polymorphisms in mutant figs

OpenAIRE

Rodrigues, M. G F; Martins, A. B G [UNESP; Bertoni, B. W.; Figueira, A.; Giuliatti, S.

2013-01-01

Fig (Ficus carica) breeding programs that use conventional approaches to develop new cultivars are rare, owing to limited genetic variability and the difficulty in obtaining plants via gamete fusion. Cytosine methylation in plants leads to gene repression, thereby affecting transcription without changing the DNA sequence. Previous studies using random amplification of polymorphic DNA and amplified fragment length polymorphism markers revealed no polymorphisms among select fig mutants that ori...

Search for methylation-sensitive amplification polymorphisms in mutant figs.

Science.gov (United States)

Rodrigues, M G F; Martins, A B G; Bertoni, B W; Figueira, A; Giuliatti, S

2013-07-08

Fig (Ficus carica) breeding programs that use conventional approaches to develop new cultivars are rare, owing to limited genetic variability and the difficulty in obtaining plants via gamete fusion. Cytosine methylation in plants leads to gene repression, thereby affecting transcription without changing the DNA sequence. Previous studies using random amplification of polymorphic DNA and amplified fragment length polymorphism markers revealed no polymorphisms among select fig mutants that originated from gamma-irradiated buds. Therefore, we conducted methylation-sensitive amplified polymorphism analysis to verify the existence of variability due to epigenetic DNA methylation among these mutant selections compared to the main cultivar 'Roxo-de-Valinhos'. Samples of genomic DNA were double-digested with either HpaII (methylation sensitive) or MspI (methylation insensitive) and with EcoRI. Fourteen primer combinations were tested, and on an average, non-methylated CCGG, symmetrically methylated CmCGG, and hemimethylated hmCCGG sites accounted for 87.9, 10.1, and 2.0%, respectively. MSAP analysis was effective in detecting differentially methylated sites in the genomic DNA of fig mutants, and methylation may be responsible for the phenotypic variation between treatments. Further analyses such as polymorphic DNA sequencing are necessary to validate these differences, standardize the regions of methylation, and analyze reads using bioinformatic tools.

Synthesis, Characterization, and Physicochemical Studies of Mixed Ligand Complexes of Inner Transition Metals with Lansoprazole and Cytosine

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Sarika Verma

2013-01-01

Full Text Available Few complexes of inner transition metals [Th(IV, Ce(IV, Nd(III, Gd(III] have been synthesized by reacting their metal salts with lansoprazole, 2-([3-methyl-4-(2,2,2-trifluoroethoxypyridin-2-yl]methylsulfinyl-1H-benzoimidazole and cytosine. All the complexes were synthesized in ethanolic medium. The yield percentage rangs from 80 to 90%. The complexes are coloured solids. The complexes were characterized through elemental analyses, conductance measurements, and spectroscopic methods (FT IR, FAB Mass, 1H NMR and UV. An IR spectrum indicates that the ligand behaves as bidentate ligands. The metal complexes have been screened for their antifungal activity towards Aspergillus niger fungi. The interaction of inner transition metals with lansoprazole, in presence of cytosine, has also been investigated potentiometrically at two different temperatures 26±1°C and 36±1°C and at 0.1 M (KNO3 ionic strength. The stability constants of ternary complexes indicate the stability order as Th(IV < Ce(IV < Gd(III < Nd(III. logK values obtained are positive and suggest greater stabilization of ternary complexes. The values of thermodynamic parameters (free energy (ΔG, enthalpy (ΔH, and entropy (ΔS are also calculated.

Epigenetics in Alzheimer's Disease: Perspective of DNA Methylation.

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Qazi, Talal Jamil; Quan, Zhenzhen; Mir, Asif; Qing, Hong

2018-02-01

Research over the years has shown that causes of Alzheimer's disease are not well understood, but over the past years, the involvement of epigenetic mechanisms in the developing memory formation either under pathological or physiological conditions has become clear. The term epigenetics represents the heredity of changes in phenotype that are independent of altered DNA sequences. Different studies validated that cytosine methylation of genomic DNA decreases with age in different tissues of mammals, and therefore, the role of epigenetic factors in developing neurological disorders in aging has been under focus. In this review, we summarized and reviewed the involvement of different epigenetic mechanisms especially the DNA methylation in Alzheimer's disease (AD), late-onset Alzheimer's disease (LOAD), familial Alzheimer's disease (FAD), and autosomal dominant Alzheimer's disease (ADAD). Down to the minutest of details, we tried to discuss the methylation patterns like mitochondrial DNA methylation and ribosomal DNA (rDNA) methylation. Additionally, we mentioned some therapeutic approaches related to epigenetics, which could provide a potential cure for AD. Moreover, we reviewed some recent studies that validate DNA methylation as a potential biomarker and its role in AD. We hope that this review will provide new insights into the understanding of AD pathogenesis from the epigenetic perspective especially from the perspective of DNA methylation.

DNA methylation in states of cell physiology and pathology.

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Lech Chyczewski

2007-10-01

Full Text Available DNA methylation is one of epigenetic mechanisms regulating gene expression. The methylation pattern is determined during embryogenesis and passed over to differentiating cells and tissues. In a normal cell, a significant degree of methylation is characteristic for extragenic DNA (cytosine within the CG dinucleotide while CpG islands located in gene promoters are unmethylated, except for inactive genes of the X chromosome and the genes subjected to genomic imprinting. The changes in the methylation pattern, which may appear as the organism age and in early stages of cancerogenesis, may lead to the silencing of over ninety endogenic genes. It has been found, that these disorders consist not only of the methylation of CpG islands, which are normally unmethylated, but also of the methylation of other dinucleotides, e.g. CpA. Such methylation has been observed in non-small cell lung cancer, in three regions of the exon 5 of the p53 gene (so-called "non-CpG" methylation. The knowledge of a normal methylation process and its aberrations appeared to be useful while searching for new markers enabling an early detection of cancer. With the application of the Real-Time PCR technique (using primers for methylated and unmethylated sequences five new genes which are potential biomarkers of lung cancer have been presented.

Winnowing DNA for rare sequences: highly specific sequence and methylation based enrichment.

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Jason D Thompson

Full Text Available Rare mutations in cell populations are known to be hallmarks of many diseases and cancers. Similarly, differential DNA methylation patterns arise in rare cell populations with diagnostic potential such as fetal cells circulating in maternal blood. Unfortunately, the frequency of alleles with diagnostic potential, relative to wild-type background sequence, is often well below the frequency of errors in currently available methods for sequence analysis, including very high throughput DNA sequencing. We demonstrate a DNA preparation and purification method that through non-linear electrophoretic separation in media containing oligonucleotide probes, achieves 10,000 fold enrichment of target DNA with single nucleotide specificity, and 100 fold enrichment of unmodified methylated DNA differing from the background by the methylation of a single cytosine residue.

Winnowing DNA for rare sequences: highly specific sequence and methylation based enrichment.

Science.gov (United States)

Thompson, Jason D; Shibahara, Gosuke; Rajan, Sweta; Pel, Joel; Marziali, Andre

2012-01-01

Rare mutations in cell populations are known to be hallmarks of many diseases and cancers. Similarly, differential DNA methylation patterns arise in rare cell populations with diagnostic potential such as fetal cells circulating in maternal blood. Unfortunately, the frequency of alleles with diagnostic potential, relative to wild-type background sequence, is often well below the frequency of errors in currently available methods for sequence analysis, including very high throughput DNA sequencing. We demonstrate a DNA preparation and purification method that through non-linear electrophoretic separation in media containing oligonucleotide probes, achieves 10,000 fold enrichment of target DNA with single nucleotide specificity, and 100 fold enrichment of unmodified methylated DNA differing from the background by the methylation of a single cytosine residue.

Identification of DNA methylation biomarkers from Infinium arrays

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Richard D Emes

2012-08-01

Full Text Available Epigenetic modifications of DNA, such as cytosine methylation are differentially abundant in diseases such as cancer. A goal for clinical research is finding sites that are differentially methylated between groups of samples to act as potential biomarkers for disease outcome. However, clinical samples are often limited in availability, represent a heterogeneous collection of cells or are of uncertain clinical class. Array based methods for identification of methylation provide a cost effective method to survey a proportion of the methylome at single base resolution. The Illumina Infinium array has become a popular and reliable high throughput method in this field and are proving useful in the identification of biomarkers for disease. Here, we compare a commonly used statistical test with a new intuitive and flexible computational approach to quickly detect differentially methylated sites. The method rapidly identifies and ranks candidate lists with greatest inter-group variability whilst controlling for intra-group variability. Intuitive and biologically relevant filters can be imposed to quickly identify sites and genes of interest.

Epigenetic editing using programmable zinc ginger proteins : inherited silencing of endogenous gene expression by targeted DNA methylation

NARCIS (Netherlands)

Stolzenburg, Sabine

2014-01-01

Cancer development is not only the result of genetic mutations but also stems from modifications in the epigenetic code leading to an aberrant expression of genes relevant for cancer. The most studied epigenetic mark is DNA methylation of cytosines in the promoters of genes, which is associated with

DNA methylation in sugarcane somaclonal variants assessed through methylation-sensitive amplified polymorphism.

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Francischini, J H M B; Kemper, E L; Costa, J B; Manechini, J R V; Pinto, L R

2017-05-04

Micropropagation is an important tool for large-scale multiplication of plant superior genotypes. However, somaclonal variation is one of the drawbacks of this process. Changes in DNA methylation have been widely reported as one of the main causes of somaclonal variations in plants. In order to investigate the occurrence of changes in the methylation pattern of sugarcane somaclonal variants, the MSAP (methylation-sensitive amplified polymorphism) technique was applied to micro-propagated plantlets sampled at the third subculture phase. The mother plant, in vitro normal plantlets, and in vitro abnormal plantlets (somaclonal variants) of four sugarcane clones were screened against 16 MSAP selective primers for EcoRI/MspI and EcoRI/HpaII restriction enzymes. A total of 1005 and 1200 MSAP-derived markers with polymorphism percentages of 28.36 and 40.67 were obtained for EcoRI/HpaII and EcoRI/MspI restriction enzyme combinations, respectively. The genetic similarity between the mother plant and the somaclonal variants ranged from 0.877 to 0.911 (EcoRI/MspI) and from 0.928 to 0.955 (EcoRI/HpaII). Most of the MASPs among mother plant and micro-propagated plantlets were derived from EcoRI/MspI restriction enzymes suggesting alteration due to gain or loss of internal cytosine methylation. A higher rate of loss of methylation (hypomethylation) than gain of methylation (hypermethylation) was observed in the abnormal in vitro sugarcane plantlets. Although changes in the methylation pattern were also observed in the in vitro normal plantlets, they were lower than those observed for the in vitro abnormal plantlets. The MASP technique proved to be a promising tool to early assessment of genetic fidelity of micro-propagated sugarcane plants.

Application of liquid chromatography/electrospray ionization ion trap tandem mass spectrometry for the evaluation of global nucleic acids: methylation in garden cress under exposure to CuO nanoparticles.

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Alcazar Magana, Armando; Wrobel, Kazimierz; Corrales Escobosa, Alma Rosa; Wrobel, Katarzyna

2016-01-15

A full understanding of the biological impact of nanomaterials demands analytical procedures suitable for the detection/quantification of epigenetic changes that occur in the exposed organisms. Here, the effect of CuO nanoparticles (NPs) on global methylation of nucleic acids in Lepidium sativum was evaluated by liquid chromatography/ion trap mass spectrometry. Enhanced selectivity toward cytosine-containing nucleosides was achieved by using their proton-bound dimers formed in positive electrospray ionization (ESI(+)) as precursor ions for multiple reaction monitoring (MRM) quantification based on one or two ion transitions. Plants were exposed to CuO NPs (0-1000 mg L(-1)); nucleic acid extracts were washed with bathocuproine disulfate; nucleosides were separated on a Luna C18 column coupled via ESI(+) to an AmaZon SL mass spectrometer (Bruker Daltonics). Cytidine, 2´-deoxycytidine, 5-methylcytidine, 5-methyl-2´-deoxycytidine and 5-hydroxymethyl-2´-deoxycytidine were quantified by MRM based on MS(3) ([2M+H](+)/[M+H](+)/[M+H-132](+) or [M+H-116](+)) and MS(2) ([2M+H](+)/[M+H](+) ). Bathocuproine disulfate, added as Cu(I) complexing agent, allowed for elimination of [2M+Cu](+) adducts from the mass spectra. Poorer instrumental detection limits were obtained for MS(3) (20-120 fmol) as compared to MS(2) (9.0-41 fmol); however, two ion transitions helped to eliminate matrix effects in plant extracts. The procedure was tested by analyzing salmon sperm DNA (Sigma) and applied for the evaluation of DNA and RNA methylation in plants; in the absence of NPs, 13.03% and 0.92% methylated cytosines were found in DNA and RNA, respectively; for NPs concentration >50 mg L(-1), DNA hypomethylation was observed with respect to unexposed plants. RNA methylation did not present significant changes upon plant exposure; 5-hydroxymethyl-2´-deoxycytidine was not detected in any sample. The MRM quantification proposed here of cytosine-containing nucleosides using their proton-bound homo

Pros and cons of methylation-based enrichment methods for ancient DNA

Science.gov (United States)

Seguin-Orlando, Andaine; Gamba, Cristina; Sarkissian, Clio Der; Ermini, Luca; Louvel, Guillaume; Boulygina, Eugenia; Sokolov, Alexey; Nedoluzhko, Artem; Lorenzen, Eline D.; Lopez, Patricio; McDonald, H. Gregory; Scott, Eric; Tikhonov, Alexei; Stafford,, Thomas W.; Alfarhan, Ahmed H.; Alquraishi, Saleh A.; Al-Rasheid, Khaled A. S.; Shapiro, Beth; Willerslev, Eske; Prokhortchouk, Egor; Orlando, Ludovic

2015-01-01

The recent discovery that DNA methylation survives in fossil material provides an opportunity for novel molecular approaches in palaeogenomics. Here, we apply to ancient DNA extracts the probe-independent Methylated Binding Domains (MBD)-based enrichment method, which targets DNA molecules containing methylated CpGs. Using remains of a Palaeo-Eskimo Saqqaq individual, woolly mammoths, polar bears and two equine species, we confirm that DNA methylation survives in a variety of tissues, environmental contexts and over a large temporal range (4,000 to over 45,000 years before present). MBD enrichment, however, appears principally biased towards the recovery of CpG-rich and long DNA templates and is limited by the fast post-mortem cytosine deamination rates of methylated epialleles. This method, thus, appears only appropriate for the analysis of ancient methylomes from very well preserved samples, where both DNA fragmentation and deamination have been limited. This work represents an essential step toward the characterization of ancient methylation signatures, which will help understanding the role of epigenetic changes in past environmental and cultural transitions. PMID:26134828

Methylation-mediated deamination of 5-methylcytosine appears to give rise to mutations causing human inherited disease in CpNpG trinucleotides, as well as in CpG dinucleotides

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Cooper David N

2010-08-01

Full Text Available Abstract The cytosine-guanine (CpG dinucleotide has long been known to be a hotspot for pathological mutation in the human genome. This hypermutability is related to its role as the major site of cytosine methylation with the attendant risk of spontaneous deamination of 5-methylcytosine (5mC to yield thymine. Cytosine methylation, however, also occurs in the context of CpNpG sites in the human genome, an unsurprising finding since the intrinsic symmetry of CpNpG renders it capable of supporting a semi-conservative model of replication of the methylation pattern. Recently, it has become clear that significant DNA methylation occurs in a CpHpG context (where H = A, C or T in a variety of human somatic tissues. If we assume that CpHpG methylation also occurs in the germline, and that 5mC deamination can occur within a CpHpG context, then we might surmise that methylated CpHpG sites could also constitute mutation hotspots causing human genetic disease. To test this postulate, 54,625 missense and nonsense mutations from 2,113 genes causing inherited disease were retrieved from the Human Gene Mutation Database http://www.hgmd.org. Some 18.2 per cent of these pathological lesions were found to be C → T and G → A transitions located in CpG dinucleotides (compatible with a model of methylation-mediated deamination of 5mC, an approximately ten-fold higher proportion than would have been expected by chance alone. The corresponding proportion for the CpHpG trinucleotide was 9.9 per cent, an approximately two-fold higher proportion than would have been expected by chance. We therefore estimate that ~5 per cent of missense/nonsense mutations causing human inherited disease may be attributable to methylation-mediated deamination of 5mC within a CpHpG context.

Involvement of a cytosine side chain in proton transfer in the rate-determining step of ribozyme self-cleavage

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Shih, I-hung; Been, Michael D.

2001-01-01

Ribozymes of hepatitis delta virus have been proposed to use an active-site cytosine as an acid-base catalyst in the self-cleavage reaction. In this study, we have examined the role of cytosine in more detail with the antigenomic ribozyme. Evidence that proton transfer in the rate-determining step involved cytosine 76 (C76) was obtained from examining cleavage activity of the wild-type and imidazole buffer-rescued C76-deleted (C76Δ) ribozymes in D2O and H2O. In both reactions, a similar kinetic isotope effect and shift in the apparent pKa indicate that the buffer is functionally substituting for the side chain in proton transfer. Proton inventory of the wild-type reaction supported a mechanism of a single proton transfer at the transition state. This proton transfer step was further characterized by exogenous base rescue of a C76Δ mutant with cytosine and imidazole analogues. For the imidazole analogues that rescued activity, the apparent pKa of the rescue reaction, measured under kcat/KM conditions, correlated with the pKa of the base. From these data a Brønsted coefficient (β) of 0.51 was determined for the base-rescued reaction of C76Δ. This value is consistent with that expected for proton transfer in the transition state. Together, these data provide strong support for a mechanism where an RNA side chain participates directly in general acid or general base catalysis of the wild-type ribozyme to facilitate RNA cleavage. PMID:11171978

Variation in whole DNA methylation in red maple (Acer rubrum) populations from a mining region: association with metal contamination and cation exchange capacity (CEC) in podzolic soils.

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Kalubi, K N; Mehes-Smith, M; Spiers, G; Omri, A

2017-04-01

Although a number of publications have provided convincing evidence that abiotic stresses such as drought and high salinity are involved in DNA methylation reports on the effects of metal contamination, pH, and cation exchange on DNA modifications are limited. The main objective of the present study is to determine the relationship between metal contamination and Cation exchange capacity (CEC) on whole DNA modifications. Metal analysis confirms that nickel and copper are the main contaminants in sampled sites within the Greater Sudbury Region (Ontario, Canada) and liming has increased soil pH significantly even after 30 years following dolomitic limestone applications. The estimated CEC values varied significantly among sites, ranging between 1.8 and 10.5 cmol(+) kg -1 , with a strong relationship being observed between CEC and pH (r = 0.96**). Cation exchange capacity, significantly lower in highly metal contaminated sites compared to both reference and less contaminated sites, was higher in the higher organic matter limed compared to unlimed sites. There was a significant variation in the level of cytosine methylation among the metal-contaminated sites. Significant and strong negative correlations between [5mdC]/[dG] and bioavailable nickel (r = -0.71**) or copper (r = -0.72**) contents were observed. The analysis of genomic DNA for adenine methylation in this study showed a very low level of [6N-mdA]/dT] in Acer rubrum plants analyzed ranging from 0 to 0.08%. Significant and very strong positive correlation was observed between [6N-mdA]/dT] and soil bioavailable nickel (r = 0.78**) and copper (r = 0.88**) content. This suggests that the increased bioavailable metal levels associated with contamination by nickel and copper particulates are associated with cytosine and adenine methylation.

DNA Methylation Dynamics of Germinal Center B Cells Are Mediated by AID

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Pilar M. Dominguez

2015-09-01

Full Text Available Changes in DNA methylation are required for the formation of germinal centers (GCs, but the mechanisms of such changes are poorly understood. Activation-induced cytidine deaminase (AID has been recently implicated in DNA demethylation through its deaminase activity coupled with DNA repair. We investigated the epigenetic function of AID in vivo in germinal center B cells (GCBs isolated from wild-type (WT and AID-deficient (Aicda−/− mice. We determined that the transit of B cells through the GC is associated with marked locus-specific loss of methylation and increased methylation diversity, both of which are lost in Aicda−/− animals. Differentially methylated cytosines (DMCs between GCBs and naive B cells (NBs are enriched in genes that are targeted for somatic hypermutation (SHM by AID, and these genes form networks required for B cell development and proliferation. Finally, we observed significant conservation of AID-dependent epigenetic reprogramming between mouse and human B cells.

DNA methylation based biomarkers: Practical considerations and applications

DEFF Research Database (Denmark)

Nielsen, Helene Myrtue; How Kit, Alexandre; Tost, Jorg

2012-01-01

of biochemical molecules such as proteins, DNA, RNA or lipids, whereby protein biomarkers have been the most extensively studied and used, notably in blood-based protein quantification tests or immunohistochemistry. The rise of interest in epigenetic mechanisms has allowed the identification of a new type...... of biomarker, DNA methylation, which is of great potential for many applications. This stable and heritable covalent modification mostly affects cytosines in the context of a CpG dinucleotide in humans. It can be detected and quantified by a number of technologies including genome-wide screening methods...... as well as locus- or gene-specific high-resolution analysis in different types of samples such as frozen tissues and FFPE samples, but also in body fluids such as urine, plasma, and serum obtained through non-invasive procedures. In some cases, DNA methylation based biomarkers have proven to be more...

DNA methylation alteration is a major consequence of genome doubling in autotetraploid Brassica rapa

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Xu Yanhao

2017-01-01

Full Text Available Polyploids are typically classified as autopolyploids or allopolyploids based on the origin of their chromosome sets. Autopolyploidy is much more common than traditionally believed. Allopolyploidization, accompanied by genomic and transcriptomic changes, has been well investigated. In this study, genetic, DNA methylation and gene expression changes in autotetraploid Brassica rapa were investigated. No genetic alteration was detected using an amplified fragment length polymorphism (AFLP approach. Using a cDNA-AFLP approach, approximately 0.58% of fragments showed changes in gene expression in autotetraploid B. rapa. The methylation-sensitive amplification polymorphism (MSAP analysis showed that approximately 1.7% of the fragments underwent DNA methylation changes upon genome doubling, with hypermethylation and demethylation changes equally affected. Fragments displaying changes in gene expression and methylation status were isolated and then sequenced and characterized, respectively. This study showed that variation in cytosine methylation is a major consequence of genome doubling in autotetraploid Brassica rapa.

Analysis of DNA methylation of maize in response to osmotic and salt stress based on methylation-sensitive amplified polymorphism.

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Tan, Ming-pu

2010-01-01

Water stress is known to alter cytosine methylation, which generally represses transcription. However, little is known about the role of methylation alteration in maize under osmotic stress. Here, methylation-sensitive amplified polymorphism (MSAP) was used to screen PEG- or NaCl-induced methylation alteration in maize seedlings. The sequences of 25 differentially amplified fragments relevant to stress were successfully obtained. Two stress-specific fragments from leaves, LP166 and LPS911, shown to be homologous to retrotransposon Gag-Pol protein genes, suggested that osmotic stress-induced methylation of retrotransposons. Three MSAP fragments, representing drought-induced or salt-induced methylation in leaves, were homologous to a maize aluminum-induced transporter. Besides these, heat shock protein HSP82, Poly [ADP-ribose] polymerase 2, Lipoxygenase, casein kinase (CK2), and dehydration-responsive element-binding (DREB) factor were also homologs of MSAP sequences from salt-treated roots. One MSAP fragment amplified from salt-treated roots, designated RS39, was homologous to the first intron of maize protein phosphatase 2C (zmPP2C), whereas - LS103, absent from salt-treated leaves, was homologous to maize glutathione S-transferases (zmGST). Expression analysis showed that salt-induced intron methylation of root zmPP2C significantly downregulated its expression, while salt-induced demethylation of leaf zmGST weakly upregulated its expression. The results suggested that salinity-induced methylation downregulated zmPP2C expression, a negative regulator of the stress response, while salinity-induced demethylation upregulated zmGST expression, a positive effecter of the stress response. Altered methylation, in response to stress, might also be involved in stress acclimation. Copyright 2009 Elsevier Masson SAS. All rights reserved.

DNA methylation in the human cerebral cortex is dynamically regulated throughout the life span and involves differentiated neurons.

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Kimberly D Siegmund

Full Text Available The role of DNA cytosine methylation, an epigenetic regulator of chromatin structure and function, during normal and pathological brain development and aging remains unclear. Here, we examined by MethyLight PCR the DNA methylation status at 50 loci, encompassing primarily 5' CpG islands of genes related to CNS growth and development, in temporal neocortex of 125 subjects ranging in age from 17 weeks of gestation to 104 years old. Two psychiatric disease cohorts--defined by chronic neurodegeneration (Alzheimer's or lack thereof (schizophrenia--were included. A robust and progressive rise in DNA methylation levels across the lifespan was observed for 8/50 loci (GABRA2, GAD1, HOXA1, NEUROD1, NEUROD2, PGR, STK11, SYK typically in conjunction with declining levels of the corresponding mRNAs. Another 16 loci were defined by a sharp rise in DNA methylation levels within the first few months or years after birth. Disease-associated changes were limited to 2/50 loci in the Alzheimer's cohort, which appeared to reflect an acceleration of the age-related change in normal brain. Additionally, methylation studies on sorted nuclei provided evidence for bidirectional methylation events in cortical neurons during the transition from childhood to advanced age, as reflected by significant increases at 3, and a decrease at 1 of 10 loci. Furthermore, the DNMT3a de novo DNA methyl-transferase was expressed across all ages, including a subset of neurons residing in layers III and V of the mature cortex. Therefore, DNA methylation is dynamically regulated in the human cerebral cortex throughout the lifespan, involves differentiated neurons, and affects a substantial portion of genes predominantly by an age-related increase.

Identification of exotic genetic components and DNA methylation pattern analysis of three cotton introgression lines from Gossypium bickii.

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He, Shou-Pu; Sun, Jun-Ling; Zhang, Chao; Du, Xiong-Ming

2011-01-01

The impact of alien DNA fragments on plant genome has been studied in many species. However, little is known about the introgression lines of Gossypium. To study the consequences of introgression in Gossypium, we investigated 2000 genomic and 800 epigenetic sites in three typical cotton introgression lines, as well as their cultivar (Gossypium hirsutum) and wild parents (Gossypium bickii), by amplified fragment length polymorphism (AFLP) and methylation-sensitive amplified polymorphism (MSAP). The results demonstrate that an average of 0.5% of exotic DNA segments from wild cotton is transmitted into the genome of each introgression line, with the addition of other forms of genetic variation. In total, an average of 0.7% of genetic variation sites is identified in introgression lines. Simultaneously, the overall cytosine methylation level in each introgression line is very close to that of the upland cotton parent (an average of 22.6%). Further dividing patterns reveal that both hypomethylation and hypermethylation occurred in introgression lines in comparison with the upland cotton parent. Sequencing of nine methylation polymorphism fragments showed that most (7 of 9) of the methylation alternations occurred in the noncoding sequences. The molecular evidence of introgression from wild cotton into introgression lines in our study is identified by AFLP. Moreover, the causes of petal variation in introgression lines are discussed.

Three-Dimensional Structure and Catalytic Mechanism of Cytosine Deaminase

Energy Technology Data Exchange (ETDEWEB)

R Hall; A Fedorov; C Xu; E Fedorov; S Almo; F Raushel

2011-12-31

Cytosine deaminase (CDA) from E. coli is a member of the amidohydrolase superfamily. The structure of the zinc-activated enzyme was determined in the presence of phosphonocytosine, a mimic of the tetrahedral reaction intermediate. This compound inhibits the deamination of cytosine with a K{sub i} of 52 nM. The zinc- and iron-containing enzymes were characterized to determine the effect of the divalent cations on activation of the hydrolytic water. Fe-CDA loses activity at low pH with a kinetic pKa of 6.0, and Zn-CDA has a kinetic pKa of 7.3. Mutation of Gln-156 decreased the catalytic activity by more than 5 orders of magnitude, supporting its role in substrate binding. Mutation of Glu-217, Asp-313, and His-246 significantly decreased catalytic activity supporting the role of these three residues in activation of the hydrolytic water molecule and facilitation of proton transfer reactions. A library of potential substrates was used to probe the structural determinants responsible for catalytic activity. CDA was able to catalyze the deamination of isocytosine and the hydrolysis of 3-oxauracil. Large inverse solvent isotope effects were obtained on k{sub cat} and k{sub cat}/K{sub m}, consistent with the formation of a low-barrier hydrogen bond during the conversion of cytosine to uracil. A chemical mechanism for substrate deamination by CDA was proposed.

Methylation of the estrogen receptor CpG island distinguishes spontaneous and plutonium-induced tumors from nitrosamine-induced lung tumors

Energy Technology Data Exchange (ETDEWEB)

Belinsky, S.A.; Baylin, S.B.; Issa, J.J. [Johns Hopkins Univ., Baltimore, MD (United States)

1995-12-01

CpG islands located in the promoter region of genes constitute one mechanism for regulating transcription. These islands are normally free of methylation, regardless of the expression state of the gene. Hypermethylation of CpG islands, the addition of a methyl group to the internal cytosine within CpG dinucleotides, can cause silencing of a gene. Hypermethylation has been detected as an early event at specific chromosome loci during the development of colon cancer and represents one mechanism used by neoplatic cells to inactivate tumor suppressor genes. Recent studies have demonstrated this mechanism in inactivation of the VHL tumor suppressor gene in 19% of sporadic renal tumors and the p16 {sup INK4a} tumor suppressor gene in 30% of non-small cell lung cancers. A recent report indicates that the estrogen receptor gene could also be inactivated through methylation. In addition, estrogen receptor CpG island methylation arises as a direct function of age in normal colonic mucosa and is present in virtually all colonic tumors. In cultured colon cancer cells, methylation-associated loss of expression of the estrogen receptor gene results in deregulated growth, suggesting a role for the estrogen receptor in colon cancer development. These results provide further evidence that gene silencing through methylation could be a predominant epigenetic mechanism underlying the development of many different types of cancer. The purpose of the current investigation was to determine whether estrogen receptor CpG island methylation is involved in the development of lung cancer. The frequency for methylation of the estrogen receptor CpG island in rodent lung tumors is summarized.

A relative quantitative Methylation-Sensitive Amplified Polymorphism (MSAP) method for the analysis of abiotic stress.

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Bednarek, Piotr T; Orłowska, Renata; Niedziela, Agnieszka

2017-04-21

We present a new methylation-sensitive amplified polymorphism (MSAP) approach for the evaluation of relative quantitative characteristics such as demethylation, de novo methylation, and preservation of methylation status of CCGG sequences, which are recognized by the isoschizomers HpaII and MspI. We applied the technique to analyze aluminum (Al)-tolerant and non-tolerant control and Al-stressed inbred triticale lines. The approach is based on detailed analysis of events affecting HpaII and MspI restriction sites in control and stressed samples, and takes advantage of molecular marker profiles generated by EcoRI/HpaII and EcoRI/MspI MSAP platforms. Five Al-tolerant and five non-tolerant triticale lines were exposed to aluminum stress using the physiologicaltest. Total genomic DNA was isolated from root tips of all tolerant and non-tolerant lines before and after Al stress following metAFLP and MSAP approaches. Based on codes reflecting events affecting cytosines within a given restriction site recognized by HpaII and MspI in control and stressed samples demethylation (DM), de novo methylation (DNM), preservation of methylated sites (MSP), and preservation of nonmethylatedsites (NMSP) were evaluated. MSAP profiles were used for Agglomerative hierarchicalclustering (AHC) based on Squared Euclidean distance and Ward's Agglomeration method whereas MSAP characteristics for ANOVA. Relative quantitative MSAP analysis revealed that both Al-tolerant and non-tolerant triticale lines subjected to Al stress underwent demethylation, with demethylation of CG predominating over CHG. The rate of de novo methylation in the CG context was ~3-fold lower than demethylation, whereas de novo methylation of CHG was observed only in Al-tolerant lines. Our relative quantitative MSAP approach, based on methylation events affecting cytosines within HpaII-MspI recognition sequences, was capable of quantifying de novo methylation, demethylation, methylation, and non-methylated status in control

Clinical implications of cytosine deletion of exon 5 of P53 gene in non small cell lung cancer patients

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Rashid Mir

2016-01-01

Full Text Available Aim: Lung cancer is considered to be the most common cancer in the world. In humans, about 50% or more cancers have a mutated tumor suppressor p53 gene thereby resulting in accumulation of p53 protein and losing its function to activate the target genes that regulate the cell cycle and apoptosis. Extensive research conducted in murine cancer models with activated p53, loss of p53, or p53 missense mutations have facilitated researchers to understand the role of this key protein. Our study was aimed to evaluate the frequency of cytosine deletion in nonsmall cell lung cancer (NSCLC patients. Methods: One hundred NSCLC patients were genotyped for P53 (exon5, codon168 cytosine deletion leading to loss of its function and activate the target genes by allele-specific polymerase chain reaction. The P53 cytosine deletion was correlated with all the clinicopathological parameters of the patients. Results and Analysis: 59% cases were carrying P53 cytosine deletion. Similarly, the significantly higher incidence of cytosine deletion was reported in current smokers (75% in comparison to exsmoker and nonsmoker. Significantly higher frequency of cytosine deletion was reported in adenocarcinoma (68.08% than squamous cell carcinoma (52.83%. Also, a significant difference was reported between p53 cytosine deletion and metastasis (64.28%. Further, the majority of the cases assessed for response carrying P53 cytosine deletion were found to show faster disease progression. Conclusion: The data suggests that there is a significant association of the P53 exon 5 deletion of cytosine in codon 168 with metastasis and staging of the disease.

Chilling-Mediated DNA Methylation Changes during Dormancy and Its Release Reveal the Importance of Epigenetic Regulation during Winter Dormancy in Apple (Malus x domestica Borkh.).

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Kumar, Gulshan; Rattan, Usha Kumari; Singh, Anil Kumar

2016-01-01

Winter dormancy is a well known mechanism adopted by temperate plants, to mitigate the chilling temperature of winters. However, acquisition of sufficient chilling during winter dormancy ensures the normal phenological traits in subsequent growing period. Thus, low temperature appears to play crucial roles in growth and development of temperate plants. Apple, being an important temperate fruit crop, also requires sufficient chilling to release winter dormancy and normal phenological traits, which are often associated with yield and quality of fruits. DNA cytosine methylation is one of the important epigenetic modifications which remarkably affect the gene expression during various developmental and adaptive processes. In present study, methylation sensitive amplified polymorphism was employed to assess the changes in cytosine methylation during dormancy, active growth and fruit set in apple, under differential chilling conditions. Under high chill conditions, total methylation was decreased from 27.2% in dormant bud to 21.0% in fruit set stage, while no significant reduction was found under low chill conditions. Moreover, the demethylation was found to be decreased, while methylation increased from dormant bud to fruit set stage under low chill as compared to high chill conditions. In addition, RNA-Seq analysis showed high expression of DNA methyltransferases and histone methyltransferases during dormancy and fruit set, and low expression of DNA glcosylases during active growth under low chill conditions, which was in accordance with changes in methylation patterns. The RNA-Seq data of 47 genes associated with MSAP fragments involved in cellular metabolism, stress response, antioxidant system and transcriptional regulation showed correlation between methylation and their expression. Similarly, bisulfite sequencing and qRT-PCR analysis of selected genes also showed correlation between gene body methylation and gene expression. Moreover, significant association

Chilling-Mediated DNA Methylation Changes during Dormancy and Its Release Reveal the Importance of Epigenetic Regulation during Winter Dormancy in Apple (Malus x domestica Borkh..

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Gulshan Kumar

Full Text Available Winter dormancy is a well known mechanism adopted by temperate plants, to mitigate the chilling temperature of winters. However, acquisition of sufficient chilling during winter dormancy ensures the normal phenological traits in subsequent growing period. Thus, low temperature appears to play crucial roles in growth and development of temperate plants. Apple, being an important temperate fruit crop, also requires sufficient chilling to release winter dormancy and normal phenological traits, which are often associated with yield and quality of fruits. DNA cytosine methylation is one of the important epigenetic modifications which remarkably affect the gene expression during various developmental and adaptive processes. In present study, methylation sensitive amplified polymorphism was employed to assess the changes in cytosine methylation during dormancy, active growth and fruit set in apple, under differential chilling conditions. Under high chill conditions, total methylation was decreased from 27.2% in dormant bud to 21.0% in fruit set stage, while no significant reduction was found under low chill conditions. Moreover, the demethylation was found to be decreased, while methylation increased from dormant bud to fruit set stage under low chill as compared to high chill conditions. In addition, RNA-Seq analysis showed high expression of DNA methyltransferases and histone methyltransferases during dormancy and fruit set, and low expression of DNA glcosylases during active growth under low chill conditions, which was in accordance with changes in methylation patterns. The RNA-Seq data of 47 genes associated with MSAP fragments involved in cellular metabolism, stress response, antioxidant system and transcriptional regulation showed correlation between methylation and their expression. Similarly, bisulfite sequencing and qRT-PCR analysis of selected genes also showed correlation between gene body methylation and gene expression. Moreover

NGSmethDB 2017: enhanced methylomes and differential methylation

Science.gov (United States)

Lebrón, Ricardo; Gómez-Martín, Cristina; Carpena, Pedro; Bernaola-Galván, Pedro; Barturen, Guillermo; Hackenberg, Michael; Oliver, José L.

2017-01-01

The 2017 update of NGSmethDB stores whole genome methylomes generated from short-read data sets obtained by bisulfite sequencing (WGBS) technology. To generate high-quality methylomes, stringent quality controls were integrated with third-part software, adding also a two-step mapping process to exploit the advantages of the new genome assembly models. The samples were all profiled under constant parameter settings, thus enabling comparative downstream analyses. Besides a significant increase in the number of samples, NGSmethDB now includes two additional data-types, which are a valuable resource for the discovery of methylation epigenetic biomarkers: (i) differentially methylated single-cytosines; and (ii) methylation segments (i.e. genome regions of homogeneous methylation). The NGSmethDB back-end is now based on MongoDB, a NoSQL hierarchical database using JSON-formatted documents and dynamic schemas, thus accelerating sample comparative analyses. Besides conventional database dumps, track hubs were implemented, which improved database access, visualization in genome browsers and comparative analyses to third-part annotations. In addition, the database can be also accessed through a RESTful API. Lastly, a Python client and a multiplatform virtual machine allow for program-driven access from user desktop. This way, private methylation data can be compared to NGSmethDB without the need to upload them to public servers. Database website: http://bioinfo2.ugr.es/NGSmethDB. PMID:27794041

Structural insight into maintenance methylation by mouse DNA methyltransferase 1 (Dnmt1)

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Takeshita, Kohei; Suetake, Isao; Yamashita, Eiki; Suga, Michihiro; Narita, Hirotaka; Nakagawa, Atsushi; Tajima, Shoji

2011-01-01

Methylation of cytosine in DNA plays a crucial role in development through inheritable gene silencing. The DNA methyltransferase Dnmt1 is responsible for the propagation of methylation patterns to the next generation via its preferential methylation of hemimethylated CpG sites in the genome; however, how Dnmt1 maintains methylation patterns is not fully understood. Here we report the crystal structure of the large fragment (291–1620) of mouse Dnmt1 and its complexes with cofactor S-adenosyl-L-methionine and its product S-adenosyl-L-homocystein. Notably, in the absence of DNA, the N-terminal domain responsible for targeting Dnmt1 to replication foci is inserted into the DNA-binding pocket, indicating that this domain must be removed for methylation to occur. Upon binding of S-adenosyl-L-methionine, the catalytic cysteine residue undergoes a conformation transition to a catalytically competent position. For the recognition of hemimethylated DNA, Dnmt1 is expected to utilize a target recognition domain that overhangs the putative DNA-binding pocket. Taking into considerations the recent report of a shorter fragment structure of Dnmt1 that the CXXC motif positions itself in the catalytic pocket and prevents aberrant de novo methylation, we propose that maintenance methylation is a multistep process accompanied by structural changes. PMID:21518897

DNA Methylation in Skeletal Muscle Stem Cell Specification, Proliferation, and Differentiation

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Rhianna C. Laker

2016-01-01

Full Text Available An unresolved and critically important question in skeletal muscle biology is how muscle stem cells initiate and regulate the genetic program during muscle development. Epigenetic dynamics are essential for cellular development and organogenesis in early life and it is becoming increasingly clear that epigenetic remodeling may also be responsible for the cellular adaptations that occur in later life. DNA methylation of cytosine bases within CpG dinucleotide pairs is an important epigenetic modification that reduces gene expression when located within a promoter or enhancer region. Recent advances in the field suggest that epigenetic regulation is essential for skeletal muscle stem cell identity and subsequent cell development. This review summarizes what is currently known about how skeletal muscle stem cells regulate the myogenic program through DNA methylation, discusses a novel role for metabolism in this process, and addresses DNA methylation dynamics in adult skeletal muscle in response to physical activity.

A novel fluorescent probe (dtpa-bis(cytosine)) for detection of Eu(III) in rare earth metal ions

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Yang, Fan; Ren, Peipei; Liu, Guanhong; Song, Youtao; Bu, Naishun; Wang, Jun

2018-03-01

In this paper, a novel fluorescent probe, dtpa-bis(cytosine), was designed and synthesized for detecting europium (Eu3 +) ion. Upon addition of Eu3 + ions into the dtpa-bis(cytosine) solution, the fluorescence intensity can strongly be enhanced. Conversely, adding other rare earth metal ions, such as Y3 +, Ce3 +, Pr3 +, Nd3 +, Sm3 +, Gd3 +, Tb3 +, Dy3 +, Ho3 +, Er3 +, Yb3 + and Lu3 +, into dtpa-bis(cytosine) solution, the fluorescence intensity is decreased slightly. Some parameters affecting the fluorescence intensity of dtpa-bis(cytosine) solution in the presence of Eu3 + ions were investigated, including solution pH value, Eu3 + ion concentration and interfering substances. The detection mechanism of Eu3 + ion using dtpa-bis(cytosine) as fluorescent probe was proposed. Under optimum conditions, the fluorescence emission intensities of EuIII-dtpa-bis(cytosine) at 375 nm in the concentration range of 0.50 × 10- 5 mol • L- 1-5.00 × 10- 5 mol • L- 1 of Eu3 + ion display a better linear relationship. The limit of detection (LOD) was determined as 8.65 × 10- 7 mol • L- 1 and the corresponding correlation coefficient (R2) of the linear equation is 0.9807. It is wished that the proposed method could be applied for sensitively and selectively detecting Eu3 + ion.

Mechanisms of Breast Cancer in Shift Workers: DNA Methylation in Five Core Circadian Genes in Nurses Working Night Shifts.

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Samulin Erdem, Johanna; Skare, Øivind; Petersen-Øverleir, Marte; Notø, Heidi Ødegaard; Lie, Jenny-Anne S; Reszka, Edyta; Pepłońska, Beata; Zienolddiny, Shanbeh

2017-01-01

Shift work has been suggested to be associated with breast cancer risk, and circadian disruption in shift workers is hypothesized as one of the mechanisms of increased cancer risk. There is, however, insufficient molecular evidence supporting this hypothesis. Using the quantitative methodology of pyrosequencing, epigenetic changes in 5-methyl cytosine (5mC) in five circadian genes CLOCK , BMAL1 , CRY1, PER1 and PER2 in female nurses working night shift work (278 breast cancer cases, 280 controls) were analyzed. In breast cancer cases, a medium exposure to night work was associated with increased methylation levels of the CLOCK (p=0.050), BMAL1 (p=0.001) and CRY1 (p=0.040) genes, compared with controls. Within the cases, analysis of the effects of shift work on the methylation patterns showed that methylation of CRY1 was lower in those who had worked night shift and had a high exposure (p=0.006) compared with cases that had worked only days. For cases with a medium exposure to night work, an increase in BMAL1 (p=0.003) and PER1 (p=0.035) methylation was observed compared with day working (unexposed) cases. The methylation levels of the five core circadian genes were also analyzed in relation to the estrogen and progesterone receptors status of the tumors in the cases, and no correlations were observed. Furthermore, nineteen polymorphisms in the five circadian genes were assessed for their effects on the methylation levels of the respective genes, but no associations were found. In summary, our data suggest that epigenetic regulation of CLOCK , BMAL1, CRY1 and PER1 may contribute to breast cancer in shift workers.

A novel CpG island set identifies tissue-specific methylation at developmental gene loci.

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Robert Illingworth

2008-01-01

Full Text Available CpG islands (CGIs are dense clusters of CpG sequences that punctuate the CpG-deficient human genome and associate with many gene promoters. As CGIs also differ from bulk chromosomal DNA by their frequent lack of cytosine methylation, we devised a CGI enrichment method based on nonmethylated CpG affinity chromatography. The resulting library was sequenced to define a novel human blood CGI set that includes many that are not detected by current algorithms. Approximately half of CGIs were associated with annotated gene transcription start sites, the remainder being intra- or intergenic. Using an array representing over 17,000 CGIs, we established that 6%-8% of CGIs are methylated in genomic DNA of human blood, brain, muscle, and spleen. Inter- and intragenic CGIs are preferentially susceptible to methylation. CGIs showing tissue-specific methylation were overrepresented at numerous genetic loci that are essential for development, including HOX and PAX family members. The findings enable a comprehensive analysis of the roles played by CGI methylation in normal and diseased human tissues.

Quantitative analysis of DNA methylation in chronic lymphocytic leukemia patients.

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Lyko, Frank; Stach, Dirk; Brenner, Axel; Stilgenbauer, Stephan; Döhner, Hartmut; Wirtz, Michaela; Wiessler, Manfred; Schmitz, Oliver J

2004-06-01

Changes in the genomic DNA methylation level have been found to be closely associated with tumorigenesis. In order to analyze the relation of aberrant DNA methylation to clinical and biological risk factors, we have determined the cytosine methylation level of 81 patients diagnosed with chronic lymphocytic leukemia (CLL). The analysis was based on DNA hydrolysis followed by derivatization of the 2'-desoxyribonucleoside-3'-monophosphates with BODIPY FL EDA. Derivatives were separated by micellar electrokinetic chromatography, and laser-induced fluorescence was used for detection. We analyzed potential correlations between DNA methylation levels and numerous patient parameters, including clinical observations and biological data. As a result, we observed a significant correlation with the immunoglobulin variable heavy chain gene (VH) mutation status. This factor has been repeatedly proposed as a reliable prognostic marker for CLL, which suggests that the methylation level might be a valuable factor in determining the prognostic outcome of CLL. We are now in the process of refining our method to broaden its application potential. In this context, we show here that the oxidation of the fluorescence marker in the samples and the evaporation of methanol in the electrolytes can be prevented by a film of paraffin oil. In summary, our results thus establish capillary electrophoresis as a valuable tool for analyzing the DNA methylation status of clinical samples.

Methylation of ribonucleic acid by the carcinogens dimethyl sulphate, N-methyl-N-nitrosourea and N-methyl-N′-nitro-N-nitrosoguanidine. Comparisons of chemical analyses at the nucleoside and base levels

Science.gov (United States)

Lawley, P. D.; Shah, S. A.

1972-01-01

1. The following methods for hydrolysis of methyl-14C-labelled RNA, and for chromatographic isolation and determination of the products, were investigated: enzymic digestion to nucleosides at pH6 or 8; alkaline hydrolysis and conversion into nucleosides; hydrolysis by acid to pyrimidine nucleotides and purine bases, or completely to bases; chromatography on Dowex 50 (NH4+ form) at pH6 or 8.9, or on Dowex 50 (H+ form), or on Sephadex G-10. 2. The suitability of the various methods for determination of methylation products was assessed. The principal product, 7-methylguanosine, was unstable under the conditions used for determinations of nucleosides. 3- and 7-Methyladenine and 3- and 7-methylguanine are best determined as bases; 1-methyladenine and 3-methylcytosine can be isolated as either nucleosides or bases; O6-methylguanine is unstable under the acid hydrolysis conditions used and can be determined as the nucleoside; 3-methyluracil was detected, but may be derived from methylation of the ionized form of uracil. 3. Differences between the patterns of methylation of RNA and homopolyribonucleotides by the N-methyl-N-nitroso compounds and dimethyl sulphate were found: the nitroso compounds were able to methylate O-6 of guanine, were relatively more reactive at N-7 of adenine and probably at N-3 of guanine, but less reactive at N-1 of adenine, N-3 of cytosine and probably at N-3 of uridine. They probably reacted more with the ribose–phosphate chain, but no products from this were identified. 4. The possible influences of these differences on biological action of the methylating agents is discussed. Nitroso compounds may differ principally in their ability to induce miscoding in the Watson–Crick sense by reaction at O-6 of guanine. Both types of agent may induce miscoding to a lesser extent through methylation at N-3 of guanine; both can methylate N atoms, presumably preventing Watson–Crick hydrogen-bonding. N-Methyl-N-nitrosourea can degrade RNA, possibly

Methyl-Donor and Cofactor Nutrient Intakes in the First 2–3 Years and Global DNA Methylation at Age 4: A Prospective Cohort Study

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Rachael M. Taylor

2018-02-01

Full Text Available Background: During the early postnatal period, the impact of nutrition on DNA methylation has not been well studied in humans. The aim was to quantify the relationship between one-carbon metabolism nutrient intake during the first three years of life and global DNA methylation levels at four years. Design: Childhood dietary intake was assessed using infant feeding questionnaires, food frequency questionnaires, 4-day weighed food records and 24-h food records. The dietary records were used to estimate the intake of methionine, folate, vitamins B2, B6 and B12 and choline. The accumulative nutrient intake specific rank from three months to three years of age was used for analysis. Global DNA methylation (%5-methyl cytosines (%5-mC was measured in buccal cells at four years of age, using an enzyme-linked immunosorbent assay (ELISA commercial kit. Linear regression models were used to quantify the statistical relationships. Results: Data were collected from 73 children recruited from the Women and their Children’s Health (WATCH study. No association was found between one-carbon metabolism nutrient intake and global DNA methylation levels (P > 0.05. Global DNA methylation levels in males were significantly higher than in females (median %5-mC: 1.82 vs. 1.03, males and females respectively, (P < 0.05. Conclusion: No association was found between the intake of one-carbon metabolism nutrients during the early postnatal period and global DNA methylation levels at age four years. Higher global DNA methylation levels in males warrants further investigation.

Methyl-Donor and Cofactor Nutrient Intakes in the First 2–3 Years and Global DNA Methylation at Age 4: A Prospective Cohort Study

Science.gov (United States)

Taylor, Rachael M.; Smith, Roger; Collins, Clare E.; Mossman, David; Wong-Brown, Michelle W.; Chan, Eng-Cheng; Evans, Tiffany-Jane; Attia, John R.; Smith, Tenele; Butler, Trent

2018-01-01

Background: During the early postnatal period, the impact of nutrition on DNA methylation has not been well studied in humans. The aim was to quantify the relationship between one-carbon metabolism nutrient intake during the first three years of life and global DNA methylation levels at four years. Design: Childhood dietary intake was assessed using infant feeding questionnaires, food frequency questionnaires, 4-day weighed food records and 24-h food records. The dietary records were used to estimate the intake of methionine, folate, vitamins B2, B6 and B12 and choline. The accumulative nutrient intake specific rank from three months to three years of age was used for analysis. Global DNA methylation (%5-methyl cytosines (%5-mC)) was measured in buccal cells at four years of age, using an enzyme-linked immunosorbent assay (ELISA) commercial kit. Linear regression models were used to quantify the statistical relationships. Results: Data were collected from 73 children recruited from the Women and their Children’s Health (WATCH) study. No association was found between one-carbon metabolism nutrient intake and global DNA methylation levels (P 0.05). Global DNA methylation levels in males were significantly higher than in females (median %5-mC: 1.82 vs. 1.03, males and females respectively, (P 0.05)). Conclusion: No association was found between the intake of one-carbon metabolism nutrients during the early postnatal period and global DNA methylation levels at age four years. Higher global DNA methylation levels in males warrants further investigation. PMID:29495543

CaMV-35S promoter sequence-specific DNA methylation in lettuce.

Science.gov (United States)

Okumura, Azusa; Shimada, Asahi; Yamasaki, Satoshi; Horino, Takuya; Iwata, Yuji; Koizumi, Nozomu; Nishihara, Masahiro; Mishiba, Kei-ichiro

2016-01-01

We found 35S promoter sequence-specific DNA methylation in lettuce. Additionally, transgenic lettuce plants having a modified 35S promoter lost methylation, suggesting the modified sequence is subjected to the methylation machinery. We previously reported that cauliflower mosaic virus 35S promoter-specific DNA methylation in transgenic gentian (Gentiana triflora × G. scabra) plants occurs irrespective of the copy number and the genomic location of T-DNA, and causes strong gene silencing. To confirm whether 35S-specific methylation can occur in other plant species, transgenic lettuce (Lactuca sativa L.) plants with a single copy of the 35S promoter-driven sGFP gene were produced and analyzed. Among 10 lines of transgenic plants, 3, 4, and 3 lines showed strong, weak, and no expression of sGFP mRNA, respectively. Bisulfite genomic sequencing of the 35S promoter region showed hypermethylation at CpG and CpWpG (where W is A or T) sites in 9 of 10 lines. Gentian-type de novo methylation pattern, consisting of methylated cytosines at CpHpH (where H is A, C, or T) sites, was also observed in the transgenic lettuce lines, suggesting that lettuce and gentian share similar methylation machinery. Four of five transgenic lettuce lines having a single copy of a modified 35S promoter, which was modified in the proposed core target of de novo methylation in gentian, exhibited 35S hypomethylation, indicating that the modified sequence may be the target of the 35S-specific methylation machinery.

Identification of a Novel Methylated Gene in Nasopharyngeal Carcinoma: TTC40

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Wajdi Ayadi

2014-01-01

Full Text Available To further explore the epigenetic changes in nasopharyngeal carcinoma (NPC, methylation-sensitive arbitrarily primed PCR was performed on NPC biopsies and nontumor nasopharyngeal samples. We have shown mainly two DNA fragments that appeared to be differentially methylated in NPCs versus nontumors. The first, defined as hypermethylated, corresponds to a CpG island at the 5′-end of the tetratricopeptide repeat domain 40 (TTC40 gene, whereas the second, defined as hypo-methylated, is located on repetitive sequences at chromosomes 16p11.1 and 13.1. Thereafter, the epigenetic alteration on the 5′-TTC40 gene was confirmed by methylation-specific PCR, showing a significant aberrant methylation in NPCs, compared to nontumors. In addition, the bisulfite sequencing analysis has shown a very high density of methylated cytosines in C15, C17, and X666 NPC xenografts. To assess whether TTC40 gene is silenced by aberrant methylation, we examined the gene expression by reverse transcription-PCR. Our analysis showed that the mRNA expression was significantly lower in tumors than in nontumors, which is associated with 5′-TTC40 gene hypermethylation. In conclusion, we found that the 5′-TTC40 gene is frequently methylated and is associated with the loss of mRNA expression in NPCs. Hypermethylation of 5′-TTC40 gene might play a role in NPC development; nevertheless, other studies are needed.

NGSmethDB 2017: enhanced methylomes and differential methylation.

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Lebrón, Ricardo; Gómez-Martín, Cristina; Carpena, Pedro; Bernaola-Galván, Pedro; Barturen, Guillermo; Hackenberg, Michael; Oliver, José L

2017-01-04

The 2017 update of NGSmethDB stores whole genome methylomes generated from short-read data sets obtained by bisulfite sequencing (WGBS) technology. To generate high-quality methylomes, stringent quality controls were integrated with third-part software, adding also a two-step mapping process to exploit the advantages of the new genome assembly models. The samples were all profiled under constant parameter settings, thus enabling comparative downstream analyses. Besides a significant increase in the number of samples, NGSmethDB now includes two additional data-types, which are a valuable resource for the discovery of methylation epigenetic biomarkers: (i) differentially methylated single-cytosines; and (ii) methylation segments (i.e. genome regions of homogeneous methylation). The NGSmethDB back-end is now based on MongoDB, a NoSQL hierarchical database using JSON-formatted documents and dynamic schemas, thus accelerating sample comparative analyses. Besides conventional database dumps, track hubs were implemented, which improved database access, visualization in genome browsers and comparative analyses to third-part annotations. In addition, the database can be also accessed through a RESTful API. Lastly, a Python client and a multiplatform virtual machine allow for program-driven access from user desktop. This way, private methylation data can be compared to NGSmethDB without the need to upload them to public servers. Database website: http://bioinfo2.ugr.es/NGSmethDB. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Infant sex-specific placental cadmium and DNA methylation associations

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Mohanty, April F., E-mail: april.mohanty@va.gov [Cardiovascular Health Research Unit, University of Washington, 1730 Minor Ave, Seattle, WA 98101 (United States); Department of Epidemiology, School of Public Health, University of Washington, Seattle, WA (United States); Farin, Fred M., E-mail: freddy@u.washington.edu [Department of Environmental and Occupational Health Sciences, School of Public Health, University of Washington, 4225 Roosevelt Way N.E., Suite #100, Seattle, WA 98105 (United States); Bammler, Theo K., E-mail: tbammler@u.washington.edu [Department of Environmental and Occupational Health Sciences, School of Public Health, University of Washington, 4225 Roosevelt Way N.E., Suite #100, Seattle, WA 98105 (United States); MacDonald, James W., E-mail: jmacdon@uw.edu [Department of Environmental and Occupational Health Sciences, School of Public Health, University of Washington, 4225 Roosevelt Way N.E., Suite #100, Seattle, WA 98105 (United States); Afsharinejad, Zahra, E-mail: zafshari@u.washington.edu [Department of Environmental and Occupational Health Sciences, School of Public Health, University of Washington, 4225 Roosevelt Way N.E., Suite #100, Seattle, WA 98105 (United States); Burbacher, Thomas M., E-mail: tmb@uw.edu [Department of Environmental and Occupational Health Sciences, School of Public Health, University of Washington, Box: 357234, 1705 N.E. Pacific Street, Seattle, WA 98195 (United States); Siscovick, David S., E-mail: dsiscovick@nyam.org [Cardiovascular Health Research Unit, University of Washington, 1730 Minor Ave, Seattle, WA 98101 (United States); Department of Epidemiology, School of Public Health, University of Washington, Seattle, WA (United States); Department of Medicine, University of Washington, Seattle, WA (United States); and others

2015-04-15

Background: Recent evidence suggests that maternal cadmium (Cd) burden and fetal growth associations may vary by fetal sex. However, mechanisms contributing to these differences are unknown. Objectives: Among 24 maternal-infant pairs, we investigated infant sex-specific associations between placental Cd and placental genome-wide DNA methylation. Methods: We used ANOVA models to examine sex-stratified associations of placental Cd (dichotomized into high/low Cd using sex-specific Cd median cutoffs) with DNA methylation at each cytosine-phosphate-guanine site or region. Statistical significance was defined using a false discovery rate cutoff (<0.10). Results: Medians of placental Cd among females and males were 5 and 2 ng/g, respectively. Among females, three sites (near ADP-ribosylation factor-like 9 (ARL9), siah E3 ubiquitin protein ligase family member 3 (SIAH3), and heparin sulfate (glucosamine) 3-O-sulfotransferase 4 (HS3ST4) and one region on chromosome 7 (including carnitine O-octanoyltransferase (CROT) and TP5S target 1 (TP53TG1)) were hypomethylated in high Cd placentas. Among males, high placental Cd was associated with methylation of three sites, two (hypomethylated) near MDS1 and EVI1 complex locus (MECOM) and one (hypermethylated) near spalt-like transcription factor 1 (SALL1), and two regions (both hypomethylated, one on chromosome 3 including MECOM and another on chromosome 8 including rho guanine nucleotide exchange factor (GEF) 10 (ARHGEF10). Differentially methylated sites were at or close to transcription start sites of genes involved in cell damage response (SIAH3, HS3ST4, TP53TG1) in females and cell differentiation, angiogenesis and organ development (MECOM, SALL1) in males. Conclusions: Our preliminary study supports infant sex-specific placental Cd-DNA methylation associations, possibly accounting for previously reported differences in Cd-fetal growth associations across fetal sex. Larger studies are needed to replicate and extend these

Infant sex-specific placental cadmium and DNA methylation associations

International Nuclear Information System (INIS)

Mohanty, April F.; Farin, Fred M.; Bammler, Theo K.; MacDonald, James W.; Afsharinejad, Zahra; Burbacher, Thomas M.; Siscovick, David S.

2015-01-01

Background: Recent evidence suggests that maternal cadmium (Cd) burden and fetal growth associations may vary by fetal sex. However, mechanisms contributing to these differences are unknown. Objectives: Among 24 maternal-infant pairs, we investigated infant sex-specific associations between placental Cd and placental genome-wide DNA methylation. Methods: We used ANOVA models to examine sex-stratified associations of placental Cd (dichotomized into high/low Cd using sex-specific Cd median cutoffs) with DNA methylation at each cytosine-phosphate-guanine site or region. Statistical significance was defined using a false discovery rate cutoff (<0.10). Results: Medians of placental Cd among females and males were 5 and 2 ng/g, respectively. Among females, three sites (near ADP-ribosylation factor-like 9 (ARL9), siah E3 ubiquitin protein ligase family member 3 (SIAH3), and heparin sulfate (glucosamine) 3-O-sulfotransferase 4 (HS3ST4) and one region on chromosome 7 (including carnitine O-octanoyltransferase (CROT) and TP5S target 1 (TP53TG1)) were hypomethylated in high Cd placentas. Among males, high placental Cd was associated with methylation of three sites, two (hypomethylated) near MDS1 and EVI1 complex locus (MECOM) and one (hypermethylated) near spalt-like transcription factor 1 (SALL1), and two regions (both hypomethylated, one on chromosome 3 including MECOM and another on chromosome 8 including rho guanine nucleotide exchange factor (GEF) 10 (ARHGEF10). Differentially methylated sites were at or close to transcription start sites of genes involved in cell damage response (SIAH3, HS3ST4, TP53TG1) in females and cell differentiation, angiogenesis and organ development (MECOM, SALL1) in males. Conclusions: Our preliminary study supports infant sex-specific placental Cd-DNA methylation associations, possibly accounting for previously reported differences in Cd-fetal growth associations across fetal sex. Larger studies are needed to replicate and extend these

DNA Methylation as a Biomarker for Preeclampsia

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Anderson, Cindy M.; Ralph, Jody L.; Wright, Michelle L.; Linggi, Bryan E.; Ohm, Joyce E.

2014-10-01

Background: Preeclampsia contributes significantly to pregnancy-associated morbidity and mortality as well as future risk of cardiovascular disease in mother and offspring, and preeclampsia in offspring. The lack of reliable methods for early detection limits the opportunities for prevention, diagnosis, and timely treatment. Purpose: The purpose of this study was to explore distinct DNA methylation patterns associated with preeclampsia in both maternal cells and fetal-derived tissue that represent potential biomarkers to predict future preeclampsia and inheritance in children. Method: A convenience sample of nulliparous women (N = 55) in the first trimester of pregnancy was recruited for this prospective study. Genome-wide DNA methylation was quantified in first-trimester maternal peripheral white blood cells and placental chorionic tissue from normotensive women and those with preeclampsia (n = 6/group). Results: Late-onset preeclampsia developed in 12.7% of women. Significant differences in DNA methylation were identified in 207 individual linked cytosine and guanine (CpG) sites in maternal white blood cells collected in the first trimester (132 sites with gain and 75 sites with loss of methylation), which were common to approximately 75% of the differentially methylated CpG sites identified in chorionic tissue of fetal origin. Conclusion: This study is the first to identify maternal epigenetic targets and common targets in fetal-derived tissue that represent putative biomarkers for early detection and heritable risk of preeclampsia. Findings may pave the way for diagnosis of preeclampsia prior to its clinical presentation and acute damaging effects, and the potential for prevention of the detrimental long-term sequelae.

Metal-mediated deamination of cytosine: experiment and DFT calculations

Czech Academy of Sciences Publication Activity Database

Šponer, Judit E.; Sanz Miguel, P. J.; Rodríguez-Santiago, L.; Erxleben, A.; Krumm, M.; Sodupe, M.; Šponer, Jiří; Lippert, B.

2004-01-01

Roč. 43, č. 40 (2004), s. 5396-5399 ISSN 1433-7851 R&D Projects: GA MŠk LN00A016 Institutional research plan: CEZ:AV0Z5004920 Keywords : cytosine * deamination * density functional calculations Subject RIV: BO - Biophysics Impact factor: 9.161, year: 2004

Methylator phenotype of malignant germ cell tumours in children identifies strong candidates for chemotherapy resistance.

Science.gov (United States)

Jeyapalan, J N; Noor, D A Mohamed; Lee, S-H; Tan, C L; Appleby, V A; Kilday, J P; Palmer, R D; Schwalbe, E C; Clifford, S C; Walker, D A; Murray, M J; Coleman, N; Nicholson, J C; Scotting, P J

2011-08-09

Yolk sac tumours (YSTs) and germinomas are the two major pure histological subtypes of germ cell tumours. To date, the role of DNA methylation in the aetiology of this class of tumour has only been analysed in adult testicular forms and with respect to only a few genes. A bank of paediatric tumours was analysed for global methylation of LINE-1 repeat elements and global methylation of regulatory elements using GoldenGate methylation arrays. Both germinomas and YSTs exhibited significant global hypomethylation of LINE-1 elements. However, in germinomas, methylation of gene regulatory regions differed little from control samples, whereas YSTs exhibited increased methylation at a large proportion of the loci tested, showing a 'methylator' phenotype, including silencing of genes associated with Caspase-8-dependent apoptosis. Furthermore, we found that the methylator phenotype of YSTs was coincident with higher levels of expression of the DNA methyltransferase, DNA (cytosine-5)-methyltransferase 3B, suggesting a mechanism underlying the phenotype. Epigenetic silencing of a large number of potential tumour suppressor genes in YSTs might explain why they exhibit a more aggressive natural history than germinomas and silencing of genes associated with Caspase-8-dependent cell death might explain the relative resistance of YSTs to conventional therapy.

Elastic electron scattering from the DNA bases: cytosine and thymine

International Nuclear Information System (INIS)

Colyer, C J; Bellm, S M; Lohmanny, B; Blanco, F; Garcia, G

2012-01-01

Relative elastic differential cross sections for elastic scattering from cytosine and thymine have been measured using the crossed beam method. The experimental data are compared with theoretical cross sections calculated by the screen corrected additivity rule method.

Instability of chromosome number and DNA methylation variation induced by hybridization and amphidiploid formation between Raphanus sativus L. and Brassica alboglabra Bailey

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Wang Yanjie

2010-09-01

Full Text Available Abstract Background Distant hybridization can result genome duplication and allopolyploid formation which may play a significant role in the origin and evolution of many plant species. It is unclear how the two or more divergent genomes coordinate in one nucleus with a single parental cytoplasm within allopolyploids. We used cytological and molecular methods to investigate the genetic and epigenetic instabilities associated with the process of distant hybridization and allopolyploid formation, measuring changes in chromosome number and DNA methylation across multiple generations. Results F1 plants from intergeneric hybridization between Raphanus sativus L. (2n = 18, RR and Brassica alboglabra Bailey (2n = 18, CC were obtained by hand crosses and subsequent embryo rescue. Random amplification of polymorphic DNA (RAPD markers were used to identify the F1 hybrid plants. The RAPD data indicated that the hybrids produced specific bands similar to those of parents and new bands that were not present in either parent. Chromosome number variation of somatic cells from allotetraploids in the F4 to F10 generations showed that intensive genetic changes occurred in the early generations of distant hybridization, leading to the formation of mixopolyploids with different chromosome numbers. DNA methylation variation was revealed using MSAP (methylation-sensitive amplification polymorphism, which showed that cytosine methylation patterns changed markedly in the process of hybridization and amphidiploid formation. Differences in cytosine methylation levels demonstrated an epigenetic instability of the allopolyploid of Raphanobrassica between the genetically stable and unstable generations. Conclusions Our results showed that chromosome instability occurred in the early generations of allopolyploidy and then the plants were reverted to largely euploidy in later generations. During this process, DNA methylation changed markedly. These results suggest that

Patterns of DNMT1 Promoter Methylation in Patients with Acute Lymphoblastic Leukemia.

Science.gov (United States)

Rahmani, Tirdad; Azad, Mehdi; Chahardouli, Bahram; Nasiri, Hajar; Vatanmakanian, Mousa; Kaviani, Saeid

2017-07-01

Background: Acute lymphoblastic leukemia (ALL) is a clonal malignant disorder characterized by an uncontrolled proliferation of immature T or B lymphocytes. Extensive studies have shown that the epigenetic changes, especially modified DNA methylation patterns in the regulatory regions through the DNA methyltransferase (DNMTs), play an important role in the development of genetic disorders and abnormal growth and maturation capacity of leukemic stem cells (LSCs).The aim of this study was to evaluate the changes in DNMT1 promoter methylation and its expression pattern in patients with ALL. Materials and Methods: In this experimental study, methylation specific PCR (MSP) was used to assess the methylation status of DNMT1 promoter regions in samples collected from ALL patients (n=45) and healthy control subjects. According to this method, un-methylated cytosine nucleotides are converted to uracil by sodium bisulfite and the proliferation of methylated and un-methylated regions are performed using specific primers for target sequences. Results: None of the patients with B and T-ALL showed methylated promoter regions of the DNMT1 gene, while the methylation pattern of both pre-B ALL patients and the control group showed a relative promoter methylation. Conclusion: Analysis of promoter methylation patterns in various subgroups of ALL has revealed the importance of DNMT1 in the regulation of gene expression. Likewise, extensive data have also highlighted the methylation-based mechanisms exerted by DNAM1 as one of the main participants regulating gene expression in B-ALL and T-ALL patients. Investigation of the overall DNA methylation pattern offers significant improvements in the prediction of disease prognosis and treatment response.

Neuronal DNA Methylation Profiling of Blast-Related Traumatic Brain Injury.

Science.gov (United States)

Haghighi, Fatemeh; Ge, Yongchao; Chen, Sean; Xin, Yurong; Umali, Michelle U; De Gasperi, Rita; Gama Sosa, Miguel A; Ahlers, Stephen T; Elder, Gregory A

2015-08-15

Long-term molecular changes in the brain resulting from blast exposure may be mediated by epigenetic changes, such as deoxyribonucleic acid (DNA) methylation, that regulate gene expression. Aberrant regulation of gene expression is associated with behavioral abnormalities, where DNA methylation bridges environmental signals to sustained changes in gene expression. We assessed DNA methylation changes in the brains of rats exposed to three 74.5 kPa blast overpressure events, conditions that have been associated with long-term anxiogenic manifestations weeks or months following the initial exposures. Rat frontal cortex eight months post-exposure was used for cell sorting of whole brain tissue into neurons and glia. We interrogated DNA methylation profiles in these cells using Expanded Reduced Representation Bisulfite Sequencing. We obtained data for millions of cytosines, showing distinct methylation profiles for neurons and glia and an increase in global methylation in neuronal versus glial cells (pDNA methylation perturbations in blast overpressure-exposed animals, compared with sham blast controls, within 458 and 379 genes in neurons and glia, respectively. Differentially methylated neuronal genes showed enrichment in cell death and survival and nervous system development and function, including genes involved in transforming growth factor β and nitric oxide signaling. Functional validation via gene expression analysis of 30 differentially methylated neuronal and glial genes showed a 1.2 fold change in gene expression of the serotonin N-acetyltransferase gene (Aanat) in blast animals (pDNA methylation induced in response to multiple blast overpressure exposures. In particular, increased methylation and decreased gene expression were observed in the Aanat gene, which is involved in converting serotonin to the circadian hormone melatonin and is implicated in sleep disturbance and depression associated with traumatic brain injury.

A nonparametric Bayesian approach for clustering bisulfate-based DNA methylation profiles.

Science.gov (United States)

Zhang, Lin; Meng, Jia; Liu, Hui; Huang, Yufei

2012-01-01

DNA methylation occurs in the context of a CpG dinucleotide. It is an important epigenetic modification, which can be inherited through cell division. The two major types of methylation include hypomethylation and hypermethylation. Unique methylation patterns have been shown to exist in diseases including various types of cancer. DNA methylation analysis promises to become a powerful tool in cancer diagnosis, treatment and prognostication. Large-scale methylation arrays are now available for studying methylation genome-wide. The Illumina methylation platform simultaneously measures cytosine methylation at more than 1500 CpG sites associated with over 800 cancer-related genes. Cluster analysis is often used to identify DNA methylation subgroups for prognosis and diagnosis. However, due to the unique non-Gaussian characteristics, traditional clustering methods may not be appropriate for DNA and methylation data, and the determination of optimal cluster number is still problematic. A Dirichlet process beta mixture model (DPBMM) is proposed that models the DNA methylation expressions as an infinite number of beta mixture distribution. The model allows automatic learning of the relevant parameters such as the cluster mixing proportion, the parameters of beta distribution for each cluster, and especially the number of potential clusters. Since the model is high dimensional and analytically intractable, we proposed a Gibbs sampling "no-gaps" solution for computing the posterior distributions, hence the estimates of the parameters. The proposed algorithm was tested on simulated data as well as methylation data from 55 Glioblastoma multiform (GBM) brain tissue samples. To reduce the computational burden due to the high data dimensionality, a dimension reduction method is adopted. The two GBM clusters yielded by DPBMM are based on data of different number of loci (P-value < 0.1), while hierarchical clustering cannot yield statistically significant clusters.

[Analysis of methylation-sensitive amplified polymorphism in wheat genome under the wheat leaf rust stress].

Science.gov (United States)

Fu, Sheng-Jie; Wang, Hui; Feng, Li-Na; Sun, Yi; Yang, Wen-Xiang; Liu, Da-Qun

2009-03-01

Intrinsic DNA methylation pattern is an integral component of the epigenetic network in many eukaryotes. DNA methylation plays an important role in regulating gene expression in eukaryotes. Biological stress in plant provides an inherent epigenetic driving force of evolution. Study of DNA methylation patterns arising from biological stress will help us fully understand the epigenetic regulation of gene expression and DNA methylation of biological functions. The wheat near-isogenic lines TcLr19 and TcLr41 were resistant to races THTT and TKTJ, respectively, and Thatcher is compatible in the interaction with Puccinia triticina THTT and TKTJ, respectively. By means of methylation-sensitive amplified polymorphism (MSAP) analysis, the patterns of cytosine methylation in TcLr19, TcLr41, and Thatcher inoculated with P. triticina THTT and TKTJ were compared with those of the untreated samples. All the DNA fragments, each representing a recognition site cleaved by each or both of isoschizomers, were amplified using 60 pairs of selective primers. The results indicated that there was no significant difference between the challenged and unchallenged plants at DNA methylation level. However, epigenetic difference between the near-isogenic line for wheat leaf rust resistance gene Lr41 and Thatcher was present.

Divergent methylation pattern in adult stage between two forms of Tetranychus urticae (Acari: Tetranychidae).

Science.gov (United States)

Yang, Si-Xia; Guo, Chao; Zhao, Xiu-Ting; Sun, Jing-Tao; Hong, Xiao-Yue

2017-02-19

The two-spotted spider mite, Tetranychus urticae Koch has two forms: green form and red form. Understanding the molecular basis of how these two forms established without divergent genetic background is an intriguing area. As a well-known epigenetic process, DNA methylation has particularly important roles in gene regulation and developmental variation across diverse organisms that do not alter genetic background. Here, to investigate whether DNA methylation could be associated with different phenotypic consequences in the two forms of T. urticae, we surveyed the genome-wide cytosine methylation status and expression level of DNA methyltransferase 3 (Tudnmt3) throughout their entire life cycle. Methylation-sensitive amplification polymorphism (MSAP) analyses of 585 loci revealed variable methylation patterns in the different developmental stages. In particular, principal coordinates analysis (PCoA) indicates a significant epigenetic differentiation between female adults of the two forms. The gene expression of Tudnmt3 was detected in all examined developmental stages, which was significantly different in the adult stage of the two forms. Together, our results reveal the epigenetic distance between the two forms of T. urticae, suggesting that DNA methylation might be implicated in different developmental demands, and contribute to different phenotypes in the adult stage of these two forms. © 2017 Institute of Zoology, Chinese Academy of Sciences.

Structure Determination of an Ag-I-Mediated Cytosine-Cytosine Base Pair within DNA Duplex in Solution with H-1/N-15/Ag-109 NMR Spectroscopy

Czech Academy of Sciences Publication Activity Database

Dairaku, T.; Furuita, K.; Sato, H.; Šebera, Jakub; Nakashima, K.; Kondo, J.; Yamanaka, D.; Kondo, Y.; Okamoto, I.; Ono, A.; Sychrovský, Vladimír; Kojima, C.; Tanaka, Y.

2016-01-01

Roč. 22, č. 37 (2016), s. 13028-13031 ISSN 0947-6539 R&D Projects: GA ČR GA13-27676S Institutional support: RVO:61388963 Keywords : NMR * Ag * cytosine * DFT Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 5.317, year: 2016

Transmission of epi-alleles with MET1-dependent dense methylation in Arabidopsis thaliana.

Directory of Open Access Journals (Sweden)

Michael Watson

Full Text Available DNA methylation in plants targets cytosines in three sequence contexts, CG, CHG and CHH (H representing A, C or T. Each of these patterns has traditionally been associated with distinct DNA methylation pathways with CHH methylation being controlled by the RNA dependent DNA methylation (RdDM pathway employing small RNAs as a guide for the de novo DOMAINS REARRANGED METHYLTRANSFERASE (DRM2, and maintenance DNA METHYLTRANSFERASE1 (MET1 being responsible for faithful propagation of CG methylation. Here we report an unusual 'dense methylation' pattern under the control of MET1, with methylation in all three sequence contexts. We identified epi-alleles of dense methylation at a non coding RNA locus (At4g15242 in Arabidopsis ecotypes, with distinct dense methylation and expression characteristics, which are stably maintained and transmitted in genetic crosses and which can be heritably altered by depletion of MET1. This suggests that, in addition to its classical CG maintenance function, at certain loci MET1 plays a role in creating transcriptional diversity based on the generation of independent epi-alleles. Database inspection identified several other loci with MET1-dependent dense methylation patterns. Arabidopsis ecotypes contain distinct epi-alleles of these loci with expression patterns that inversely correlate with methylation density, predominantly within the transcribed region. In Arabidopsis, dense methylation appears to be an exception as it is only found at a small number of loci. Its presence does, however, highlight the potential for MET1 as a contributor to epigenetic diversity, and it will be interesting to investigate the representation of dense methylation in other plant species.

N3 and O2 Protonated Conformers of the Cytosine Mononucleotides Coexist in the Gas Phase

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Wu, R. R.; Hamlow, L. A.; He, C. C.; Nei, Y.-w.; Berden, G.; Oomens, J.; Rodgers, M. T.

2017-08-01

The gas-phase conformations of the protonated forms of the DNA and RNA cytosine mononucleotides, [pdCyd+H]+ and [pCyd+H]+, are examined by infrared multiple photon dissociation (IRMPD) action spectroscopy over the IR fingerprint and hydrogen-stretching regions complemented by electronic structure calculations. The low-energy conformations of [pdCyd+H]+ and [pCyd+H]+ and their relative stabilities are computed at the B3LYP/6-311+G(2d,2p)//B3LYP/6-311+G(d,p) and MP2(full)/6-311+G(2d,2p)//B3LYP/6-311+G(d,p) levels of theory. Comparisons of the measured IRMPD action spectra and B3LYP/6-311+G(d,p) linear IR spectra computed for the low-energy conformers allow the conformers present in the experiments to be determined. Similar to that found in previous IRMPD action spectroscopy studies of the protonated forms of the cytosine nucleosides, [dCyd+H]+ and [Cyd+H]+, both N3 and O2 protonated cytosine mononucleotides exhibiting an anti orientation of cytosine are found to coexist in the experimental population. The 2'-hydroxyl substituent does not significantly influence the most stable conformations of [pCyd+H]+ versus those of [pdCyd+H]+, as the IRMPD spectral profiles of [pdCyd+H]+ and [pCyd+H]+ are similar. However, the presence of the 2'-hydroxyl substituent does influence the relative intensities of the measured IRMPD bands. Comparisons to IRMPD spectroscopy studies of the deprotonated forms of the cytosine mononucleotides, [pdCyd-H]- and [pCyd-H]-, provide insight into the effects of protonation versus deprotonation on the conformational features of the nucleobase and sugar moieties. Likewise, comparisons to results of IRMPD spectroscopy studies of the protonated cytosine nucleosides provide insight into the influence of the phosphate moiety on structure. Comparison with previous ion mobility results shows the superiority of IRMPD spectroscopy for distinguishing various protonation sites.

IRMPD Action Spectroscopy of Alkali Metal Cation-Cytosine Complexes: Effects of Alkali Metal Cation Size on Gas Phase Conformation

NARCIS (Netherlands)

Yang, B.; Wu, R.R.; Polfer, N.C.; Berden, G.; Oomens, J.; Rodgers, M.T.

2013-01-01

The gas-phase structures of alkali metal cation-cytosine complexes generated by electrospray ionization are probed via infrared multiple photon dissociation (IRMPD) action spectroscopy and theoretical calculations. IRMPD action spectra of five alkali metal cation-cytosine complexes exhibit both

On-Chip Evaluation of DNA Methylation with Electrochemical Combined Bisulfite Restriction Analysis Utilizing a Carbon Film Containing a Nanocrystalline Structure.

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Kurita, Ryoji; Yanagisawa, Hiroyuki; Kamata, Tomoyuki; Kato, Dai; Niwa, Osamu

2017-06-06

This paper reports an on-chip electrochemical assessment of the DNA methylation status in genomic DNA on a conductive nanocarbon film electrode realized with combined bisulfite restriction analysis (COBRA). The film electrode consists of sp 2 and sp 3 hybrid bonds and is fabricated with an unbalanced magnetron (UBM) sputtering method. First, we studied the effect of the sp 2 /sp 3 ratio of the UBM nanocarbon film electrode with p-aminophenol, which is a major electro-active product of the labeling enzyme from p-aminophenol phosphate. The signal current for p-aminophenol increases as the sp 2 content in the UBM nanocarbon film electrode increases because of the π-π interaction between aromatic p-aminophenol and the graphene-like sp 2 structure. Furthermore, the capacitative current at the UBM nanocarbon film electrode was successfully reduced by about 1 order of magnitude thanks to the angstrom-level surface flatness. Therefore, a high signal-to-noise ratio was achieved compared with that of conventional electrodes. Then, after performing an ELISA-like hybridization assay with a restriction enzyme, we undertook an electrochemical evaluation of the cytosine methylation status in DNA by measuring the oxidation current derived from p-aminophenol. When the target cytosine in the analyte sequence is methylated (unmethylated), the restriction enzyme of HpyCH4IV is able (unable) to cleave the sequence, that is, the detection probe cannot (can) hybridize. We succeeded in estimating the methylation ratio at a site-specific CpG site from the peak current of a cyclic voltammogram obtained from a PCR product solution ranging from 0.01 to 1 nM.

Role of methionine on epigenetic modification of DNA methylation and gene expression in animals

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Naifeng Zhang

2018-03-01

Full Text Available DNA methylation is one of the main epigenetic phenomena affecting gene expression. It is an important mechanism for the development of embryo, growth and health of animals. As a key nutritional factor limiting the synthesis of protein, methionine serves as the precursor of S-adenosylmethionine (SAM in the hepatic one-carbon metabolism. The dietary fluctuation of methionine content can alter the levels of metabolic substrates in one-carbon metabolism, e.g., the SAM, S-adenosylhomocysteine (SAH, and change the expression of genes related to the growth and health of animals by DNA methylation reactions. The ratio of SAM to SAH is called ‘methylation index’ but it should be carefully explained because the complexity of methylation reaction. Alterations of methylation in a specific cytosine-guanine (CpG site, rather than the whole promoter region, might be enough to change gene expression. Aberrant methionine cycle may provoke molecular changes of one-carbon metabolism that results in deregulation of cellular hemostasis and health problems. The importance of DNA methylation has been underscored but the mechanisms of methionine affecting DNA methylation are poorly understood. Nutritional epigenomics provides a promising insight into the targeting epigenetic changes in animals from a nutritional standpoint, which will deepen and expand our understanding of genes, molecules, tissues, and animals in which methionine alteration influences DNA methylation and gene expression. Keywords: Epigenetics, Methionine, DNA methylation, Gene expression, Epigenetic modification

Transitive RNA silencing signals induce cytosine methylation of a transgenic but not an endogenous target

Czech Academy of Sciences Publication Activity Database

Vermeersch, L.; De Winne, N.; Nolf, J.; Bleys, A.; Kovařík, Aleš; Depicker, A.

2013-01-01

Roč. 74, č. 5 (2013), s. 867-879 ISSN 0960-7412 R&D Projects: GA ČR GBP501/12/G090 Institutional research plan: CEZ:AV0Z50040702 Institutional support: RVO:68081707 Keywords : DIRECTED DNA METHYLATION * POTATO-VIRUS-X * DOUBLE-STRANDED-RNA Subject RIV: BO - Biophysics Impact factor: 6.815, year: 2013

Global DNA methylation in earthworms: a candidate biomarker of epigenetic risks related to the presence of metals/metalloids in terrestrial environments.

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Santoyo, María Maldonado; Flores, Crescencio Rodríguez; Torres, Adolfo Lopez; Wrobel, Kazimierz; Wrobel, Katarzyna

2011-10-01

In this work, possible relationships between global DNA methylation and metal/metalloid concentrations in earthworms have been explored. Direct correlation was observed between soil and tissue As, Se, Sb, Zn, Cu, Mn, Ag, Co, Hg, Pb (p< 0.05). Speciation results obtained for As and Hg hint at the capability of earthworms for conversion of inorganic element forms present in soil to methylated species. Inverse correlation was observed between the percentage of methylated DNA cytosines and total tissue As, As + Hg, As + Hg + Se + Sb (β = -0.8456, p = 0.071; β = -0.9406, p = 0.017; β = -0.9526, p = 0.012 respectively), as well as inorganic As + Hg (β = -0.8807, p = 0.049). It was concluded that earthworms would be particularly helpful as bioindicators of elements undergoing in vivo methylation and might also be used to assess the related risk of epigenetic changes in DNA methylation. Copyright © 2011 Elsevier Ltd. All rights reserved.

Suicidal function of DNA methylation in age-related genome disintegration.

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Mazin, Alexander L

2009-10-01

This article is dedicated to the 60th anniversary of 5-methylcytosine discovery in DNA. Cytosine methylation can affect genetic and epigenetic processes, works as a part of the genome-defense system and has mutagenic activity; however, the biological functions of this enzymatic modification are not well understood. This review will put forward the hypothesis that the host-defense role of DNA methylation in silencing and mutational destroying of retroviruses and other intragenomic parasites was extended during evolution to most host genes that have to be inactivated in differentiated somatic cells, where it acquired a new function in age-related self-destruction of the genome. The proposed model considers DNA methylation as the generator of 5mC>T transitions that induce 40-70% of all spontaneous somatic mutations of the multiple classes at CpG and CpNpG sites and flanking nucleotides in the p53, FIX, hprt, gpt human genes and some transgenes. The accumulation of 5mC-dependent mutations explains: global changes in the structure of the vertebrate genome throughout evolution; the loss of most 5mC from the DNA of various species over their lifespan and the Hayflick limit of normal cells; the polymorphism of methylation sites, including asymmetric mCpNpN sites; cyclical changes of methylation and demethylation in genes. The suicidal function of methylation may be a special genetic mechanism for increasing DNA damage and the programmed genome disintegration responsible for cell apoptosis and organism aging and death.

Identification of body fluid-specific DNA methylation markers for use in forensic science.

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Park, Jong-Lyul; Kwon, Oh-Hyung; Kim, Jong Hwan; Yoo, Hyang-Sook; Lee, Han-Chul; Woo, Kwang-Man; Kim, Seon-Young; Lee, Seung-Hwan; Kim, Yong Sung

2014-11-01

DNA methylation, which occurs at the 5'-position of the cytosine in CpG dinucleotides, has great potential for forensic identification of body fluids, because tissue-specific patterns of DNA methylation have been demonstrated, and DNA is less prone to degradation than proteins or RNA. Previous studies have reported several body fluid-specific DNA methylation markers, but DNA methylation differences are sometimes low in saliva and vaginal secretions. Moreover, specific DNA methylation markers in four types of body fluids (blood, saliva, semen, and vaginal secretions) have not been investigated with genome-wide profiling. Here, we investigated novel DNA methylation markers for identification of body fluids for use in forensic science using the Illumina HumanMethylation 450K bead array, which contains over 450,000 CpG sites. Using methylome data from 16 samples of blood, saliva, semen, and vaginal secretions, we first selected 2986 hypermethylated or hypomethylated regions that were specific for each type of body fluid. We then selected eight CpG sites as novel, forensically relevant DNA methylation markers: cg06379435 and cg08792630 for blood, cg26107890 and cg20691722 for saliva, cg23521140 and cg17610929 for semen, and cg01774894 and cg14991487 for vaginal secretions. These eight selected markers were evaluated in 80 body fluid samples using pyrosequencing, and all showed high sensitivity and specificity for identification of the target body fluid. We suggest that these eight DNA methylation markers may be good candidates for developing an effective molecular assay for identification of body fluids in forensic science. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Environmental chemicals and DNA methylation in adults: a systematic review of the epidemiologic evidence

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Current evidence supports the notion that environmental exposures are associated with DNA-methylation and expression changes that can impact human health. Our objective was to conduct a systematic review of epidemiologic studies evaluating the association between environmental chemicals with DNA met...

DNA methylation and temperature stress in an Antarctic polychaete, Spiophanes tcherniai.

Science.gov (United States)

Marsh, Adam G; Pasqualone, Annamarie A

2014-01-01

Epigenetic modifications of DNA and histones are a primary mechanism by which gene expression activities may be modified in response to environmental stimuli. Here we characterize patterns of methyl-cytosine composition in the marine polychaete Spiophanes tcherniai from McMurdo Sound, Antarctica. We cultured adult worms at two temperatures, -1.5°C (ambient control) and +4°C (warm treatment), for 4 weeks. We observed a rapid capacity for S. tcherniai organismal respiration rates and underlying catalytic rates of citrate synthase at +4°C to return to control levels in less than 4 weeks. We profiled changes in the methylation states of CpG sites in these treatments using an NGS strategy to computationally reconstruct and quantify methylation status across the genome. In our analysis we recovered 120,000 CpG sites in assembled contigs from both treatments. Of those, we were able to align 28,000 CpG sites in common between the two sample groups. In comparing these aligned sites between treatments, only 3000 (11%) evidenced a change in methylation state, but over 85% of changes involved a gain of a 5-methyl group on a CpG site (net increase in methyation). The ability to score CpG sites as partially methylated among gDNA copies in a sample opens up a new avenue for assessing DNA methylation responses to changing environments. By quantitatively distinguishing a "mixed" population of copies of one CpG site, we can begin to identify dynamic, non-binary, continuous-response reactions in DNA methylation intensity or density that previously may have been overlooked as noise.

DNA Methylation and Temperature Stress in an Antarctic Polychaete, Spiophanes tcherniai

Directory of Open Access Journals (Sweden)

Adam G. Marsh

2014-05-01

Full Text Available Epigenetic modifications of DNA and histones are a primary mechanism by which gene expression activities may be modified in response to environmental stimuli. Here we characterize patterns of methyl-cytosine composition in the marine polychaete emph{Spiophanes tcherniai} from McMurdo Sound, Antarctica. We cultured adult worms at two temperatures, -1.5 C (ambient control and +4 C (warm treatment, for four weeks. We observed a rapid capacity for emph{S. tcherniai} organismal respiration rates and underlying catalytic rates of citrate synthase to acclimate at +4 C and return to control levels. We profiled changes in the methylation states of CpG sites in these treatments using an NGS strategy to computationally reconstruct and quantify methylation status across the genome. In our analysis we recovered 120,000 CpG sites in assembled contigs from both treatments. Of those, we were able to align 28,000 CpG sites in common between the two sample groups. In comparing these aligned sites between treatments, only 3,000 (11% evidenced a change in methylation state, but over 85% of changes involved a gain of a 5-methyl group on a CpG site (net increase in methyation. The ability to score CpG sites as partially methylated among gDNA copies in a sample opens up a new avenue for assessing DNA methylation responses to changing environments. By quantitatively distinguishing a ``mixed'' population of copies of one CpG site, we can begin to identify dynamic, non-binary, continuous-response reactions in DNA methylation intensity or density that previously may have been overlooked as noise.

Whole Genome DNA Methylation Analysis of Obstructive Sleep Apnea: IL1R2, NPR2, AR, SP140 Methylation and Clinical Phenotype.

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Chen, Yung-Che; Chen, Ting-Wen; Su, Mao-Chang; Chen, Chung-Jen; Chen, Kuang-Den; Liou, Chia-Wei; Tang, Petrus; Wang, Ting-Ya; Chang, Jen-Chieh; Wang, Chin-Chou; Lin, Hsin-Ching; Chin, Chien-Hung; Huang, Kuo-Tung; Lin, Meng-Chih; Hsiao, Chang-Chun

2016-04-01

We hypothesized that DNA methylation patterns may contribute to disease severity or the development of hypertension and excessive daytime sleepiness (EDS) in patients with obstructive sleep apnea (OSA). Illumina's (San Diego, CA, USA) DNA methylation 27-K assay was used to identify differentially methylated loci (DML). DNA methylation levels were validated by pyrosequencing. A discovery cohort of 15 patients with OSA and 6 healthy subjects, and a validation cohort of 72 patients with sleep disordered breathing (SDB). Microarray analysis identified 636 DMLs in patients with OSA versus healthy subjects, and 327 DMLs in patients with OSA and hypertension versus those without hypertension. In the validation cohort, no significant difference in DNA methylation levels of six selected genes was found between the primary snoring subjects and OSA patients (primary outcome). However, a secondary outcome analysis showed that interleukin-1 receptor 2 (IL1R2) promoter methylation (-114 cytosine followed by guanine dinucleotide sequence [CpG] site) was decreased and IL1R2 protein levels were increased in the patients with SDB with an oxygen desaturation index > 30. Androgen receptor (AR) promoter methylation (-531 CpG site) and AR protein levels were both increased in the patients with SDB with an oxygen desaturation index > 30. Natriuretic peptide receptor 2 (NPR2) promoter methylation (-608/-618 CpG sites) were decreased, whereas levels of both NPR2 and serum C type natriuretic peptide protein were increased in the SDB patients with EDS. Speckled protein 140 (SP140) promoter methylation (-194 CpG site) was increased, and SP140 protein levels were decreased in the patients with SDB and EDS. IL1R2 hypomethylation and AR hypermethylation may constitute an important determinant of disease severity, whereas NPR2 hypomethylation and SP140 hypermethylation may provide a biomarker for vulnerability to EDS in OSA. A commentary on this article appears in this issue on page 723. © 2016

Variation of DNA methylation patterns associated with gene expression in rice (Oryza sativa) exposed to cadmium.

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Feng, Sheng Jun; Liu, Xue Song; Tao, Hua; Tan, Shang Kun; Chu, Shan Shan; Oono, Youko; Zhang, Xian Duo; Chen, Jian; Yang, Zhi Min

2016-12-01

We report genome-wide single-base resolution maps of methylated cytosines and transcriptome change in Cd-exposed rice. Widespread differences were identified in CG and non-CG methylation marks between Cd-exposed and Cd-free rice genomes. There are 2320 non-redundant differentially methylated regions detected in the genome. RNA sequencing revealed 2092 DNA methylation-modified genes differentially expressed under Cd exposure. More genes were found hypermethylated than those hypomethylated in CG, CHH and CHG (where H is A, C or T) contexts in upstream, gene body and downstream regions. Many of the genes were involved in stress response, metal transport and transcription factors. Most of the DNA methylation-modified genes were transcriptionally altered under Cd stress. A subset of loss of function mutants defective in DNA methylation and histone modification activities was used to identify transcript abundance of selected genes. Compared with wide type, mutation of MET1 and DRM2 resulted in general lower transcript levels of the genes under Cd stress. Transcripts of OsIRO2, OsPR1b and Os09g02214 in drm2 were significantly reduced. A commonly used DNA methylation inhibitor 5-azacytidine was employed to investigate whether DNA demethylation affected physiological consequences. 5-azacytidine provision decreased general DNA methylation levels of selected genes, but promoted growth of rice seedlings and Cd accumulation in rice plant. © 2016 John Wiley & Sons Ltd.

Promoter Methylation Analysis of IDH Genes in Human Gliomas

International Nuclear Information System (INIS)

Flanagan, Simon; Lee, Maggie; Li, Cheryl C. Y.; Suter, Catherine M.; Buckland, Michael E.

2012-01-01

Mutations in isocitrate dehydrogenase (IDH)-1 or -2 are found in the majority of WHO grade II and III astrocytomas and oligodendrogliomas, and secondary glioblastomas. Almost all described mutations are heterozygous missense mutations affecting a conserved arginine residue in the substrate binding site of IDH1 (R132) or IDH2 (R172). But the exact mechanism of IDH mutations in neoplasia is not understood. It has been proposed that IDH mutations impart a “toxic gain-of-function” to the mutant protein, however a dominant-negative effect of mutant IDH has also been described, implying that IDH may function as a tumor suppressor gene. As most, if not all, tumor suppressor genes are inactivated by epigenetic silencing, in a wide variety of tumors, we asked if IDH1 or IDH2 carry the epigenetic signature of a tumor suppressor by assessing cytosine methylation at their promoters. Methylation was quantified in 68 human brain tumors, including both IDH-mutant and IDH wildtype, by bisulfite pyrosequencing. In all tumors examined, CpG methylation levels were less than 8%. Our data demonstrate that inactivation of IDH function through promoter hypermethylation is not common in human gliomas and other brain tumors. These findings do not support a tumor suppressor role for IDH genes in human gliomas.

Promoter methylation inhibits BRD7 expression in human nasopharyngeal carcinoma cells

International Nuclear Information System (INIS)

Liu, Huaying; Li, Guiyuan; Zhang, Liming; Niu, Zhaoxia; Zhou, Ming; Peng, Cong; Li, Xiayu; Deng, Tan; Shi, Lei; Tan, Yixin

2008-01-01

Nasopharyngeal carcinoma (NPC) is a head and neck malignancy with high occurrence in South-East Asia and Southern China. Recent findings suggest that epigenetic inactivation of multiple tumor suppressor genes plays an important role in the tumourigenesis of NPC. BRD7 is a NPC-associated bromodomain gene that exhibits a much higher-level of mRNA expression in normal than in NPC biopsies and cell lines. In this study, we explored the role of DNA methylation in regulation of BRD7 transcription. The presence of CpG islands within BRD7 promoter was predicted by EMBOSS CpGplot and Softberry CpGFinder, respectively. Nested methylation-specific PCR and RT-PCR were employed to detect the methylation status of BRD7 promoter and the mRNA expression of BRD7 gene in tumor cell lines as well as clinical samples. Electrophoretic mobility shift assays (EMSA) and luciferase assay were used to detect the effects of cytosine methylation on the nuclear protein binding to BRD7 promoter. We found that DNA methylation suppresses BRD7 expression in NPC cells. In vitro DNA methylation in NPC cells silenced BRD7 promoter activity and inhibited the binding of the nuclear protein (possibly Sp1) to Sp1 binding sites in the BRD7 promoter. In contrast, inhibition of DNA methylation augments induction of endogenous BRD7 mRNA in NPC cells. We also found that methylation frequency of BRD7 promoter is much higher in the tumor and matched blood samples from NPC patients than in the blood samples from normal individuals. BRD7 promoter demethylation is a prerequisite for high level induction of BRD7 gene expression. DNA methylation of BRD7 promoter might serve as a diagnostic marker in NPC

Loss of maintenance DNA methylation results in abnormal DNA origin firing during DNA replication.

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Haruta, Mayumi; Shimada, Midori; Nishiyama, Atsuya; Johmura, Yoshikazu; Le Tallec, Benoît; Debatisse, Michelle; Nakanishi, Makoto

2016-01-22

The mammalian maintenance methyltransferase DNMT1 [DNA (cytosine-5-)-methyltransferase 1] mediates the inheritance of the DNA methylation pattern during replication. Previous studies have shown that depletion of DNMT1 causes a severe growth defect and apoptosis in differentiated cells. However, the detailed mechanisms behind this phenomenon remain poorly understood. Here we show that conditional ablation of Dnmt1 in murine embryonic fibroblasts (MEFs) resulted in an aberrant DNA replication program showing an accumulation of late-S phase replication and causing severely defective growth. Furthermore, we found that the catalytic activity and replication focus targeting sequence of DNMT1 are required for a proper DNA replication program. Taken together, our findings suggest that the maintenance of DNA methylation by DNMT1 plays a critical role in proper regulation of DNA replication in mammalian cells. Copyright © 2015 Elsevier Inc. All rights reserved.

Identifying DNA Methylation Biomarkers for Non-Endoscopic Detection of Barrett’s Esophagus

Science.gov (United States)

Moinova, Helen R.; LaFramboise, Thomas; Lutterbaugh, James D.; Chandar, Apoorva Krishna; Dumot, John; Faulx, Ashley; Brock, Wendy; De la Cruz Cabrera, Omar; Guda, Kishore; Barnholtz-Sloan, Jill S.; Iyer, Prasad G.; Canto, Marcia I.; Wang, Jean S.; Shaheen, Nicholas J.; Thota, Prashanti N.; Willis, Joseph E.; Chak, Amitabh; Markowitz, Sanford D.

2018-01-01

We report a biomarker-based non-endoscopic method for detecting Barrett’s esophagus (BE), based on detecting methylated DNAs retrieved via a swallowable balloon-based esophageal sampling device. BE is the precursor of, and a major recognized risk factor for, developing esophageal adenocarcinoma (EAC). Endoscopy, the current standard for BE detection, is not cost-effective for population screening. We performed genome-wide screening to ascertain regions targeted for recurrent aberrant cytosine methylation in BE, identifying high-frequency methylation within the CCNA1 locus. We tested CCNA1 DNA methylation as a BE biomarker in cytology brushings of the distal esophagus from 173 individuals with or without BE. CCNA1 DNA methylation demonstrated an area under the curve (AUC)=0.95 for discriminating BE-related metaplasia and neoplasia cases versus normal individuals, performing identically to methylation of VIM DNA, an established BE biomarker. When combined, the resulting two biomarker panel was 95% sensitive and 91% specific. These results were replicated in an independent validation cohort of 149 individuals, who were assayed using the same cutoff values for test positivity established in the training population. To progress toward non-endoscopic esophageal screening, we engineered a well-tolerated, swallowable, encapsulated balloon device able to selectively sample the distal esophagus within 5 minutes. In balloon samples from 86 individuals, tests of CCNA1 plus VIM DNA methylation detected BE metaplasia with 90.3% sensitivity and 91.7% specificity. Combining the balloon sampling device with molecular assays of CCNA1 plus VIM DNA methylation enables an efficient, well-tolerated, sensitive, and specific method of screening at-risk populations for BE. PMID:29343623

Electron Attachment to the Gas Phase DNA Bases Cytosine and Thymine

Czech Academy of Sciences Publication Activity Database

Denifl, S.; Ptasiňska, S.; Probst, M.; Hrušák, Jan; Scheier, P.; Märk, T. D.

2004-01-01

Roč. 108, č. 31 (2004), s. 6562-6569 ISSN 1089-5639 R&D Projects: GA ČR GA203/02/0737 Institutional research plan: CEZ:AV0Z4040901 Keywords : gas-phase * cytosine * thymine Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 2.639, year: 2004

Determination of epigenetic inheritance, genetic inheritance, and estimation of genome DNA methylation in a full-sib family of Cupressus sempervirens L.

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Avramidou, Evangelia V; Doulis, Andreas G; Aravanopoulos, Filippos A

2015-05-15

Genetic inheritance and epigenetic inheritance are significant determinants of plant evolution, adaptation and plasticity. We studied inheritance of restriction site polymorphisms by the f-AFLP method and epigenetic DNA cytosine methylation inheritance by the f-MSAP technique. The study involved parents and 190 progeny of a Cupressus sempervirens L. full-sib family. Results from AFLP genetic data revealed that 71.8% of the fragments studied are under Mendelian genetic control, whereas faithful Mendelian inheritance for the MSAP fragments was low (4.29%). Further, MSAP fragment analysis showed that total methylation presented a mean of 28.2%, which was higher than the midparent value, while maternal inheritance was higher (5.65%) than paternal (3.01%). Interestingly de novo methylation in the progeny was high (19.65%) compared to parental methylation. Genetic and epigenetic distances for parents and offspring were not correlated (R(2)=0.0005). Furthermore, we studied correlation of total relative methylation and CG methylation with growth (height, diameter). We found CG/CNG methylation (N: A, C, T) to be positively correlated with height and diameter, while total relative methylation and CG methylation were positively correlated with height. Results are discussed in light of further research needed and of their potential application in breeding. Copyright © 2015 Elsevier B.V. All rights reserved.

Persistent and plastic effects of temperature on DNA methylation across the genome of threespine stickleback (Gasterosteus aculeatus).

Science.gov (United States)

Metzger, David C H; Schulte, Patricia M

2017-10-11

Epigenetic mechanisms such as changes in DNA methylation have the potential to affect the resilience of species to climate change, but little is known about the response of the methylome to changes in environmental temperature in animals. Using reduced representation bisulfite sequencing, we assessed the effects of development temperature and adult acclimation temperature on DNA methylation levels in threespine stickleback ( Gasterosteus aculeatus ). Across all treatments, we identified 2130 differentially methylated cytosines distributed across the genome. Both increases and decreases in temperature during development and with thermal acclimation in adults increased global DNA methylation levels. Approximately 25% of the differentially methylated regions (DMRs) responded to both developmental temperature and adult thermal acclimation, and 50 DMRs were common to all treatments, demonstrating a core response of the epigenome to thermal change at multiple time scales. We also identified differentially methylated loci that were specific to a particular developmental or adult thermal response, which could facilitate the accumulation of epigenetic variation between natural populations that experience different thermal regimes. These data demonstrate that thermal history can have long-lasting effects on the epigenome, highlighting the role of epigenetic modifications in the response to temperature change across multiple time scales. © 2017 The Author(s).

An RNA polymerase II-and AGO4-associated protein acts in RNA-directed DNA methylation

KAUST Repository

Gao, Zhihuan

2010-04-21

DNA methylation is an important epigenetic mark in many eukaryotes. In plants, 24-nucleotide small interfering RNAs (siRNAs) bound to the effector protein, Argonaute 4 (AGO4), can direct de novo DNA methylation by the methyltransferase DRM2 (refs 2, 4-6). Here we report a new regulator of RNA-directed DNA methylation (RdDM) in Arabidopsis: RDM1. Loss-of-function mutations in the RDM1 gene impair the accumulation of 24-nucleotide siRNAs, reduce DNA methylation, and release transcriptional gene silencing at RdDM target loci. RDM1 encodes a small protein that seems to bind single-stranded methyl DNA, and associates and co-localizes with RNA polymerase II (Pol II, also known as NRPB), AGO4 and DRM2 in the nucleus. Our results indicate that RDM1 is a component of the RdDM effector complex and may have a role in linking siRNA production with pre-existing or de novo cytosine methylation. Our results also indicate that, although RDM1 and Pol V (also known as NRPE) may function together at some RdDM target sites in the peri-nucleolar siRNA processing centre, Pol II rather than Pol V is associated with the RdDM effector complex at target sites in the nucleoplasm. © 2010 Macmillan Publishers Limited. All rights reserved.

In vivo control of CpG and non-CpG DNA methylation by DNA methyltransferases.

Directory of Open Access Journals (Sweden)

Julia Arand

2012-06-01

Full Text Available The enzymatic control of the setting and maintenance of symmetric and non-symmetric DNA methylation patterns in a particular genome context is not well understood. Here, we describe a comprehensive analysis of DNA methylation patterns generated by high resolution sequencing of hairpin-bisulfite amplicons of selected single copy genes and repetitive elements (LINE1, B1, IAP-LTR-retrotransposons, and major satellites. The analysis unambiguously identifies a substantial amount of regional incomplete methylation maintenance, i.e. hemimethylated CpG positions, with variant degrees among cell types. Moreover, non-CpG cytosine methylation is confined to ESCs and exclusively catalysed by Dnmt3a and Dnmt3b. This sequence position-, cell type-, and region-dependent non-CpG methylation is strongly linked to neighboring CpG methylation and requires the presence of Dnmt3L. The generation of a comprehensive data set of 146,000 CpG dyads was used to apply and develop parameter estimated hidden Markov models (HMM to calculate the relative contribution of DNA methyltransferases (Dnmts for de novo and maintenance DNA methylation. The comparative modelling included wild-type ESCs and mutant ESCs deficient for Dnmt1, Dnmt3a, Dnmt3b, or Dnmt3a/3b, respectively. The HMM analysis identifies a considerable de novo methylation activity for Dnmt1 at certain repetitive elements and single copy sequences. Dnmt3a and Dnmt3b contribute de novo function. However, both enzymes are also essential to maintain symmetrical CpG methylation at distinct repetitive and single copy sequences in ESCs.

Genome-wide tracking of unmethylated DNA Alu repeats in normal and cancer cells

DEFF Research Database (Denmark)

Rodriguez, Jairo; Vives, Laura; Jordà, Mireia

2008-01-01

Methylation of the cytosine is the most frequent epigenetic modification of DNA in mammalian cells. In humans, most of the methylated cytosines are found in CpG-rich sequences within tandem and interspersed repeats that make up to 45% of the human genome, being Alu repeats the most common family....

[Triplet expansion cytosine-guanine-guanine: Three cases of OMIM syndrome in the same family].

Science.gov (United States)

González-Pérez, Jesús; Izquierdo-Álvarez, Silvia; Fuertes-Rodrigo, Cristina; Monge-Galindo, Lorena; Peña-Segura, José Luis; López-Pisón, Francisco Javier

2016-04-01

The dynamic increase in the number of triplet repeats of cytosine-guanine-guanine (CGG) in the FMR1 gene mutation is responsible for three OMIM syndromes with a distinct clinical phenotype: Fragile X syndrome (FXS) and two pathologies in adult carriers of the premutation (55-200 CGG repeats): Primary ovarian insufficiency (FXPOI) and tremor-ataxia syndrome (FXTAS) associated with FXS. CGG mutation dynamics of the FMR1 gene were studied in DNA samples from peripheral blood from the index case and other relatives of first, second and third degree by TP-PCR, and the percentage methylation. Diagnosis of FXS was confirmed in three patients (21.4%), eight patients (57.1%) were confirmed in the premutation range transmitters, one male patient with full mutation/permutation mosaicism (7.1%) and two patients (14.3%) with normal study. Of the eight permutated patients, three had FXPOI and one male patient had FXTAS. Our study suggests the importance of making an early diagnosis of SXF in order to carry out a family study and genetic counselling, which allow the identification of new cases or premutated patients with FMR1 gene- associated syndromes (FXTAS, FXPOI). Copyright © 2015 Elsevier España, S.L.U. All rights reserved.

Origin and fate of 4-methyl steroid hydrocarbons. I. Diagenesis of 4-methyl sterenes

Energy Technology Data Exchange (ETDEWEB)

Wolff, G.A.; Lamb, N.A.; Maxwell, J.R.

1986-03-01

Treatment of 4-methylcholest-4-ene under mild acid conditions at low temperatures gives chemical evidence for certain features seen in the distributions of sedimentary 4-methyl steroid hydrocarbons, and further indicates that many low temperature diagenetic reactions of steroids are explicable in terms of acid catalyzed rearrangements. Specifically, the results provide: (i) Indirect evidence that the 4-ene skeleton is a key intermediate in the dehydration of 4-methyl stanols in sediments. (ii) An explanation for the distribution of 4-methyl sterenes and A-nor sterenes in the lacustrine Messel shale (Eocene). (iii) An explanation for the presence of 4..beta..-methyl steranes in relatively immature sedimentary rocks, despite the precursor stanols having the 4..cap alpha..-methyl configuration. With increasing maturity in the Paris Basin shales (Lower Toarcian), the less stable 4..beta..-methyl steranes decrease gradually in abundance relative to their 4..cap alpha..-methyl counterparts, at a rate fairly similar to the change in pristane stereochemistry.

DNA methylation in metabolic disorders

DEFF Research Database (Denmark)

Barres, Romain; Zierath, Juleen R

2011-01-01

DNA methylation is a major epigenetic modification that controls gene expression in physiologic and pathologic states. Metabolic diseases such as diabetes and obesity are associated with profound alterations in gene expression that are caused by genetic and environmental factors. Recent reports...... have provided evidence that environmental factors at all ages could modify DNA methylation in somatic tissues, which suggests that DNA methylation is a more dynamic process than previously appreciated. Because of the importance of lifestyle factors in metabolic disorders, DNA methylation provides...... a mechanism by which environmental factors, including diet and exercise, can modify genetic predisposition to disease. This article considers the current evidence that defines a role for DNA methylation in metabolic disorders....

Evaluation of Global Genomic DNA Methylation in Human Whole Blood by Capillary Electrophoresis UV Detection

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Angelo Zinellu

2017-01-01

Full Text Available Alterations in global DNA methylation are implicated in various pathophysiological processes. The development of simple and quick, yet robust, methods to assess DNA methylation is required to facilitate its measurement and interpretation in clinical practice. We describe a highly sensitive and reproducible capillary electrophoresis method with UV detection for the separation and detection of cytosine and methylcytosine, after formic acid hydrolysis of DNA extracted from human whole blood. Hydrolysed samples were dried and resuspended with water and directly injected into the capillary without sample derivatization procedures. The use of a run buffer containing 50 mmol/L BIS-TRIS propane (BTP phosphate buffer at pH 3.25 and 60 mmol/L sodium acetate buffer at pH 3.60 (4 : 1, v/v allowed full analyte identification within 11 min. Precision tests indicated an elevated reproducibility with an interassay CV of 1.98% when starting from 2 μg of the extracted DNA. The method was successfully tested by measuring the DNA methylation degree both in healthy volunteers and in reference calf thymus DNA.

L1 retrotransposition is activated by Ten-eleven-translocation protein 1 and repressed by methyl-CpG binding proteins.

Science.gov (United States)

Zhang, Peng; Ludwig, Anne K; Hastert, Florian D; Rausch, Cathia; Lehmkuhl, Anne; Hellmann, Ines; Smets, Martha; Leonhardt, Heinrich; Cardoso, M Cristina

2017-09-03

One of the major functions of DNA methylation is the repression of transposable elements, such as the long-interspersed nuclear element 1 (L1). The underlying mechanism(s), however, are unclear. Here, we addressed how retrotransposon activation and mobilization are regulated by methyl-cytosine modifying ten-eleven-translocation (Tet) proteins and how this is modulated by methyl-CpG binding domain (MBD) proteins. We show that Tet1 activates both, endogenous and engineered L1 retrotransposons. Furthermore, we found that Mecp2 and Mbd2 repress Tet1-mediated activation of L1 by preventing 5hmC formation at the L1 promoter. Finally, we demonstrate that the methyl-CpG binding domain, as well as the adjacent non-sequence specific DNA binding domain of Mecp2 are each sufficient to mediate repression of Tet1-induced L1 mobilization. Our study reveals a mechanism how L1 elements get activated in the absence of Mecp2 and suggests that Tet1 may contribute to Mecp2/Mbd2-deficiency phenotypes, such as the Rett syndrome. We propose that the balance between methylation "reader" and "eraser/writer" controls L1 retrotransposition.

The origin and fate of 4-methyl steroid hydrocarbons. I. Diagenesis of 4-methyl sterenes

Science.gov (United States)

Wolff, George A.; Lamb, Neil A.; Maxwell, James R.

1986-03-01

Treatment of 4-methylcholest-4-ene under mild acid conditions at low temperatures gives chemical evidence for certain features seen in the distributions of sedimentary 4-methyl steroid hydrocarbons, and further indicates that many low temperature diagenetic reactions of steroids are explicable in terms of acid catalysed rearrangements. Specifically, the results provide: (i) Indirect evidence that the 4-ene skeleton is a key intermediate in the dehydration of 4-methyl stanols in sediments. (ii) An explanation for the distribution of 4-methyl sterenes and A-nor sterenes in the lacustrine Messel shale (Eocene). (iii) An explanation for the presence of 4β-methyl steranes in relatively immature sedimentary rocks, despite the precursor stanols having the 4α-methyl configuration. With increasing maturity in the Paris Basin shales (Lower Toarcian), the less stable 4β-methyl steranes decrease gradually in abundance relative to their 4α-methyl counterparts, at a rate fairly similar to the change in pristane stereochemistry.

Methylated Host Cell Gene Promoters and Human Papillomavirus Type 16 and 18 Predicting Cervical Lesions and Cancer.

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Nina Milutin Gašperov

Full Text Available Change in the host and/or human papillomavirus (HPV DNA methylation profile is probably one of the main factors responsible for the malignant progression of cervical lesions to cancer. To investigate those changes we studied 173 cervical samples with different grades of cervical lesion, from normal to cervical cancer. The methylation status of nine cellular gene promoters, CCNA1, CDH1, C13ORF18, DAPK1, HIC1, RARβ2, hTERT1, hTERT2 and TWIST1, was investigated by Methylation Specific Polymerase Chain Reaction (MSP. The methylation of HPV18 L1-gene was also investigated by MSP, while the methylated cytosines within four regions, L1, 5'LCR, enhancer, and promoter of the HPV16 genome covering 19 CpG sites were evaluated by bisulfite sequencing. Statistically significant methylation biomarkers distinguishing between cervical precursor lesions from normal cervix were primarily C13ORF18 and secondly CCNA1, and those distinguishing cervical cancer from normal or cervical precursor lesions were CCNA1, C13ORF18, hTERT1, hTERT2 and TWIST1. In addition, the methylation analysis of individual CpG sites of the HPV16 genome in different sample groups, notably the 7455 and 7694 sites, proved to be more important than the overall methylation frequency. The majority of HPV18 positive samples contained both methylated and unmethylated L1 gene, and samples with L1-gene methylated forms alone had better prognosis when correlated with the host cell gene promoters' methylation profiles. In conclusion, both cellular and viral methylation biomarkers should be used for monitoring cervical lesion progression to prevent invasive cervical cancer.

Global DNA methylation in earthworms: A candidate biomarker of epigenetic risks related to the presence of metals/metalloids in terrestrial environments

Energy Technology Data Exchange (ETDEWEB)

Maldonado Santoyo, Maria; Rodriguez Flores, Crescencio; Lopez Torres, Adolfo; Wrobel, Kazimierz [Department of Chemistry, University of Guanajuato, L de Retana No 5, 36000 Guanajuato (Mexico); Wrobel, Katarzyna, E-mail: katarzyn@quijote.ugto.mx [Department of Chemistry, University of Guanajuato, L de Retana No 5, 36000 Guanajuato (Mexico)

2011-10-15

In this work, possible relationships between global DNA methylation and metal/metalloid concentrations in earthworms have been explored. Direct correlation was observed between soil and tissue As, Se, Sb, Zn, Cu, Mn, Ag, Co, Hg, Pb (p < 0.05). Speciation results obtained for As and Hg hint at the capability of earthworms for conversion of inorganic element forms present in soil to methylated species. Inverse correlation was observed between the percentage of methylated DNA cytosines and total tissue As, As + Hg, As + Hg + Se + Sb ({beta} = -0.8456, p = 0.071; {beta} = -0.9406, p = 0.017; {beta} = -0.9526, p = 0.012 respectively), as well as inorganic As + Hg ({beta} = -0.8807, p = 0.049). It was concluded that earthworms would be particularly helpful as bioindicators of elements undergoing in vivo methylation and might also be used to assess the related risk of epigenetic changes in DNA methylation. - Graphical abstract: Display Omitted Highlights: > Several metals and metalloids contribute to epigenetic gene regulation. > As, Hg, Se, Sb inversely correlated with global DNA methylation in earthworms. > Biomethylation of the above elements in worms suggested. > Elements biomethylation apparently competes with DNA methylation. > DNA methylation a biomarker of epigenetic risks related to soil metals/metalloids. - Biomethylation of As, Hg in earthworms versus DNA methylation - a candidate biomarker of epigenetic risks related to the presence of metals/metalloids in soil.

Differential DNA methylation may contribute to temporal and spatial regulation of gene expression and the development of mycelia and conidia in entomopathogenic fungus Metarhizium robertsii.

Science.gov (United States)

Li, Wanzhen; Wang, Yulong; Zhu, Jianyu; Wang, Zhangxun; Tang, Guiliang; Huang, Bo

2017-03-01

Conidia and mycelia are two important developmental stages in the asexual life cycle of entomopathogenic fungus Metarhizium. Despite the crucial role that DNA methylation plays in many biological processes, its role in regulation of gene expression and development in fungi is not yet fully understood. We performed genome-wide analysis of DNA methylation patterns of an M. robertsii strain with single base pair resolution. Specifically, we examined for changes in methylation patterns between the conidia and mycelia stages. The results showed that approximately 0.38 % of cytosines are methylated in conidia, which is lower than the DNA methylation level (0.42 %) in mycelia. We found that DNA methylation undergoes genome-wide reprogramming during fungal development in M. robertsii. 132 differentially methylated regions (DMRs), which were mostly distributed in gene regions, were identified. KEGG analysis revealed that the DMR-associated genes belong to metabolic pathways. Intriguingly, in contrast to most other eukaryotes, promoter activities in M. robertsii seemed differentially modulated by DNA methylation levels. We found that transcription tended to be enhanced in genes with moderate promoter methylation, while gene expression was decreased in genes with high or low promoter methylation. Copyright © 2017 British Mycological Society. Published by Elsevier Ltd. All rights reserved.

DNA methylation and memory formation.

Science.gov (United States)

Day, Jeremy J; Sweatt, J David

2010-11-01

Memory formation and storage require long-lasting changes in memory-related neuronal circuits. Recent evidence indicates that DNA methylation may serve as a contributing mechanism in memory formation and storage. These emerging findings suggest a role for an epigenetic mechanism in learning and long-term memory maintenance and raise apparent conundrums and questions. For example, it is unclear how DNA methylation might be reversed during the formation of a memory, how changes in DNA methylation alter neuronal function to promote memory formation, and how DNA methylation patterns differ between neuronal structures to enable both consolidation and storage of memories. Here we evaluate the existing evidence supporting a role for DNA methylation in memory, discuss how DNA methylation may affect genetic and neuronal function to contribute to behavior, propose several future directions for the emerging subfield of neuroepigenetics, and begin to address some of the broader implications of this work.

Fingerprints of Both Watson-Crick and Hoogsteen Isomers of the Isolated (Cytosine-Guanine)H+ Pair.

Science.gov (United States)

Cruz-Ortiz, Andrés F; Rossa, Maximiliano; Berthias, Francis; Berdakin, Matías; Maitre, Philippe; Pino, Gustavo A

2017-11-16

 Gas phase protonated guanine-cytosine (CGH + ) pair was generated using an electrospray ionization source from solutions at two different pH (5.8 and 3.2). Consistent evidence from MS/MS fragmentation patterns and differential ion mobility spectra (DIMS) point toward the presence of two isomers of the CGH + pair, whose relative populations depend strongly on the pH of the solution. Gas phase infrared multiphoton dissociation (IRMPD) spectroscopy in the 900-1900 cm -1 spectral range further confirms that the Watson-Crick isomer is preferentially produced (91%) at pH = 5.8, while the Hoogsteen isomer predominates (66%) at pH = 3.2). These fingerprint signatures are expected to be useful for the development of new analytical methodologies and to trigger isomer selective photochemical studies of protonated DNA base pairs.

Allelic Dropout During Polymerase Chain Reaction due to G-Quadruplex Structures and DNA Methylation Is Widespread at Imprinted Human Loci

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Aaron J. Stevens

2017-03-01

Full Text Available Loss of one allele during polymerase chain reaction (PCR amplification of DNA, known as allelic dropout, can be caused by a variety of mechanisms. Allelic dropout during PCR may have profound implications for molecular diagnostic and research procedures that depend on PCR and assume biallelic amplification has occurred. Complete allelic dropout due to the combined effects of cytosine methylation and G-quadruplex formation was previously described for a differentially methylated region of the human imprinted gene, MEST. We now demonstrate that this parent-of-origin specific allelic dropout can potentially occur at several other genomic regions that display genomic imprinting and have propensity for G-quadruplex formation, including AIM1, BLCAP, DNMT1, PLAGL1, KCNQ1, and GRB10. These findings demonstrate that systematic allelic dropout during PCR is a general phenomenon for regions of the genome where differential allelic methylation and G-quadruplex motifs coincide, and suggest that great care must be taken to ensure biallelic amplification is occurring in such situations.

Diversification, evolution and methylation of short interspersed nuclear element families in sugar beet and related Amaranthaceae species.

Science.gov (United States)

Schwichtenberg, Katrin; Wenke, Torsten; Zakrzewski, Falk; Seibt, Kathrin M; Minoche, André; Dohm, Juliane C; Weisshaar, Bernd; Himmelbauer, Heinz; Schmidt, Thomas

2016-01-01

Short interspersed nuclear elements (SINEs) are non-autonomous non-long terminal repeat retrotransposons which are widely distributed in eukaryotic organisms. While SINEs have been intensively studied in animals, only limited information is available about plant SINEs. We analysed 22 SINE families from seven genomes of the Amaranthaceae family and identified 34 806 SINEs, including 19 549 full-length copies. With the focus on sugar beet (Beta vulgaris), we performed a comparative analysis of the diversity, genomic and chromosomal organization and the methylation of SINEs to provide a detailed insight into the evolution and age of Amaranthaceae SINEs. The lengths of consensus sequences of SINEs range from 113 nucleotides (nt) up to 224 nt. The SINEs show dispersed distribution on all chromosomes but were found with higher incidence in subterminal euchromatic chromosome regions. The methylation of SINEs is increased compared with their flanking regions, and the strongest effect is visible for cytosines in the CHH context, indicating an involvement of asymmetric methylation in the silencing of SINEs. © 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd.

Gamma irradiation does not induce detectable changes in DNA methylation directly following exposure of human cells.

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Christoph Lahtz

Full Text Available Environmental chemicals and radiation have often been implicated in producing alterations of the epigenome thus potentially contributing to cancer and other diseases. Ionizing radiation, released during accidents at nuclear power plants or after atomic bomb explosions, is a potentially serious health threat for the exposed human population. This type of high-energy radiation causes DNA damage including single- and double-strand breaks and induces chromosomal rearrangements and mutations, but it is not known if ionizing radiation directly induces changes in the epigenome of irradiated cells. We treated normal human fibroblasts and normal human bronchial epithelial cells with different doses of γ-radiation emitted from a cesium 137 ((137Cs radiation source. After a seven-day recovery period, we analyzed global DNA methylation patterns in the irradiated and control cells using the methylated-CpG island recovery assay (MIRA in combination with high-resolution microarrays. Bioinformatics analysis revealed only a small number of potential methylation changes with low fold-difference ratios in the irradiated cells. These minor methylation differences seen on the microarrays could not be verified by COBRA (combined bisulfite restriction analysis or bisulfite sequencing of selected target loci. Our study shows that acute γ-radiation treatment of two types of human cells had no appreciable direct effect on DNA cytosine methylation patterns in exposed cells.

The concerted impact of domestication and transposon insertions on methylation patterns between dogs and gray wolves

Science.gov (United States)

Koch, Ilana Janowitz; Clark, Michelle M.; Thompson, Michael J.; Deere-Machemer, Kerry A.; Wang, Jun; Duarte, Lionel; Gnanadesikan, Gitanjali E.; McCoy, Eskender L.; Rubbi, Liudmilla; Stahler, Daniel R.; Pellegrini, Matteo; Ostrander, Elaine A.; Wayne, Robert K.; Sinsheimer, Janet S.; vonHoldt, Bridgett M.

2015-01-01

The process of domestication can exert intense trait-targeted selection on genes and regulatory regions. Specifically, rapid shifts in the structure and sequence of genomic regulatory elements could provide an explanation for the extensive, and sometimes extreme, variation in phenotypic traits observed in domesticated species. Here, we explored methylation differences from >24,000 cytosines distributed across the genomes of the domesticated dog (Canis familiaris) and the gray wolf (C. lupus). PCA and model-based cluster analyses identified two primary groups, domestic versus wild canids. A scan for significantly differentially methylated sites (DMSs) revealed species-specific patterns at 68 sites after correcting for cell heterogeneity, with weak yet significant hyper-methylation typical of purebred dogs when compared to wolves (59% and 58%, p66%) of differentially methylated regions contained or were associated with repetitive elements, indicative of a genotype-mediated trend. However, DMSs were also often linked to functionally relevant genes (e.g. neurotransmitters). Finally, we utilized known genealogical relationships among Yellowstone wolves to survey transmission stability of methylation marks, from which we found a substantial fraction that demonstrated high heritability (both H2 and h2>0.99). These analyses provide a unique epigenetic insight into the molecular consequences of recent selection and radiation of our most ancient domesticated companion, the dog. These findings suggest selection has acted on methylation patterns, providing a new genomic perspective on phenotypic diversification in domesticated species. PMID:27112634

Formulation, quality control and shelf life of the experimental cytostatic drug cyclopentenyl cytosine

NARCIS (Netherlands)

Schimmel, Kirsten; Guchelaar, Henk-Jan; van Kan, Erik

2006-01-01

This paper describes the formulation and quality control of an aqueous sterilized formulation of the experimental cytostatic drug cyclopentenyl cytosine (CPEC) to be used in Phase I/II clinical trials. The raw drug substance was extensively tested. A High Pressure Liquid Chromotography (HPLC) method

Chilling- and Freezing-Induced Alterations in Cytosine Methylation and Its Association with the Cold Tolerance of an Alpine Subnival Plant, Chorispora bungeana.

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Yuan Song

Full Text Available Chilling (0-18°C and freezing (<0°C are two distinct types of cold stresses. Epigenetic regulation can play an important role in plant adaptation to abiotic stresses. However, it is not yet clear whether and how epigenetic modification (i.e., DNA methylation mediates the adaptation to cold stresses in nature (e.g., in alpine regions. Especially, whether the adaptation to chilling and freezing is involved in differential epigenetic regulations in plants is largely unknown. Chorispora bungeana is an alpine subnival plant that is distributed in the freeze-thaw tundra in Asia, where chilling and freezing frequently fluctuate daily (24 h. To disentangle how C. bungeana copes with these intricate cold stresses through epigenetic modifications, plants of C. bungeana were treated at 4°C (chilling and -4°C (freezing over five periods of time (0-24 h. Methylation-sensitive amplified fragment-length polymorphism markers were used to investigate the variation in DNA methylation of C. bungeana in response to chilling and freezing. It was found that the alterations in DNA methylation of C. bungeana largely occurred over the period of chilling and freezing. Moreover, chilling and freezing appeared to gradually induce distinct DNA methylation variations, as the treatment went on (e.g., after 12 h. Forty-three cold-induced polymorphic fragments were randomly selected and further analyzed, and three of the cloned fragments were homologous to genes encoding alcohol dehydrogenase, UDP-glucosyltransferase and polygalacturonase-inhibiting protein. These candidate genes verified the existence of different expressive patterns between chilling and freezing. Our results showed that C. bungeana responded to cold stresses rapidly through the alterations of DNA methylation, and that chilling and freezing induced different DNA methylation changes. Therefore, we conclude that epigenetic modifications can potentially serve as a rapid and flexible mechanism for C. bungeana

Cytosine Arabinoside Influx and Nucleoside Transport Sites in Acute Leukemia

OpenAIRE

Wiley, J. S.; Jones, S. P.; Sawyer, W. H.; Paterson, A. R. P.

1982-01-01

Although cytosine arabinoside (araC) can induce a remission in a majority of patients presenting with acute myeloblastic leukemia (AML), a minority fail to respond and moreover the drug has less effect in acute lymphoblastic leukemia (ALL). The carrier-mediated influx of araC into purified blasts from patients with AML, ALL, and acute undifferentiated leukemia (AUL) has been compared to that of normal lymphocytes and polymorphs. Blasts showed a larger mediated influx of araC than mature cells...

Biophysical and molecular docking insight into the interaction of cytosine β-D arabinofuranoside with human serum albumin

International Nuclear Information System (INIS)

Alam, Parvez; Chaturvedi, Sumit Kumar; Anwar, Tamanna; Siddiqi, Mohammad Khursheed; Ajmal, Mohd Rehan; Badr, Gamal; Mahmoud, Mohamed H.; Hasan Khan, Rizwan

2015-01-01

Interaction of pharmacologically important anticancer drug cytosine β-D arabinofuranoside with human serum albumin (HSA) at physiological pH 7.4 has been studied by utilizing various spectroscopic and molecular docking strategies. Fluorescence results revealed that cytosine β-D arabinofuranoside interacts with HSA through static quenching mechanism with binding affinity of 2.4×10 3 M −1 . The average binding distance between drug and Trp 214 of HSA was found to be 2.23 nm on the basis of the theory of Förster's energy transfer. Synchronous fluorescence data indicated that interaction of drug with HSA changed the microenvironment around the tryptophan residue. UV–visible spectroscopy and circular dichroism results deciphered the complex formation and conformational alterations in the HSA respectively. Dynamic light scattering was utilized to understand the topology of protein in absence and presence of drug. Thermodynamic parameters obtained from isothermal titration calorimetry (ΔH=−26.01 kJ mol −1 and TΔS=6.5 kJ mol −1 ) suggested the involvement of van der Waal interaction and hydrogen bonding. Molecular docking and displacement study with site specific markers suggested that cytosine β-D arabinofuranoside binds to subdomain IB of HSA which is also known as the hemin binding site. This study will be helpful to understand the binding mechanism of cytosine β-D arabinofuranoside with HSA and associated alterations. - Highlights: • Comprehensive insight into the interaction of CBDA with HSA. • The interaction process is spontaneous and exothermic. • The main governing forces for stabilizing HSA–CBDA complex are van der Waal interaction and hydrogen bonding. • CBDA binds at subdomain IB on HSA

Cytosine deletion at AP2-box region of HSP70 promoter and its ...

Indian Academy of Sciences (India)

Cytosine deletion at AP2-box region of HSP70 promoter and its influence on semen quality traits in crossbred bulls ... Laboratory, ICAR-Central Institute for Research on Cattle, Meerut 250 001, India; School of Atmospheric Stress Management, ICAR-National Institute of Abiotic Stress Management, Baramati 413 115, India ...

Functional characterization of a rice de novo DNA methyltransferase, OsDRM2, expressed in Escherichia coli and yeast

Energy Technology Data Exchange (ETDEWEB)

Pang, Jinsong, E-mail: pangjs542@nenu.edu.cn [Key Laboratory of Molecular Epigenetics of the Ministry of Education, Northeast Normal University, Changchun, Jilin 130024 (China); Dong, Mingyue; Li, Ning; Zhao, Yanli [Key Laboratory of Molecular Epigenetics of the Ministry of Education, Northeast Normal University, Changchun, Jilin 130024 (China); Liu, Bao, E-mail: baoliu@nenu.edu.cn [Key Laboratory of Molecular Epigenetics of the Ministry of Education, Northeast Normal University, Changchun, Jilin 130024 (China)

2013-03-01

Highlights: ► A rice de novo DNA methyltransferase OsDRM2 was cloned. ► In vitro methylation activity of OsDRM2 was characterized with Escherichia coli. ► Assays of OsDRM2 in vivo methylation were done with Saccharomyces cerevisiae. ► OsDRM2 methylation activity is not preferential to any type of cytosine context. ► The activity of OsDRM2 is independent of RdDM pathway. - Abstract: DNA methylation of cytosine nucleotides is an important epigenetic modification that occurs in most eukaryotic organisms and is established and maintained by various DNA methyltransferases together with their co-factors. There are two major categories of DNA methyltransferases: de novo and maintenance. Here, we report the isolation and functional characterization of a de novo methyltransferase, named OsDRM2, from rice (Oryza sativa L.). The full-length coding region of OsDRM2 was cloned and transformed into Escherichia coli and Saccharomyces cerevisiae. Both of these organisms expressed the OsDRM2 protein, which exhibited stochastic de novo methylation activity in vitro at CG, CHG, and CHH di- and tri-nucleotide patterns. Two lines of evidence demonstrated the de novo activity of OsDRM2: (1) a 5′-CCGG-3′ containing DNA fragment that had been pre-treated with OsDRM2 protein expressed in E. coli was protected from digestion by the CG-methylation-sensitive isoschizomer HpaII; (2) methylation-sensitive amplified polymorphism (MSAP) analysis of S. cerevisiae genomic DNA from transformants that had been introduced with OsDRM2 revealed CG and CHG methylation levels of 3.92–9.12%, and 2.88–6.93%, respectively, whereas the mock control S. cerevisiae DNA did not exhibit cytosine methylation. These results were further supported by bisulfite sequencing of the 18S rRNA and EAF5 genes of the transformed S. cerevisiae, which exhibited different DNA methylation patterns, which were observed in the genomic DNA. Our findings establish that OsDRM2 is an active de novo DNA

Functional characterization of a rice de novo DNA methyltransferase, OsDRM2, expressed in Escherichia coli and yeast

International Nuclear Information System (INIS)

Pang, Jinsong; Dong, Mingyue; Li, Ning; Zhao, Yanli; Liu, Bao

2013-01-01

Highlights: ► A rice de novo DNA methyltransferase OsDRM2 was cloned. ► In vitro methylation activity of OsDRM2 was characterized with Escherichia coli. ► Assays of OsDRM2 in vivo methylation were done with Saccharomyces cerevisiae. ► OsDRM2 methylation activity is not preferential to any type of cytosine context. ► The activity of OsDRM2 is independent of RdDM pathway. - Abstract: DNA methylation of cytosine nucleotides is an important epigenetic modification that occurs in most eukaryotic organisms and is established and maintained by various DNA methyltransferases together with their co-factors. There are two major categories of DNA methyltransferases: de novo and maintenance. Here, we report the isolation and functional characterization of a de novo methyltransferase, named OsDRM2, from rice (Oryza sativa L.). The full-length coding region of OsDRM2 was cloned and transformed into Escherichia coli and Saccharomyces cerevisiae. Both of these organisms expressed the OsDRM2 protein, which exhibited stochastic de novo methylation activity in vitro at CG, CHG, and CHH di- and tri-nucleotide patterns. Two lines of evidence demonstrated the de novo activity of OsDRM2: (1) a 5′-CCGG-3′ containing DNA fragment that had been pre-treated with OsDRM2 protein expressed in E. coli was protected from digestion by the CG-methylation-sensitive isoschizomer HpaII; (2) methylation-sensitive amplified polymorphism (MSAP) analysis of S. cerevisiae genomic DNA from transformants that had been introduced with OsDRM2 revealed CG and CHG methylation levels of 3.92–9.12%, and 2.88–6.93%, respectively, whereas the mock control S. cerevisiae DNA did not exhibit cytosine methylation. These results were further supported by bisulfite sequencing of the 18S rRNA and EAF5 genes of the transformed S. cerevisiae, which exhibited different DNA methylation patterns, which were observed in the genomic DNA. Our findings establish that OsDRM2 is an active de novo DNA

The Effects of Lycopene on the Methylation of the GSTP1 Promoter and Global Methylation in Prostatic Cancer Cell Lines PC3 and LNCaP

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Li-Juan Fu

2014-01-01

Full Text Available DNA (cytosine-5- methylation silencing of GSTP1 function occurs in prostate adenocarcinoma (PCa. Previous studies have shown that there is an inverse relationship between dietary lycopene intake and the risk of PCa. However, it is unknown whether lycopene reactivates the tumor suppressor gene glutathioneS-transferase-π (GSTP1 by demethylation of the hypermethylated CpGs that act to silence the GSTP1 promoter. Here, we demonstrated that lycopene treatment significantly decreased the methylation levels of the GSTP1 promoter and increased the mRNA and protein levels of GSTP1 in an androgen-independent PC-3 cell line. In contrast, lycopene treatment did not demethylate the GSTP1 promoter or increase GSTP1 expression in the androgen-dependent LNCaP cell line. DNA methyltransferase (DNMT 3A protein levels were downregulated in PC-3 cells following lycopene treatment; however, DNMT1 and DNMT3B levels were unchanged. Furthermore, the long interspersed element (LINE-1 and short interspersed element ALU were not demethylated when treated by lycopene. In LNCaP cells, lycopene treatment did not affect any detected DNMT protein expression, and the methylation levels of LINE-1 and ALU were decreased. These results indicated that the protective effect of lycopene on the prostate is different between androgen-dependent and androgen-independent derived PCa cells. Further, in vivo studies should be conducted to confirm these promising results and to evaluate the potential role of lycopene in the protection of the prostate.

An N-Glycosidase from Escherichia coli That Releases Free Uracil from DNA Containing Deaminated Cytosine Residues

Science.gov (United States)

Lindahl, Tomas

1974-01-01

An enzyme that liberates uracil from single-stranded and double-stranded DNA containing deaminated cytosine residues and from deoxycytidylate-deoxyuridylate copolymers in the absence of Mg++ has been purified 30-fold from cell extracts of E. coli. The enzyme does not release uracil from deoxyuridine, dUMP, uridine, or RNA, nor does it liberate the normally occurring pyrimidine bases, cytosine and thymine, from DNA. The enzymatic cleavage of N-glycosidic bonds in DNA occurs without concomitant cleavage of phosphodiester bonds, resulting in the formation of free uracil and DNA strands of unaltered chain length that contain apyrimidinic sites as reaction products. The enzyme may be active in DNA repair, converting deaminated dCMP residues to an easily repairable form. PMID:4610583

The concerted impact of domestication and transposon insertions on methylation patterns between dogs and grey wolves.

Science.gov (United States)

Janowitz Koch, Ilana; Clark, Michelle M; Thompson, Michael J; Deere-Machemer, Kerry A; Wang, Jun; Duarte, Lionel; Gnanadesikan, Gitanjali E; McCoy, Eskender L; Rubbi, Liudmilla; Stahler, Daniel R; Pellegrini, Matteo; Ostrander, Elaine A; Wayne, Robert K; Sinsheimer, Janet S; vonHoldt, Bridgett M

2016-04-01

The process of domestication can exert intense trait-targeted selection on genes and regulatory regions. Specifically, rapid shifts in the structure and sequence of genomic regulatory elements could provide an explanation for the extensive, and sometimes extreme, variation in phenotypic traits observed in domesticated species. Here, we explored methylation differences from >24 000 cytosines distributed across the genomes of the domesticated dog (Canis familiaris) and the grey wolf (Canis lupus). PCA and model-based cluster analyses identified two primary groups, domestic vs. wild canids. A scan for significantly differentially methylated sites (DMSs) revealed species-specific patterns at 68 sites after correcting for cell heterogeneity, with weak yet significant hypermethylation typical of purebred dogs when compared to wolves (59% and 58%, P 66%) of differentially methylated regions contained or were associated with repetitive elements, indicative of a genotype-mediated trend. However, DMSs were also often linked to functionally relevant genes (e.g. neurotransmitters). Finally, we utilized known genealogical relationships among Yellowstone wolves to survey transmission stability of methylation marks, from which we found a substantial fraction that demonstrated high heritability (both H(2) and h(2 ) > 0.99). These analyses provide a unique epigenetic insight into the molecular consequences of recent selection and radiation of our most ancient domesticated companion, the dog. These findings suggest selection has acted on methylation patterns, providing a new genomic perspective on phenotypic diversification in domesticated species. © 2015 John Wiley & Sons Ltd.

Biophysical and molecular docking insight into the interaction of cytosine β-D arabinofuranoside with human serum albumin

Energy Technology Data Exchange (ETDEWEB)

Alam, Parvez; Chaturvedi, Sumit Kumar [Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh 202002, UP (India); Anwar, Tamanna [Center of Bioinformatics Research and Technology, Aligarh 202002 (India); Siddiqi, Mohammad Khursheed; Ajmal, Mohd Rehan [Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh 202002, UP (India); Badr, Gamal [Laboratory of Immunology & Molecular Physiology, Zoology Department, Faculty of Science, Assiut University, 71516 Assiut (Egypt); Mahmoud, Mohamed H. [Food Science and Nutrition Department, National Research Center, Dokki, Cairo (Egypt); Deanship of Scientific Research, King Saud University, Riyadh (Saudi Arabia); Hasan Khan, Rizwan, E-mail: rizwanhkhan@hotmail.com [Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh 202002, UP (India)

2015-08-15

Interaction of pharmacologically important anticancer drug cytosine β-D arabinofuranoside with human serum albumin (HSA) at physiological pH 7.4 has been studied by utilizing various spectroscopic and molecular docking strategies. Fluorescence results revealed that cytosine β-D arabinofuranoside interacts with HSA through static quenching mechanism with binding affinity of 2.4×10{sup 3} M{sup −1}. The average binding distance between drug and Trp{sup 214} of HSA was found to be 2.23 nm on the basis of the theory of Förster's energy transfer. Synchronous fluorescence data indicated that interaction of drug with HSA changed the microenvironment around the tryptophan residue. UV–visible spectroscopy and circular dichroism results deciphered the complex formation and conformational alterations in the HSA respectively. Dynamic light scattering was utilized to understand the topology of protein in absence and presence of drug. Thermodynamic parameters obtained from isothermal titration calorimetry (ΔH=−26.01 kJ mol{sup −1} and TΔS=6.5 kJ mol{sup −1}) suggested the involvement of van der Waal interaction and hydrogen bonding. Molecular docking and displacement study with site specific markers suggested that cytosine β-D arabinofuranoside binds to subdomain IB of HSA which is also known as the hemin binding site. This study will be helpful to understand the binding mechanism of cytosine β-D arabinofuranoside with HSA and associated alterations. - Highlights: • Comprehensive insight into the interaction of CBDA with HSA. • The interaction process is spontaneous and exothermic. • The main governing forces for stabilizing HSA–CBDA complex are van der Waal interaction and hydrogen bonding. • CBDA binds at subdomain IB on HSA.

Elastic electron scattering from the DNA bases cytosine and thymine

International Nuclear Information System (INIS)

Colyer, C. J.; Bellm, S. M.; Lohmann, B.; Blanco, F.; Garcia, G.

2011-01-01

Cross-section data for electron scattering from biologically relevant molecules are important for the modeling of energy deposition in living tissue. Relative elastic differential cross sections have been measured for cytosine and thymine using the crossed-beam method. These measurements have been performed for six discrete electron energies between 60 and 500 eV and for detection angles between 15 deg. and 130 deg. Calculations have been performed via the screen-corrected additivity rule method and are in good agreement with the present experiment.

Allelic Dropout During Polymerase Chain Reaction due to G-Quadruplex Structures and DNA Methylation Is Widespread at Imprinted Human Loci.

Science.gov (United States)

Stevens, Aaron J; Taylor, Millie G; Pearce, Frederick Grant; Kennedy, Martin A

2017-03-10

Loss of one allele during polymerase chain reaction (PCR) amplification of DNA, known as allelic dropout, can be caused by a variety of mechanisms. Allelic dropout during PCR may have profound implications for molecular diagnostic and research procedures that depend on PCR and assume biallelic amplification has occurred. Complete allelic dropout due to the combined effects of cytosine methylation and G-quadruplex formation was previously described for a differentially methylated region of the human imprinted gene, MEST We now demonstrate that this parent-of-origin specific allelic dropout can potentially occur at several other genomic regions that display genomic imprinting and have propensity for G-quadruplex formation, including AIM1 , BLCAP , DNMT1 , PLAGL1 , KCNQ1 , and GRB10 These findings demonstrate that systematic allelic dropout during PCR is a general phenomenon for regions of the genome where differential allelic methylation and G-quadruplex motifs coincide, and suggest that great care must be taken to ensure biallelic amplification is occurring in such situations. Copyright © 2017 Stevens et al.

Stress-induced gene expression and behavior are controlled by DNA methylation and methyl donor availability in the dentate gyrus

Science.gov (United States)

Saunderson, Emily A.; Spiers, Helen; Gutierrez-Mecinas, Maria; Trollope, Alexandra F.; Shaikh, Abeera; Mill, Jonathan; Reul, Johannes M. H. M.

2016-01-01

Stressful events evoke long-term changes in behavioral responses; however, the underlying mechanisms in the brain are not well understood. Previous work has shown that epigenetic changes and immediate-early gene (IEG) induction in stress-activated dentate gyrus (DG) granule neurons play a crucial role in these behavioral responses. Here, we show that an acute stressful challenge [i.e., forced swimming (FS)] results in DNA demethylation at specific CpG (5′-cytosine–phosphate–guanine-3′) sites close to the c-Fos (FBJ murine osteosarcoma viral oncogene homolog) transcriptional start site and within the gene promoter region of Egr-1 (early growth response protein 1) specifically in the DG. Administration of the (endogenous) methyl donor S-adenosyl methionine (SAM) did not affect CpG methylation and IEG gene expression at baseline. However, administration of SAM before the FS challenge resulted in an enhanced CpG methylation at the IEG loci and suppression of IEG induction specifically in the DG and an impaired behavioral immobility response 24 h later. The stressor also specifically increased the expression of the de novo DNA methyltransferase Dnmt3a [DNA (cytosine-5-)-methyltransferase 3 alpha] in this hippocampus region. Moreover, stress resulted in an increased association of Dnmt3a enzyme with the affected CpG loci within the IEG genes. No effects of SAM were observed on stress-evoked histone modifications, including H3S10p-K14ac (histone H3, phosphorylated serine 10 and acetylated lysine-14), H3K4me3 (histone H3, trimethylated lysine-4), H3K9me3 (histone H3, trimethylated lysine-9), and H3K27me3 (histone H3, trimethylated lysine-27). We conclude that the DNA methylation status of IEGs plays a crucial role in FS-induced IEG induction in DG granule neurons and associated behavioral responses. In addition, the concentration of available methyl donor, possibly in conjunction with Dnmt3a, is critical for the responsiveness of dentate neurons to environmental

Electron attachment to the guanine-cytosine nucleic acid base pair and the effects of monohydration and proton transfer.

Science.gov (United States)

Gupta, Ashutosh; Jaeger, Heather M; Compaan, Katherine R; Schaefer, Henry F

2012-05-17

The guanine-cytosine (GC) radical anion and its interaction with a single water molecule is studied using ab initio and density functional methods. Z-averaged second-order perturbation theory (ZAPT2) was applied to GC radical anion for the first time. Predicted spin densities show that the radical character is localized on cytosine. The Watson-Crick monohydrated GC anion is compared to neutral GC·H2O, as well as to the proton-transferred analogue on the basis of structural and energetic properties. In all three systems, local minima are identified that correspond to water positioned in the major and minor grooves of macromolecular DNA. On the anionic surface, two novel structures have water positioned above or below the GC plane. On the neutral and anionic surfaces, the global minimum can be described as water interacting with the minor groove. These structures are predicted to have hydration energies of 9.7 and 11.8 kcal mol(-1), respectively. Upon interbase proton-transfer (PT), the anionic global minimum has water positioned in the major groove, and the hydration energy increases to 13.4 kcal mol(-1). PT GC·H2O(•-) has distonic character; the radical character resides on cytosine, while the negative charge is localized on guanine. The effects of proton transfer are further investigated through the computed adiabatic electron affinities (AEA) of GC and monohydrated GC, and the vertical detachment energies (VDE) of the corresponding anions. Monohydration increases the AEAs and VDEs by only 0.1 eV, while proton-transfer increases the VDEs substantially (0.8 eV). The molecular charge distribution of monohydrated guanine-cytosine radical anion depends heavily on interbase proton transfer.

Experimental mitochondria-targeted DNA methylation identifies GpC methylation, not CpG methylation, as potential regulator of mitochondrial gene expression

NARCIS (Netherlands)

van der Wijst, Monique G. P.; van Tilburg, Amanda Y.; Ruiters, Marcel H. J.; Rots, Marianne G.

2017-01-01

Like the nucleus, mitochondria contain their own DNA and recent reports provide accumulating evidence that also the mitochondrial DNA (mtDNA) is subjective to DNA methylation. This evidence includes the demonstration of mitochondria-localised DNA methyltransferases and demethylases, and the

Epigenetic Diversity of Clonal White Poplar (Populus alba L. Populations: Could Methylation Support the Success of Vegetative Reproduction Strategy?

Directory of Open Access Journals (Sweden)

Francesco Guarino

Full Text Available The widespread poplar populations of Sardinia are vegetatively propagated and live in different natural environments forming large monoclonal stands. The main goals of the present study were: i to investigate/measure the epigenetic diversity of the poplar populations by determining their DNA methylation status; ii to assess if and how methylation status influences population clustering; iii to shed light on the changes that occur in the epigenome of ramets of the same poplar clone. To these purposes, 83 white poplar trees were sampled at different locations on the island of Sardinia. Methylation sensitive amplified polymorphism analysis was carried out on the genomic DNA extracted from leaves at the same juvenile stage. The study showed that the genetic biodiversity of poplars is quite limited but it is counterbalanced by epigenetic inter-population molecular variability. The comparison between MspI and HpaII DNA fragmentation profiles revealed that environmental conditions strongly influence hemi-methylation of the inner cytosine. The variable epigenetic status of Sardinian white poplars revealed a decreased number of population clusters. Landscape genetics analyses clearly demonstrated that ramets of the same clone were differentially methylated in relation to their geographic position. Therefore, our data support the notion that studies on plant biodiversity should no longer be restricted to genetic aspects, especially in the case of vegetatively propagated plant species.

Methylation changes associated with early maturation stages in the Atlantic salmon

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Pérez-Figueroa Andrés

2011-10-01

Full Text Available Abstract Background Early maturation in the Atlantic salmon is an interesting subject for numerous research lines. Prior to sea migration, parr can reach sexual maturation and successfully fertilize adult female eggs during the reproductive season. These individuals are known as precocious parr, mature parr or "sneakers". Reasons for early maturation are unknown and this transitory stage is usually considered to be a threshold trait. Here, we compare methylation patterns between mature and immature salmon parr from two different rivers in order to infer if such methylation differences may be related to their maturation condition. First we analyzed genetic differences between rivers by means of AFLPs. Then, we compared the DNA methylation differences between mature and immature parrs, using a Methylation-Sensitive Amplified Polymorphism (MSAP, which is a modification of the AFLPs method by making use of the differential sensitivity of a pair of restriction enzymes isoschizomeres to cytosine methylation. The tissues essayed included brain, liver and gonads. Results AFLPs statistical analysis showed that there was no significant differentiation between rivers or a significant differentiation between maturation states in each river. MSAP statistical analysis showed that among the three tissues sampled, the gonads had the highest number of significant single-locus variation among populations with 74 loci followed by brain with 70 and finally liver with only 12. Principal components analysis (PCA of the MSAP profiles revealed different profiles among different tissues (liver, brain and testis clearly separating maturation states in the testis tissue when compared to the liver. Conclusions Our results reveal that genetically-similar mature and immature salmon parr present high levels of DNA methylation variation in two of the three analyzed tissues. We hypothesize that early maturation may be mostly mediated by epigenetic processes rather than by

Patterns of DNA methylation in development, division of labor and hybridization in an ant with genetic caste determination.

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Chris R Smith

Full Text Available BACKGROUND: DNA methylation is a common regulator of gene expression, including acting as a regulator of developmental events and behavioral changes in adults. Using the unique system of genetic caste determination in Pogonomyrmex barbatus, we were able to document changes in DNA methylation during development, and also across both ancient and contemporary hybridization events. METHODOLOGY/PRINCIPAL FINDINGS: Sodium bisulfite sequencing demonstrated in vivo methylation of symmetric CG dinucleotides in P. barbatus. We also found methylation of non-CpG sequences. This validated two bioinformatics methods for predicting gene methylation, the bias in observed to expected ratio of CpG dinucleotides and the density of CpG/TpG single nucleotide polymorphisms (SNP. Frequencies of genomic DNA methylation were determined for different developmental stages and castes using ms-AFLP assays. The genetic caste determination system (GCD is probably the product of an ancestral hybridization event between P. barbatus and P. rugosus. Two lineages obligately co-occur within a GCD population, and queens are derived from intra-lineage matings whereas workers are produced from inter-lineage matings. Relative DNA methylation levels of queens and workers from GCD lineages (contemporary hybrids were not significantly different until adulthood. Virgin queens had significantly higher relative levels of DNA methylation compared to workers. Worker DNA methylation did not vary among developmental stages within each lineage, but was significantly different between the currently hybridizing lineages. Finally, workers of the two genetic caste determination lineages had half as many methylated cytosines as workers from the putative parental species, which have environmental caste determination. CONCLUSIONS/SIGNIFICANCE: These results suggest that DNA methylation may be a conserved regulatory mechanism moderating division of labor in both bees and ants. Current and historic

Journal of Genetics | Indian Academy of Sciences

Indian Academy of Sciences (India)

Keywords. cytosine methylation; DNA methylation mechanisms; DNA demethylation mechanisms; Darwinian-cum-Lamarckian evolution; epialleles; epigenetic modifications; genetic recombination; heritable induced defence; mutational hotspots; transgenerational inheritance.

Nanoswitches based on DNA base pairs: why adenine-thymine is less suitable than guanine-cytosine

NARCIS (Netherlands)

Fonseca Guerra, C.; van der Wijst, T.; Bickelhaupt, F.M.

2006-01-01

Substituted Watson-Crick guanine-cytosine (GC) base pairs were recently shown to yield robust three-state nanoswitches. Here, we address the question: Can such supramolecular switches also be based on Watson-Crick adenine-thymine (AT) base pairs? We have theoretically analyzed AT pairs in which

The value of SHOX2 methylation test in peripheral blood samples used for the differential diagnosis of lung cancer and other lung disorders.

Science.gov (United States)

Konecny, M; Markus, J; Waczulikova, I; Dolesova, L; Kozlova, R; Repiska, V; Novosadova, H; Majer, I

2016-01-01

Methylation of the cytosine residues within the CpG dinucleotides plays an important role in the fundamental cellular processes, human diseases and even cancer. The DNA methylation represents a very stable sign and therefore may be used as a valuable marker for cancer screening. Epigenetic cancer biomarkers are independent of classical morphology and thus show extensive potential to overcome the limitations of cytology. Several epigenetic cancer markers have been reported to be detectable in body fluids such as bronchial aspirate, sputum, plasma and serum.Short stature homeobox gene 2 (SHOX2) encodes a homeo-domain transcription factor, which has been identified as a close homologue of the SHOX gene and both genes are involved in skeletogenesis and heart development. Methylation of SHOX2 gene has been shown to be present at high prevalence in carcinomas of lung, however may also be used to identify other tumour entities.In the presented study, we have compared suitability of two types of material associated with lung cancer for the detection of SHOX2 methylation. We have confirmed that methylation of SHOX2 gene represents reliable marker of lung malignancies. The parallel tests in the blood plasma revealed that it may represent a good alternative material for testing of the SHOX2 methylation, making the test available to patients who are unable to undergo bronchoscopy.

Model-based clustering of DNA methylation array data: a recursive-partitioning algorithm for high-dimensional data arising as a mixture of beta distributions

Directory of Open Access Journals (Sweden)

Wiemels Joseph

2008-09-01

Full Text Available Abstract Background Epigenetics is the study of heritable changes in gene function that cannot be explained by changes in DNA sequence. One of the most commonly studied epigenetic alterations is cytosine methylation, which is a well recognized mechanism of epigenetic gene silencing and often occurs at tumor suppressor gene loci in human cancer. Arrays are now being used to study DNA methylation at a large number of loci; for example, the Illumina GoldenGate platform assesses DNA methylation at 1505 loci associated with over 800 cancer-related genes. Model-based cluster analysis is often used to identify DNA methylation subgroups in data, but it is unclear how to cluster DNA methylation data from arrays in a scalable and reliable manner. Results We propose a novel model-based recursive-partitioning algorithm to navigate clusters in a beta mixture model. We present simulations that show that the method is more reliable than competing nonparametric clustering approaches, and is at least as reliable as conventional mixture model methods. We also show that our proposed method is more computationally efficient than conventional mixture model approaches. We demonstrate our method on the normal tissue samples and show that the clusters are associated with tissue type as well as age. Conclusion Our proposed recursively-partitioned mixture model is an effective and computationally efficient method for clustering DNA methylation data.

Towards an understanding of CG methylation in DNA transcription

International Nuclear Information System (INIS)

Chela-Flores, J.; Migoni, R.L.

1989-09-01

A simple model of DNA is considered in which the nucleotides cytosine (C) and guanine (G) are not assumed to be identical, and in which macroscopic thermodynamic quantities may be calculated exactly. The H bonds between the C and G nucleotides are assumed to be Morse potentials. We discuss the statistical mechanics of the DNA molecule in the configuration (5'...GGG...3'; 3'...CCC...5'), which may be copied by RNA polymerase into a messenger RNA (mRNA) strand (5'...CCC...3'). This model suggests that replacements of C by 5-methylcytosine (5mC) may be a secondary effect in the inhibition of genetic expression, not interfering directly with the formation of an open state. An experimental test is suggested. The implications of this result are discussed for a related system, in which the enzyme methylase is known to methylate almost exclusively those Cs that are followed by Gs as a regulatory strategy employed by some eukaryotes. (author). 14 refs, 2 figs

An optimized rapid bisulfite conversion method with high recovery of cell-free DNA.

Science.gov (United States)

Yi, Shaohua; Long, Fei; Cheng, Juanbo; Huang, Daixin

2017-12-19

Methylation analysis of cell-free DNA is a encouraging tool for tumor diagnosis, monitoring and prognosis. Sensitivity of methylation analysis is a very important matter due to the tiny amounts of cell-free DNA available in plasma. Most current methods of DNA methylation analysis are based on the difference of bisulfite-mediated deamination of cytosine between cytosine and 5-methylcytosine. However, the recovery of bisulfite-converted DNA based on current methods is very poor for the methylation analysis of cell-free DNA. We optimized a rapid method for the crucial steps of bisulfite conversion with high recovery of cell-free DNA. A rapid deamination step and alkaline desulfonation was combined with the purification of DNA on a silica column. The conversion efficiency and recovery of bisulfite-treated DNA was investigated by the droplet digital PCR. The optimization of the reaction results in complete cytosine conversion in 30 min at 70 °C and about 65% of recovery of bisulfite-treated cell-free DNA, which is higher than current methods. The method allows high recovery from low levels of bisulfite-treated cell-free DNA, enhancing the analysis sensitivity of methylation detection from cell-free DNA.

Cord blood PRF1 methylation patterns and risk of lower respiratory tract infections in infants: findings from the Ulm Birth Cohort.

Science.gov (United States)

Elgizouli, Magdeldin; Logan, Chad; Nieters, Alexandra; Brenner, Hermann; Rothenbacher, Dietrich

2015-01-01

Lower respiratory tract infections (LRTIs) are a major cause of morbidity in children. DNA methylation provides a mechanism for transmitting environmental effects on the genome, but its potential role in LRTIs is not well studied. We investigated the methylation pattern of an enhancer region of the immune effector gene perforin-1 (PRF1), which encodes a cytolytic molecule of cytotoxic T lymphocytes (CTLs) and natural killer cells (NK), in cord blood DNA of children recruited in a German birth cohort in association with LRTIs in the first year of life.Pyrosequencing was used to determine the methylation levels of target cytosine-phosphate-guanines (CpGs) in a 2-stage case-control design. Cases were identified as children who developed ≥2 episodes of physician-recorded LRTIs during the first year of life and controls as children who had none. Discovery (n = 87) and replication (n = 90) sets were arranged in trios of 1 case and 2 controls matched for sex and season of birth.Logistic regression analysis revealed higher levels of methylation at a CpG that corresponds to a signal transducer and activator of transcription 5 (STAT5) responsive enhancer in the discovery (odds ratio [OR] per 1% methylation difference 1.24, 95% confidence interval [CI] 1.03-1.50) and replication (OR per 1% methylation difference 1.25, 95% CI 1.04-1.50) sets. Adjustment for having siblings blood PRF1 enhancer methylation patterns and subsequent risk of LRTIs in infants. Methylation levels at specific CpGs of the PRF1 enhancer varied according to maternal and family environmental factors suggesting a role for DNA methylation in mediating environmental influences on gene function.

Liberation of methyl acrylate from metallalactone complexes via M-O ring opening (M = Ni, Pd) with methylation agents

KAUST Repository

Lee, S. Y Tina; Ghani, Amylia Abdul; D'Elia, Valerio; Cokoja, Mirza; Herrmann, Wolfgang A.; Basset, Jean-Marie; Kü hn, Fritz

2013-01-01

Ring opening of various nickela- and palladalactones induced by the cleavage of the M-O bond by methyl trifluoromethanesulfonate (MeOTf) and methyl iodide (MeI) is examined. Experimental evidence supports the mechanism of ring opening by the alkylating agent followed by β-H elimination leading to methyl acrylate and a metal-hydride species. MeOTf shows by far higher efficiency in the lactone ring opening than any other methylating agent including the previously reported methyl iodide. © 2013 The Royal Society of Chemistry and the Centre National de la Recherche Scientifique.

Cyclopentenyl cytosine induces apoptosis and increases cytarabine-induced apoptosis in a T-lymphoblastic leukemic cell-line

NARCIS (Netherlands)

Verschuur, A. C.; Brinkman, J.; van Gennip, A. H.; Leen, R.; Vet, R. J.; Evers, L. M.; Voûte, P. A.; van Kuilenburg, A. B.

2001-01-01

Cyclopentenyl cytosine (CPEC) is a nucleoside-analogue that decreases the concentrations of cytidine triphosphate (CTP) and deoxycytidine triphosphate (dCTP) in leukemic cells by inhibiting the enzyme CTP synthetase, resulting in a decreased synthesis of RNA and DNA. Low concentrations of dCTP

Suppression of prolactin gene expression in GH cells correlates with site-specific DNA methylation.

Science.gov (United States)

Zhang, Z X; Kumar, V; Rivera, R T; Pasion, S G; Chisholm, J; Biswas, D K

1989-10-01

Prolactin- (PRL) producing and nonproducing subclones of the GH line of (rat) pituitary tumor cells have been compared to elucidate the regulatory mechanisms of PRL gene expression. Particular emphasis was placed on delineating the molecular basis of the suppressed state of the PRL gene in the prolactin-nonproducing (PRL-) GH subclone (GH(1)2C1). We examined six methylatable cytosine residues (5, -CCGG- and 1, -GCGC-) within the 30-kb region of the PRL gene in these subclones. This analysis revealed that -CCGG-sequences of the transcribed region, and specifically, one in the fourth exon of the PRL gene, were heavily methylated in the PRL-, GH(1)2C1 cells. Furthermore, the inhibition of PRL gene expression in GH(1)2C1 was reversed by short-term treatment of the cells with a sublethal concentration of azacytidine (AzaC), an inhibitor of DNA methylation. The reversion of PRL gene expression by AzaC was correlated with the concurrent demethylation of the same -CCGG- sequences in the transcribed region of PRL gene. An inverse correlation between PRL gene expression and the level of methylation of the internal -C- residues in the specific -CCGG-sequence of the transcribed region of the PRL gene was demonstrated. The DNase I sensitivity of these regions of the PRL gene in PRL+, PRL-, and AzaC-treated cells was also consistent with an inverse relationship between methylation state, a higher order of structural modification, and gene expression.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of phenobarbital on the methylation level of the p16 promoter region in rat liver

International Nuclear Information System (INIS)

Kostka, Grazyna; Urbanek, Katarzyna; Ludwicki, Jan K.

2007-01-01

It has been suggested that non-genotoxic carcinogens (NGCs) may cause modification of the DNA methylation status. We studied the effects of phenobarbital (PB) - a non-genotoxic rodent liver carcinogen - on the methylation level of the promoter region of the p16 suppressor gene, as well as on hepatomegaly, DNA synthesis, and DNA-methyltransferase (DNMTs) activity in the rat liver. Male Wistar rats received PB in 1, 3 or 14 daily oral doses (at 24-h intervals), each equivalent to 1/10 of the LD 50 value. The study showed that PB has caused persistent elevation in relative liver weight (RLW) as well as a transient increase in DNA synthesis. This suggests that the PB-induced increase in RLW was due to a combination of both hyperplasia and hypertrophy of liver cells. The effect of PB on DNA synthesis corresponded to an increase in the methylation pattern of the p16 promoter sequence. Methylation of cytosine in the analyzed CpG sites of the p16 gene was found after short exposure of the animals to PB. Treatment of rats with PB for 1 and 3 days also produced an increase in nuclear DNMTs activity. After prolonged administration (14 days), DNA synthesis declined, returning to the control level. No changes in methylation of the p16 gene nor in DNMTs activity were observed. The reversibility of early induced changes in target tissues is a mark characteristic of tumor promoters. Thus, transient changes in methylation of the p16 gene, although their direct role in the mechanisms of PB toxicity, including its carcinogenic action, remains doubtful, may therefore be a significant element of such processes

Effects of Unpredictable Variable Prenatal Stress (UVPS) on Bdnf DNA Methylation and Telomere Length in the Adult Rat Brain

Science.gov (United States)

Blaze, Jennifer; Asok, A.; Moyer, E. L.; Roth, T. L.; Ronca, A. E.

2015-01-01

In utero exposure to stress can shape neurobiological and behavioral outcomes in offspring, producing vulnerability to psychopathology later in life. Animal models of prenatal stress likewise have demonstrated long-­-term alterations in brain function and behavioral deficits in offspring. For example, using a rodent model of unpredictable variable prenatal stress (UVPS), in which dams are exposed to unpredictable, variable stress across pregnancy, we have found increased body weight and anxiety-­-like behavior in adult male, but not female, offspring. DNA methylation (addition of methyl groups to cytosines which normally represses gene transcription) and changes in telomere length (TTAGGG repeats on the ends of chromosomes) are two molecular modifications that result from stress and could be responsible for the long-­-term effects of UVPS. Here, we measured methylation of brain-­-derived neurotrophic factor (bdnf), a gene important in development and plasticity, and telomere length in the brains of adult offspring from the UVPS model. Results indicate that prenatally stressed adult males have greater methylation in the medial prefrontal cortex (mPFC) compared to non-­-stressed controls, while females have greater methylation in the ventral hippocampus compared to controls. Further, prenatally stressed males had shorter telomeres than controls in the mPFC. These findings demonstrate the ability of UVPS to produce epigenetic alterations and changes in telomere length across behaviorally-­-relevant brain regions, which may have linkages to the phenotypic outcomes.

Simultaneous determination of cytosine arabinoside, daunorubicin and etoposide in human plasma

DEFF Research Database (Denmark)

Krogh-Madsen, Mikkel; Hansen, Steen Honoré; Honoré, Per Hartvig

2010-01-01

A method for simultaneous bioanalysis of the three cytotoxic drugs cytosine arabinoside, daunorubicin and etoposide in human plasma was developed and validated. A HPLC method with ultra-violet and fluorescence detection, preceded by mixed-mode cation-exchange solid phase extraction sample....... The overall precision (% relative standard deviation) was within 0.2-13.5% and the recovery ranged between 86.1% and 110.1% for the three drugs at all concentrations tested. Plasma samples were stable for at least two months when stored at -20 degrees C. The method was successfully applied to quantification...

Folate, colorectal cancer and the involvement of DNA methylation.

Science.gov (United States)

Williams, Elizabeth A

2012-11-01

Diet is a major factor in the aetiology of colorectal cancer (CRC). Epidemiological evidence suggests that folate confers a modest protection against CRC risk. However, the relationship is complex, and evidence from human intervention trials and animal studies suggests that a high-dose of folic acid supplementation may enhance the risk of colorectal carcinogenesis in certain circumstances. The molecular mechanisms underlying the apparent dual modulatory effect of folate on colorectal carcinogenesis are not fully understood. Folate is central to C1 metabolism and is needed for both DNA synthesis and DNA methylation, providing plausible biological mechanisms through which folate could modulate cancer risk. Aberrant DNA methylation is an early event in colorectal carcinogenesis and is typically associated with the transcriptional silencing of tumour suppressor genes. Folate is required for the production of S-adenosyl methionine, which serves as a methyl donor for DNA methylation events; thereby folate availability is proposed to modulate DNA methylation status. The evidence for an effect of folate on DNA methylation in the human colon is limited, but a modulation of DNA methylation in response to folate has been demonstrated. More research is required to clarify the optimum intake of folate for CRC prevention and to elucidate the effect of folate availability on DNA methylation and the associated impact on CRC biology.

Loss of maintenance DNA methylation results in abnormal DNA origin firing during DNA replication

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Haruta, Mayumi [Department of Cell Biology, Graduate School of Medical Sciences, Nagoya City University, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467-8601 (Japan); Shimada, Midori, E-mail: midorism@med.nagoya-cu.ac.jp [Department of Cell Biology, Graduate School of Medical Sciences, Nagoya City University, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467-8601 (Japan); Nishiyama, Atsuya; Johmura, Yoshikazu [Department of Cell Biology, Graduate School of Medical Sciences, Nagoya City University, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467-8601 (Japan); Le Tallec, Benoît; Debatisse, Michelle [Institut Curie, Centre de Recherche, 26 rue d’Ulm, CNRS UMR 3244, 75248 ParisCedex 05 (France); Nakanishi, Makoto, E-mail: mkt-naka@med.nagoya-cu.ac.jp [Department of Cell Biology, Graduate School of Medical Sciences, Nagoya City University, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467-8601 (Japan)

2016-01-22

The mammalian maintenance methyltransferase DNMT1 [DNA (cytosine-5-)-methyltransferase 1] mediates the inheritance of the DNA methylation pattern during replication. Previous studies have shown that depletion of DNMT1 causes a severe growth defect and apoptosis in differentiated cells. However, the detailed mechanisms behind this phenomenon remain poorly understood. Here we show that conditional ablation of Dnmt1 in murine embryonic fibroblasts (MEFs) resulted in an aberrant DNA replication program showing an accumulation of late-S phase replication and causing severely defective growth. Furthermore, we found that the catalytic activity and replication focus targeting sequence of DNMT1 are required for a proper DNA replication program. Taken together, our findings suggest that the maintenance of DNA methylation by DNMT1 plays a critical role in proper regulation of DNA replication in mammalian cells. - Highlights: • DNMT1 depletion results in an abnormal DNA replication program. • Aberrant DNA replication is independent of the DNA damage checkpoint in DNMT1cKO. • DNMT1 catalytic activity and RFT domain are required for proper DNA replication. • DNMT1 catalytic activity and RFT domain are required for cell proliferation.

Loss of maintenance DNA methylation results in abnormal DNA origin firing during DNA replication

International Nuclear Information System (INIS)

Haruta, Mayumi; Shimada, Midori; Nishiyama, Atsuya; Johmura, Yoshikazu; Le Tallec, Benoît; Debatisse, Michelle; Nakanishi, Makoto

2016-01-01

The mammalian maintenance methyltransferase DNMT1 [DNA (cytosine-5-)-methyltransferase 1] mediates the inheritance of the DNA methylation pattern during replication. Previous studies have shown that depletion of DNMT1 causes a severe growth defect and apoptosis in differentiated cells. However, the detailed mechanisms behind this phenomenon remain poorly understood. Here we show that conditional ablation of Dnmt1 in murine embryonic fibroblasts (MEFs) resulted in an aberrant DNA replication program showing an accumulation of late-S phase replication and causing severely defective growth. Furthermore, we found that the catalytic activity and replication focus targeting sequence of DNMT1 are required for a proper DNA replication program. Taken together, our findings suggest that the maintenance of DNA methylation by DNMT1 plays a critical role in proper regulation of DNA replication in mammalian cells. - Highlights: • DNMT1 depletion results in an abnormal DNA replication program. • Aberrant DNA replication is independent of the DNA damage checkpoint in DNMT1cKO. • DNMT1 catalytic activity and RFT domain are required for proper DNA replication. • DNMT1 catalytic activity and RFT domain are required for cell proliferation.

Identification of a highly conserved domain in the EcoRII methyltransferase which can be photolabeled with S-adenosyl-L-[methyl-3H]methionine. Evidence for UV-induced transmethylation of cysteine 186

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Som, S.; Friedman, S.

1991-01-01

DNA methyltransferases can be photolabeled with S-adenosyl-L-methionine (AdoMet). Specific incorporation of radioactivity has been demonstrated after photolabeling with either [methyl-3H]AdoMet or [35S]AdoMet. The labeling is believed to occur at the AdoMet binding site. With the purpose of localizing the site responsible for [methyl-3H]AdoMet photolabeling, we cleaved the labeled EcoRII methyltransferase by chemical and enzymatic reactions and isolated the radiolabeled peptides by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and high pressure liquid chromatography. The labeled peptides were identified by amino-terminal sequencing. A common region was localized which accounted for 65-70% of the total label. This region includes a highly conserved core sequence present in all DNA (cytosine 5)-methyltransferases. One such fragment was digested further with chymotrypsin, and amino acid analysis of the resulting 3H-labeled peptide was consistent with the sequence Ala-Gly-Phe-Pro-(Cys)-Gln-Pro-Phe-Ser-Leu. However, the cysteine residue was not recovered as carboxymethylcysteine. The Pro-Cys bond was found to be protected from cleavage at cysteine residues after cyanylation. These results suggest that the cysteine residue is modified by the labeling reaction. The chymotryptic fragment was hydrolyzed enzymatically to single amino acids, and the labeled amino acid was identified as S-methylcysteine by thin layer chromatography. These results indicate that the cysteine residue is located at or close to the AdoMet binding site of EcoRII methyltransferase

DNA methyltransferases and stress-related genes expression in zebrafish larvae after exposure to heat and copper during reprogramming of DNA methylation.

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Dorts, Jennifer; Falisse, Elodie; Schoofs, Emilie; Flamion, Enora; Kestemont, Patrick; Silvestre, Frédéric

2016-10-12

DNA methylation, a well-studied epigenetic mark, is important for gene regulation in adulthood and for development. Using genetic and epigenetic approaches, the present study aimed at evaluating the effects of heat stress and copper exposure during zebrafish early embryogenesis when patterns of DNA methylation are being established, a process called reprogramming. Embryos were exposed to 325 μg Cu/L from fertilization (<1 h post fertilization - hpf) to 4 hpf at either 26.5 °C or 34 °C, followed by incubation in clean water at 26.5 °C till 96 hpf. Significant increased mortality rates and delayed hatching were observed following exposure to combined high temperature and Cu. Secondly, both stressors, alone or in combination, significantly upregulated the expression of de novo DNA methyltransferase genes (dnmt3) along with no differences in global cytosine methylation level. Finally, Cu exposure significantly increased the expression of metallothionein (mt2) and heat shock protein (hsp70), the latter being also increased following exposure to high temperature. These results highlighted the sensitivity of early embryogenesis and more precisely of the reprogramming period to environmental challenges, in a realistic situation of combined stressors.

Planarizing cytosine: The S1 state structure, vibrations, and nonradiative dynamics of jet-cooled 5,6-trimethylenecytosine

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Trachsel, Maria A.; Lobsiger, Simon; Schär, Tobias; Blancafort, Lluís; Leutwyler, Samuel

2017-06-01

We measure the S0 → S1 spectrum and time-resolved S1 state nonradiative dynamics of the "clamped" cytosine derivative 5,6-trimethylenecytosine (TMCyt) in a supersonic jet, using two-color resonant two-photon ionization (R2PI), UV/UV holeburning, and ns time-resolved pump/delayed ionization. The experiments are complemented with spin-component scaled second-order approximate coupled cluster (SCS-CC2), time-dependent density functional theory, and multi-state second-order perturbation-theory (MS-CASPT2) ab initio calculations. While the R2PI spectrum of cytosine breaks off ˜500 cm-1 above its 000 band, that of TMCyt extends up to +4400 cm-1 higher, with over a hundred resolved vibronic bands. Thus, clamping the cytosine C5-C6 bond allows us to explore the S1 state vibrations and S0 → S1 geometry changes in detail. The TMCyt S1 state out-of-plane vibrations ν1', ν3', and ν5' lie below 420 cm-1, and the in-plane ν11', ν12', and ν23' vibrational fundamentals appear at 450, 470, and 944 cm-1. S0 → S1 vibronic simulations based on SCS-CC2 calculations agree well with experiment if the calculated ν1', ν3', and ν5' frequencies are reduced by a factor of 2-3. MS-CASPT2 calculations predict that the ethylene-type S1 ⇝ S0 conical intersection (CI) increases from +366 cm-1 in cytosine to >6000 cm-1 in TMCyt, explaining the long lifetime and extended S0 → S1 spectrum. The lowest-energy S1 ⇝ S0 CI of TMCyt is the "amino out-of-plane" (OPX) intersection, calculated at +4190 cm-1. The experimental S1 ⇝ S0 internal conversion rate constant at the S1(v'=0 ) level is kI C=0.98 -2.2 ṡ1 08 s-1, which is ˜10 times smaller than in 1-methylcytosine and cytosine. The S1(v'=0 ) level relaxes into the T1(3π π *) state by intersystem crossing with kI S C=0.41 -1.6 ṡ1 08 s-1. The T1 state energy is measured to lie 24 580 ±560 cm-1 above the S0 state. The S1(v'=0 ) lifetime is τ =2.9 ns, resulting in an estimated fluorescence quantum yield of Φf l=24 %. Intense

GPU-BSM: a GPU-based tool to map bisulfite-treated reads.

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Andrea Manconi

Full Text Available Cytosine DNA methylation is an epigenetic mark implicated in several biological processes. Bisulfite treatment of DNA is acknowledged as the gold standard technique to study methylation. This technique introduces changes in the genomic DNA by converting cytosines to uracils while 5-methylcytosines remain nonreactive. During PCR amplification 5-methylcytosines are amplified as cytosine, whereas uracils and thymines as thymine. To detect the methylation levels, reads treated with the bisulfite must be aligned against a reference genome. Mapping these reads to a reference genome represents a significant computational challenge mainly due to the increased search space and the loss of information introduced by the treatment. To deal with this computational challenge we devised GPU-BSM, a tool based on modern Graphics Processing Units. Graphics Processing Units are hardware accelerators that are increasingly being used successfully to accelerate general-purpose scientific applications. GPU-BSM is a tool able to map bisulfite-treated reads from whole genome bisulfite sequencing and reduced representation bisulfite sequencing, and to estimate methylation levels, with the goal of detecting methylation. Due to the massive parallelization obtained by exploiting graphics cards, GPU-BSM aligns bisulfite-treated reads faster than other cutting-edge solutions, while outperforming most of them in terms of unique mapped reads.

Methylation status of imprinted genes DLK1-GTL2, MEST (PEG1), ZAC (PLAGL1), and LINE-1 elements in spermatozoa of normozoospermic men, unlike H19 imprinting control regions, is not associated with idiopathic recurrent spontaneous miscarriages.

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Ankolkar, Mandar; Salvi, Vinita; Warke, Himangi; Vundinti, Babu Rao; Balasinor, N H

2013-05-01

To study methylation aberrations in spermatozoa at developmentally important imprinted regions to ascertain their role in early embryo loss in idiopathic recurrent spontaneous miscarriages (RSM). Case-control study. Academic research setting at National Institute for Research in Reproductive Health, Parel, Mumbai. Male partners of couples with a history of RSM and male partners of couples with proven fertility (control group). None. DNA methylation levels at imprinting control regions of DLK1-GTL2, MEST (PEG1), and ZAC (PLAGL1) by Epityper Massarray and global methylation levels as measured by LINE-1 methylation and anti-5-methyl cytosine antibody in spermatozoa of 23 men in control group and 23 men in RSM group. We did not observe any aberration in the total methylation levels in any of the imprinted genes or global methylation analyzed. Our results indicate that paternal methylation aberrations at imprinting control regions of DLK1-GTL2, MEST (PEG1), and ZAC (PLAGL1) and global methylation levels are not associated with idiopathic RSM and may not be good epigenetic markers (unlike the H-19 imprinting control region) for diagnosis of idiopathic RSM. Copyright © 2013 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Quantitative analysis of DNA methylation at all human imprinted regions reveals preservation of epigenetic stability in adult somatic tissue

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Woodfine Kathryn

2011-01-01

Full Text Available Abstract Background Genes subject to genomic imprinting are mono-allelically expressed in a parent-of-origin dependent manner. Each imprinted locus has at least one differentially methylated region (DMR which has allele specific DNA methylation and contributes to imprinted gene expression. Once DMRs are established, they are potentially able to withstand normal genome reprogramming events that occur during cell differentiation and germ-line DMRs are stably maintained throughout development. These DMRs, in addition to being either maternally or paternally methylated, have differences in whether methylation was acquired in the germ-line or post fertilization and are present in a variety of genomic locations with different Cytosine-phosphate guanine (CpG densities and CTCF binding capacities. We therefore examined the stability of maintenance of DNA methylation imprints and determined the normal baseline DNA methylation levels in several adult tissues for all imprinted genes. In order to do this, we first developed and validated 50 highly specific, quantitative DNA methylation pyrosequencing assays for the known DMRs associated with human imprinted genes. Results Remarkable stability of the DNA methylation imprint was observed in all germ-line DMRs and paternally methylated somatic DMRs (which maintained average methylation levels of between 35% - 65% in all somatic tissues, independent of gene expression. Maternally methylated somatic DMRs were found to have more variation with tissue specific methylation patterns. Most DMRs, however, showed some intra-individual variability for DNA methylation levels in peripheral blood, suggesting that more than one DMR needs to be examined in order to get an overall impression of the epigenetic stability in a tissue. The plasticity of DNA methylation at imprinted genes was examined in a panel of normal and cancer cell lines. All cell lines showed changes in DNA methylation, especially at the paternal germ

Depleted uranium induces sex- and tissue-specific methylation patterns in adult zebrafish

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Gombeau, Kewin; Pereira, Sandrine; Ravanat, Jean-Luc; Camilleri, Virginie; Cavalie, Isabelle; Bourdineaud, Jean-Paul; Adam-Guillermin, Christelle

2016-01-01

We examined the effects of chronic exposure to different concentrations (2 and 20 μg L"−"1) of environmentally relevant waterborne depleted uranium (DU) on the DNA methylation patterns both at HpaII restriction sites (5′-CCGG-3′) and across the whole genome in the zebrafish brain, gonads, and eyes. We first identified sex-dependent differences in the methylation level of HpaII sites after exposure. In males, these effects were present as early as 7 days after exposure to 20 μg L"−"1 DU, and were even more pronounced in the brain, gonads, and eyes after 24 days. However, in females, hypomethylation was only observed in the gonads after exposure to 20 μg L"−"1 DU for 24 days. Sex-specific effects of DU were also apparent at the whole-genome level, because in males, exposure to 20 μg L"−"1 DU for 24 days resulted in cytosine hypermethylation in the brain and eyes and hypomethylation in the gonads. In contrast, in females, hypermethylation was observed in the brain after exposure to both concentrations of DU for 7 days. Based on our current knowledge of uranium toxicity, several hypotheses are proposed to explain these findings, including the involvement of oxidative stress, alteration of demethylation enzymes and the calcium signaling pathway. This study reports, for the first time, the sex- and tissue-specific epigenetic changes that occur in a nonhuman organism after exposure to environmentally relevant concentrations of uranium, which could induce transgenerational epigenetic effects. - Highlights: • This study demonstrates a sex-related effect of DU exposure on DNA methylation patterns. • Impacts on DNA methylation patterns revealed a tissue-specific effect of DU exposure. • The MS–AFLP and HPLC–MS/MS sensitively and complementarily demonstrated the responses to environmental concentrations of DU.

Experimental vapor pressures (from 1 Pa to 100 kPa) of six saturated Fatty Acid Methyl Esters (FAMEs): Methyl hexanoate, methyl octanoate, methyl decanoate, methyl dodecanoate, methyl tetradecanoate and methyl hexadecanoate

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Sahraoui, Lakhdar; Khimeche, Kamel; Dahmani, Abdallah; Mokbel, Ilham; Jose, Jacques

2016-01-01

Highlight: • Vapor-liquid equilibria, Enthalpy of Vaporization, saturated Fatty Acid Methyl Ester. - Abstract: Vapor pressures of six saturated Fatty Acid Methyl Esters (FAMEs), methyl hexanoate (or methyl caproate), methyl octanoate (or methyl caprylate), Methyl decanoate (or methyl caprate), methyl dodecanoate (or methyl laurate), methyl tetradecanoate (or methyl myristate), and methyl hexadecanoate (or methyl palmitate) were measured from 1 Pa to 100 kPa and at temperature range between 262 and 453 K using a static apparatus. The experimental data (P-T) were compared with the available literature data.

Functional analyses of PtRDM1 gene overexpression in poplars and evaluation of its effect on DNA methylation and response to salt stress.

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Movahedi, Ali; Zhang, Jiaxin; Sun, Weibo; Mohammadi, Kourosh; Almasi Zadeh Yaghuti, Amir; Wei, Hui; Wu, Xiaolong; Yin, Tongming; Zhuge, Qiang

2018-06-01

Epigenetic modification by DNA methylation is necessary for all cellular processes, including genetic expression events, DNA repair, genomic imprinting and regulation of tissue development. It occurs almost exclusively at the C5 position of symmetric CpG and asymmetric CpHpG and CpHpH sites in genomic DNA. The RNA-directed DNA methylation (RDM1) gene is crucial for heterochromatin and DNA methylation. We overexpressed PtRDM1 gene from Populus trichocarpa to amplify transcripts of orthologous RDM1 in 'Nanlin895' (P. deltoides × P. euramericana 'Nanlin895'). This overexpression resulted in increasing RDM1 transcript levels: by ∼150% at 0 mM NaCl treatment and by ∼300% at 60 mM NaCl treatment compared to WT (control) poplars. Genomic cytosine methylation was monitored within 5.8S rDNA and histone H3 loci by bisulfite sequencing. In total, transgenic poplars revealed more DNA methylation than WT plants. In our results, roots revealed more methylated CG contexts than stems and leaves whereas, histone H3 presented more DNA methylation than 5.8S rDNA in both WT and transgenic poplars. The NaCl stresses enhanced more DNA methylation in transgenic poplars than WT plants through histone H3 and 5.8 rDNA loci. Also, the overexpression of PtRDM1 resulted in hyper-methylation, which affected plant phenotype. Transgenic poplars revealed significantly more regeneration of roots than WT poplars via NaCl treatments. Our results proved that RDM1 protein enhanced the DNA methylation by chromatin remodeling (e.g. histone H3) more than repetitive DNA sequences (e.g. 5.8S rDNA). Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Effect of maternal gestational weight gain on offspring DNA methylation: a follow-up to the ALSPAC cohort study.

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Bohlin, Jon; Andreassen, Bettina K; Joubert, Bonnie R; Magnus, Maria C; Wu, Michael C; Parr, Christine L; Håberg, Siri E; Magnus, Per; Reese, Sarah E; Stoltenberg, Camilla; London, Stephanie J; Nystad, Wenche

2015-07-29

Several epidemiologic studies indicate that maternal gestational weight gain (GWG) influences health outcomes in offspring. Any underlying mechanisms have, however, not been established. A recent study of 88 children based on the Avon Longitudinal Study of Parents and Children (ALSPAC) cohort examined the methylation levels at 1,505 Cytosine-Guanine methylation (CpG) loci and found several to be significantly associated with maternal weight gain between weeks 0 and 18 of gestation. Since these results could not be replicated we wanted to examine associations between 0 and 18 week GWG and genome-wide methylation levels using the Infinium HumanMethylation450 BeadChip (450K) platform on a larger sample size, i.e. 729 newborns sampled from the Norwegian Mother and Child Cohort Study (MoBa). We found no CpG loci associated with 0-18 week GWG after adjusting for the set of covariates used in the ALSPAC study (i.e. child's sex and maternal age) and for multiple testing (q > 0.9, both 1,505 and 473,731 tests). Hence, none of the CpG loci linked with the genes found significantly associated with 0-18 week GWG in the ALSPAC study were significant in our study. The inconsistency in the results with the ALSPAC study with regards to the 0-18 week GWG model may arise for several reasons: sampling from different populations, dissimilar methylome coverage, sample size and/or false positive findings.

Two-dimensional condensation of nucleobases: A comparative study of halogen derivatives of cytosine

Czech Academy of Sciences Publication Activity Database

Fojt, Lukáš; Vetterl, Vladimír; Doneux, T.

2009-01-01

Roč. 74, 11-12 (2009), s. 1611-1622 ISSN 0010-0765 R&D Projects: GA AV ČR(CZ) KAN200040651; GA MŠk(CZ) LC06035; GA ČR(CZ) GA202/08/1688 Grant - others:GA MŠk(CZ) 1M0528 Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : hanging mercury drop electrode * cytosine * 2D condensation Subject RIV: BO - Biophysics Impact factor: 0.856, year: 2009

Normal breast tissue DNA methylation differences at regulatory elements are associated with the cancer risk factor age.

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Johnson, Kevin C; Houseman, E Andres; King, Jessica E; Christensen, Brock C

2017-07-10

The underlying biological mechanisms through which epidemiologically defined breast cancer risk factors contribute to disease risk remain poorly understood. Identification of the molecular changes associated with cancer risk factors in normal tissues may aid in determining the earliest events of carcinogenesis and informing cancer prevention strategies. Here we investigated the impact cancer risk factors have on the normal breast epigenome by analyzing DNA methylation genome-wide (Infinium 450 K array) in cancer-free women from the Susan G. Komen Tissue Bank (n = 100). We tested the relation of established breast cancer risk factors, age, body mass index, parity, and family history of disease, with DNA methylation adjusting for potential variation in cell-type proportions. We identified 787 cytosine-guanine dinucleotide (CpG) sites that demonstrated significant associations (Q value breast cancer risk factors. Age-related DNA methylation changes are primarily increases in methylation enriched at breast epithelial cell enhancer regions (P = 7.1E-20), and binding sites of chromatin remodelers (MYC and CTCF). We validated the age-related associations in two independent populations, using normal breast tissue samples (n = 18) and samples of normal tissue adjacent to tumor tissue (n = 97). The genomic regions classified as age-related were more likely to be regions altered in both pre-invasive (n = 40, P = 3.0E-03) and invasive breast tumors (n = 731, P = 1.1E-13). DNA methylation changes with age occur at regulatory regions, and are further exacerbated in cancer, suggesting that age influences breast cancer risk in part through its contribution to epigenetic dysregulation in normal breast tissue.

Why do results conflict regarding the prognostic value of the methylation status in colon cancers? the role of the preservation method

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Tournier Benjamin

2012-01-01

Full Text Available Abstract Background In colorectal carcinoma, extensive gene promoter hypermethylation is called the CpG island methylator phenotype (CIMP. Explaining why studies on CIMP and survival yield conflicting results is essential. Most experiments to measure DNA methylation rely on the sodium bisulfite conversion of unmethylated cytosines into uracils. No study has evaluated the performance of bisulfite conversion and methylation levels from matched cryo-preserved and Formalin-Fixed Paraffin Embedded (FFPE samples using pyrosequencing. Methods Couples of matched cryo-preserved and FFPE samples from 40 colon adenocarcinomas were analyzed. Rates of bisulfite conversion and levels of methylation of LINE-1, MLH1 and MGMT markers were measured. Results For the reproducibility of bisulfite conversion, the mean of bisulfite-to-bisulfite standard deviation (SD was 1.3%. The mean of run-to-run SD of PCR/pyrosequencing was 0.9%. Of the 40 DNA couples, only 67.5%, 55.0%, and 57.5% of FFPE DNA were interpretable for LINE-1, MLH1, and MGMT markers, respectively, after the first analysis. On frozen samples the proportion of well converted samples was 95.0%, 97.4% and 87.2% respectively. For DNA showing a total bisulfite conversion, 8 couples (27.6% for LINE-1, 4 couples (15.4% for MLH1 and 8 couples (25.8% for MGMT displayed significant differences in methylation levels. Conclusions Frozen samples gave reproducible results for bisulfite conversion and reliable methylation levels. FFPE samples gave unsatisfactory and non reproducible bisulfite conversions leading to random results for methylation levels. The use of FFPE collections to assess DNA methylation by bisulfite methods must not be recommended. This can partly explain the conflicting results on the prognosis of CIMP colon cancers.

CpG island methylator phenotype-low (CIMP-low) colorectal cancer shows not only few methylated CIMP-high-specific CpG islands, but also low-level methylation at individual loci.

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Kawasaki, Takako; Ohnishi, Mutsuko; Nosho, Katsuhiko; Suemoto, Yuko; Kirkner, Gregory J; Meyerhardt, Jeffrey A; Fuchs, Charles S; Ogino, Shuji

2008-03-01

The CpG island methylator phenotype (CIMP or CIMP-high) with widespread promoter methylation is a distinct phenotype in colorectal cancer. However, the concept of CIMP-low with less extensive CpG island methylation is still evolving. Our aim is to examine whether density of methylation in individual CpG islands was different between CIMP-low and CIMP-high tumors. Utilizing MethyLight technology and 889 population-based colorectal cancers, we quantified DNA methylation (methylation index, percentage of methylated reference) at 14 CpG islands, including 8 CIMP-high-specific loci (CACNA1G, CDKN2A (p16), CRABP1, IGF2, MLH1, NEUROG1, RUNX3 and SOCS1). Methylation positivity in each locus was defined as methylation index>4. Low-level methylation (methylation index>0, CIMP-high-specific locus was significantly more common in 340 CIMP-low tumors (1/8-5/8 methylation-positive loci) than 133 CIMP-high tumors (> or =6/8 methylation-positive loci) and 416 CIMP-0 tumors (0/8 methylation-positive loci) (PCIMP-high, low-level methylation, was not persistently more prevalent in CIMP-low tumors. In conclusion, compared to CIMP-high and CIMP-0 tumors, CIMP-low colorectal cancers show not only few methylated CIMP-high-specific CpG islands, but also more frequent low-level methylation at individual loci. Our data may provide supporting evidence for a difference in pathogenesis of DNA methylation between CIMP-low and CIMP-high tumors.

Targeted and genome-scale methylomics reveals gene body signatures in human cell lines

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Ball, Madeleine Price; Li, Jin Billy; Gao, Yuan; Lee, Je-Hyuk; LeProust, Emily; Park, In-Hyun; Xie, Bin; Daley, George Q.; Church, George M.

2012-01-01

Cytosine methylation, an epigenetic modification of DNA, is a target of growing interest for developing high throughput profiling technologies. Here we introduce two new, complementary techniques for cytosine methylation profiling utilizing next generation sequencing technology: bisulfite padlock probes (BSPPs) and methyl sensitive cut counting (MSCC). In the first method, we designed a set of ~10,000 BSPPs distributed over the ENCODE pilot project regions to take advantage of existing expression and chromatin immunoprecipitation data. We observed a pattern of low promoter methylation coupled with high gene body methylation in highly expressed genes. Using the second method, MSCC, we gathered genome-scale data for 1.4 million HpaII sites and confirmed that gene body methylation in highly expressed genes is a consistent phenomenon over the entire genome. Our observations highlight the usefulness of techniques which are not inherently or intentionally biased in favor of only profiling particular subsets like CpG islands or promoter regions. PMID:19329998

Protection against de novo methylation is instrumental in maintaining parent-of-origin methylation inherited from the gametes.

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Proudhon, Charlotte; Duffié, Rachel; Ajjan, Sophie; Cowley, Michael; Iranzo, Julian; Carbajosa, Guillermo; Saadeh, Heba; Holland, Michelle L; Oakey, Rebecca J; Rakyan, Vardhman K; Schulz, Reiner; Bourc'his, Déborah

2012-09-28

Identifying loci with parental differences in DNA methylation is key to unraveling parent-of-origin phenotypes. By conducting a MeDIP-Seq screen in maternal-methylation free postimplantation mouse embryos (Dnmt3L-/+), we demonstrate that maternal-specific methylation exists very scarcely at midgestation. We reveal two forms of oocyte-specific methylation inheritance: limited to preimplantation, or with longer duration, i.e. maternally imprinted loci. Transient and imprinted maternal germline DMRs (gDMRs) are indistinguishable in gametes and preimplantation embryos, however, de novo methylation of paternal alleles at implantation delineates their fates and acts as a major leveling factor of parent-inherited differences. We characterize two new imprinted gDMRs, at the Cdh15 and AK008011 loci, with tissue-specific imprinting loss, again by paternal methylation gain. Protection against demethylation after fertilization has been emphasized as instrumental in maintaining parent-of-origin methylation inherited from the gametes. Here we provide evidence that protection against de novo methylation acts as an equal major pivot, at implantation and throughout life. Copyright © 2012 Elsevier Inc. All rights reserved.

DNA methylation of the IGF2/H19 imprinting control region and adiposity distribution in young adults

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Huang Rae-Chi

2012-11-01

Full Text Available Abstract Background The insulin-like growth factor 2 (IGF2 and H19 imprinted genes control growth and body composition. Adverse in-utero environments have been associated with obesity-related diseases and linked with altered DNA methylation at the IGF2/H19 locus. Postnatally, methylation at the IGF2/H19 imprinting control region (ICR has been linked with cerebellum weight. We aimed to investigate whether decreased IGF2/H19 ICR methylation is associated with decreased birth and childhood anthropometry and increased contemporaneous adiposity. DNA methylation in peripheral blood (n = 315 at 17 years old was measured at 12 cytosine-phosphate-guanine sites (CpGs, analysed as Sequenom MassARRAY EpiTYPER units within the IGF2/H19 ICR. Birth size, childhood head circumference (HC at six time-points and anthropometry at age 17 years were measured. DNA methylation was investigated for its association with anthropometry using linear regression. Results The principal component of IGF2/H19 ICR DNA methylation (representing mean methylation across all CpG units positively correlated with skin fold thickness (at four CpG units (P-values between 0.04 to 0.001 and subcutaneous adiposity (P = 0.023 at age 17, but not with weight, height, BMI, waist circumference or visceral adiposity. IGF2/H19 methylation did not associate with birth weight, length or HC, but CpG unit 13 to 14 methylation was negatively associated with HC between 1 and 10 years. β-coefficients of four out of five remaining CpG units also estimated lower methylation with increasing childhood HC. Conclusions As greater IGF2/H19 methylation was associated with greater subcutaneous fat measures, but not overall, visceral or central adiposity, we hypothesize that obesogenic pressures in youth result in excess fat being preferentially stored in peripheral fat depots via the IGF2/H19 domain. Secondly, as IGF2/H19 methylation was not associated with birth size but negatively with early childhood HC, we

Reasons of carcinogenesis indicate a big-bang inside: a hypothesis for the aberration of DNA methylation.

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Roy, A; Roy Chattopadhyay, N

2013-07-01

Cancer involves various sets of altered gene functions which embrace all the three basic mechanisms of regulation of gene expression. However, no common mechanism is inferred till date for this versatile disease and thus no full proof remedy can be offered. Here we show that the basic mechanisms are interlinked and indicate towards one of those mechanisms as being the superior one; the methylation of cytosines in specific DNA sequences, for the initiation and maintenance of carcinogenesis. The analyses of the previous reports and the nucleotide sequences of the DNA methyltransferases strongly support the assumption that the mutation(s) in the DNA-binding site(s) of DNA-methyltransferases acts as a master regulator; though it continues the cycle from mutation to repair to methylation. We anticipate that our hypothesis will start a line of study for the proposal of a treatment regime for cancers by introducing wild type methyltransferases in the diseased cells and/or germ cells, and/or by targeting ligands to the altered binding domain(s) where a mutation in the concerned enzyme(s) is seen. Copyright © 2013. Published by Elsevier Ltd.

Bridging the gap between protein carboxyl methylation and phospholipid methylation to understand glucose-stimulated insulin secretion from the pancreatic beta cell.

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Kowluru, Anjaneyulu

2008-01-15

Recent findings have implicated post-translational modifications at C-terminal cysteines [e.g., methylation] of specific proteins [e.g., G-proteins] in glucose-stimulated insulin secretion [GSIS]. Furthermore, methylation at the C-terminal leucine of the catalytic subunit of protein phosphatase 2A [PP2Ac] has also been shown to be relevant for GSIS. In addition to these two classes of protein methyl transferases, a novel class of glucose-activated phospholipid methyl transferases have also been identified in the beta cell. These enzymes catalyze three successive methylations of phosphatidylethanolamine to yield phosphatidylcholine. The "newly formed" phosphatidylcholine is felt to induce alterations in the membrane fluidity, which might favor vesicular fusion with the plasma membrane for the exocytosis of insulin. The objectives of this commentary are to: (i) review the existing evidence on the regulation, by glucose and other insulin secretagogues, of post-translational carboxylmethylation [CML] of specific proteins in the beta cell; (ii) discuss the experimental evidence, which implicates regulation, by glucose and other insulin secretagogues, of phosphatidylethanolamine methylation in the islet beta cell; (iii) propose a model for potential cross-talk between the protein and lipid methylation pathways in the regulation of GSIS and (iv) highlight potential avenues for future research, including the development of specific pharmacological inhibitors to further decipher regulatory roles for these methylation reactions in islet beta cell function.

Aberrant TET1 Methylation Closely Associated with CpG Island Methylator Phenotype in Colorectal Cancer.

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Ichimura, Norihisa; Shinjo, Keiko; An, Byonggu; Shimizu, Yasuhiro; Yamao, Kenji; Ohka, Fumiharu; Katsushima, Keisuke; Hatanaka, Akira; Tojo, Masayuki; Yamamoto, Eiichiro; Suzuki, Hiromu; Ueda, Minoru; Kondo, Yutaka

2015-08-01

Inactivation of methylcytosine dioxygenase, ten-eleven translocation (TET) is known to be associated with aberrant DNA methylation in cancers. Tumors with a CpG island methylator phenotype (CIMP), a distinct subgroup with extensive DNA methylation, show characteristic features in the case of colorectal cancer. The relationship between TET inactivation and CIMP in colorectal cancers is not well understood. The expression level of TET family genes was compared between CIMP-positive (CIMP-P) and CIMP-negative (CIMP-N) colorectal cancers. Furthermore, DNA methylation profiling, including assessment of the TET1 gene, was assessed in colorectal cancers, as well as colon polyps. The TET1 was silenced by DNA methylation in a subset of colorectal cancers as well as cell lines, expression of which was reactivated by demethylating agent. TET1 methylation was more frequent in CIMP-P (23/55, 42%) than CIMP-N (2/113, 2%, P CIMP-P, 16/40, 40%; CIMP-N, 2/24, 8%; P = 0.002), suggesting that TET1 methylation is an early event in CIMP tumorigenesis. TET1 methylation was significantly associated with BRAF mutation but not with hMLH1 methylation in the CIMP-P colorectal cancers. Colorectal cancers with TET1 methylation have a significantly greater number of DNA methylated genes and less pathological metastasis compared to those without TET1 methylation (P = 0.007 and 0.045, respectively). Our data suggest that TET1 methylation may contribute to the establishment of a unique pathway in respect to CIMP-mediated tumorigenesis, which may be incidental to hMLH1 methylation. In addition, our findings provide evidence that TET1 methylation may be a good biomarker for the prediction of metastasis in colorectal cancer. ©2015 American Association for Cancer Research.

Automated quantum chemistry based molecular dynamics simulations of electron ionization induced fragmentations of the nucleobases Uracil, Thymine, Cytosine, and Guanine.

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Grimme, Stefan; Bauer, Christopher Alexander

2015-01-01

The gas-phase decomposition pathways of electron ionization (EI)-induced radical cations of the nucleobases uracil, thymine, cytosine, and guanine are investigated by means of mixed quantum-classical molecular dynamics. No preconceived fragmentation channels are used in the calculations. The results compare well to a plethora of experimental and theoretical data for these important biomolecules. With our combined stochastic and dynamic approach, one can access in an unbiased way the energetically available decomposition mechanisms. Additionally, we are able to separate the EI mass spectra of different tautomers of cytosine and guanine. Our method (previously termed quantum chemistry electron ionization mass spectra) reproduces free nucleobase experimental mass spectra well and provides detailed mechanistic in-sight into high-energy unimolecular decomposition processes.

High resolution melt curve analysis based on methylation status for human semen identification.

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Fachet, Caitlyn; Quarino, Lawrence; Karnas, K Joy

2017-03-01

A high resolution melt curve assay to differentiate semen from blood, saliva, urine, and vaginal fluid based on methylation status at the Dapper Isoform 1 (DACT1) gene was developed. Stains made from blood, saliva, urine, semen, and vaginal fluid were obtained from volunteers and DNA was isolated using either organic extraction (saliva, urine, and vaginal fluid) or Chelex ® 100 extraction (blood and semen). Extracts were then subjected to bisulfite modification in order to convert unmethylated cytosines to uracil, consequently creating sequences whose amplicons have melt curves that vary depending on their initial methylation status. When primers designed to amplify the promoter region of the DACT1 gene were used, DNA from semen samples was distinguishable from other fluids by a having a statistically significant lower melting temperature. The assay was found to be sperm-significant since semen from a vasectomized man produced a melting temperature similar to the non-semen body fluids. Blood and semen stains stored up to 5 months and tested at various intervals showed little variation in melt temperature indicating the methylation status was stable during the course of the study. The assay is a more viable method for forensic science practice than most molecular-based methods for body fluid stain identification since it is time efficient and utilizes instrumentation common to forensic biology laboratories. In addition, the assay is advantageous over traditional presumptive chemical methods for body fluid identification since results are confirmatory and the assay offers the possibility of multiplexing which may test for multiple body fluids simultaneously.

DNA (cytosine-5-methyltransferase 3B (DNMT 3B polymorphism and risk of Down syndrome offspring

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Cláudia Melo de Moura

2018-01-01

Full Text Available Down syndrome (DS is the most common form of human genetic mental retardation. Several polymorphisms in genes coding folic acid cycle enzymes have been associated to the risk of bearing a DS child; however, the results are controversial. S-adenosyl-l-methionine (SAM is an important intermediate of folic acid pathway and acts as methyl donor and substrate for DNA (cytosine-5-methyltransferase 3B (DNMT3B – EC 2.1.1.37 de novo methylation processes during embryogenesis. Recent studies suggest that a functional polymorphism of DNMT 3B in maternal genotype may be associated with a decreased risk of having a DS child. We herein investigate the association of this polymorphism with the occurrence of DS in a Brazilian population. We have genotyped 111 mothers of DS infants (MDS and 212 control mothers (CM through PCR-RFLP. The observed genotypic frequencies were CC = 0.22; CT = 0.49 and TT = 0.29 in CM, and CC = 0.30; CT = 0.52 and TT = 0.18 in MDS. Allelic frequencies were C = 0.47 and T = 0.53 in CM and C = 0.56 and T = 0.44 in MDS. No deviation of HWE was observed, and both DNMT 3B rs2424913 genotype (χ2 = 4.53; DF = 1; P = 0.03 and allelic (χ2 = 4.90; DF = 1; P = 0.03 frequencies show significant differences between MDS and CM. The presence of the mutant DNMT 3B T allele decreases 30% the risk of bearing a DS child (OR = 0.69; 95% CI: 0.50–0.96; P = 0.03, and the risk is diminished up to 45% in association with the homozygous genotype (OR = 0.54; 95% CI: 0.31–0.96; P = 0.04. Our results suggest that women harboring the single nucleotide polymorphism DNMT 3B rs2424913 have a decreased risk of a DS pregnancy, and further studies are necessary to confirm this protective effect.

Aberrant regulation of DNA methylation in amyotrophic lateral sclerosis: a new target of disease mechanisms.

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Martin, Lee J; Wong, Margaret

2013-10-01

Amyotrophic lateral sclerosis (ALS) is the third most common adult-onset neurodegenerative disease. A diagnosis is fatal owing to degeneration of motor neurons in brain and spinal cord that control swallowing, breathing, and movement. ALS can be inherited, but most cases are not associated with a family history of the disease. The mechanisms causing motor neuron death in ALS are still unknown. Given the suspected complex interplay between multiple genes, the environment, metabolism, and lifestyle in the pathogenesis of ALS, we have hypothesized that the mechanisms of disease in ALS involve epigenetic contributions that can drive motor neuron degeneration. DNA methylation is an epigenetic mechanism for gene regulation engaged by DNA methyltransferase (Dnmt)-catalyzed methyl group transfer to carbon-5 in cytosine residues in gene regulatory promoter and nonpromoter regions. Recent genome-wide analyses have found differential gene methylation in human ALS. Neuropathologic assessments have revealed that motor neurons in human ALS show significant abnormalities in Dnmt1, Dnmt3a, and 5-methylcytosine. Similar changes are seen in mice with motor neuron degeneration, and Dnmt3a was found abundantly at synapses and in mitochondria. During apoptosis of cultured motor neuron-like cells, Dnmt1 and Dnmt3a protein levels increase, and 5-methylcytosine accumulates. Enforced expression of Dnmt3a, but not Dnmt1, induces degeneration of cultured neurons. Truncation mutation of the Dnmt3a catalytic domain and Dnmt3a RNAi blocks apoptosis of cultured neurons. Inhibition of Dnmt catalytic activity with small molecules RG108 and procainamide protects motor neurons from excessive DNA methylation and apoptosis in cell culture and in a mouse model of ALS. Thus, motor neurons can engage epigenetic mechanisms to cause their degeneration, involving Dnmts and increased DNA methylation. Aberrant DNA methylation in vulnerable cells is a new direction for discovering mechanisms of ALS

Cross-Talk between Dnmt2-Dependent tRNA Methylation and Queuosine Modification

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Ann E. Ehrenhofer-Murray

2017-02-01

Full Text Available Enzymes of the Dnmt2 family of methyltransferases have yielded a number of unexpected discoveries. The first surprise came more than ten years ago when it was realized that, rather than being DNA methyltransferases, Dnmt2 enzymes actually are transfer RNA (tRNA methyltransferases for cytosine-5 methylation, foremost C38 (m5C38 of tRNAAsp. The second unanticipated finding was our recent discovery of a nutritional regulation of Dnmt2 in the fission yeast Schizosaccharomyces pombe. Significantly, the presence of the nucleotide queuosine in tRNAAsp strongly stimulates Dnmt2 activity both in vivo and in vitro in S. pombe. Queuine, the respective base, is a hypermodified guanine analog that is synthesized from guanosine-5’-triphosphate (GTP by bacteria. Interestingly, most eukaryotes have queuosine in their tRNA. However, they cannot synthesize it themselves, but rather salvage it from food or from gut microbes. The queuine obtained from these sources comes from the breakdown of tRNAs, where the queuine ultimately was synthesized by bacteria. Queuine thus has been termed a micronutrient. This review summarizes the current knowledge of Dnmt2 methylation and queuosine modification with respect to translation as well as the organismal consequences of the absence of these modifications. Models for the functional cooperation between these modifications and its wider implications are discussed.

Adolescent binge-pattern alcohol exposure alters genome-wide DNA methylation patterns in the hypothalamus of alcohol-naïve male offspring.

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Asimes, AnnaDorothea; Torcaso, Audrey; Pinceti, Elena; Kim, Chun K; Zeleznik-Le, Nancy J; Pak, Toni R

2017-05-01

Teenage binge drinking is a major health concern in the United States, with 21% of teenagers reporting binge-pattern drinking behavior in the previous 30 days. Recently, our lab showed that alcohol-naïve offspring of rats exposed to alcohol during adolescence exhibited altered gene expression profiles in the hypothalamus, a brain region involved in stress regulation. We employed Enhanced Reduced Representation Bisulfite Sequencing as an unbiased approach to test the hypothesis that parental exposure to binge-pattern alcohol during adolescence alters DNA methylation profiles in their alcohol-naïve offspring. Wistar rats were administered a repeated binge-ethanol exposure paradigm during early (postnatal day (PND) 37-44) and late (PND 67-74) adolescent development. Animals were mated 24 h after the last ethanol dose and subsequent offspring were produced. Analysis of male PND7 offspring revealed that offspring of alcohol-exposed parents exhibited differential DNA methylation patterns in the hypothalamus. The differentially methylated cytosines (DMCs) were distinct between offspring depending on which parent was exposed to ethanol. Moreover, novel DMCs were observed when both parents were exposed to ethanol and many DMCs from single parent ethanol exposure were not recapitulated with dual parent exposure. We also measured mRNA expression of several differentially methylated genes and some, but not all, showed correlative changes in expression. Importantly, methylation was not a direct predictor of expression levels, underscoring the complexity of transcriptional regulation. Overall, we demonstrate that adolescent binge ethanol exposure causes altered genome-wide DNA methylation patterns in the hypothalamus of alcohol-naïve offspring. Copyright © 2016 Elsevier Inc. All rights reserved.

Supramolecular Switches Based on the Guanine–Cytosine (GC) Watson–Crick Pair: Effect of Neutral and Ionic Substituents

NARCIS (Netherlands)

Guerra, C.F.; van der Wijst, T.; Bickelhaupt, F.M.

2006-01-01

We have theoretically analyzed Watson–Crick guanine–cytosine (GC) base pairs in which purine-C8 and/or pyrimidine-C6 positions carry a substituent X = NH−, NH2, NH3+ (N series), O−, OH, or OH2+ (O series), using the generalized gradient approximation (GGA) of density functional theory at the

Quantitative analysis of the experimental cytotoxic drug cyclopentenyl cytosine and its metabolite in plasma with HPLC tandem mass spectrometry

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Schimmel, Kirsten; van Lenthe, Henk; Leen, Rene; Kulik, Willem; Verschuur, Arnauld; Guchelaar, Henk-Jan; van Kuilenburg, André

2008-01-01

The cytotoxic drug cyclopentenyl cytosine (CPEC) is currently being investigated in early clinical trials. Monitoring of plasma levels is required for pharmacokinetic analysis and management of toxicity. This paper describes the analysis of CPEC and cyclopentenyl uracil (CPEU) in plasma by

MeCP2 Expression and Promoter Methylation of Cyclin D1 Gene Are Associated with Cyclin D1 Expression in Developing Rat Epididymal Duct

International Nuclear Information System (INIS)

Darwanto, Agus; Kitazawa, Riko; Mori, Kiyoshi; Kondo, Takeshi; Kitazawa, Sohei

2008-01-01

Hypermethylation-dependent silencing of the gene is achieved by recruiting methyl-CpG binding proteins (MeCPs). Among the MeCPs, MeCP2 is the most abundantly and ubiquitously expressed in various types of cells. We first screened the distribution and expression pattern of MeCP2 in adult and developing rat tissues and found strong MeCP2 expression, albeit rather ubiquitously among normal tissues, in ganglion cells and intestinal epithelium in the small intestine, in Purkinje cells and neurons in the brain, in spermatogonia and in epithelial cells in the epididymal duct of the testis. We then assessed the expression and the methylation pattern of the promoter region of cyclin D1 by immunohistochemistry and sodium bisulfite mapping, and found that cyclin D1 expression in the epididymal duct decreased rapidly during rat development: strong in newborn rats and very weak or almost negative in 7-day-old rats. Mirroring the decrease of cyclin D1 expression, methylated cytosine at both CpG and non-CpG loci in the cyclin D1 promoter was frequently observed in the epididymal duct of 7-day-old rats but not in that of newborn rats. Interestingly, MeCP2 expression also increased concomitant with the increase of methylation. Cyclin D1 expression in the epididymal duct may be efficiently regulated by the epigenetic mechanism of the cooperative increase of MeCP2 expression and promoter methylation

DNA methylation changes in Mexican children exposed to arsenic from two historic mining areas in San Luis potosí.

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Alegría-Torres, Jorge Alejandro; Carrizales-Yánez, Leticia; Díaz-Barriga, Fernando; Rosso-Camacho, Fernando; Motta, Valeria; Tarantini, Letizia; Bollati, Valentina

2016-12-01

Arsenic is a carcinogen and epimutagen that threatens the health of exposed populations worldwide. In this study, we examined the methylation status of Alu and long interspersed nucleotide elements (LINE-1) and their association with levels of urinary arsenic in 84 Mexican children between 6 and 12 years old from two historic mining areas in the State of San Luis Potosí, Mexico. Urinary arsenic levels were determined by atomic absorption spectrophotometry and DNA methylation analysis was performed in peripheral blood leukocytes by bisulfite-pyrosequencing. The geometric mean of urinary arsenic was 26.44 µg/g Cr (range 1.93-139.35). No significant differences in urinary arsenic or methylation patterns due to gender were observed. A positive correlation was found between urinary arsenic and the mean percentage of methylated cytosines in Alu sequences (Spearman correlation coefficient r = 0.532, P < 0.001), and a trend of LINE-1 hypomethylation was also observed (Spearman correlation coefficient r = -0.232, P = 0.038) after adjustment for sex and age. A linear regression model showed an association with log-normalized urinary arsenic for Alu (β = 1.05, 95% CI: 0.67; 1.43, P < 0.001) and LINE-1 (β = -0.703, 95% CI: -1.36; -0.38, P = 0.038). Despite the low-level arsenic exposure, a subtle epigenetic imbalance measured as DNA methylation was detected in the leukocytes of Mexican children living in two historic mining areas. Environ. Mol. Mutagen. 57:717-723, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Transcription factors as readers and effectors of DNA methylation.

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Zhu, Heng; Wang, Guohua; Qian, Jiang

2016-08-01

Recent technological advances have made it possible to decode DNA methylomes at single-base-pair resolution under various physiological conditions. Many aberrant or differentially methylated sites have been discovered, but the mechanisms by which changes in DNA methylation lead to observed phenotypes, such as cancer, remain elusive. The classical view of methylation-mediated protein-DNA interactions is that only proteins with a methyl-CpG binding domain (MBD) can interact with methylated DNA. However, evidence is emerging to suggest that transcription factors lacking a MBD can also interact with methylated DNA. The identification of these proteins and the elucidation of their characteristics and the biological consequences of methylation-dependent transcription factor-DNA interactions are important stepping stones towards a mechanistic understanding of methylation-mediated biological processes, which have crucial implications for human development and disease.

Evolution of DNA Methylation across Insects.

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Bewick, Adam J; Vogel, Kevin J; Moore, Allen J; Schmitz, Robert J

2017-03-01

DNA methylation contributes to gene and transcriptional regulation in eukaryotes, and therefore has been hypothesized to facilitate the evolution of plastic traits such as sociality in insects. However, DNA methylation is sparsely studied in insects. Therefore, we documented patterns of DNA methylation across a wide diversity of insects. We predicted that underlying enzymatic machinery is concordant with patterns of DNA methylation. Finally, given the suggestion that DNA methylation facilitated social evolution in Hymenoptera, we tested the hypothesis that the DNA methylation system will be associated with presence/absence of sociality among other insect orders. We found DNA methylation to be widespread, detected in all orders examined except Diptera (flies). Whole genome bisulfite sequencing showed that orders differed in levels of DNA methylation. Hymenopteran (ants, bees, wasps and sawflies) had some of the lowest levels, including several potential losses. Blattodea (cockroaches and termites) show all possible patterns, including a potential loss of DNA methylation in a eusocial species whereas solitary species had the highest levels. Species with DNA methylation do not always possess the typical enzymatic machinery. We identified a gene duplication event in the maintenance DNA methyltransferase 1 (DNMT1) that is shared by some Hymenoptera, and paralogs have experienced divergent, nonneutral evolution. This diversity and nonneutral evolution of underlying machinery suggests alternative DNA methylation pathways may exist. Phylogenetically corrected comparisons revealed no evidence that supports evolutionary association between sociality and DNA methylation. Future functional studies will be required to advance our understanding of DNA methylation in insects. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Oxytocin receptor gene methylation: converging multilevel evidence for a role in social anxiety.

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Ziegler, Christiane; Dannlowski, Udo; Bräuer, David; Stevens, Stephan; Laeger, Inga; Wittmann, Hannah; Kugel, Harald; Dobel, Christian; Hurlemann, René; Reif, Andreas; Lesch, Klaus-Peter; Heindel, Walter; Kirschbaum, Clemens; Arolt, Volker; Gerlach, Alexander L; Hoyer, Jürgen; Deckert, Jürgen; Zwanzger, Peter; Domschke, Katharina

2015-05-01

Social anxiety disorder (SAD) is a commonly occurring and highly disabling disorder. The neuropeptide oxytocin and its receptor (OXTR) have been implicated in social cognition and behavior. This study-for the first time applying a multilevel epigenetic approach-investigates the role of OXTR gene methylation in categorical, dimensional, and intermediate neuroendocrinological/neural network phenotypes of social anxiety. A total of 110 unmedicated patients with SAD and matched 110 controls were analyzed for OXTR methylation by direct sequencing of sodium bisulfite-converted DNA extracted from whole blood. Furthermore, OXTR methylation was investigated regarding SAD-related traits (Social Phobia Scale (SPS) and Social Interaction Anxiety Scale (SIAS)), salivary cortisol response during the Trier social stress test (TSST), and amygdala responsiveness to social phobia related verbal stimuli using fMRI. Significantly decreased OXTR methylation particularly at CpG Chr3: 8 809 437 was associated with (1) the categorical phenotype of SAD (psocial phobia-related word processing (right: p(corr)<0.001; left: p(corr)=0.005). Assuming that decreased OXTR methylation confers increased OXTR expression, the present finding may reflect a compensatory upregulation for pathologically reduced oxytocin levels or a causally relevant increased OXTR activation in SAD and related traits. OXTR methylation patterns might thus serve as peripheral surrogates of oxytocin tone and aid in establishing accessible biomarkers of SAD risk allowing for indicated preventive interventions and personalized treatment approaches targeting the oxytocin system.

DNA methylation signatures in cord blood associated with maternal gestational weight gain: results from the ALSPAC cohort.

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Morales, Eva; Groom, Alexandra; Lawlor, Debbie A; Relton, Caroline L

2014-05-02

Epigenetic changes could mediate the association of maternal pre-pregnancy body mass index (BMI) and gestational weight gain (GWG) with adverse offspring outcomes. However, studies in humans are lacking. Here, we examined the association of maternal pre-pregnancy BMI and GWG in different periods of pregnancy with cytosine-guanine (CpG) dinucleotide site methylation differences in newborn cord blood DNA from 88 participants in the Avon Longitudinal Study of Parents and Children (ALSPAC) cohort using the Illumina GoldenGate Panel I. Pyrosequencing was used for validation of the top associated locus and for replication in 170 non-overlapping mother-offspring pairs from the ALSPAC cohort. After correction for multiple testing greater GWG in early pregnancy (between 0 to 18 weeks of gestation) was associated with increased DNA methylation levels in four CpG sites at MMP7, KCNK4, TRPM5 and NFKB1 genes (difference in methylation >5% per 400 g/week greater GWG) (q values 0.023 -0.065). Pre-pregnancy BMI and GWG in mid- or late pregnancy were not associated with differential DNA methylation at any CpG site. Pyrosequencing showed that greater GWG in early pregnancy was associated with increased DNA methylation levels at the top associated CpG site at MMP7, although association did not reach statistical significance (p = 0.302). Greater GWG in mid- (p = 0.167) and late-pregnancy (p = 0.037) were also associated with increased DNA methylation levels at the MMP7 CpG site. In addition, newborns of mothers who exceeded the IoM-recommended GWG had higher DNA methylation levels at the MMP7 CpG site than those of mothers with IoM-recommended GWG (p = 0.080). We failed to replicate findings. Greater GWG in early pregnancy was associated with increased methylation at CpG sites at MMP7, KCNK4, TRPM5 and NFKB1 genes in offspring cord blood DNA. The specific association of GWG in early pregnancy with the top associated CpG site at MMP7 was not validated using

Genome-wide methylation analysis identifies differentially methylated CpG loci associated with severe obesity in childhood.

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Huang, R C; Garratt, E S; Pan, H; Wu, Y; Davis, E A; Barton, S J; Burdge, G C; Godfrey, K M; Holbrook, J D; Lillycrop, K A

2015-01-01

Childhood obesity is a major public health issue. Here we investigated whether differential DNA methylation was associated with childhood obesity. We studied DNA methylation profiles in whole blood from 78 obese children (mean BMI Z-score: 2.6) and 71 age- and sex-matched controls (mean BMI Z-score: 0.1). DNA samples from obese and control groups were pooled and analyzed using the Infinium HumanMethylation450 BeadChip array. Comparison of the methylation profiles between obese and control subjects revealed 129 differentially methylated CpG (DMCpG) loci associated with 80 unique genes that had a greater than 10% difference in methylation (P-value obesity were validated using sodium bisulfite pyrosequencing across loci within the FYN, PIWIL4, and TAOK3 genes in individual subjects. Three CpG loci within FYN were hypermethylated in obese individuals (all P obesity was associated with lower methylation of CpG loci within PIWIL4 (P = 0.003) and TAOK3 (P = 0.001). After building logistic regression models, we determined that a 1% increase in methylation in TAOK3, multiplicatively decreased the odds of being obese by 0.91 (95% CI: 0.86 - 0.97), and an increase of 1% methylation in FYN CpG3, multiplicatively increased the odds of being obese by 1.03 (95% CI: 0.99 - 1.07). In conclusion, these findings provide evidence that childhood obesity is associated with specific DNA methylation changes in whole blood, which may have utility as biomarkers of obesity risk.

ESR1 gene promoter region methylation in free circulating DNA and its correlation with estrogen receptor protein expression in tumor tissue in breast cancer patients

International Nuclear Information System (INIS)

Martínez-Galán, Joaquina; Ríos, Sandra; Delgado, Juan Ramón; Torres-Torres, Blanca; Núñez, María Isabel; López-Peñalver, Jesús; Del Moral, Rosario; Ruiz De Almodóvar, José Mariano; Menjón, Salomón; Concha, Ángel; Chamorro, Clara

2014-01-01

Tumor expression of estrogen receptor (ER) is an important marker of prognosis, and is predictive of response to endocrine therapy in breast cancer. Several studies have observed that epigenetic events, such methylation of cytosines and deacetylation of histones, are involved in the complex mechanisms that regulate promoter transcription. However, the exact interplay of these factors in transcription activity is not well understood. In this study, we explored the relationship between ER expression status in tumor tissue samples and the methylation of the 5′ CpG promoter region of the estrogen receptor gene (ESR1) isolated from free circulating DNA (fcDNA) in plasma samples from breast cancer patients. Patients (n = 110) with non-metastatic breast cancer had analyses performed of ER expression (luminal phenotype in tumor tissue, by immunohistochemistry method), and the ESR1-DNA methylation status (fcDNA in plasma, by quantitative methylation specific PCR technique). Our results showed a significant association between presence of methylated ESR1 in patients with breast cancer and ER negative status in the tumor tissue (p = 0.0179). There was a trend towards a higher probability of ESR1-methylation in those phenotypes with poor prognosis i.e. 80% of triple negative patients, 60% of HER2 patients, compared to 28% and 5.9% of patients with better prognosis such as luminal A and luminal B, respectively. Silencing, by methylation, of the promoter region of the ESR1 affects the expression of the estrogen receptor protein in tumors of breast cancer patients; high methylation of ESR1-DNA is associated with estrogen receptor negative status which, in turn, may be implicated in the patient’s resistance to hormonal treatment in breast cancer. As such, epigenetic markers in plasma may be of interest as new targets for anticancer therapy, especially with respect to endocrine treatment

[Association of etheno-DNA adduct and DNA methylation level among workers exposed to diesel engine exhaust].

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Shen, M L; He, Z N; Zhang, X; Duan, H W; Niu, Y; Bin, P; Ye, M; Meng, T; Dai, Y F; Yu, S F; Chen, W; Zheng, Y X

2017-06-06

Objective: To investigate the association between etheno-DNA adduct and the promoter of DNA methylation levels of cyclin dependent kinase inhibitor 2A (P16), Ras association domain family 1 (RASSF1A) and O-6-methylguanine-DNA methyltransferase (MGMT) in workers with occupational exposure to diesel engine exhaust (DEE). Methods: We recruited 124 diesel engine testing workers as DEE exposure group and 112 water pump operator in the same area as control group in Henan province in 2012 using cluster sampling. The demographic data were obtained by questionnaire survey; urine after work and venous blood samples were collected from each subject. The urinary etheno-DNA adducts were detected using UPLC-MS/MS, including 1,N6-etheno-2'-deoxyadenosine (εdA) and 3,N4-etheno-2'-deoxycytidine(εdC). The DNA methylation levels of P16, RASSF1A, and MGMT were evaluated using bisulfite-pyrosequencing assay. The percentage of methylation was expressed as the 5-methylcytosine (5mC) over the sum of cytosines (%5mC). Spearman correlation and multiple linear regression were applied to analyze the association between etheno-DNA adducts and DNA methylation of P16, RASSF1A, and MGMT. Results: The median ( P (25)- P (75)) of urinary εdA level was 230.00 (98.04-470.91) pmol/g creatinine in DEE exposure group, and 102.10 (49.95-194.48) creatinine in control group. The level of εdA was higher in DEE exposure group than control group ( P 0.05) . Multiple linear regression confirmed the negative correlation between εdA and DNA methylation levels of P16, RASSF1A, and MGMT in non-smoking group (β (95 %CI ) was -0.068 (-0.132--0.003), -0.082 (-0.159--0.004) and -0.048 (-0.090--0.007), P values were 0.039, 0.039 and 0.024, respectively). Moreover, εdC was negative associated with DNA methylation level of MGMT in non-smoking group (β (95 %CI ) was -0.094 (-0.179--0.008), P= 0.032). Conclusion: DEE exposure could induce the increased of εdA and decreased of DNA methylation levels of P16, RASSF1A

Labelling of the thymidine and deoxyctidine bases of DNA by (2-14C) deoxycytidine in cultured L1210 cells

International Nuclear Information System (INIS)

Karle, J.M.; Hoeraut, R.M.; Cysyk, R.L.

1983-01-01

Exposure of cultured L1210 cells to (2- 14 C) deoxycytidine and analysis of radioactivity incorporated into DNA-pyrimidines revealed that 2.7-5.5-fold more radioactivity is incorporated into DNA-thymine than into cytosine bases. Thus, the pathway involving deamination of deoxycytidylate to deoxyuridylate and methylation to thymidylate is highly favoured over successive phosphorlation to dCTP. Several modified and endogenous pyrimidines altered the labelling of DNA-thymine and DNA-cytosine with (2- 14 C)-deoxycytidine. 3-deazauridine at 0.1 mM caused a 56% increase in the labelling of DNA-thymine. Both thymidine and 3-deazauridine (>=10 μM) increased the specific activity to DNA-cytosine by 4-fold. Cytosine arabinoside (ara-C) (>= 10 μM) reduced the labelling of both DNA-cytosine and DNA-thymine. Excess cytidine (0.1 mM) reduced the labelling of DNA-cytosine by 40%. Tetrahydrouridine at concentrations up to 1 mM had no effect. (author)

Prognostic DNA Methylation Markers for Prostate Cancer

Directory of Open Access Journals (Sweden)

Siri H. Strand

2014-09-01

Full Text Available Prostate cancer (PC is the most commonly diagnosed neoplasm and the third most common cause of cancer-related death amongst men in the Western world. PC is a clinically highly heterogeneous disease, and distinction between aggressive and indolent disease is a major challenge for the management of PC. Currently, no biomarkers or prognostic tools are able to accurately predict tumor progression at the time of diagnosis. Thus, improved biomarkers for PC prognosis are urgently needed. This review focuses on the prognostic potential of DNA methylation biomarkers for PC. Epigenetic changes are hallmarks of PC and associated with malignant initiation as well as tumor progression. Moreover, DNA methylation is the most frequently studied epigenetic alteration in PC, and the prognostic potential of DNA methylation markers for PC has been demonstrated in multiple studies. The most promising methylation marker candidates identified so far include PITX2, C1orf114 (CCDC181 and the GABRE~miR-452~miR-224 locus, in addition to the three-gene signature AOX1/C1orf114/HAPLN3. Several other biomarker candidates have also been investigated, but with less stringent clinical validation and/or conflicting evidence regarding their possible prognostic value available at this time. Here, we review the current evidence for the prognostic potential of DNA methylation markers in PC.

Possible Involvement of Hydrosulfide in B12-Dependent Methyl Group Transfer

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John I. Toohey

2017-04-01

Full Text Available Evidence from several fields of investigation lead to the hypothesis that the sulfur atom is involved in vitamin B12-dependent methyl group transfer. To compile the evidence, it is necessary to briefly review the following fields: methylation, the new field of sulfane sulfur/hydrogen sulfide (S°/H2S, hydrosulfide derivatives of cobalamins, autoxidation of hydrosulfide radical, radical S-adenosylmethionine methyl transfer (RSMT, and methionine synthase (MS. Then, new reaction mechanisms for B12-dependent methyl group transfer are proposed; the mechanisms are facile and overcome difficulties that existed in previously-accepted mechanisms. Finally, the theory is applied to the effect of S°/H2S in nerve tissue involving the “hypomethylation theory” that was proposed 50 years ago to explain the neuropathology resulting from deficiency of vitamin B12 or folic acid. The conclusions are consistent with emerging evidence that sulfane sulfur/hydrogen sulfide may be beneficial in treating Alzheimer’s disease.

Methyl-Analyzer--whole genome DNA methylation profiling.

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Xin, Yurong; Ge, Yongchao; Haghighi, Fatemeh G

2011-08-15

Methyl-Analyzer is a python package that analyzes genome-wide DNA methylation data produced by the Methyl-MAPS (methylation mapping analysis by paired-end sequencing) method. Methyl-MAPS is an enzymatic-based method that uses both methylation-sensitive and -dependent enzymes covering >80% of CpG dinucleotides within mammalian genomes. It combines enzymatic-based approaches with high-throughput next-generation sequencing technology to provide whole genome DNA methylation profiles. Methyl-Analyzer processes and integrates sequencing reads from methylated and unmethylated compartments and estimates CpG methylation probabilities at single base resolution. Methyl-Analyzer is available at http://github.com/epigenomics/methylmaps. Sample dataset is available for download at http://epigenomicspub.columbia.edu/methylanalyzer_data.html. fgh3@columbia.edu Supplementary data are available at Bioinformatics online.

Modification of the cerebral perfusion during a chemotherapy by arabinoside cytosine (A.R.A.C.) among patients suffering of an acute myelo-blastic leukemia (A.M.L.); Modification de la perfusion cerebrale au cours d'une chimiotherapie par cytosine arabinoside (ARAC) chez les patients atteints d'une leucemie aigue myeloblastique (LAM)

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Modzelewski, R.; Vera, P. [Universite de Medecine de Rouen, QUANT.I.F-LITIS EA4108, departement de medecine nucleaire, 76 (France); Lepretre, S.; Tilly, H. [Centre Henri-Becquerel, departement d' hematologie, 76 - Rouen (France); Martinaud, O.; Hannequin, D. [CHU de Rouen, departement de neurologie, 76 (France); Habert, M.O. [CHU de la Pitie-Salpetriere, departement de medecine nucleaire, 75 - Paris (France)

2010-07-01

Cytosine arabinoside in high doses is a major treatment in acute myelo-blastic leukemia (A.M.L.). This treatment leads to neurological complications in 3-16% of cases, but the EEG, CT or MRI are normal.This prospective study examines brain perfusion in single photon emission tomography (SPECT) for patients receiving high dose arabinoside cytosine (H.D. A.R.A.C.). The SPECT of perfusion with hexamethyl propylene amine oxime (H.M.P.A.O.) for patients suffering of A.M.L. allowed to show a reduction of perfusion at the cerebellum level, of the occipito-parietal cortex and thalami, after conventional doses of A.R.A.C., while the patients had not any neurological accidents. (N.C.)

Structure-wise discrimination of cytosine, thymine, and uracil by proteins in terms of their nonbonded interactions.

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Usha, S; Selvaraj, S

2014-01-01

The molecular recognition and discrimination of very similar ligand moieties by proteins are important subjects in protein-ligand interaction studies. Specificity in the recognition of molecules is determined by the arrangement of protein and ligand atoms in space. The three pyrimidine bases, viz. cytosine, thymine, and uracil, are structurally similar, but the proteins that bind to them are able to discriminate them and form interactions. Since nonbonded interactions are responsible for molecular recognition processes in biological systems, our work attempts to understand some of the underlying principles of such recognition of pyrimidine molecular structures by proteins. The preferences of the amino acid residues to contact the pyrimidine bases in terms of nonbonded interactions; amino acid residue-ligand atom preferences; main chain and side chain atom contributions of amino acid residues; and solvent-accessible surface area of ligand atoms when forming complexes are analyzed. Our analysis shows that the amino acid residues, tyrosine and phenyl alanine, are highly involved in the pyrimidine interactions. Arginine prefers contacts with the cytosine base. The similarities and differences that exist between the interactions of the amino acid residues with each of the three pyrimidine base atoms in our analysis provide insights that can be exploited in designing specific inhibitors competitive to the ligands.

Modified-cytosine restriction-system-induced recombinant cloning artefacts in Escherichia coli

DEFF Research Database (Denmark)

Williamson, M R; Doherty, J P; Woodcock, D M

1993-01-01

.HpaII plus M.HhaI) or (2) methylated with M.SssI which methylates at all CpG dinucleotides. These two protocols generated theoretical levels of DNA methylation in the central fragment of 10.5% and 33%, respectively. The construct was transformed into a series of isogenic (recA+) bacterial strains that were...

Intrauterine Reprogramming of the Polycystic Ovary Syndrome: Evidence from a Pilot Study of Cord Blood Global Methylation Analysis

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Luca Lambertini

2017-12-01

Full Text Available Polycystic ovary syndrome (PCOS affects 5–15% of women. PCOS is a heterogeneous disorder displaying endocrine, metabolic, and reproductive dysfunction and cardiovascular risk manifestations. Evidence of heritability exists, but only a portion of the genetic transmission has been identified by genome-wide association studies and linkage studies, suggesting epigenetic phenomena may play a role. Evidence implicates intrauterine influences in the genesis of PCOS. This was a pilot study that aimed at identifying an epigenetic PCOS reprogramming signature by profiling the methylation of the DNA extracted from umbilical cord blood (UCB from 12 subjects undergoing in vitro fertilization. Six subjects were anovulatory PCOS women diagnosed by Rotterdam criteria and six ovulatory non-PCOS women matched for age and body mass index. UCB was collected at delivery of the placenta; the DNA was extracted and submitted to methylation analysis. A differential methylation picture of prevalent hypomethylation affecting 918 genes was detected. Of these, 595 genes (64.8% carried single or multiple hypomethylated CpG dinucleotides and 323 genes (35.2% single or multiple hypermethylated CpG dinucleotides. The Ingenuity Pathway Analysis (IPA online platform enlisted 908 of the 918 input genes and clustered 794 of them into 21 gene networks. Key features of the primary networks scored by IPA included carbohydrate and lipid metabolism, neurotransmitter signaling, cardiovascular system development and function, glycosaminoglycan signaling regulation and control of amino acid biosynthesis. Central to the network activities were genes controlling hormonal regulation (ESR1, mitochondrial activity (APP, PARK2, and glucose metabolism (INS. Regulatory pathways such as G-protein coupled receptor signaling, inositol metabolism, and inflammatory response were also highlighted. These data suggested the existence of a putative “PCOS epigenomic superpathway” with three main

SINE transcription by RNA polymerase III is suppressed by histone methylation but not by DNA methylation

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Varshney, Dhaval; Vavrova-Anderson, Jana; Oler, Andrew J.; Cowling, Victoria H.; Cairns, Bradley R.; White, Robert J.

2015-01-01

Short interspersed nuclear elements (SINEs), such as Alu, spread by retrotransposition, which requires their transcripts to be copied into DNA and then inserted into new chromosomal sites. This can lead to genetic damage through insertional mutagenesis and chromosomal rearrangements between non-allelic SINEs at distinct loci. SINE DNA is heavily methylated and this was thought to suppress its accessibility and transcription, thereby protecting against retrotransposition. Here we provide several lines of evidence that methylated SINE DNA is occupied by RNA polymerase III, including the use of high-throughput bisulphite sequencing of ChIP DNA. We find that loss of DNA methylation has little effect on accessibility of SINEs to transcription machinery or their expression in vivo. In contrast, a histone methyltransferase inhibitor selectively promotes SINE expression and occupancy by RNA polymerase III. The data suggest that methylation of histones rather than DNA plays a dominant role in suppressing SINE transcription. PMID:25798578

Interaction of Cu+ with cytosine and formation of i-motif-like C-M+-C complexes: alkali versus coinage metals

NARCIS (Netherlands)

Gao, J.; Berden, G.; Rodgers, M.T.; Oomens, J.

2016-01-01

The Watson-Crick structure of DNA is among the most well-known molecular structures of our time. However, alternative base-pairing motifs are also known to occur, often depending on base sequence, pH, or the presence of cations. Pairing of cytosine (C) bases induced by the sharing of a single proton

Epigenetic differentiation and relationship to adaptive genetic divergence in discrete populations of the violet Viola cazorlensis.

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Herrera, Carlos M; Bazaga, Pilar

2010-08-01

*In plants, epigenetic variations based on DNA methylation are often heritable and could influence the course of evolution. Before this hypothesis can be assessed, fundamental questions about epigenetic variation remain to be addressed in a real-world context, including its magnitude, structuring within and among natural populations, and autonomy in relation to the genetic context. *Extent and patterns of cytosine methylation, and the relationship to adaptive genetic divergence between populations, were investigated for wild populations of the southern Spanish violet Viola cazorlensis (Violaceae) using the methylation-sensitive amplified polymorphism (MSAP) technique, a modification of the amplified fragment length polymorphism method (AFLP) based on the differential sensitivity of isoschizomeric restriction enzymes to site-specific cytosine methylation. *The genome of V. cazorlensis plants exhibited extensive levels of methylation, and methylation-based epigenetic variation was structured into distinct between- and within- population components. Epigenetic differentiation of populations was correlated with adaptive genetic divergence revealed by a Bayesian population-genomic analysis of AFLP data. Significant associations existed at the individual genome level between adaptive AFLP loci and the methylation state of methylation-susceptible MSAP loci. *Population-specific, divergent patterns of correlated selection on epigenetic and genetic individual variation could account for the coordinated epigenetic-genetic adaptive population differentiation revealed by this study.

Methylation on the Circadian Gene BMAL1 Is Associated with the Effects of a Weight Loss Intervention on Serum Lipid Levels.

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Samblas, Mirian; Milagro, Fermin I; Gómez-Abellán, Purificación; Martínez, J Alfredo; Garaulet, Marta

2016-06-01

The circadian clock system has been linked to the onset and development of obesity and some accompanying comorbidities. Epigenetic mechanisms, such as DNA methylation, are putatively involved in the regulation of the circadian clock system. The aim of this study was to investigate the influence of a weight loss intervention based on an energy-controlled Mediterranean dietary pattern in the methylation levels of 3 clock genes, BMAL1, CLOCK, and NR1D1, and the association between the methylation levels and changes induced in the serum lipid profile with the weight loss treatment. The study sample enrolled 61 women (body mass index = 28.6 ± 3.4 kg/m(2); age: 42.2 ± 11.4 years), who followed a nutritional program based on a Mediterranean dietary pattern. DNA was isolated from whole blood obtained at the beginning and end point. Methylation levels at different CpG sites of BMAL1, CLOCK, and NR1D1 were analyzed by Sequenom's MassArray. The energy-restricted intervention modified the methylation levels of different CpG sites in BMAL1 (CpGs 5, 6, 7, 9, 11, and 18) and NR1D1 (CpGs 1, 10, 17, 18, 19, and 22). Changes in cytosine methylation in the CpG 5 to 9 region of BMAL1 with the intervention positively correlated with the eveningness profile (p = 0.019). The baseline methylation of the CpG 5 to 9 region in BMAL1 positively correlated with energy (p = 0.047) and carbohydrate (p = 0.017) intake and negatively correlated with the effect of the weight loss intervention on total cholesterol (p = 0.032) and low-density lipoprotein cholesterol (p = 0.005). Similar significant and positive correlations were found between changes in methylation levels in the CpG 5 to 9 region of BMAL1 due to the intervention and changes in serum lipids (p < 0.05). This research describes apparently for the first time an association between changes in the methylation of the BMAL1 gene with the intervention and the effects of a weight loss intervention on blood lipids levels. © 2016 The Author(s).

DNA Methylation Biomarkers: Cancer and Beyond

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Thomas Mikeska

2014-09-01

Full Text Available Biomarkers are naturally-occurring characteristics by which a particular pathological process or disease can be identified or monitored. They can reflect past environmental exposures, predict disease onset or course, or determine a patient’s response to therapy. Epigenetic changes are such characteristics, with most epigenetic biomarkers discovered to date based on the epigenetic mark of DNA methylation. Many tissue types are suitable for the discovery of DNA methylation biomarkers including cell-based samples such as blood and tumor material and cell-free DNA samples such as plasma. DNA methylation biomarkers with diagnostic, prognostic and predictive power are already in clinical trials or in a clinical setting for cancer. Outside cancer, strong evidence that complex disease originates in early life is opening up exciting new avenues for the detection of DNA methylation biomarkers for adverse early life environment and for estimation of future disease risk. However, there are a number of limitations to overcome before such biomarkers reach the clinic. Nevertheless, DNA methylation biomarkers have great potential to contribute to personalized medicine throughout life. We review the current state of play for DNA methylation biomarkers, discuss the barriers that must be crossed on the way to implementation in a clinical setting, and predict their future use for human disease.

Aberrant methylation and associated transcriptional mobilization of Alu elements contributes to genomic instability in hypoxia.

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Pal, Arnab; Srivastava, Tapasya; Sharma, Manish K; Mehndiratta, Mohit; Das, Prerna; Sinha, Subrata; Chattopadhyay, Parthaprasad

2010-11-01

Hypoxia is an integral part of tumorigenesis and contributes extensively to the neoplastic phenotype including drug resistance and genomic instability. It has also been reported that hypoxia results in global demethylation. Because a majority of the cytosine-phosphate-guanine (CpG) islands are found within the repeat elements of DNA, and are usually methylated under normoxic conditions, we suggested that retrotransposable Alu or short interspersed nuclear elements (SINEs) which show altered methylation and associated changes of gene expression during hypoxia, could be associated with genomic instability. U87MG glioblastoma cells were cultured in 0.1% O₂ for 6 weeks and compared with cells cultured in 21% O₂ for the same duration. Real-time PCR analysis showed a significant increase in SINE and reverse transcriptase coding long interspersed nuclear element (LINE) transcripts during hypoxia. Sequencing of bisulphite treated DNA as well as the Combined Bisulfite Restriction Analysis (COBRA) assay showed that the SINE loci studied underwent significant hypomethylation though there was patchy hypermethylation at a few sites. The inter-alu PCR profile of DNA from cells cultured under 6-week hypoxia, its 4-week revert back to normoxia and 6-week normoxia showed several changes in the band pattern indicating increased alu mediated genomic alteration. Our results show that aberrant methylation leading to increased transcription of SINE and reverse transcriptase associated LINE elements could lead to increased genomic instability in hypoxia. This might be a cause of genetic heterogeneity in tumours especially in variegated hypoxic environment and lead to a development of foci of more aggressive tumour cells. © 2009 The Authors Journal compilation © 2010 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd.

SNP rs16906252C>T is an expression and methylation quantitative trait locus associated with an increased risk of developing MGMT-methylated colorectal cancer

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Kuroiwa-Trzmielina, Joice; Wang, Fan; Rapkins, Robert W.; Ward, Robyn L.; Buchanan, Daniel D.; Win, Aung Ko; Clendenning, Mark; Rosty, Christophe; Southey, Melissa C.; Winship, Ingrid M.; Hopper, John L.; Jenkins, Mark A.; Olivier, Jake; Hawkins, Nicholas J.; Hitchins, Megan P.

2016-01-01

Purpose Methylation of the MGMT promoter is the major cause of O6-methylguanine methyltransferase deficiency in cancer and has been associated with the T variant of the promoter-enhancer SNP rs16906252C>T. We sought evidence for an association between the rs16906252C>T genotype and increased risk of developing a subtype of colorectal cancer (CRC) featuring MGMT methylation, mediated by genotype-dependent epigenetic silencing within normal tissues. Experimental design By applying a molecular pathological epidemiology case-control study design, associations between rs16906252C>T and risk for CRC overall, and CRC stratified by MGMT methylation status, were estimated using multinomial logistic regression in two independent retrospective series of CRC cases and controls. The test sample comprised 1054 CRC cases and 451 controls from Sydney, Australia. The validation sample comprised 612 CRC cases and 245 controls from the Australasian Colon Cancer Family Registry (ACCFR). To determine if rs16906252C>T was linked to a constitutively altered epigenetic state, quantitative allelic expression and methylation analyses were performed in normal tissues. Results An association between rs16906252C>T and increased risk of developing MGMT-methylated CRC in the Sydney sample was observed (OR 3.3; 95%CI=2.0–5.3; PT represents an expression and methylation quantitative trait locus. Conclusions We provide evidence that rs16906252C>T is associated with elevated risk for MGMT-methylated CRC, likely mediated by constitutive epigenetic repression of the T allele. PMID:27267851

DHA-rich n-3 fatty acid supplementation decreases DNA methylation in blood leukocytes: the OmegAD study.

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Karimi, Mohsen; Vedin, Inger; Freund Levi, Yvonne; Basun, Hans; Faxén Irving, Gerd; Eriksdotter, Maria; Wahlund, Lars-Olof; Schultzberg, Marianne; Hjorth, Erik; Cederholm, Tommy; Palmblad, Jan

2017-10-01

Background: Dietary fish oils, rich in long-chain n-3 (ω-3) fatty acids (FAs) [e.g., docosahexaenoic acid (DHA, 22:6n-3) and eicosapentaenoic acid (EPA, 20:5n-3)], modulate inflammatory reactions through various mechanisms, including gene expression, which is measured as messenger RNA concentration. However, the effects of long-term treatment of humans with DHA and EPA on various epigenetic factors-such as DNA methylation, which controls messenger RNA generation-are poorly described. Objective: We wanted to determine the effects of 6 mo of dietary supplementation with an n-3 FA preparation rich in DHA on global DNA methylation of peripheral blood leukocytes (PBLs) and the relation to plasma EPA and DHA concentrations in Alzheimer disease (AD) patients. Design: In the present study, DNA methylation in four 5'-cytosine-phosphate-guanine-3' (CpG) sites of long interspersed nuclear element-1 repetitive sequences was assessed in a group of 63 patients (30 given the n-3 FA preparation and 33 given placebo) as an estimation of the global DNA methylation in blood cells. Patients originated from the randomized, double-blind, placebo-controlled OmegAD study, in which 174 AD patients received either 1.7 g DHA and 0.6 g EPA (the n-3 FA group) or placebo daily for 6 mo. Results: At 6 mo, the n-3 FA group displayed marked increases in DHA and EPA plasma concentrations (2.6- and 3.5-fold), as well as decreased methylation in 2 out of 4 CpG sites ( P DHA concentration, and were not related to apolipoprotein E-4 allele frequency. Conclusion: Supplementation with n-3 FA for 6 mo was associated with global DNA hypomethylation in PBLs. Our data may be of importance in measuring various effects of marine oils, including gene expression, in patients with AD and in other patients taking n-3 FA supplements. This trial was registered at clinicaltrials.gov as NCT00211159. © 2017 American Society for Nutrition.

Allele specific expression and methylation in the bumblebee, Bombus terrestris

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Zoë Lonsdale

2017-09-01

Full Text Available The social hymenoptera are emerging as models for epigenetics. DNA methylation, the addition of a methyl group, is a common epigenetic marker. In mammals and flowering plants methylation affects allele specific expression. There is contradictory evidence for the role of methylation on allele specific expression in social insects. The aim of this paper is to investigate allele specific expression and monoallelic methylation in the bumblebee, Bombus terrestris. We found nineteen genes that were both monoallelically methylated and monoallelically expressed in a single bee. Fourteen of these genes express the hypermethylated allele, while the other five express the hypomethylated allele. We also searched for allele specific expression in twenty-nine published RNA-seq libraries. We found 555 loci with allele-specific expression. We discuss our results with reference to the functional role of methylation in gene expression in insects and in the as yet unquantified role of genetic cis effects in insect allele specific methylation and expression.

Increased expression and altered methylation of HERVWE1 in the human placentas of smaller fetuses from monozygotic, dichorionic, discordant twins.

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Yu Gao

Full Text Available BACKGROUND: The human endogenous retroviral family W, Env(C7, member 1 gene (HERVWE1 is thought to participate in trophoblast cell fusion, and its expression is diminished in the placentas of singleton intrauterine growth-retarded pregnancies. However, there is limited information about the role of HERVWE1 in discordant fetal growth in twins. This study was to compare HERVWE1 gene expression between the placentas of discordant monozygotic twins and to identify its regulation by methylation. METHODOLOGY/PRINCIPAL FINDINGS: Fetuses from twenty-one pairs of monozygotic, dichorionic, discordant twins were marked as "smaller" or "larger" according to birth weight. Placental HERVWE1 mRNA and protein expression profiles were analyzed using quantitative RT-PCR and immunohistochemistry (IHC staining. Methylation profiles of the HERVWE1 promoter region were analyzed using a pyrosequencing assay. DNA methyltransferase (DNMT transcript levels were analyzed by RT-PCR. 5-methyl cytosine (5-MC was stained using an immunohistochemical assay. There was a significant negative correlation between HERVWE1 mRNA levels and birth weight in twins (P0.05. The DNMT3b3 mRNA levels in the smaller group were significantly downregulated compared with the larger group in discordant twins(P<0.05, whereas the DNMT3b7 mRNA levels in the smaller group were significantly upregulated compared with the larger group in discordant twins(P<0.05. CONCLUSIONS/SIGNIFICANCE: In discordant, monozygotic, dichorionic twins, HERVWE1 expression was higher in smaller fetuses and lower in larger fetuses. Methylation of the HERVWE1 gene promoter region may participate in the regulation of HERVWE1 gene expression in discordant twin pregnancies.

Increased Expression and Altered Methylation of HERVWE1 in the Human Placentas of Smaller Fetuses from Monozygotic, Dichorionic, Discordant Twins

Science.gov (United States)

Wang, Zilian; Luo, Yanmin; Sun, Hongyu; Zhou, Yi; Huang, Linhuan; Li, Manchao; Fang, Qun; Jiang, Shiwen

2012-01-01

Background The human endogenous retroviral family W, Env(C7), member 1 gene (HERVWE1) is thought to participate in trophoblast cell fusion, and its expression is diminished in the placentas of singleton intrauterine growth-retarded pregnancies. However, there is limited information about the role of HERVWE1 in discordant fetal growth in twins. This study was to compare HERVWE1 gene expression between the placentas of discordant monozygotic twins and to identify its regulation by methylation. Methodology/Principal Findings Fetuses from twenty-one pairs of monozygotic, dichorionic, discordant twins were marked as “smaller” or “larger” according to birth weight. Placental HERVWE1 mRNA and protein expression profiles were analyzed using quantitative RT-PCR and immunohistochemistry (IHC) staining. Methylation profiles of the HERVWE1 promoter region were analyzed using a pyrosequencing assay. DNA methyltransferase (DNMT) transcript levels were analyzed by RT-PCR. 5-methyl cytosine (5-MC) was stained using an immunohistochemical assay. There was a significant negative correlation between HERVWE1 mRNA levels and birth weight in twins (P0.05). The DNMT3b3 mRNA levels in the smaller group were significantly downregulated compared with the larger group in discordant twins(P<0.05), whereas the DNMT3b7 mRNA levels in the smaller group were significantly upregulated compared with the larger group in discordant twins(P<0.05). Conclusions/Significance In discordant, monozygotic, dichorionic twins, HERVWE1 expression was higher in smaller fetuses and lower in larger fetuses. Methylation of the HERVWE1 gene promoter region may participate in the regulation of HERVWE1 gene expression in discordant twin pregnancies. PMID:22457770

Transition-transversion bias is not universal: a counter example from grasshopper pseudogenes.

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Irene Keller

2007-02-01

Full Text Available Comparisons of the DNA sequences of metazoa show an excess of transitional over transversional substitutions. Part of this bias is due to the relatively high rate of mutation of methylated cytosines to thymine. Postmutation processes also introduce a bias, particularly selection for codon-usage bias in coding regions. It is generally assumed, however, that there is a universal bias in favour of transitions over transversions, possibly as a result of the underlying chemistry of mutation. Surprisingly, this underlying trend has been evaluated only in two types of metazoan, namely Drosophila and the Mammalia. Here, we investigate a third group, and find no such bias. We characterize the point substitution spectrum in Podisma pedestris, a grasshopper species with a very large genome. The accumulation of mutations was surveyed in two pseudogene families, nuclear mitochondrial and ribosomal DNA sequences. The cytosine-guanine (CpG dinucleotides exhibit the high transition frequencies expected of methylated sites. The transition rate at other cytosine residues is significantly lower. After accounting for this methylation effect, there is no significant difference between transition and transversion rates. These results contrast with reports from other taxa and lead us to reject the hypothesis of a universal transition/transversion bias. Instead we suggest fundamental interspecific differences in point substitution processes.

Genome-Wide Analysis of DNA Methylation and Fine Particulate Matter Air Pollution in Three Study Populations: KORA F3, KORA F4, and the Normative Aging Study.

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Panni, Tommaso; Mehta, Amar J; Schwartz, Joel D; Baccarelli, Andrea A; Just, Allan C; Wolf, Kathrin; Wahl, Simone; Cyrys, Josef; Kunze, Sonja; Strauch, Konstantin; Waldenberger, Melanie; Peters, Annette

2016-07-01

Epidemiological studies have reported associations between particulate matter (PM) concentrations and cancer and respiratory and cardiovascular diseases. DNA methylation has been identified as a possible link but so far it has only been analyzed in candidate sites. We studied the association between DNA methylation and short- and mid-term air pollution exposure using genome-wide data and identified potential biological pathways for additional investigation. We collected whole blood samples from three independent studies-KORA F3 (2004-2005) and F4 (2006-2008) in Germany, and the Normative Aging Study (1999-2007) in the United States-and measured genome-wide DNA methylation proportions with the Illumina 450k BeadChip. PM concentration was measured daily at fixed monitoring stations and three different trailing averages were considered and regressed against DNA methylation: 2-day, 7-day and 28-day. Meta-analysis was performed to pool the study-specific results. Random-effect meta-analysis revealed 12 CpG (cytosine-guanine dinucleotide) sites as associated with PM concentration (1 for 2-day average, 1 for 7-day, and 10 for 28-day) at a genome-wide Bonferroni significance level (p ≤ 7.5E-8); 9 out of these 12 sites expressed increased methylation. Through estimation of I2 for homogeneity assessment across the studies, 4 of these sites (annotated in NSMAF, C1orf212, MSGN1, NXN) showed p > 0.05 and I2 F4, and the Normative Aging Study. Environ Health Perspect 124:983-990; http://dx.doi.org/10.1289/ehp.1509966.

Methylation of the S f locus in almond is associated with S-RNase loss of function.

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Fernández i Martí, Angel; Gradziel, Thomas M; Socias i Company, Rafel

2014-12-01

Self-compatibility in almond (Prunus dulcis) is attributed to the presence of the S f haplotype, allelic to and dominant over the series of S-alleles controlling self-incompatibility. Some forms of the S f haplotype, however, are phenotypically self-incompatible even though their nucleotide sequences are identical. DNA from leaves and styles from genetically diverse almond samples was cloned and sequenced and then analyzed for changes affecting S f -RNase variants. Epigenetic changes in several cytosine residues were detected in a fragment of 4,700 bp of the 5' upstream region of all self-compatible samples of the S f -RNases, differentiating them from all self-incompatible samples of S f -RNases analyzed. This is the first report of DNA methylation in a Rosaceae species and appears to be strongly associated with inactivation of the S f allele. Results facilitate an understanding of the evolution of self-compatibility/self-incompatibility in almond and other Prunus species, and suggest novel approaches for future crop improvement.

Methylation pattern of IFNG in periapical granulomas and radicular cysts.

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Campos, Kelma; Gomes, Carolina Cavaliéri; de Fátima Correia-Silva, Jeane; Farias, Lucyana Conceição; Fonseca-Silva, Thiago; Bernardes, Vanessa Fátima; Pereira, Cláudia Maria; Gomez, Ricardo Santiago

2013-04-01

Interferon-γ plays an important role in the pathogenesis of periapical lesions, and the methylation of IFNG has been associated with transcriptional inactivation. The purpose of the present study was to investigate IFNG promoter methylation in association with gene transcription and protein levels in periapical granulomas and radicular cysts. Methylation-specific polymerase chain reaction was used to assess the DNA methylation pattern of the IFNG gene in 16 periapical granulomas and 13 radicular cyst samples. The transcription levels of IFNG mRNA were verified by quantitative real-time polymerase chain reaction, and protein expression was evaluated by immunohistochemistry. All the periapical lesion samples exhibited partial or total methylation of the IFNG gene. In addition, an increased methylation profile was found in radicular cysts compared with periapical granulomas. Increased IFNG mRNA expression was observed in the partially methylated periapical lesion samples relative to the samples that were completely methylated. The present study provides the first evidence of the possible impact of IFNG methylation on IFNG transcription in periapical lesions. Copyright © 2013 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

Polymorphism and methylation of the MC4R gene in obese and non-obese dogs.

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Mankowska, Monika; Nowacka-Woszuk, Joanna; Graczyk, Aneta; Ciazynska, Paulina; Stachowiak, Monika; Switonski, Marek

2017-08-01

The dog is considered to be a useful biomedical model for human diseases and disorders, including obesity. One of the numerous genes associated with human polygenic obesity is MC4R, encoding the melanocortin 4 receptor. The aim of our study was to analyze polymorphisms and methylation of the canine MC4R in relation to adiposity. Altogether 270 dogs representing four breeds predisposed to obesity: Labrador Retriever (n = 187), Golden Retriever (n = 38), Beagle (n = 28) and Cocker Spaniel (n = 17), were studied. The dogs were classified into three groups: lean, overweight and obese, according to the 5-point Body Condition Score (BCS) scale. In the cohort of Labradors a complete phenotypic data (age, sex, neutering status, body weight and BCS) were collected for 127 dogs. The entire coding sequence as well as 5' and 3'-flanking regions of the studied gene were sequenced and six polymorphic sites were reported. Genotype frequencies differed considerably between breeds and Labrador Retrievers appeared to be the less polymorphic. Moreover, distribution of some polymorphic variants differed significantly (P C, c.868C>T and c.*33C>G) and Beagles (c.-435T>C and c.637G>T). On the contrary, in Labradors no association between the studied polymorphisms and BCS or body weight was observed. Methylation analysis, using bisulfite DNA conversion followed by Sanger sequencing, was carried out for 12 dogs with BCS = 3 and 12 dogs with BCS = 5. Two intragenic CpG islands, containing 19 cytosines, were analyzed and the methylation profile did not differ significantly between lean and obese animals. We conclude that an association of the MC4R gene polymorphism with dog obesity or body weight is unlikely, in spite of the fact that some associations were found in small cohorts of Beagles and Golden Retrievers. Also methylation level of this gene is not related with dog adiposity.

Exclusion of the GNAS locus in PHP-Ib patients with broad GNAS methylation changes: evidence for an autosomal recessive form of PHP-Ib?

Science.gov (United States)

Fernández-Rebollo, Eduardo; Pérez de Nanclares, Guiomar; Lecumberri, Beatriz; Turan, Serap; Anda, Emma; Pérez-Nanclares, Gustavo; Feig, Denice; Nik-Zainal, Serena; Bastepe, Murat; Jüppner, Harald

2011-08-01

Most patients with autosomal dominant pseudohypoparathyroidism type Ib (AD-PHP-Ib) carry maternally inherited microdeletions upstream of GNAS that are associated with loss of methylation restricted to GNAS exon A/B. Only few AD-PHP-Ib patients carry microdeletions within GNAS that are associated with loss of all maternal methylation imprints. These epigenetic changes are often indistinguishable from those observed in patients affected by an apparently sporadic PHP-Ib form that has not yet been defined genetically. We have now investigated six female patients affected by PHP-Ib (four unrelated and two sisters) with complete or almost complete loss of GNAS methylation, whose healthy children (11 in total) showed no epigenetic changes at this locus. Analysis of several microsatellite markers throughout the 20q13 region made it unlikely that PHP-Ib is caused in these patients by large deletions involving GNAS or by paternal uniparental isodisomy or heterodisomy of chromosome 20 (patUPD20). Microsatellite and single-nucleotide variation (SNV) data revealed that the two affected sisters share their maternally inherited GNAS alleles with unaffected relatives that lack evidence for abnormal GNAS methylation, thus excluding linkage to this locus. Consistent with these findings, healthy children of two unrelated sporadic PHP-Ib patients had inherited different maternal GNAS alleles, also arguing against linkage to this locus. Based on our data, it appears plausible that some forms of PHP-Ib are caused by homozygous or compound heterozygous mutation(s) in an unknown gene involved in establishing or maintaining GNAS methylation. Copyright © 2011 American Society for Bone and Mineral Research.

Methylation of DNA Ligase 1 by G9a/GLP Recruits UHRF1 to Replicating DNA and Regulates DNA Methylation.

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Ferry, Laure; Fournier, Alexandra; Tsusaka, Takeshi; Adelmant, Guillaume; Shimazu, Tadahiro; Matano, Shohei; Kirsh, Olivier; Amouroux, Rachel; Dohmae, Naoshi; Suzuki, Takehiro; Filion, Guillaume J; Deng, Wen; de Dieuleveult, Maud; Fritsch, Lauriane; Kudithipudi, Srikanth; Jeltsch, Albert; Leonhardt, Heinrich; Hajkova, Petra; Marto, Jarrod A; Arita, Kyohei; Shinkai, Yoichi; Defossez, Pierre-Antoine

2017-08-17

DNA methylation is an essential epigenetic mark in mammals that has to be re-established after each round of DNA replication. The protein UHRF1 is essential for this process; it has been proposed that the protein targets newly replicated DNA by cooperatively binding hemi-methylated DNA and H3K9me2/3, but this model leaves a number of questions unanswered. Here, we present evidence for a direct recruitment of UHRF1 by the replication machinery via DNA ligase 1 (LIG1). A histone H3K9-like mimic within LIG1 is methylated by G9a and GLP and, compared with H3K9me2/3, more avidly binds UHRF1. Interaction with methylated LIG1 promotes the recruitment of UHRF1 to DNA replication sites and is required for DNA methylation maintenance. These results further elucidate the function of UHRF1, identify a non-histone target of G9a and GLP, and provide an example of a histone mimic that coordinates DNA replication and DNA methylation maintenance. Copyright © 2017 Elsevier Inc. All rights reserved.

4'-Methyl derivatives of 5-MOP and 5-MOA: synthesis, photoreactivity, and photobiological activity.

Science.gov (United States)

Gia, O; Anselmo, A; Conconi, M T; Antonello, C; Uriarte, E; Caffieri, S

1996-10-25

The synthesis and photobiological activity of four new 4'-methyl derivatives of 5-MOP (5-methoxypsoralen) and 5-MOA (5-methoxyangelicin), i.e., 4,4'-dimethyl-5-methoxypsoralen, 3,4'-dimethyl-5-methoxypsoralen, 4,4'-dimethyl-5-methoxyangelicin, and 3,4'-dimethyl-5-methoxyangelicin, are described. All these compounds photobind efficiently to DNA. The DNA-photobinding process was investigated using various nucleic acid structures such as double-helix DNA, bacterial DNA, and synthetic polydeoxyribonucleotides. Photoreaction experiments showed that, unlike 8-MOP (8-methoxypsoralen) and 5-MOP, both angular derivatives bind thymine and cytosine with the same efficiency. The principal nucleoside-psoralen monoadducts were isolated and characterized after enzymatic digestion or acid hydrolysis. Biological activity studies revealed a good correlation with the extent of covalent photoaddition. Moreover, the two angular derivatives and the 4,4'-dimethyl-5-methoxypsoralen were unable to induce skin erythema, in striking contrast with the reference drugs, 8-MOP and 5-MOP; only the 3,4'-dimethyl-5-methoxypsoralen caused erythema, although to a substantially lower extent than that induced by the two parent compounds.

Effects of temperature and isotopic substitution on electron attachment dynamics of guanine–cytosine base pair: Ring-polymer and classical molecular dynamics simulations

International Nuclear Information System (INIS)

Minoshima, Yusuke; Seki, Yusuke; Takayanagi, Toshiyuki; Shiga, Motoyuki

2016-01-01

Highlights: • Dynamics of excess electron attachment to guanine–cytosine base pair. • Ring-polymer and classical molecular dynamics simulations are performed. • Temperature and isotope substitution effects are investigated. - Abstract: The dynamical process of electron attachment to a guanine–cytosine pair in the normal (h-GC) and deuterated (d-GC) forms has been studied theoretically by semiclassical ring-polymer molecular dynamics (RPMD) simulations using the empirical valence bond model. The initially formed dipole-bound anion is converted rapidly to the valence-bound anion within about 0.1 ps in both h-GC and d-GC. However, the subsequent proton transfer in h-GC occurs with a rate five times greater than the deuteron transfer in d-GC. The change of rates with isotopic substitution and temperature variation in the RPMD simulations are quantitatively and qualitatively different from those in the classical molecular dynamics (MD) simulations, demonstrating the importance of nuclear quantum effects on the dynamics of this system.

Effects of temperature and isotopic substitution on electron attachment dynamics of guanine–cytosine base pair: Ring-polymer and classical molecular dynamics simulations

Energy Technology Data Exchange (ETDEWEB)

Minoshima, Yusuke; Seki, Yusuke [Department of Chemistry, Saitama University, 255 Shimo-Okubo, Sakura-ku, Saitama City, Saitama 338-8570 (Japan); Takayanagi, Toshiyuki, E-mail: tako@mail.saitama-u.ac.jp [Department of Chemistry, Saitama University, 255 Shimo-Okubo, Sakura-ku, Saitama City, Saitama 338-8570 (Japan); Shiga, Motoyuki [Center for Computational Science and E-Systems, Japan Atomic Energy Agency, 148-4, Kashiwanoha Campus, 178-4 Wakashiba, Kashiwa, Chiba 277-0871 (Japan)

2016-06-15

Highlights: • Dynamics of excess electron attachment to guanine–cytosine base pair. • Ring-polymer and classical molecular dynamics simulations are performed. • Temperature and isotope substitution effects are investigated. - Abstract: The dynamical process of electron attachment to a guanine–cytosine pair in the normal (h-GC) and deuterated (d-GC) forms has been studied theoretically by semiclassical ring-polymer molecular dynamics (RPMD) simulations using the empirical valence bond model. The initially formed dipole-bound anion is converted rapidly to the valence-bound anion within about 0.1 ps in both h-GC and d-GC. However, the subsequent proton transfer in h-GC occurs with a rate five times greater than the deuteron transfer in d-GC. The change of rates with isotopic substitution and temperature variation in the RPMD simulations are quantitatively and qualitatively different from those in the classical molecular dynamics (MD) simulations, demonstrating the importance of nuclear quantum effects on the dynamics of this system.

Genome-wide methylation profiling identifies an essential role of reactive oxygen species in pediatric glioblastoma multiforme and validates a methylome specific for H3 histone family 3A with absence of G-CIMP/isocitrate dehydrogenase 1 mutation.

Science.gov (United States)

Jha, Prerana; Pia Patric, Irene Rosita; Shukla, Sudhanshu; Pathak, Pankaj; Pal, Jagriti; Sharma, Vikas; Thinagararanjan, Sivaarumugam; Santosh, Vani; Suri, Vaishali; Sharma, Mehar Chand; Arivazhagan, Arimappamagan; Suri, Ashish; Gupta, Deepak; Somasundaram, Kumaravel; Sarkar, Chitra

2014-12-01

Pediatric glioblastoma multiforme (GBM) is rare, and there is a single study, a seminal discovery showing association of histone H3.3 and isocitrate dehydrogenase (IDH)1 mutation with a DNA methylation signature. The present study aims to validate these findings in an independent cohort of pediatric GBM, compare it with adult GBM, and evaluate the involvement of important functionally altered pathways. Genome-wide methylation profiling of 21 pediatric GBM cases was done and compared with adult GBM data (GSE22867). We performed gene mutation analysis of IDH1 and H3 histone family 3A (H3F3A), status evaluation of glioma cytosine-phosphate-guanine island methylator phenotype (G-CIMP), and Gene Ontology analysis. Experimental evaluation of reactive oxygen species (ROS) association was also done. Distinct differences were noted between methylomes of pediatric and adult GBM. Pediatric GBM was characterized by 94 hypermethylated and 1206 hypomethylated cytosine-phosphate-guanine (CpG) islands, with 3 distinct clusters, having a trend to prognostic correlation. Interestingly, none of the pediatric GBM cases showed G-CIMP/IDH1 mutation. Gene Ontology analysis identified ROS association in pediatric GBM, which was experimentally validated. H3F3A mutants (36.4%; all K27M) harbored distinct methylomes and showed enrichment of processes related to neuronal development, differentiation, and cell-fate commitment. Our study confirms that pediatric GBM has a distinct methylome compared with that of adults. Presence of distinct clusters and an H3F3A mutation-specific methylome indicate existence of epigenetic subgroups within pediatric GBM. Absence of IDH1/G-CIMP status further indicates that findings in adult GBM cannot be simply extrapolated to pediatric GBM and that there is a strong need for identification of separate prognostic markers. A possible role of ROS in pediatric GBM pathogenesis is demonstrated for the first time and needs further evaluation. © The Author(s) 2014

Intramolecular tautomerisation and the conformational variability of some classical mutagens – cytosine derivatives: quantum chemical study

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Hovorun D. M.

2011-04-01

Full Text Available Aim. To determine the lifetime of the mutagenic cytosine derivatives through the investigation of the physicochemical mechanisms of their intramolecular proton transfer. Methods. Non-empirical quantum chemistry, the analysis of the electron density by means of Bader’s atoms in molecules (AIM theory and physicochemical kinetics were used. Results. It is shown that the modification of all investigated compounds, except DCyt, prevents their pairing in both mutagenic and canonical tautomeric forms with a base which is an interacting partner. This effect can inhibit their mutagenic potential. It is also established that Watson-Crick tautomeric hypothesis can be formally expanded for the investigated molecules so far as a lifetime of the mutagenic tautomers much more exceeds characteristic time for the incorporation of one nucleotides pair by DNA biosynthesis machinery. It seems that just within the frame of this hypothesis it will be possible to give an adequate explanation of the mechanisms of mutagenic action of N4-aminocytosine, N4-methoxycytosine, N4-hydroxycytosine and N4dehydrocytosine, which have much more energy advantageous imino form in comparison with amino form. Conclusions. For the first time the comprehensive conformational analysis of a number of classical mutagens, namely cytosine derivatives, has been performed using the methods of non-empirical quantum chemistry at the MP2/6-311++G (2df,pd//B3LYP/6-311++G(d,p level of theory

Bacillus halodurans Strain C125 Encodes and Synthesizes Enzymes from Both Known Pathways To Form dUMP Directly from Cytosine Deoxyribonucleotides

DEFF Research Database (Denmark)

Oehlenschlæger, Christian Berg; Løvgreen, Monika Nøhr; Reinauer, Eva

2015-01-01

Analysis of the genome of Bacillus halodurans strain C125 indicated that two pathways leading from a cytosine deoxyribonucleotide to dUMP, used for dTMP synthesis, were encoded by the genome of the bacterium. The genes that were responsible, the comEB gene and the dcdB gene, encoding dCMP deaminase...

Cyclopentenyl cytosine has biological and anti-tumour activity, but does not enhance the efficacy of gemcitabine and radiation in two animal tumour models

NARCIS (Netherlands)

van Bree, Chris; Barten-van Rijbroek, Angeliqué D.; Leen, René; Rodermond, Hans M.; van Kuilenburg, André B. P.; Kal, Henk B.

2009-01-01

Cyclopentenyl cytosine (CPEC), targetting the de novo biosynthesis of cytidine triphosphate (CTP), increases the cytotoxicity of gemcitabine (2',2'-difluoro-2'-deoxycytidine, dFdC) alone and in combination with irradiation in several human tumour cells in vitro. We investigated whether OPEC enhances

Somatic mutations, allele loss, and DNA methylation of the Cub and Sushi Multiple Domains 1 (CSMD1 gene reveals association with early age of diagnosis in colorectal cancer patients.

Directory of Open Access Journals (Sweden)

Austin Y Shull

Full Text Available The Cub and Sushi Multiple Domains 1 (CSMD1 gene, located on the short arm of chromosome 8, codes for a type I transmembrane protein whose function is currently unknown. CSMD1 expression is frequently lost in many epithelial cancers. Our goal was to characterize the relationships between CSMD1 somatic mutations, allele imbalance, DNA methylation, and the clinical characteristics in colorectal cancer patients.We sequenced the CSMD1 coding regions in 54 colorectal tumors using the 454FLX pyrosequencing platform to interrogate 72 amplicons covering the entire coding sequence. We used heterozygous SNP allele ratios at multiple CSMD1 loci to determine allelic balance and infer loss of heterozygosity. Finally, we performed methylation-specific PCR on 76 colorectal tumors to determine DNA methylation status for CSMD1 and known methylation targets ALX4, RUNX3, NEUROG1, and CDKN2A.Using 454FLX sequencing and confirming with Sanger sequencing, 16 CSMD1 somatic mutations were identified in 6 of the 54 colorectal tumors (11%. The nonsynonymous to synonymous mutation ratio of the 16 somatic mutations was 15:1, a ratio significantly higher than the expected 2:1 ratio (p = 0.014. This ratio indicates a presence of positive selection for mutations in the CSMD1 protein sequence. CSMD1 allelic imbalance was present in 19 of 37 informative cases (56%. Patients with allelic imbalance and CSMD1 mutations were significantly younger (average age, 41 years than those without somatic mutations (average age, 68 years. The majority of tumors were methylated at one or more CpG loci within the CSMD1 coding sequence, and CSMD1 methylation significantly correlated with two known methylation targets ALX4 and RUNX3. C:G>T:A substitutions were significantly overrepresented (47%, suggesting extensive cytosine methylation predisposing to somatic mutations.Deep amplicon sequencing and methylation-specific PCR reveal that CSMD1 alterations can correlate with earlier clinical

Ionophoretic method in the study of mixed ligand ternary chelates of UO2(II), Ni(II) and Zn(II) involving nitrilotriacetate and cytosine as ligands

International Nuclear Information System (INIS)

Mishra, A.P.; Mishra, S.K.; Yadava, K.L.

1987-01-01

A novel electrophoretic technique is described for the assessment of the equilibria in mixed-ligand complex system in solution. It is based on the movement of spot of the metal ion under an electric field with the complexants added in the background electrolyte at fixed pH. The concentration of primary ligand nitrilotriacetate was constant while that of secondary ligand (cytosine) was varied. The plot of log (cytosine) against mobility was used to obtain information on the formation of the mixed complexes and to calculate its stability constants. Experimentally obtained logK values are as 5.62, 4.55 and 4.42 for mixed complexes of UO 2 (II), Ni(II) and Zn(II) respectively at μ=0.1 and temp.=35 +- 01.degC. (author). 10 refs

Pretreatment long interspersed element (LINE-1 methylation levels, not early hypomethylation under treatment, predict hematological response to azacitidine in elderly patients with acute myeloid leukemia

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Cross M

2013-06-01

Full Text Available Michael Cross,1 Enrica Bach,1 Thao Tran,1 Rainer Krahl,1 Nadja Jaekel,1 Dietger Niederwieser,1 Christian Junghanss,2 Georg Maschmeyer,3 Haifa Kathrin Al-Ali11Division of Hematology and Oncology, University of Leipzig, Leipzig, Germany; 2Clinic for Hematology, Oncology and Palliative Medicine, University of Rostock, Rostock, Germany; 3Clinic for Hematology, Oncology and Palliative Medicine, Ernst-von-Bergmann Clinic, Potsdam, GermanyBackground: Epigenetic modulations, including changes in DNA cytosine methylation, are implicated in the pathogenesis and progression of acute myeloid leukemia (AML. Azacitidine is a hypomethylating agent that is incorporated into RNA as well as DNA. Thus, there is a rationale to its use in patients with AML. We determined whether baseline and/or early changes in the methylation of long interspersed element (LINE-1 or CDH13 correlate with bone marrow blast clearance, hematological response, or survival in patients with AML treated with azacitidine.Methods: An open label, phase I/II trial was performed in 40 AML patients (median bone marrow blast count was 42% unfit for intensive chemotherapy treated with azacitidine 75 mg/m2/day subcutaneously for 5 days every 4 weeks. Bone marrow mononuclear cell samples were taken on day 0 (pretreatment and day 15 during the first treatment cycle; LINE-1 and CDH13 methylation levels were quantified by methylation-specific, semiquantitative, real-time polymerase chain reaction.Results: Treatment with azacitidine significantly reduced LINE-1 but not CDH13 methylation levels over the first cycle (P < 0.0001. Absolute LINE-1 methylation levels tended to be lower on day 0 (P = 0.06 and day 15 of cycle 1 (P = 0.03 in patients who went on to achieve subsequent complete remission, partial remission or hematological improvement versus patients with stable disease. However, the decrease in LINE-1 methylation over the first treatment cycle did not correlate with subsequent response (P = 0

Comprehensive analysis of preeclampsia-associated DNA methylation in the placenta.

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Tianjiao Chu

Full Text Available A small number of recent reports have suggested that altered placental DNA methylation may be associated with early onset preeclampsia. It is important that further studies be undertaken to confirm and develop these findings. We therefore undertook a systematic analysis of DNA methylation patterns in placental tissue from 24 women with preeclampsia and 24 with uncomplicated pregnancy outcome.We analyzed the DNA methylation status of approximately 27,000 CpG sites in placental tissues in a massively parallel fashion using an oligonucleotide microarray. Follow up analysis of DNA methylation at specific CpG loci was performed using the Epityper MassArray approach and high-throughput bisulfite sequencing.Preeclampsia-specific DNA methylation changes were identified in placental tissue samples irrespective of gestational age of delivery. In addition, we identified a group of CpG sites within specific gene sequences that were only altered in early onset-preeclampsia (EOPET although these DNA methylation changes did not correlate with altered mRNA transcription. We found evidence that fetal gender influences DNA methylation at autosomal loci but could find no clear association between DNA methylation and gestational age.Preeclampsia is associated with altered placental DNA methylation. Fetal gender should be carefully considered during the design of future studies in which placental DNA is analyzed at the level of DNA methylation. Further large-scale analyses of preeclampsia-associated DNA methylation are necessary.

The Fine LINE: Methylation Drawing the Cancer Landscape

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Isabelle R. Miousse

2015-01-01

Full Text Available LINE-1 (L1 is the most abundant mammalian transposable element that comprises nearly 20% of the genome, and nearly half of the mammalian genome has stemmed from L1-mediated mobilization. Expression and retrotransposition of L1 are suppressed by complex mechanisms, where the key role belongs to DNA methylation. Alterations in L1 methylation may lead to aberrant expression of L1 and have been described in numerous diseases. Accumulating evidence clearly indicates that loss of global DNA methylation observed in cancer development and progression is tightly associated with hypomethylation of L1 elements. Significant progress achieved in the last several years suggests that such parameters as L1 methylation status can be potentially utilized as clinical biomarkers for determination of the disease stage and in predicting the disease-free survival in cancer patients. In this paper, we summarize the current knowledge on L1 methylation, with specific emphasis given to success and challenges on the way of introduction of L1 into clinical practice.

Mutation Processes in 293-Based Clones Overexpressing the DNA Cytosine Deaminase APOBEC3B.

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Monica K Akre

Full Text Available Molecular, cellular, and clinical studies have combined to demonstrate a contribution from the DNA cytosine deaminase APOBEC3B (A3B to the overall mutation load in breast, head/neck, lung, bladder, cervical, ovarian, and other cancer types. However, the complete landscape of mutations attributable to this enzyme has yet to be determined in a controlled human cell system. We report a conditional and isogenic system for A3B induction, genomic DNA deamination, and mutagenesis. Human 293-derived cells were engineered to express doxycycline-inducible A3B-eGFP or eGFP constructs. Cells were subjected to 10 rounds of A3B-eGFP exposure that each caused 80-90% cell death. Control pools were subjected to parallel rounds of non-toxic eGFP exposure, and dilutions were done each round to mimic A3B-eGFP induced population fluctuations. Targeted sequencing of portions of TP53 and MYC demonstrated greater mutation accumulation in the A3B-eGFP exposed pools. Clones were generated and microarray analyses were used to identify those with the greatest number of SNP alterations for whole genome sequencing. A3B-eGFP exposed clones showed global increases in C-to-T transition mutations, enrichments for cytosine mutations within A3B-preferred trinucleotide motifs, and more copy number aberrations. Surprisingly, both control and A3B-eGFP clones also elicited strong mutator phenotypes characteristic of defective mismatch repair. Despite this additional mutational process, the 293-based system characterized here still yielded a genome-wide view of A3B-catalyzed mutagenesis in human cells and a system for additional studies on the compounded effects of simultaneous mutation mechanisms in cancer cells.

Endogenous 5-methylcytosine protects neighboring guanines from N7 and O6-methylation and O6-pyridyloxobutylation by the tobacco carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone.

Science.gov (United States)

Ziegel, Rebecca; Shallop, Anthony; Upadhyaya, Pramod; Jones, Roger; Tretyakova, Natalia

2004-01-20

All CG dinucleotides along exons 5-8 of the p53 tumor suppressor gene contain endogenous 5-methylcytosine (MeC). These same sites (e.g., codons 157, 158, 245, 248, and 273) are mutational hot spots in smoking-induced lung cancer. Several groups used the UvrABC endonuclease incision assay to demonstrate that methylated CG dinucleotides of the p53 gene are the preferred binding sites for the diol epoxides of bay region polycyclic aromatic hydrocarbons (PAH). In contrast, effects of endogenous cytosine methylation on the distribution of DNA lesions induced by tobacco-specific nitrosamines, e.g., 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), have not been elucidated. In the work presented here, a stable isotope labeling HPLC-ESI-MS/MS approach was employed to analyze the reactivity of the N7 and O6 positions of guanines within hemimethylated and fully methylated CG dinucleotides toward NNK-derived methylating and pyridyloxobutylating species. 15N3-labeled guanine bases were placed within synthetic DNA sequences representing endogenously methylated p53 codons 154, 157, and 248, followed by treatment with acetylated precursors to NNK diazohydroxides. HPLC-ESI-MS/MS analysis was used to determine the relative yields of N7- and O6-guanine adducts at the 15N3-labeled position. In all cases, the presence of MeC inhibited the formation of N7-methylguanine, O6-methylguanine, and O6-pyridyloxobutylguanine at a neighboring G, with the greatest decrease observed in fully methylated dinucleotides and at guanines preceded by MeC. Furthermore, the O6-Me-dG/N7-Me-G molar ratios were decreased in the presence of the 5'-neighboring MeC, suggesting that the observed decline in O6-alkylguanine adduct yields is, at least partially, a result of an altered reactivity pattern in methylated CG dinucleotides. These results indicate that, unlike N2-guanine adducts of PAH diol epoxides, NNK-induced N7- and O6-alkylguanine adducts are not preferentially formed at the endogenously

Fluorescent strategy based on cationic conjugated polymer fluorescence resonance energy transfer for the quantification of 5-(hydroxymethyl)cytosine in genomic DNA.

Science.gov (United States)

Hong, Tingting; Wang, Tianlu; Guo, Pu; Xing, Xiwen; Ding, Fei; Chen, Yuqi; Wu, Jinjun; Ma, Jingwei; Wu, Fan; Zhou, Xiang

2013-11-19

DNA methylation is dynamically reprogrammed during early embryonic development in mammals. It can be explained partially by the discovery of 5-(hydroxymethyl)cytosine (5-hmC), 5-formylcytosine (5-fC), and 5-carboxylcytosine (5-caC), which are identified as key players involved in both active and passive demethylation pathways. As one of the ten-eleven translocation oxidation products, 5-hmC was found relatively abundant in neuron cells and embryonic stem cells. Herein we report a new method for 5-hmC quantification in genomic DNA based on CCP-FRET (cationic conjugated polymers act as the energy donor and induce fluorescence resonance energy transfer) assay combined with KRuO4 oxidation. 5-hmC in genomic DNA can be selectively transformed into 5-fC by the oxidation of KRuO4 and then labeled with hydroxylamine-BODIPY (BODIPY = 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene) fluorophore through the reaction between 5-fC and hydroxylamine-BODIPY. After the fluorescently labeled DNA was captured by CCP through electrostatic interactions, a significant FRET between CCP and hydroxylamine-BODIPY fluorophore was observed. This CCP-FRET-based assay benefits from light-harvesting, large Stokes shift, and optical signal amplification properties of the CCP. Furthermore, this CCP-FRET-based assay was quite successfully demonstrated for the 5-hmC quantification in three types of cells (mESc, HeLa, HEK 293T), providing a much more convenient choice for 5-hmC quantification in genomic DNA.

Dynamics of self-assembled cytosine nucleobases on graphene

Science.gov (United States)

Saikia, Nabanita; Johnson, Floyd; Waters, Kevin; Pandey, Ravindra

2018-05-01

Molecular self-assembly of cytosine (C n ) bases on graphene was investigated using molecular dynamics methods. For free-standing C n bases, simulation conditions (gas versus aqueous) determine the nature of self-assembly; the bases prefer to aggregate in the gas phase and are stabilized by intermolecular H-bonds, while in the aqueous phase, the water molecules disrupt base-base interactions, which facilitate the formation of π-stacked domains. The substrate-induced effects, on the other hand, find the polarity and donor-acceptor sites of the bases to govern the assembly process. For example, in the gas phase, the assembly of C n bases on graphene displays short-range ordered linear arrays stabilized by the intermolecular H-bonds. In the aqueous phase, however, there are two distinct configurations for the C n bases assembly on graphene. For the first case corresponding to low surface coverage, the bases are dispersed on graphene and are isolated. The second configuration archetype is disordered linear arrays assembled with medium and high surface coverage. The simulation results establish the role of H-bonding, vdW π-stacking, and the influence of graphene surface towards the self-assembly. The ability to regulate the assembly into well-defined patterns can aid in the design of self-assembled nanostructures for the next-generation DNA based biosensors and nanoelectronic devices.

Histone Lysine Methylation in Diabetic Nephropathy

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Guang-dong Sun

2014-01-01

Full Text Available Diabetic nephropathy (DN belongs to debilitating microvascular complications of diabetes and is the leading cause of end-stage renal diseases worldwide. Furthermore, outcomes from the DCCT/EDIC study showed that DN often persists and progresses despite intensive glucose control in many diabetes patients, possibly as a result of prior episode of hyperglycemia, which is called “metabolic memory.” The underlying mechanisms responsible for the development and progression of DN remain poorly understood. Activation of multiple signaling pathways and key transcription factors can lead to aberrant expression of DN-related pathologic genes in target renal cells. Increasing evidence suggests that epigenetic mechanisms in chromatin such as DNA methylation, histone acetylation, and methylation can influence the pathophysiology of DN and metabolic memory. Exciting researches from cell culture and experimental animals have shown that key histone methylation patterns and the related histone methyltransferases and histone demethylases can play important roles in the regulation of inflammatory and profibrotic genes in renal cells under diabetic conditions. Because histone methylation is dynamic and potentially reversible, it can provide a window of opportunity for the development of much-needed novel therapeutic potential for DN in the future. In this minireview, we discuss recent advances in the field of histone methylation and its roles in the pathogenesis and progression of DN.

Interaction of Cu(+) with cytosine and formation of i-motif-like C-M(+)-C complexes: alkali versus coinage metals.

Science.gov (United States)

Gao, Juehan; Berden, Giel; Rodgers, M T; Oomens, Jos

2016-03-14

The Watson-Crick structure of DNA is among the most well-known molecular structures of our time. However, alternative base-pairing motifs are also known to occur, often depending on base sequence, pH, or the presence of cations. Pairing of cytosine (C) bases induced by the sharing of a single proton (C-H(+)-C) may give rise to the so-called i-motif, which occurs primarily in expanded trinucleotide repeats and the telomeric region of DNA, particularly at low pH. At physiological pH, silver cations were recently found to stabilize C dimers in a C-Ag(+)-C structure analogous to the hemiprotonated C-dimer. Here we use infrared ion spectroscopy in combination with density functional theory calculations at the B3LYP/6-311G+(2df,2p) level to show that copper in the 1+ oxidation state induces an analogous formation of C-Cu(+)-C structures. In contrast to protons and these transition metal ions, alkali metal ions induce a different dimer structure, where each ligand coordinates the alkali metal ion in a bidentate fashion in which the N3 and O2 atoms of both cytosine ligands coordinate to the metal ion, sacrificing hydrogen-bonding interactions between the ligands for improved chelation of the metal cation.

Whole-genome methylation caller designed for methyl- DNA ...

African Journals Online (AJOL)

etchie

2013-02-20

Feb 20, 2013 ... Our method uses a single-CpG-resolution, whole-genome methylation ... Key words: Methyl-DNA immunoprecipitation, next-generation sequencing, ...... methylation is prevalent in embryonic stem cells andmaybe mediated.

Global DNA methylation of ischemic stroke subtypes.

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Carolina Soriano-Tárraga

Full Text Available Ischemic stroke (IS, a heterogeneous multifactorial disorder, is among the leading causes of mortality and long-term disability in the western world. Epidemiological data provides evidence for a genetic component to the disease, but its epigenetic involvement is still largely unknown. Epigenetic mechanisms, such as DNA methylation, change over time and may be associated with aging processes and with modulation of the risk of various pathologies, such as cardiovascular disease and stroke. We analyzed 2 independent cohorts of IS patients. Global DNA methylation was measured by luminometric methylation assay (LUMA of DNA blood samples. Univariate and multivariate regression analyses were used to assess the methylation differences between the 3 most common IS subtypes, large-artery atherosclerosis (LAA, small-artery disease (SAD, and cardio-aortic embolism (CE. A total of 485 IS patients from 2 independent hospital cohorts (n = 281 and n = 204 were included, distributed across 3 IS subtypes: LAA (78/281, 59/204, SAD (97/281, 53/204, and CE (106/281, 89/204. In univariate analyses, no statistical differences in LUMA levels were observed between the 3 etiologies in either cohort. Multivariate analysis, adjusted by age, sex, hyperlipidemia, and smoking habit, confirmed the lack of differences in methylation levels between the analyzed IS subtypes in both cohorts. Despite differences in pathogenesis, our results showed no global methylation differences between LAA, SAD, and CE subtypes of IS. Further work is required to establish whether the epigenetic mechanism of methylation might play a role in this complex disease.

Epigenetic switches of tobacco transgenes associate with transient redistribution of histone marks in callus culture

Czech Academy of Sciences Publication Activity Database

Křížová, Kateřina; Depicker, A.; Kovařík, Aleš

2013-01-01

Roč. 8, č. 6 (2013), s. 666-676 ISSN 1559-2294 R&D Projects: GA ČR(CZ) GPP501/11/P667; GA ČR(CZ) GBP501/12/G090; GA ČR(CZ) GA13-10057S Institutional support: RVO:68081707 Keywords : JMJC DOMAIN PROTEIN * DNA METHYLATION * CYTOSINE METHYLATION Subject RIV: BO - Biophysics Impact factor: 5.108, year: 2013

Compromised telomere maintenance in hypomethylated Arabidopsis thaliana plants

Czech Academy of Sciences Publication Activity Database

Ogrocká, A.; Polanská, P.; Majerová, E.; Janeba, Zlatko; Fajkus, Jiří; Fojtová, M.

2014-01-01

Roč. 42, č. 5 (2014), s. 2919-2931 ISSN 0305-1048 Grant - others:GA ČR(CZ) GAP501/11/0596; GA MŠk(CZ) ED1.1.00/02.0068 Program:ED Institutional support: RVO:61388963 ; RVO:68081707 Keywords : DNA methylation * cytosine methylation * mammalian telomeres Subject RIV: CE - Biochemistry; BO - Biophysics (BFU-R) Impact factor: 9.112, year: 2014

Identification of endometrial cancer methylation features using combined methylation analysis methods.

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Michael P Trimarchi

Full Text Available DNA methylation is a stable epigenetic mark that is frequently altered in tumors. DNA methylation features are attractive biomarkers for disease states given the stability of DNA methylation in living cells and in biologic specimens typically available for analysis. Widespread accumulation of methylation in regulatory elements in some cancers (specifically the CpG island methylator phenotype, CIMP can play an important role in tumorigenesis. High resolution assessment of CIMP for the entire genome, however, remains cost prohibitive and requires quantities of DNA not available for many tissue samples of interest. Genome-wide scans of methylation have been undertaken for large numbers of tumors, and higher resolution analyses for a limited number of cancer specimens. Methods for analyzing such large datasets and integrating findings from different studies continue to evolve. An approach for comparison of findings from a genome-wide assessment of the methylated component of tumor DNA and more widely applied methylation scans was developed.Methylomes for 76 primary endometrial cancer and 12 normal endometrial samples were generated using methylated fragment capture and second generation sequencing, MethylCap-seq. Publically available Infinium HumanMethylation 450 data from The Cancer Genome Atlas (TCGA were compared to MethylCap-seq data.Analysis of methylation in promoter CpG islands (CGIs identified a subset of tumors with a methylator phenotype. We used a two-stage approach to develop a 13-region methylation signature associated with a "hypermethylator state." High level methylation for the 13-region methylation signatures was associated with mismatch repair deficiency, high mutation rate, and low somatic copy number alteration in the TCGA test set. In addition, the signature devised showed good agreement with previously described methylation clusters devised by TCGA.We identified a methylation signature for a "hypermethylator phenotype" in

Methyl gallate is a natural constituent of maple (Genus Acer) leaves.

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Abou-Zaid, Mamdouh M; Lombardo, Domenic A; Nozzolillo, Constance

2009-01-01

Methyl gallate was found in ethanolic extracts of red maple (Acer rubrum L.), silver maple (A. saccharinum L.) and sugar maple (A. saccharum Marsh) leaves, but more was present in methanolic extracts. The increased amount of methyl gallate in methanolic extracts was accompanied by a disappearance of m-digallate. It is concluded that only some of the methyl gallate detected in methanolic extracts is an artefact as a result of methanolysis of m-digallate. Its presence in ethanolic extracts is evidence that it is also a natural constituent of maple leaves.

Effect of alginate microencapsulation on the catalytic efficiency and in vitro enzyme-prodrug therapeutic efficacy of cytosine deaminase and of recombinant E. coli expressing cytosine deaminase.

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Funaro, Michael G; Nemani, Krishnamurthy V; Chen, Zhihang; Bhujwalla, Zaver M; Griswold, Karl E; Gimi, Barjor

2016-02-01

Cytosine deaminase (CD) catalyses the enzymatic conversion of the non-toxic prodrug 5-fluorocytosine (5-FC) to the potent chemotherapeutic form, 5-fluorouracil (5-FU). Intratumoral delivery of CD localises chemotherapy dose while reducing systemic toxicity. Encapsulation in biocompatible microcapsules immunoisolates CD and protects it from degradation. We report on the effect of alginate encapsulation on the catalytic and functional activity of isolated CD and recombinant E. coli engineered to express CD (E. coli(CD)). Alginate microcapsules containing either CD or Escherichia coli(CD) were prepared using ionotropic gelation. Conversion of 5-FC to 5-FU was quantitated in unencapsulated and encapsulated CD/E. coli(CD) using spectrophotometry, with a slower rate of conversion observed following encapsulation. Both encapsulated CD/5-FC and E. coli(CD)/5-FC resulted in cell kill and reduced proliferation of 9 L rat glioma cells, which was comparable to direct 5-FU treatment. Our results show that encapsulation preserves the therapeutic potential of CD and E. coli(CD) is equally effective for enzyme-prodrug therapy.

Epigenetic variability in the genetically uniform forest tree species Pinus pinea L.

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Sáez-Laguna, Enrique; Guevara, María-Ángeles; Díaz, Luis-Manuel; Sánchez-Gómez, David; Collada, Carmen; Aranda, Ismael; Cervera, María-Teresa

2014-01-01

There is an increasing interest in understanding the role of epigenetic variability in forest species and how it may contribute to their rapid adaptation to changing environments. In this study we have conducted a genome-wide analysis of cytosine methylation pattern in Pinus pinea, a species characterized by very low levels of genetic variation and a remarkable degree of phenotypic plasticity. DNA methylation profiles of different vegetatively propagated trees from representative natural Spanish populations of P. pinea were analyzed with the Methylation Sensitive Amplified Polymorphism (MSAP) technique. A high degree of cytosine methylation was detected (64.36% of all scored DNA fragments). Furthermore, high levels of epigenetic variation were observed among the studied individuals. This high epigenetic variation found in P. pinea contrasted with the lack of genetic variation based on Amplified Fragment Length Polymorphism (AFLP) data. In this manner, variable epigenetic markers clearly discriminate individuals and differentiates two well represented populations while the lack of genetic variation revealed with the AFLP markers fail to differentiate at both, individual or population levels. In addition, the use of different replicated trees allowed identifying common polymorphic methylation sensitive MSAP markers among replicates of a given propagated tree. This set of MSAPs allowed discrimination of the 70% of the analyzed trees.

Epigenetic variability in the genetically uniform forest tree species Pinus pinea L.

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Enrique Sáez-Laguna

Full Text Available There is an increasing interest in understanding the role of epigenetic variability in forest species and how it may contribute to their rapid adaptation to changing environments. In this study we have conducted a genome-wide analysis of cytosine methylation pattern in Pinus pinea, a species characterized by very low levels of genetic variation and a remarkable degree of phenotypic plasticity. DNA methylation profiles of different vegetatively propagated trees from representative natural Spanish populations of P. pinea were analyzed with the Methylation Sensitive Amplified Polymorphism (MSAP technique. A high degree of cytosine methylation was detected (64.36% of all scored DNA fragments. Furthermore, high levels of epigenetic variation were observed among the studied individuals. This high epigenetic variation found in P. pinea contrasted with the lack of genetic variation based on Amplified Fragment Length Polymorphism (AFLP data. In this manner, variable epigenetic markers clearly discriminate individuals and differentiates two well represented populations while the lack of genetic variation revealed with the AFLP markers fail to differentiate at both, individual or population levels. In addition, the use of different replicated trees allowed identifying common polymorphic methylation sensitive MSAP markers among replicates of a given propagated tree. This set of MSAPs allowed discrimination of the 70% of the analyzed trees.

Methylation of food commodities during fumigation with methyl bromide

International Nuclear Information System (INIS)

Starratt, A.N.; Bond, E.J.

1990-01-01

Sites of methylation in several commodities (wheat, oatmeal, peanuts, almonds, apples, oranges, maize, alfalfa and potatoes) during fumigation with 14 C-methyl bromide were studied. Differences were observed in levels of the major volatiles: methanol, dimethyl sulphide and methyl mercaptan, products of O- and S-methylation, resulting from treatment of the fumigated materials with 1N sodium hydroxide. In studies of maize and wheat, histidine was the amino acid which underwent the highest level of N-methylation. (author). 24 refs, 3 tabs

Detection of hypoxanthine, xanthine and uric acid in γ-irradiated aqueous solution of cytosine

International Nuclear Information System (INIS)

Kobayashi, Tsuya; Shirai, Kazuo

1979-01-01

The aqueous solution of cytosine of 3.6 x 10 -2 M was irradiated with gamma -ray (60 megarad) in nitrogen-saturated glass ampules, and freeze-dried, then the residue obtained was changed to trimethylsilylacid, and this was analyzed by paper chromatography, UV spectrometry, and/or gas-liquid chromatography. Hypoxanthine, xanthine and uric acid were detected in this solution, in addition to some other compounds already known to be produced by gamma -irradiation, e.g., TMS-uracil, TMS-6-hydroxyuracil and TMS-hypoxanthine. It was presumed that these compounds were formed by the recombination of the primary radiolytic products. Uric acid formation by this mechanism was confirmed by gamma -irradiation of the mixture that contained urea, and 5- and 6-hydroxyuracil. (Kaihara, S.)

Epigenetics and Epigenomics of Plants.

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Yadav, Chandra Bhan; Pandey, Garima; Muthamilarasan, Mehanathan; Prasad, Manoj

2018-01-23

The genetic material DNA in association with histone proteins forms the complex structure called chromatin, which is prone to undergo modification through certain epigenetic mechanisms including cytosine DNA methylation, histone modifications, and small RNA-mediated methylation. Alterations in chromatin structure lead to inaccessibility of genomic DNA to various regulatory proteins such as transcription factors, which eventually modulates gene expression. Advancements in high-throughput sequencing technologies have provided the opportunity to study the epigenetic mechanisms at genome-wide levels. Epigenomic studies using high-throughput technologies will widen the understanding of mechanisms as well as functions of regulatory pathways in plant genomes, which will further help in manipulating these pathways using genetic and biochemical approaches. This technology could be a potential research tool for displaying the systematic associations of genetic and epigenetic variations, especially in terms of cytosine methylation onto the genomic region in a specific cell or tissue. A comprehensive study of plant populations to correlate genotype to epigenotype and to phenotype, and also the study of methyl quantitative trait loci (QTL) or epiGWAS, is possible by using high-throughput sequencing methods, which will further accelerate molecular breeding programs for crop improvement. Graphical Abstract.

Effect of anionic surfactants on the process of Fenton degradation of methyl orange.

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Yang, C W; Wang, D

2009-01-01

Fenton process has been shown to be very successful to remove dyes from water. However, the influence of other constituents in dyeing industry wastewater, such as Sodium Dodecyl Sulphate (SDS) surfactants, has not been investigated. In this study, the effect of SDS surfactant on the kinetics of Methyl Orange degradation undergoing Fenton process was investigated. Results show that Methyl Orange degradation rate decreased as SDS concentration increased, which was attributed to the consumption of hydroxyl radicals (OH) by surfactants and the formation of Methyl Orange-SDS complex. No evidence was found that the Methyl Orange degradation pathway was affected by the presence of SDS. The kinetics modelling indicates the reaction was the first-order reaction to Methyl Orange.

Diet and Asthma: Vitamins and Methyl Donors

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Han, Yueh-Ying; Blatter, Josh; Brehm, John M.; Forno, Erick; Litonjua, Augusto A; Celedón, Juan C.

2014-01-01

SUMMARY Dietary changes may partly explain the high burden of asthma in industrialized nations. Experimental studies have motivated a significant number of observational studies of the relation between vitamins (A, C, D, and E) or nutrients acting as methyl donors (folate, vitamin B12, and choline) and asthma. Because observational studies are susceptible to several sources of bias, well-conducted randomized controlled trials (RCTs) remain the “gold standard” to determine whether a vitamin or nutrient has an effect on asthma. Evidence from observational studies and/or relatively few RCTs most strongly justify ongoing and future RCTs of: 1) vitamin D to prevent or treat asthma, 2) choline supplementation as adjuvant treatment for asthma, and 3) vitamin E to prevent the detrimental effects of air pollution in subjects with asthma. At this time, there is insufficient evidence to recommend supplementation with any vitamin or nutrient acting as a methyl donor to prevent or treat asthma. PMID:24461761

Effect of D-valine and cytosine arabinoside on [3H]thymidine incorporation in rat and rabbit epididymal epithelial cell cultures

International Nuclear Information System (INIS)

Orgebin-Crist, M.C.; Jonas-Davies, J.; Storey, P.; Olson, G.E.

1984-01-01

Epithelial cell enriched primary cultures were established from the rat and the rabbit epididymis. Epithelial cell aggregates, obtained after pronase digestion of minced epididymis, attached to the culture dish and after 72 h in vitro spread out to form discrete patches of cells. These cells have an epithelioid morphology and form a monolayer of closely apposed polygonal cells where DNA synthesis, as judged by [ 3 H]thymidine uptake, is very low. In L-valine medium the nonepithelial cell contamination was no more than 10% in rat and rabbit epididymal primary cultures. The labeling index of rat epididymal cells cultured in D-valine medium was significantly lower than that of cells cultured in L-valine medium. In contrast, the labeling index of rabbit epididymal cells cultured in D-valine medium was significantly higher than that of cells cultured in L-valine medium. Cytosine arabinoside decreased the number of labeled cells in both L-valine and D-valine cultures. From these results, it appears that D-valine is a selective agent for rat epididymal epithelial cells, but not for rabbit epithelial cells, and that cytosine arabinoside is a simple and effective means to control the proliferation of fibroblast-like cells in both rat and rabbit epididymal cell cultures

The dynamics of smoking-related disturbed methylation: a two time-point study of methylation change in smokers, non-smokers and former smokers.

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Wilson, Rory; Wahl, Simone; Pfeiffer, Liliane; Ward-Caviness, Cavin K; Kunze, Sonja; Kretschmer, Anja; Reischl, Eva; Peters, Annette; Gieger, Christian; Waldenberger, Melanie

2017-10-18

The evidence for epigenome-wide associations between smoking and DNA methylation continues to grow through cross-sectional studies. However, few large-scale investigations have explored the associations using observations for individuals at multiple time-points. Here, through the use of the Illumina 450K BeadChip and data collected at two time-points separated by approximately 7 years, we investigate changes in methylation over time associated with quitting smoking or remaining a former smoker, and those associated with continued smoking. Our results indicate that after quitting smoking the most rapid reversion of altered methylation occurs within the first two decades, with reversion rates related to the initial differences in methylation. For 52 CpG sites, the change in methylation from baseline to follow-up is significantly different for former smokers relative to the change for never smokers (lowest p-value 3.61 x 10 -39 for cg26703534, gene AHRR). Most of these sites' respective regions have been previously implicated in smoking-associated diseases. Despite the early rapid change, dynamism of methylation appears greater in former smokers vs never smokers even four decades after cessation. Furthermore, our study reveals the heterogeneous effect of continued smoking: the methylation levels of some loci further diverge between smokers and non-smokers, while others re-approach. Though intensity of smoking habit appears more significant than duration, results remain inconclusive. This study improves the understanding of the dynamic link between cigarette smoking and methylation, revealing the continued fluctuation of methylation levels decades after smoking cessation and demonstrating that continuing smoking can have an array of effects. The results can facilitate insights into the molecular mechanisms behind smoking-induced disturbed methylation, improving the possibility for development of biomarkers of past smoking behavior and increasing the understanding of

Morphological and cytosine DNA methylation changes induced by a ...

African Journals Online (AJOL)

Boron (B) toxicity is one of the abiotic stresses limiting plant growth in arid and semi arid regions globally. Although studies have been conducted on the combined effect of B and sodium chloride (NaCl) toxicity on overall plant growth revealing an antagonistic relationship, the morphology and epigenetic interactions have ...

The Dynamics of Smoking-Related Disturbed Methylation: A Two Time-Point Study of Methylation Change in Smokers, Non-Smokers and Former Smokers

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BACKGROUND: The evidence for epigenome-wide associations between smoking and DNA methylation continues to grow through cross-sectional studies. However, few large­ scale investigations have explored the associations using observations for individuals at multiple time-points. ...

MethylMix 2.0: an R package for identifying DNA methylation genes.

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Cedoz, Pierre-Louis; Prunello, Marcos; Brennan, Kevin; Gevaert, Olivier

2018-04-14

DNA methylation is an important mechanism regulating gene transcription, and its role in carcinogenesis has been extensively studied. Hyper and hypomethylation of genes is a major mechanism of gene expression deregulation in a wide range of diseases. At the same time, high-throughput DNA methylation assays have been developed generating vast amounts of genome wide DNA methylation measurements. We developed MethylMix, an algorithm implemented in R to identify disease specific hyper and hypomethylated genes. Here we present a new version of MethylMix that automates the construction of DNA-methylation and gene expression datasets from The Cancer Genome Atlas (TCGA). More precisely, MethylMix 2.0 incorporates two major updates: the automated downloading of DNA methylation and gene expression datasets from TCGA and the automated preprocessing of such datasets: value imputation, batch correction and CpG sites clustering within each gene. The resulting datasets can subsequently be analyzed with MethylMix to identify transcriptionally predictive methylation states. We show that the Differential Methylation Values created by MethylMix can be used for cancer subtyping. olivier.gevaert@stanford.edu. https://bioconductor.org/packages/release/bioc/manuals/MethylMix/man/MethylMix.pdf. MethylMix 2.0 was implemented as an R package and is available in bioconductor.

Global DNA methylation analysis using methyl-sensitive amplification polymorphism (MSAP).

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Yaish, Mahmoud W; Peng, Mingsheng; Rothstein, Steven J

2014-01-01

DNA methylation is a crucial epigenetic process which helps control gene transcription activity in eukaryotes. Information regarding the methylation status of a regulatory sequence of a particular gene provides important knowledge of this transcriptional control. DNA methylation can be detected using several methods, including sodium bisulfite sequencing and restriction digestion using methylation-sensitive endonucleases. Methyl-Sensitive Amplification Polymorphism (MSAP) is a technique used to study the global DNA methylation status of an organism and hence to distinguish between two individuals based on the DNA methylation status determined by the differential digestion pattern. Therefore, this technique is a useful method for DNA methylation mapping and positional cloning of differentially methylated genes. In this technique, genomic DNA is first digested with a methylation-sensitive restriction enzyme such as HpaII, and then the DNA fragments are ligated to adaptors in order to facilitate their amplification. Digestion using a methylation-insensitive isoschizomer of HpaII, MspI is used in a parallel digestion reaction as a loading control in the experiment. Subsequently, these fragments are selectively amplified by fluorescently labeled primers. PCR products from different individuals are compared, and once an interesting polymorphic locus is recognized, the desired DNA fragment can be isolated from a denaturing polyacrylamide gel, sequenced and identified based on DNA sequence similarity to other sequences available in the database. We will use analysis of met1, ddm1, and atmbd9 mutants and wild-type plants treated with a cytidine analogue, 5-azaC, or zebularine to demonstrate how to assess the genetic modulation of DNA methylation in Arabidopsis. It should be noted that despite the fact that MSAP is a reliable technique used to fish for polymorphic methylated loci, its power is limited to the restriction recognition sites of the enzymes used in the genomic

New Insights into 5hmC DNA Modification: Generation, Distribution and Function

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Dong-Qiao Shi

2017-07-01

Full Text Available Dynamic DNA modifications, such as methylation/demethylation on cytosine, are major epigenetic mechanisms to modulate gene expression in both eukaryotes and prokaryotes. In addition to the common methylation on the 5th position of the pyrimidine ring of cytosine (5mC, other types of modifications at the same position, such as 5-hydroxymethyl (5hmC, 5-formyl (5fC, and 5-carboxyl (5caC, are also important. Recently, 5hmC, a product of 5mC demethylation by the Ten-Eleven Translocation family proteins, was shown to regulate many cellular and developmental processes, including the pluripotency of embryonic stem cells, neuron development, and tumorigenesis in mammals. Here, we review recent advances on the generation, distribution, and function of 5hmC modification in mammals and discuss its potential roles in plants.

Phosphatidylcholine synthesis in the rat: The substrate for methylation and regulation by choline

International Nuclear Information System (INIS)

Datko, A.H.; Aksamit, R.R.; Mudd, S.H.

1990-01-01

Two lines of evidence led us to reexamine the possibility that methylation of phosphoethanolamine and its partially methylated derivatives, in addition to methylation of the corresponding phosphatidyl derivatives, plays a role in mammalian phosphatidylcholine biosynthesis: (a) Results obtained by Salerno and Beeler with rat appear to strongly support such a role for methylation of phosphobases; (b) Such reactions have recently been shown to play major roles in phosphatidylcholine synthesis by higher plants. We found that, following continuous labeling of rat liver with L-[methyl-3H]methionine for 10.4 min (intraperitoneal administration) or for 0.75 min (intraportal administration), virtually no 3H was detected in methylated derivatives of phosphoethanolamine, but readily detectable amounts of 3H were present in the base moiety of each methylated derivative of phosphatidylethanolamine. Thus, there was no indication that phospho-base methylation makes a significant contribution. Studies of cultured rat hepatoma cells showed definitively for the first time in a mammalian system that choline deprivation up-regulates the rate of flow of methyl groups originating in methionine into phosphatidylethanolamine and derivatives. Even under these conditions, methylation of phosphoethanolamine bases appeared to play a negligible role

Rationalizing the structural variability of the exocyclic amino groups in nucleobases and their metal complexes: cytosine and adenine.

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Fonseca Guerra, Célia; Sanz Miguel, Pablo J; Cebollada, Andrea; Bickelhaupt, F Matthias; Lippert, Bernhard

2014-07-28

The exocyclic amino groups of cytosine and adenine nucleobases are normally almost flat, with the N atoms essentially sp(2) hybridized and the lone pair largely delocalized into the heterocyclic rings. However, a change to marked pyramidality of the amino group (N then sp(3) hybridized, lone pair essentially localized at N) occurs during i) involvement of an amino proton in strong hydrogen bonding donor conditions or ii) with monofunctional metal coordination following removal of one of the two protons. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The inhibition of DNA repair by aphidicolin or cytosine arabinoside in X-irradiated normal and xeroderma pigmentosum fibroblasts

International Nuclear Information System (INIS)

Waters, R.; Crocombe, K.; Mirzayans, R.

1981-01-01

Normal and excision-deficient xeroderma pigmentosum fibroblasts were X-irradiated and the influence on DNA repair of either the repair inhibitor cytosine arabinoside or the specific inhibitor of DNA polymerase α, aphidicolin, investigated. The data indicated that the repair of a certain fraction of X-ray-induced lesions can be inhibited in both cell lines by both compounds. Thus, as aphidicolin blocks the operation of polymerase α, this enzyme must be involved in an excision repair pathway operating in both normal and excision-deficient xeroderma pigmentosum cells. (orig.)

Hypermethylation of E-Cadherin and Estrogen Receptor-a Gene Promoter and Its Association with Clinicopathological Features of Breast Cancer in Iranian Patients

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Mozhgan Rasti

2009-06-01

Full Text Available Background: Aberrant methylation of cytosine-guanine dinucleotideislands leads to inactivation of tumor suppressorgenes in breast cancer. Tumor suppressor genes are unmethylatedin normal tissue and often become hypermethylatedduring tumor formation, leading to gene silencing. We investigatedthe association between E-cadherin (CDH1 and estrogenreceptor-α (ESRα gene promoter methylation andmajor clinical and pathological features of breast cancer inIranian women.Methods: DNA was extracted from 67 primary breast tumorsand gene promoter methylation was analyzed by methylationspecificpolymerase chain reaction method.Results: Fifty percent of the samples showed aberrant methylationin at least one of the two tested loci. We detectedCDH1 hypermethylation in 41% of invasive tumors and receptor-α gene hypermethylation in 18% of invasive tumorsamples. We found no association between CDH1 and receptor-α gene hypermethylation (P=0.45. There was a correlationbetween hypermethylation of CDH1 locus and tumorsize ≥5 cm (P=0.019.Conclusion: Our data suggest that the malignant progressionof human ductal and lobular breast carcinoma in Iranianwomen involves a heterogeneous pattern of cytosine-guaninedinucleotide island hypermethylation of the CDH1 gene.

Age-associated sperm DNA methylation alterations: possible implications in offspring disease susceptibility.

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Jenkins, Timothy G; Aston, Kenneth I; Pflueger, Christian; Cairns, Bradley R; Carrell, Douglas T

2014-07-01

Recent evidence demonstrates a role for paternal aging on offspring disease susceptibility. It is well established that various neuropsychiatric disorders (schizophrenia, autism, etc.), trinucleotide expansion associated diseases (myotonic dystrophy, Huntington's, etc.) and even some forms of cancer have increased incidence in the offspring of older fathers. Despite strong epidemiological evidence that these alterations are more common in offspring sired by older fathers, in most cases the mechanisms that drive these processes are unclear. However, it is commonly believed that epigenetics, and specifically DNA methylation alterations, likely play a role. In this study we have investigated the impact of aging on DNA methylation in mature human sperm. Using a methylation array approach we evaluated changes to sperm DNA methylation patterns in 17 fertile donors by comparing the sperm methylome of 2 samples collected from each individual 9-19 years apart. With this design we have identified 139 regions that are significantly and consistently hypomethylated with age and 8 regions that are significantly hypermethylated with age. A representative subset of these alterations have been confirmed in an independent cohort. A total of 117 genes are associated with these regions of methylation alterations (promoter or gene body). Intriguingly, a portion of the age-related changes in sperm DNA methylation are located at genes previously associated with schizophrenia and bipolar disorder. While our data does not establish a causative relationship, it does raise the possibility that the age-associated methylation of the candidate genes that we observe in sperm might contribute to the increased incidence of neuropsychiatric and other disorders in the offspring of older males. However, further study is required to determine whether, and to what extent, a causative relationship exists.

MECP2 promoter methylation and X chromosome inactivation in autism.

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Nagarajan, Raman P; Patzel, Katherine A; Martin, Michelle; Yasui, Dag H; Swanberg, Susan E; Hertz-Picciotto, Irva; Hansen, Robin L; Van de Water, Judy; Pessah, Isaac N; Jiang, Ruby; Robinson, Wendy P; LaSalle, Janine M

2008-06-01

Epigenetic mechanisms have been proposed to play a role in the etiology of autism. This hypothesis is supported by the discovery of increased MECP2 promoter methylation associated with decreased MeCP2 protein expression in autism male brain. To further understand the influence of female X chromosome inactivation (XCI) and neighboring methylation patterns on aberrant MECP2 promoter methylation in autism, multiple methylation analyses were peformed on brain and blood samples from individuals with autism. Bisulfite sequencing analyses of a region 0.6 kb upstream of MECP2 in brain DNA samples revealed an abrupt transition from a highly methylated region in both sexes to a region unmethylated in males and subject to XCI in females. Chromatin immunoprecipitation analysis demonstrated that the CCTC-binding factor (CTCF) bound to this transition region in neuronal cells, consistent with a chromatin boundary at the methylation transition. Male autism brain DNA samples displayed a slight increase in methylation in this transition region, suggesting a possible aberrant spreading of methylation into the MECP2 promoter in autism males across this boundary element. In addition, autistic female brain DNA samples showed evidence for aberrant MECP2 promoter methylation as an increase in the number of bisulfite sequenced clones with undefined XCI status for MECP2 but not androgen receptor (AR). To further investigate the specificity of MECP2 methylation alterations in autism, blood DNA samples from females and mothers of males with autism were also examined for XCI skewing at AR, but no significant increase in XCI skewing was observed compared to controls. These results suggest that the aberrant MECP2 methylation in autism brain DNA samples is due to locus-specific rather than global X chromosome methylation changes.

Arsenic (+3 oxidation state) methyltransferase and the inorganic arsenic methylation phenotype

International Nuclear Information System (INIS)

Li Jiaxin; Waters, Stephen B.; Drobna, Zuzana; Devesa, Vicenta; Styblo, Miroslav; Thomas, David J.

2005-01-01

Inorganic arsenic is enzymatically methylated; hence, its ingestion results in exposure to the parent compound and various methylated arsenicals. Both experimental and epidemiological evidences suggest that some of the adverse health effects associated with chronic exposure to inorganic arsenic may be mediated by these methylated metabolites. If i As methylation is an activation process, then the phenotype for inorganic arsenic methylation may determine risk associated with exposure to this metalloid. We examined inorganic arsenic methylation phenotypes and arsenic (+3 oxidation state) methyltransferase genotypes in four species: three that methylate inorganic arsenic (human (Homo sapiens), rat (Rattus norwegicus), and mouse (Mus musculus)) and one that does not methylate inorganic arsenic (chimpanzee, Pan troglodytes). The predicted protein products from arsenic (+3 oxidation state) methyltransferase are similar in size for rat (369 amino acid residues), mouse (376 residues), and human (375 residues). By comparison, a 275-nucleotide deletion beginning at nucleotide 612 in the chimpanzee gene sequence causes a frameshift that leads to a nonsense mutation for a premature stop codon after amino acid 205. The null phenotype for inorganic arsenic methylation in the chimpanzee is likely due to the deletion in the gene for arsenic (+3 oxidation state) methyltransferase that yields an inactive truncated protein. This lineage-specific loss of function caused by the deletion event must have occurred in the Pan lineage after Homo-Pan divergence about 5 million years ago

miR-125b targets DNMT3b and mediates p53 DNA methylation involving in the vascular smooth muscle cells proliferation induced by homocysteine

Energy Technology Data Exchange (ETDEWEB)

Cao, ChengJian [Key Laboratory of Basic Research in Cardio-Cerebral Vascular Diseases, Ningxia Medical University, Yinchuan (China); Zhang, HuiPing [Department of Prenatal Diagnosis Center, General Hospital of Ningxia Medical University, Yinchuan (China); Zhao, Li [Department of Medical Laboratory, Ningxia Medical University, Yinchuan (China); Zhou, Longxia [Department of Basic Medicine, Ningxia Medical University, Yinchuan (China); Zhang, Minghao; Xu, Hua [Key Laboratory of Basic Research in Cardio-Cerebral Vascular Diseases, Ningxia Medical University, Yinchuan (China); Department of Basic Medicine, Ningxia Medical University, Yinchuan (China); Han, Xuebo [Department of Medical Laboratory, Ningxia Medical University, Yinchuan (China); Li, Guizhong; Yang, Xiaoling [Key Laboratory of Basic Research in Cardio-Cerebral Vascular Diseases, Ningxia Medical University, Yinchuan (China); Department of Basic Medicine, Ningxia Medical University, Yinchuan (China); Jiang, YiDeng, E-mail: jyjcyxy@yeah.net [Key Laboratory of Basic Research in Cardio-Cerebral Vascular Diseases, Ningxia Medical University, Yinchuan (China); Department of Basic Medicine, Ningxia Medical University, Yinchuan (China)

2016-09-10

MicroRNAs (miRNAs) are short non-coding RNA and play crucial roles in a wide array of biological processes, including cell proliferation, differentiation and apoptosis. Our previous studies found that homocysteine(Hcy) can stimulate the proliferation of vascular smooth muscle cells (VSMCs), however, the underlying mechanisms were not fully elucidated. Here, we found proliferation of VSMCs induced by Hcy was of correspondence to the miR-125b expression reduced both in vitro and in the ApoE knockout mice, the hypermethylation of p53, its decreased expression, and DNA (cytosine-5)-methyltransferase 3b (DNMT3b) up-regulated. And, we found DNMT3b is a target of miR-125b, which was verified by the Dual-Luciferase reporter assay and western blotting. Besides, the siRNA interference for DNMT3b significantly decreased the methylation level of p53, which unveiled the causative role of DNMT3b in p53 hypermethylation. miR-125b transfection further confirmed its regulative roles on p53 gene methylation status and the VSMCs proliferation. Our data suggested that a miR-125b-DNMT3b-p53 signal pathway may exist in the VSMCs proliferation induced by Hcy.

miR-125b targets DNMT3b and mediates p53 DNA methylation involving in the vascular smooth muscle cells proliferation induced by homocysteine

International Nuclear Information System (INIS)

Cao, ChengJian; Zhang, HuiPing; Zhao, Li; Zhou, Longxia; Zhang, Minghao; Xu, Hua; Han, Xuebo; Li, Guizhong; Yang, Xiaoling; Jiang, YiDeng

2016-01-01

MicroRNAs (miRNAs) are short non-coding RNA and play crucial roles in a wide array of biological processes, including cell proliferation, differentiation and apoptosis. Our previous studies found that homocysteine(Hcy) can stimulate the proliferation of vascular smooth muscle cells (VSMCs), however, the underlying mechanisms were not fully elucidated. Here, we found proliferation of VSMCs induced by Hcy was of correspondence to the miR-125b expression reduced both in vitro and in the ApoE knockout mice, the hypermethylation of p53, its decreased expression, and DNA (cytosine-5)-methyltransferase 3b (DNMT3b) up-regulated. And, we found DNMT3b is a target of miR-125b, which was verified by the Dual-Luciferase reporter assay and western blotting. Besides, the siRNA interference for DNMT3b significantly decreased the methylation level of p53, which unveiled the causative role of DNMT3b in p53 hypermethylation. miR-125b transfection further confirmed its regulative roles on p53 gene methylation status and the VSMCs proliferation. Our data suggested that a miR-125b-DNMT3b-p53 signal pathway may exist in the VSMCs proliferation induced by Hcy.

Epigenomic profiling of DNA methylation in paired prostate cancer versus adjacent benign tissue.

Science.gov (United States)

Geybels, Milan S; Zhao, Shanshan; Wong, Chao-Jen; Bibikova, Marina; Klotzle, Brandy; Wu, Michael; Ostrander, Elaine A; Fan, Jian-Bing; Feng, Ziding; Stanford, Janet L

2015-12-01

Aberrant DNA methylation may promote prostate carcinogenesis. We investigated epigenome-wide DNA methylation profiles in prostate cancer (PCa) compared to adjacent benign tissue to identify differentially methylated CpG sites. The study included paired PCa and adjacent benign tissue samples from 20 radical prostatectomy patients. Epigenetic profiling was done using the Infinium HumanMethylation450 BeadChip. Linear models that accounted for the paired study design and False Discovery Rate Q-values were used to evaluate differential CpG methylation. mRNA expression levels of the genes with the most differentially methylated CpG sites were analyzed. In total, 2,040 differentially methylated CpG sites were identified in PCa versus adjacent benign tissue (Q-value Cancer Genome Atlas (TCGA) data provided confirmatory evidence for our findings. This study of PCa versus adjacent benign tissue showed many differentially methylated CpGs and regions in and outside gene promoter regions, which may potentially be used for the development of future epigenetic-based diagnostic tests or as therapeutic targets. © 2015 Wiley Periodicals, Inc.

Systemic effects of chronically administered methyl prednisolonate and methyl 17-deoxyprednisolonate.

Science.gov (United States)

Olejniczak, E; Lee, H J

1984-06-01

The systemic activities of methyl prednisolonate and methyl 17-deoxyprednisolonate (1) were studied in rats. Methyl 17-deoxyprednisolonate produced significant changes in the amount of sodium ion (decreased) and potassium ion (increased) in urine; however, methyl prednisolonate had no effect on electrolyte balance. Both methyl prednisolonate and methyl 17-deoxyprednisolonate had no effect on liver glycogen content, plasma corticosterone level and relative adrenal weight. In contrast, the parent compound prednisolone caused a significant decrease in liver glycogen content, plasma corticosterone level and relative adrenal weight.

Metformin regulates global DNA methylation via mitochondrial one-carbon metabolism.

Science.gov (United States)

Cuyàs, E; Fernández-Arroyo, S; Verdura, S; García, R Á-F; Stursa, J; Werner, L; Blanco-González, E; Montes-Bayón, M; Joven, J; Viollet, B; Neuzil, J; Menendez, J A

2018-02-15

The anti-diabetic biguanide metformin may exert health-promoting effects via metabolic regulation of the epigenome. Here we show that metformin promotes global DNA methylation in non-cancerous, cancer-prone and metastatic cancer cells by decreasing S-adenosylhomocysteine (SAH), a strong feedback inhibitor of S-adenosylmethionine (SAM)-dependent DNA methyltransferases, while promoting the accumulation of SAM, the universal methyl donor for cellular methylation. Using metformin and a mitochondria/complex I (mCI)-targeted analog of metformin (norMitoMet) in experimental pairs of wild-type and AMP-activated protein kinase (AMPK)-, serine hydroxymethyltransferase 2 (SHMT2)- and mCI-null cells, we provide evidence that metformin increases the SAM:SAH ratio-related methylation capacity by targeting the coupling between serine mitochondrial one-carbon flux and CI activity. By increasing the contribution of one-carbon units to the SAM from folate stores while decreasing SAH in response to AMPK-sensed energetic crisis, metformin can operate as a metabolo-epigenetic regulator capable of reprogramming one of the key conduits linking cellular metabolism to the DNA methylation machinery.

A structure-based approach to ligand discovery for 2C-methyl-D-erythritol-2,4-cyclodiphosphate synthase: a target for antimicrobial therapy.

Science.gov (United States)

Ramsden, Nicola L; Buetow, Lori; Dawson, Alice; Kemp, Lauris A; Ulaganathan, Venkatsubramanian; Brenk, Ruth; Klebe, Gerhard; Hunter, William N

2009-04-23

The nonmevalonate route to isoprenoid biosynthesis is essential in Gram-negative bacteria and apicomplexan parasites. The enzymes of this pathway are absent from mammals, contributing to their appeal as chemotherapeutic targets. One enzyme, 2C-methyl-d-erythritol-2,4-cyclodiphosphate synthase (IspF), has been validated as a target by genetic approaches in bacteria. Virtual screening against Escherichia coli IspF (EcIspF) was performed by combining a hierarchical filtering methodology with molecular docking. Docked compounds were inspected and 10 selected for experimental validation. A surface plasmon resonance assay was developed and two weak ligands identified. Crystal structures of EcIspF complexes were determined to support rational ligand development. Cytosine analogues and Zn(2+)-binding moieties were characterized. One of the putative Zn(2+)-binding compounds gave the lowest measured K(D) to date (1.92 +/- 0.18 muM). These data provide a framework for the development of IspF inhibitors to generate lead compounds of therapeutic potential against microbial pathogens.

Direct observation of vibrational energy dispersal via methyl torsions.

Science.gov (United States)

Gardner, Adrian M; Tuttle, William D; Whalley, Laura E; Wright, Timothy G

2018-02-28

Explicit evidence for the role of methyl rotor levels in promoting energy dispersal is reported. A set of coupled zero-order vibration/vibration-torsion (vibtor) levels in the S 1 state of para -fluorotoluene ( p FT) are investigated. Two-dimensional laser-induced fluorescence (2D-LIF) and two-dimensional zero-kinetic-energy (2D-ZEKE) spectra are reported, and the assignment of the main features in both sets of spectra reveals that the methyl torsion is instrumental in providing a route for coupling between vibrational levels of different symmetry classes. We find that there is very localized, and selective, dissipation of energy via doorway states, and that, in addition to an increase in the density of states, a critical role of the methyl group is a relaxation of symmetry constraints compared to direct vibrational coupling.

Synthesis of Fischer carbene complexes of iridium by C-H bond activation of methyl and cyclic ethers: Evidence for reversible {alpha}-hydrogen migration

Energy Technology Data Exchange (ETDEWEB)

Luecke, H.F.; Arndtsen, B.A.; Burger, P.; Bergman, R.G. [Lawrence Berkeley Lab., CA (United States)]|[Univ. of California, Berkeley, CA (United States)

1996-03-13

We report here a mild and versatile route to Fischer carbene complexes of iridium via the activation of C-H bonds of methyl and cyclic ethers, along with our preliminary studies of this rare family of carbene complexes. Theoretical studies suggest that {alpha}-hydrogen migrations can be kinetically favorable if a coordinatively unsaturated species can be accessed. Thus, the lability of the triflate ligand presumably facilitates this process. Further evidence for the rapidity, as well as reversibility, of this rearrangement was obtained by NMR analysis. 20 refs.

DNA methylation changes at infertility genes in newborn twins conceived by in vitro fertilisation.

Science.gov (United States)

Castillo-Fernandez, Juan E; Loke, Yuk Jing; Bass-Stringer, Sebastian; Gao, Fei; Xia, Yudong; Wu, Honglong; Lu, Hanlin; Liu, Yuan; Wang, Jun; Spector, Tim D; Saffery, Richard; Craig, Jeffrey M; Bell, Jordana T

2017-03-24

The association of in vitro fertilisation (IVF) and DNA methylation has been studied predominantly at regulatory regions of imprinted genes and at just thousands of the ~28 million CpG sites in the human genome. We investigated the links between IVF and DNA methylation patterns in whole cord blood cells (n = 98) and cord blood mononuclear cells (n = 82) from newborn twins using genome-wide methylated DNA immunoprecipitation coupled with deep sequencing. At a false discovery rate (FDR) of 5%, we identified one significant whole blood DNA methylation change linked to conception via IVF, which was located ~3 kb upstream of TNP1, a gene previously linked to male infertility. The 46 most strongly associated signals (FDR of 25%) included a second region in a gene also previously linked to infertility, C9orf3, suggesting that our findings may in part capture the effect of parental subfertility. Using twin modelling, we observed that individual-specific environmental factors appear to be the main overall contributors of methylation variability at the FDR 25% IVF-associated differentially methylated regions, although evidence for methylation heritability was also obtained at several of these regions. We replicated previous findings of differential methylation associated with IVF at the H19/IGF2 region in cord blood mononuclear cells, and we validated the signal at C9orf3 in monozygotic twins. We also explored the impact of intracytoplasmic sperm injection on the FDR 25% signals for potential effects specific to male or female infertility factors. To our knowledge, this is the most comprehensive study of DNA methylation profiles at birth and IVF conception to date, and our results show evidence for epigenetic modifications that may in part reflect parental subfertility.

Age-associated sperm DNA methylation alterations: possible implications in offspring disease susceptibility.

Directory of Open Access Journals (Sweden)

Timothy G Jenkins

2014-07-01

Full Text Available Recent evidence demonstrates a role for paternal aging on offspring disease susceptibility. It is well established that various neuropsychiatric disorders (schizophrenia, autism, etc., trinucleotide expansion associated diseases (myotonic dystrophy, Huntington's, etc. and even some forms of cancer have increased incidence in the offspring of older fathers. Despite strong epidemiological evidence that these alterations are more common in offspring sired by older fathers, in most cases the mechanisms that drive these processes are unclear. However, it is commonly believed that epigenetics, and specifically DNA methylation alterations, likely play a role. In this study we have investigated the impact of aging on DNA methylation in mature human sperm. Using a methylation array approach we evaluated changes to sperm DNA methylation patterns in 17 fertile donors by comparing the sperm methylome of 2 samples collected from each individual 9-19 years apart. With this design we have identified 139 regions that are significantly and consistently hypomethylated with age and 8 regions that are significantly hypermethylated with age. A representative subset of these alterations have been confirmed in an independent cohort. A total of 117 genes are associated with these regions of methylation alterations (promoter or gene body. Intriguingly, a portion of the age-related changes in sperm DNA methylation are located at genes previously associated with schizophrenia and bipolar disorder. While our data does not establish a causative relationship, it does raise the possibility that the age-associated methylation of the candidate genes that we observe in sperm might contribute to the increased incidence of neuropsychiatric and other disorders in the offspring of older males. However, further study is required to determine whether, and to what extent, a causative relationship exists.

Elucidaton of DNA methylation changes in response to ionizng radiation induced double strand breaks

Energy Technology Data Exchange (ETDEWEB)

Herrlitz, Maren Linda

2014-07-04

Recent studies indicate that epigenetic modifications like DNA methylation are involved in the DNA damage response to ionizing radiation (IR). In this doctoral thesis DNA methylation changes after treatment with IR within one replication cycle time frame (≤ 24 hours) were investigated and a decrease in DNA methylation at short times after IR was observed. This fast decrease cannot be explained by a passive, replication-dependent mechanism due to reduced DNMT (DNA methyltransferase) expression. Rather it is conceivable that changing the enzymatic activity of enzymes may lead to changes in DNA methylation as a response to IR. In this context especially TET (ten eleven translocation) enzymes might play a role. These oxidize Methylcytosine (5mC) to Hydroxymethylcytosine (5hmC) and further to formylcytosine (5fC) and carboxylcytosine (5caC), being eventually replaced by cytosine via base excision repair (BER) mechanism. For the analysis of a TET-dependent DNA demethylation an appropriate experimental cellular system was established. Therefore, global 5mC levels, TET2 expression levels and 5hmC levels in different cell lines were investigated. An anti-correlation between 5mC levels and TET2 expression was shown for all cell lines, while 5hmC and 5mC levels were correlated only in a part of the investigated cell lines and no correlation between 5hmC and TET2 expression was observed. NIH/3T3 cells, showing no TET2 expression, were chosen for experiments in which the catalytic domain of TET2 (TET2CD-GFP) was overexpressed. Using two different techniques (immunofluorescence and ELISA-based colorimetric analysis), an increase in 5hmC abundance was demonstrated as a response to TET2CD-GFP overexpression, indicating that ectopically expressed TET2 is active. Similar results were obtained when TET2CD-GFP was overexpressed in U-2 OS cells, which have a high expression level of TET2. TET2CD-GFP overexpression also led to its accumulation in nucleoli. Whether this observation

Elucidaton of DNA methylation changes in response to ionizng radiation induced double strand breaks

International Nuclear Information System (INIS)

Herrlitz, Maren Linda

2014-01-01

Recent studies indicate that epigenetic modifications like DNA methylation are involved in the DNA damage response to ionizing radiation (IR). In this doctoral thesis DNA methylation changes after treatment with IR within one replication cycle time frame (≤ 24 hours) were investigated and a decrease in DNA methylation at short times after IR was observed. This fast decrease cannot be explained by a passive, replication-dependent mechanism due to reduced DNMT (DNA methyltransferase) expression. Rather it is conceivable that changing the enzymatic activity of enzymes may lead to changes in DNA methylation as a response to IR. In this context especially TET (ten eleven translocation) enzymes might play a role. These oxidize Methylcytosine (5mC) to Hydroxymethylcytosine (5hmC) and further to formylcytosine (5fC) and carboxylcytosine (5caC), being eventually replaced by cytosine via base excision repair (BER) mechanism. For the analysis of a TET-dependent DNA demethylation an appropriate experimental cellular system was established. Therefore, global 5mC levels, TET2 expression levels and 5hmC levels in different cell lines were investigated. An anti-correlation between 5mC levels and TET2 expression was shown for all cell lines, while 5hmC and 5mC levels were correlated only in a part of the investigated cell lines and no correlation between 5hmC and TET2 expression was observed. NIH/3T3 cells, showing no TET2 expression, were chosen for experiments in which the catalytic domain of TET2 (TET2CD-GFP) was overexpressed. Using two different techniques (immunofluorescence and ELISA-based colorimetric analysis), an increase in 5hmC abundance was demonstrated as a response to TET2CD-GFP overexpression, indicating that ectopically expressed TET2 is active. Similar results were obtained when TET2CD-GFP was overexpressed in U-2 OS cells, which have a high expression level of TET2. TET2CD-GFP overexpression also led to its accumulation in nucleoli. Whether this observation

Transcriptional similarity in couples reveals the impact of shared environment and lifestyle on gene regulation through modified cytosines

Directory of Open Access Journals (Sweden)

Ke Tang

2016-06-01

Full Text Available Gene expression is a complex and quantitative trait that is influenced by both genetic and non-genetic regulators including environmental factors. Evaluating the contribution of environment to gene expression regulation and identifying which genes are more likely to be influenced by environmental factors are important for understanding human complex traits. We hypothesize that by living together as couples, there can be commonly co-regulated genes that may reflect the shared living environment (e.g., diet, indoor air pollutants, behavioral lifestyle. The lymphoblastoid cell lines (LCLs derived from unrelated couples of African ancestry (YRI, Yoruba people from Ibadan, Nigeria from the International HapMap Project provided a unique model for us to characterize gene expression pattern in couples by comparing gene expression levels between husbands and wives. Strikingly, 778 genes were found to show much smaller variances in couples than random pairs of individuals at a false discovery rate (FDR of 5%. Since genetic variation between unrelated family members in a general population is expected to be the same assuming a random-mating society, non-genetic factors (e.g., epigenetic systems are more likely to be the mediators for the observed transcriptional similarity in couples. We thus evaluated the contribution of modified cytosines to those genes showing transcriptional similarity in couples as well as the relationships these CpG sites with other gene regulatory elements, such as transcription factor binding sites (TFBS. Our findings suggested that transcriptional similarity in couples likely reflected shared common environment partially mediated through cytosine modifications.

Evolutionary Transition of Promoter and Gene Body DNA Methylation across Invertebrate-Vertebrate Boundary.

Science.gov (United States)

Keller, Thomas E; Han, Priscilla; Yi, Soojin V

2016-04-01

Genomes of invertebrates and vertebrates exhibit highly divergent patterns of DNA methylation. Invertebrate genomes tend to be sparsely methylated, and DNA methylation is mostly targeted to a subset of transcription units (gene bodies). In a drastic contrast, vertebrate genomes are generally globally and heavily methylated, punctuated by the limited local hypo-methylation of putative regulatory regions such as promoters. These genomic differences also translate into functional differences in DNA methylation and gene regulation. Although promoter DNA methylation is an important regulatory component of vertebrate gene expression, its role in invertebrate gene regulation has been little explored. Instead, gene body DNA methylation is associated with expression of invertebrate genes. However, the evolutionary steps leading to the differentiation of invertebrate and vertebrate genomic DNA methylation remain unresolved. Here we analyzed experimentally determined DNA methylation maps of several species across the invertebrate-vertebrate boundary, to elucidate how vertebrate gene methylation has evolved. We show that, in contrast to the prevailing idea, a substantial number of promoters in an invertebrate basal chordate Ciona intestinalis are methylated. Moreover, gene expression data indicate significant, epigenomic context-dependent associations between promoter methylation and expression in C. intestinalis. However, there is no evidence that promoter methylation in invertebrate chordate has been evolutionarily maintained across the invertebrate-vertebrate boundary. Rather, body-methylated invertebrate genes preferentially obtain hypo-methylated promoters among vertebrates. Conversely, promoter methylation is preferentially found in lineage- and tissue-specific vertebrate genes. These results provide important insights into the evolutionary origin of epigenetic regulation of vertebrate gene expression. © The Author(s) 2015. Published by Oxford University Press on behalf

Sexual Polyploidization in Medicago sativa L.: Impact on the Phenotype, Gene Transcription, and Genome Methylation.

Science.gov (United States)

Rosellini, Daniele; Ferradini, Nicoletta; Allegrucci, Stefano; Capomaccio, Stefano; Zago, Elisa Debora; Leonetti, Paola; Balech, Bachir; Aversano, Riccardo; Carputo, Domenico; Reale, Lara; Veronesi, Fabio

2016-04-07

Polyploidization as the consequence of 2n gamete formation is a prominent mechanism in plant evolution. Studying its effects on the genome, and on genome expression, has both basic and applied interest. We crossed two diploid (2n = 2x = 16) Medicago sativa plants, a subsp. falcata seed parent, and a coerulea × falcata pollen parent that form a mixture of n and 2n eggs and pollen, respectively. Such a cross produced full-sib diploid and tetraploid (2n = 4x = 32) hybrids, the latter being the result of bilateral sexual polyploidization (BSP). These unique materials allowed us to investigate the effects of BSP, and to separate the effect of intraspecific hybridization from those of polyploidization by comparing 2x with 4x full sib progeny plants. Simple sequence repeat marker segregation demonstrated tetrasomic inheritance for all chromosomes but one, demonstrating that these neotetraploids are true autotetraploids. BSP brought about increased biomass, earlier flowering, higher seed set and weight, and larger leaves with larger cells. Microarray analyses with M. truncatula gene chips showed that several hundred genes, related to diverse metabolic functions, changed their expression level as a consequence of polyploidization. In addition, cytosine methylation increased in 2x, but not in 4x, hybrids. Our results indicate that sexual polyploidization induces significant transcriptional novelty, possibly mediated in part by DNA methylation, and phenotypic novelty that could underpin improved adaptation and reproductive success of tetraploid M. sativa with respect to its diploid progenitor. These polyploidy-induced changes may have promoted the adoption of tetraploid alfalfa in agriculture. Copyright © 2016 Rosellini et al.

Sexual Polyploidization in Medicago sativa L.: Impact on the Phenotype, Gene Transcription, and Genome Methylation

Directory of Open Access Journals (Sweden)

Daniele Rosellini

2016-04-01

Full Text Available Polyploidization as the consequence of 2n gamete formation is a prominent mechanism in plant evolution. Studying its effects on the genome, and on genome expression, has both basic and applied interest. We crossed two diploid (2n = 2x = 16 Medicago sativa plants, a subsp. falcata seed parent, and a coerulea × falcata pollen parent that form a mixture of n and 2n eggs and pollen, respectively. Such a cross produced full-sib diploid and tetraploid (2n = 4x = 32 hybrids, the latter being the result of bilateral sexual polyploidization (BSP. These unique materials allowed us to investigate the effects of BSP, and to separate the effect of intraspecific hybridization from those of polyploidization by comparing 2x with 4x full sib progeny plants. Simple sequence repeat marker segregation demonstrated tetrasomic inheritance for all chromosomes but one, demonstrating that these neotetraploids are true autotetraploids. BSP brought about increased biomass, earlier flowering, higher seed set and weight, and larger leaves with larger cells. Microarray analyses with M. truncatula gene chips showed that several hundred genes, related to diverse metabolic functions, changed their expression level as a consequence of polyploidization. In addition, cytosine methylation increased in 2x, but not in 4x, hybrids. Our results indicate that sexual polyploidization induces significant transcriptional novelty, possibly mediated in part by DNA methylation, and phenotypic novelty that could underpin improved adaptation and reproductive success of tetraploid M. sativa with respect to its diploid progenitor. These polyploidy-induced changes may have promoted the adoption of tetraploid alfalfa in agriculture.

Ultraviolet and chemical induced DNA repair in human cells assayed by bromodeoxyuridine photolysis or cytosine arabinoside arrest

International Nuclear Information System (INIS)

Regan, J.D.; Dunn, W.C.

1979-01-01

The bromodeoxyuridine photolysis assay of DNA damage in human cells permits an estimate of both the number of repaired regions in the DNA and the size of the average repaired region - the patch size. The antineoplastic agent arabinofuranosyl cytosine (ara-C) can also be employed to assay the magnitude of repair since this agent appears to block rejoining of single-strand incisions made in the DNA during the initial step of repair. Thus, the number of incisions can be accumulated. The ara-C effect is dependent on the presence of hydroxyurea. Both assays can be employed for the study of physical or chemical DNA damages. Results comparing these assays are presented

Anti-N-methyl-D-aspartate receptor encephalitis with an imaging-invisible ovarian teratoma: a case report.

Science.gov (United States)

Abdul-Rahman, Zainab M; Panegyres, Peter K; Roeck, Margareta; Hawkins, David; Bharath, Jude; Grolman, Paul; Neppe, Cliffe; Palmer, David

2016-10-24

Anti-N-methyl-D-aspartate receptor encephalitis is a recently discovered disease entity of paraneoplastic limbic encephalitis. It largely affects young women and is often associated with an ovarian teratoma. It is a serious yet treatable condition if diagnosed early. Its remedy involves immunotherapy and surgical removal of the teratoma of the ovaries. This case of anti-N-methyl-D-aspartate receptor encephalitis involves an early surgical intervention with bilateral oophorectomy, despite negative imaging evidence of a teratoma. A 25-year-old white woman with anti-N-methyl-D-aspartate receptor encephalitis presented with behavioral changes and seizures that were confirmed to be secondary to anti-N-methyl-D-aspartate receptor encephalitis. She required an admission to our intensive care unit for ventilator support and received a number of immunological therapies. Multiple imaging investigations showed no evidence of an ovarian teratoma; she had a bilateral oophorectomy 29 days after admission. Ovarian histology confirmed the presence of a teratoma with neuronal cells. A few days after the operation she began to show signs of improvement and, apart from mild short-term memory loss, she returned to normal function. Our patient is an example of teratoma-associated anti-N-methyl-D-aspartate receptor encephalitis, in which the teratoma was identified only microscopically. Her case highlights that even with negative imaging evidence of a teratoma, ovarian pathology should still be considered and explored.

Pathogenic mechanisms of intracellular bacteria.

Science.gov (United States)

Niller, Hans Helmut; Masa, Roland; Venkei, Annamária; Mészáros, Sándor; Minarovits, Janos

2017-06-01

We wished to overview recent data on a subset of epigenetic changes elicited by intracellular bacteria in human cells. Reprogramming the gene expression pattern of various host cells may facilitate bacterial growth, survival, and spread. DNA-(cytosine C5)-methyltransferases of Mycoplasma hyorhinis targeting cytosine-phosphate-guanine (CpG) dinucleotides and a Mycobacterium tuberculosis methyltransferase targeting non-CpG sites methylated the host cell DNA and altered the pattern of gene expression. Gene silencing by CpG methylation and histone deacetylation, mediated by cellular enzymes, also occurred in M. tuberculosis-infected macrophages. M. tuberculosis elicited cell type-specific epigenetic changes: it caused increased DNA methylation in macrophages, but induced demethylation, deposition of euchromatic histone marks and activation of immune-related genes in dendritic cells. A secreted transposase of Acinetobacter baumannii silenced a cellular gene, whereas Mycobacterium leprae altered the epigenotype, phenotype, and fate of infected Schwann cells. The 'keystone pathogen' oral bacterium Porphyromonas gingivalis induced local DNA methylation and increased the level of histone acetylation in host cells. These epigenetic changes at the biofilm-gingiva interface may contribute to the development of periodontitis. Epigenetic regulators produced by intracellular bacteria alter the epigenotype and gene expression pattern of host cells and play an important role in pathogenesis.

Sequential Oral Hydroxyurea and Intravenous Cytosine Arabinoside in Refractory Childhood Acute Leukemia: A Pediatric Oncology Group Phase I Study

OpenAIRE

Dubowy, Ronald; Graham, Michael; Hakami, Nasrollah; Kletzel, Morris; Mahoney, Donald; Newman, Edward; Ravindranath, Yaddanapudi; Camitta, Bruce

2008-01-01

At concentrations >0.1 mM, Hydroxyurea (HU) enhances the accumulation of cytosine arabinoside (ara-C) in leukemia cells in vitro. This study of children with refractory acute leukemia was designed to take advantage of this biochemical modulation. A fixed dose of HU and an escalating dose of ara-C were used. Oral HU, 1200 mg/m2 was followed 2 hours later by ara-C, 250-3100 mg/m2 intravenously in 15 minutes. The combination was given on days 1,2,3 and 8,9,10. Thirty-three children (26 ALL, 7 AN...

Application of multiplex nested methylated specific PCR in early diagnosis of epithelial ovarian cancer.

Science.gov (United States)

Wang, Bi; Yu, Lei; Yang, Guo-Zhen; Luo, Xin; Huang, Lin

2015-01-01

To explore the application of multiplex nested methylated specific polymerase chain reaction (PCR) in the early diagnosis of epithelial ovarian carcinoma (EOC). Serum and fresh tissue samples were collected from 114 EOC patients. RUNX3, TFPI2 and OPCML served as target genes. Methylation levels of tissues were assessed by multiplex nested methylated specific PCR, the results being compared with those for carcinoma antigen 125 (CA125). The serum free deoxyribose nucleic acid (DNA) methylation spectrum of EOC patients was completely contained in the DNA spectrum of cancer tissues, providing an accurate reflection of tumor DNA methylation conditions. Serum levels of CA125 and free DNA methylation in the EOC group were evidently higher than those in benign lesion and control groups (p0.05). The sensitivity, specificity and positive predicative value (PPV) of multiplex nested methylated specific PCR were significantly higher for detection of all patients and those with early EOC than those for CA125 (pnested methylated specific PCR (p>0.05), but there was no significant difference in sensitivity (p>0.05). Serum free DNA methylation can be used as a biological marker for EOC and multiplex nested methylated specific PCR should be considered for early diagnosis since it can accurately determine tumor methylation conditions.

Neural Tube Defects, Folic Acid and Methylation

Science.gov (United States)

Imbard, Apolline; Benoist, Jean-François; Blom, Henk J.

2013-01-01

Neural tube defects (NTDs) are common complex congenital malformations resulting from failure of the neural tube closure during embryogenesis. It is established that folic acid supplementation decreases the prevalence of NTDs, which has led to national public health policies regarding folic acid. To date, animal studies have not provided sufficient information to establish the metabolic and/or genomic mechanism(s) underlying human folic acid responsiveness in NTDs. However, several lines of evidence suggest that not only folates but also choline, B12 and methylation metabolisms are involved in NTDs. Decreased B12 vitamin and increased total choline or homocysteine in maternal blood have been shown to be associated with increased NTDs risk. Several polymorphisms of genes involved in these pathways have also been implicated in risk of development of NTDs. This raises the question whether supplementation with B12 vitamin, betaine or other methylation donors in addition to folic acid periconceptional supplementation will further reduce NTD risk. The objective of this article is to review the role of methylation metabolism in the onset of neural tube defects. PMID:24048206

Common DNA methylation alterations in multiple brain regions in autism.

Science.gov (United States)

Ladd-Acosta, C; Hansen, K D; Briem, E; Fallin, M D; Kaufmann, W E; Feinberg, A P

2014-08-01

Autism spectrum disorders (ASD) are increasingly common neurodevelopmental disorders defined clinically by a triad of features including impairment in social interaction, impairment in communication in social situations and restricted and repetitive patterns of behavior and interests, with considerable phenotypic heterogeneity among individuals. Although heritability estimates for ASD are high, conventional genetic-based efforts to identify genes involved in ASD have yielded only few reproducible candidate genes that account for only a small proportion of ASDs. There is mounting evidence to suggest environmental and epigenetic factors play a stronger role in the etiology of ASD than previously thought. To begin to understand the contribution of epigenetics to ASD, we have examined DNA methylation (DNAm) in a pilot study of postmortem brain tissue from 19 autism cases and 21 unrelated controls, among three brain regions including dorsolateral prefrontal cortex, temporal cortex and cerebellum. We measured over 485,000 CpG loci across a diverse set of functionally relevant genomic regions using the Infinium HumanMethylation450 BeadChip and identified four genome-wide significant differentially methylated regions (DMRs) using a bump hunting approach and a permutation-based multiple testing correction method. We replicated 3/4 DMRs identified in our genome-wide screen in a different set of samples and across different brain regions. The DMRs identified in this study represent suggestive evidence for commonly altered methylation sites in ASD and provide several promising new candidate genes.

Cytosine hypomethylation at CHG and CHH sites in the pleiotropic ...

Indian Academy of Sciences (India)

Keywords. bisulphite sequencing; CMT3; DNA demethylases; DNA methyl transferases; demethylation; excision repair; mutagenic hot spots; phylogenetic relationships; 5S rDNA; 18S rDNA; RdDM; spontaneous mutagenesis.

DNA methylation analysis reveals distinct methylation signatures in pediatric germ cell tumors

International Nuclear Information System (INIS)

Amatruda, James F; Frazier, A Lindsay; Poynter, Jenny N; Ross, Julie A; Christensen, Brock; Fustino, Nicholas J; Chen, Kenneth S; Hooten, Anthony J; Nelson, Heather; Kuriger, Jacquelyn K; Rakheja, Dinesh

2013-01-01

Aberrant DNA methylation is a prominent feature of many cancers, and may be especially relevant in germ cell tumors (GCTs) due to the extensive epigenetic reprogramming that occurs in the germ line during normal development. We used the Illumina GoldenGate Cancer Methylation Panel to compare DNA methylation in the three main histologic subtypes of pediatric GCTs (germinoma, teratoma and yolk sac tumor (YST); N = 51) and used recursively partitioned mixture models (RPMM) to test associations between methylation pattern and tumor and demographic characteristics. We identified genes and pathways that were differentially methylated using generalized linear models and Ingenuity Pathway Analysis. We also measured global DNA methylation at LINE1 elements and evaluated methylation at selected imprinted loci using pyrosequencing. Methylation patterns differed by tumor histology, with 18/19 YSTs forming a distinct methylation class. Four pathways showed significant enrichment for YSTs, including a human embryonic stem cell pluripotency pathway. We identified 190 CpG loci with significant methylation differences in mature and immature teratomas (q < 0.05), including a number of CpGs in stem cell and pluripotency-related pathways. Both YST and germinoma showed significantly lower methylation at LINE1 elements compared with normal adjacent tissue while there was no difference between teratoma (mature and immature) and normal tissue. DNA methylation at imprinted loci differed significantly by tumor histology and location. Understanding methylation patterns may identify the developmental stage at which the GCT arose and the at-risk period when environmental exposures could be most harmful. Further, identification of relevant genetic pathways could lead to the development of new targets for therapy

Formation of O2-methylthymine in poly(dA-dT) on methylation with N-methyl-N-nitrosourea and dimethyl sulphate. Evidence that O2-methylthymine does not miscode during DNA synthesis.

Science.gov (United States)

Saffhill, R; Abbott, P J

1978-01-01

The alternating co-polymer has been methylated with either N methyl-N-nitrosourea (MNU) or dimethyl sulphate (DMS) and the levels of the various methylated thymidines (O2-methylthymidine, 3-methylthymidine and O4-methylthymidine) measured. MNU produced all three compounds whereas DMS only produced 3-methylthymidine and O2-methylthymidine at detectable levels. These results have been combined with our earlier results concerning the misincorporation of dGMP with E. coli DNA polymerase using MNU-methylated poly(dA-dT). These results indicate that O2-methylthymidine does not miscode during DNA synthesis. PMID:353735

A probabilistic generative model for quantification of DNA modifications enables analysis of demethylation pathways.

Science.gov (United States)

Äijö, Tarmo; Huang, Yun; Mannerström, Henrik; Chavez, Lukas; Tsagaratou, Ageliki; Rao, Anjana; Lähdesmäki, Harri

2016-03-14

We present a generative model, Lux, to quantify DNA methylation modifications from any combination of bisulfite sequencing approaches, including reduced, oxidative, TET-assisted, chemical-modification assisted, and methylase-assisted bisulfite sequencing data. Lux models all cytosine modifications (C, 5mC, 5hmC, 5fC, and 5caC) simultaneously together with experimental parameters, including bisulfite conversion and oxidation efficiencies, as well as various chemical labeling and protection steps. We show that Lux improves the quantification and comparison of cytosine modification levels and that Lux can process any oxidized methylcytosine sequencing data sets to quantify all cytosine modifications. Analysis of targeted data from Tet2-knockdown embryonic stem cells and T cells during development demonstrates DNA modification quantification at unprecedented detail, quantifies active demethylation pathways and reveals 5hmC localization in putative regulatory regions.

Modeling of the oxidation of methyl esters—Validation for methyl hexanoate, methyl heptanoate, and methyl decanoate in a jet-stirred reactor

Science.gov (United States)

Glaude, Pierre Alexandre; Herbinet, Olivier; Bax, Sarah; Biet, Joffrey; Warth, Valérie; Battin-Leclerc, Frédérique

2013-01-01

The modeling of the oxidation of methyl esters was investigated and the specific chemistry, which is due to the presence of the ester group in this class of molecules, is described. New reactions and rate parameters were defined and included in the software EXGAS for the automatic generation of kinetic mechanisms. Models generated with EXGAS were successfully validated against data from the literature (oxidation of methyl hexanoate and methyl heptanoate in a jet-stirred reactor) and a new set of experimental results for methyl decanoate. The oxidation of this last species was investigated in a jet-stirred reactor at temperatures from 500 to 1100 K, including the negative temperature coefficient region, under stoichiometric conditions, at a pressure of 1.06 bar and for a residence time of 1.5 s: more than 30 reaction products, including olefins, unsaturated esters, and cyclic ethers, were quantified and successfully simulated. Flow rate analysis showed that reactions pathways for the oxidation of methyl esters in the low-temperature range are similar to that of alkanes. PMID:23710076

Modeling of the oxidation of methyl esters-Validation for methyl hexanoate, methyl heptanoate, and methyl decanoate in a jet-stirred reactor.

Science.gov (United States)

Glaude, Pierre Alexandre; Herbinet, Olivier; Bax, Sarah; Biet, Joffrey; Warth, Valérie; Battin-Leclerc, Frédérique

2010-11-01

The modeling of the oxidation of methyl esters was investigated and the specific chemistry, which is due to the presence of the ester group in this class of molecules, is described. New reactions and rate parameters were defined and included in the software EXGAS for the automatic generation of kinetic mechanisms. Models generated with EXGAS were successfully validated against data from the literature (oxidation of methyl hexanoate and methyl heptanoate in a jet-stirred reactor) and a new set of experimental results for methyl decanoate. The oxidation of this last species was investigated in a jet-stirred reactor at temperatures from 500 to 1100 K, including the negative temperature coefficient region, under stoichiometric conditions, at a pressure of 1.06 bar and for a residence time of 1.5 s: more than 30 reaction products, including olefins, unsaturated esters, and cyclic ethers, were quantified and successfully simulated. Flow rate analysis showed that reactions pathways for the oxidation of methyl esters in the low-temperature range are similar to that of alkanes.

LINE1 CpG-DNA Hypomethylation in Granulosa Cells and Blood Leukocytes Is Associated With PCOS and Related Traits.

Science.gov (United States)

Sagvekar, Pooja; Mangoli, Vijay; Desai, Sadhana; Patil, Anushree; Mukherjee, Srabani

2017-04-01

Altered global DNA methylation is indicative of epigenomic instability concerning chronic diseases. Investigating its incidence and association with polycystic ovary syndrome (PCOS) is essential to understand the etiopathogenesis of this disorder. We assessed global DNA methylation differences in peripheral blood leukocytes (PBLs) and cumulus granulosa cells (CGCs) of controls and women with PCOS; and their association with PCOS and its traits. This study included a total of 102 controls and women with PCOS. Forty-one women undergoing controlled ovarian hyperstimulation (COH) and 61 women not undergoing COH were recruited from in vitro fertilization (IVF) and infertility clinics. DNA methylation was measured by ELISA for 5'-methyl-cytosine content and bisulfite sequencing of 5'-untranslated region (5'-UTR) of long interspersed nucleotide element-1 (LINE1/L1). Total 5'-methyl-cytosine and L1 methylation levels in PBLs and CGCs were similar between controls and women with PCOS. Methylation assessed at CpG sites of L1 5'-UTR revealed a single CpG-site (CpG-4) to be consistently hypomethylated in PBLs of both PCOS groups and CGCs of stimulated PCOS group. In unstimulated women, hypomethylation at CpG-4 was strongly associated with PCOS susceptibility, whereas in stimulated group it showed strong associations with PCOS and its hormonal traits. Furthermore, CGCs demonstrated consistent global and CpG-DNA hypomethylation relative to PBLs, irrespective of normal or disease states. Our study revealed strong association of single hypomethylated CpG-site with PCOS. Identification and characterization of more such methyl-CpG signatures in repetitive elements in larger study populations would provide valuable epigenetic insights into PCOS. Copyright © 2017 by the Endocrine Society

DNA methylation of amino acid transporter genes in the human placenta.

Science.gov (United States)

Simner, C; Novakovic, B; Lillycrop, K A; Bell, C G; Harvey, N C; Cooper, C; Saffery, R; Lewis, R M; Cleal, J K

2017-12-01

Placental transfer of amino acids via amino acid transporters is essential for fetal growth. Little is known about the epigenetic regulation of amino acid transporters in placenta. This study investigates the DNA methylation status of amino acid transporters and their expression across gestation in human placenta. BeWo cells were treated with 5-aza-2'-deoxycytidine to inhibit methylation and assess the effects on amino acid transporter gene expression. The DNA methylation levels of amino acid transporter genes in human placenta were determined across gestation using DNA methylation array data. Placental amino acid transporter gene expression across gestation was also analysed using data from publically available Gene Expression Omnibus data sets. The expression levels of these transporters at term were established using RNA sequencing data. Inhibition of DNA methylation in BeWo cells demonstrated that expression of specific amino acid transporters can be inversely associated with DNA methylation. Amino acid transporters expressed in term placenta generally showed low levels of promoter DNA methylation. Transporters with little or no expression in term placenta tended to be more highly methylated at gene promoter regions. The transporter genes SLC1A2, SLC1A3, SLC1A4, SLC7A5, SLC7A11 and SLC7A10 had significant changes in enhancer DNA methylation across gestation, as well as gene expression changes across gestation. This study implicates DNA methylation in the regulation of amino acid transporter gene expression. However, in human placenta, DNA methylation of these genes remains low across gestation and does not always play an obvious role in regulating gene expression, despite clear evidence for differential expression as gestation proceeds. Copyright © 2017. Published by Elsevier Ltd.

DNA methylation polymorphism in flue-cured tobacco and candidate markers for tobacco mosaic virus resistance*

Science.gov (United States)

Zhao, Jie-hong; Zhang, Ji-shun; Wang, Yi; Wang, Ren-gang; Wu, Chun; Fan, Long-jiang; Ren, Xue-liang

2011-01-01

DNA methylation plays an important role in the epigenetic regulation of gene expression during plant growth, development, and polyploidization. However, there is still no distinct evidence in tobacco regarding the distribution of the methylation pattern and whether it contributes to qualitative characteristics. We studied the levels and patterns of methylation polymorphism at CCGG sites in 48 accessions of allotetraploid flue-cured tobacco, Nicotiana tabacum, using a methylation-sensitive amplified polymorphism (MSAP) technique. The results showed that methylation existed at a high level among tobacco accessions, among which 49.3% sites were methylated and 69.9% allelic sites were polymorphic. A cluster analysis revealed distinct patterns of geography-specific groups. In addition, three polymorphic sites significantly related to tobacco mosaic virus (TMV) resistance were explored. This suggests that tobacco breeders should pay more attention to epigenetic traits. PMID:22042659

DNA methylation polymorphism in flue-cured tobacco and candidate markers for tobacco mosaic virus resistance.

Science.gov (United States)

Zhao, Jie-hong; Zhang, Ji-shun; Wang, Yi; Wang, Ren-gang; Wu, Chun; Fan, Long-jiang; Ren, Xue-liang

2011-11-01

DNA methylation plays an important role in the epigenetic regulation of gene expression during plant growth, development, and polyploidization. However, there is still no distinct evidence in tobacco regarding the distribution of the methylation pattern and whether it contributes to qualitative characteristics. We studied the levels and patterns of methylation polymorphism at CCGG sites in 48 accessions of allotetraploid flue-cured tobacco, Nicotiana tabacum, using a methylation-sensitive amplified polymorphism (MSAP) technique. The results showed that methylation existed at a high level among tobacco accessions, among which 49.3% sites were methylated and 69.9% allelic sites were polymorphic. A cluster analysis revealed distinct patterns of geography-specific groups. In addition, three polymorphic sites significantly related to tobacco mosaic virus (TMV) resistance were explored. This suggests that tobacco breeders should pay more attention to epigenetic traits.

No evidence for promoter region methylation of the succinate dehydrogenase and fumarate hydratase tumour suppressor genes in breast cancer

Directory of Open Access Journals (Sweden)

Dobrovic Alexander

2009-09-01

Full Text Available Abstract Background Succinate dehydrogenase (SDH and fumarate hydratase (FH are tricarboxylic acid (TCA cycle enzymes that are also known to act as tumour suppressor genes. Increased succinate or fumarate levels as a consequence of SDH and FH deficiency inhibit hypoxia inducible factor-1α (HIF-1α prolyl hydroxylases leading to sustained HIF-1α expression in tumours. Since HIF-1α is frequently expressed in breast carcinomas, DNA methylation at the promoter regions of the SDHA, SDHB, SDHC and SDHD and FH genes was evaluated as a possible mechanism in silencing of SDH and FH expression in breast carcinomas. Findings No DNA methylation was identified in the promoter regions of the SDHA, SDHB, SDHC, SDHD and FH genes in 72 breast carcinomas and 10 breast cancer cell lines using methylation-sensitive high resolution melting which detects both homogeneous and heterogeneous methylation. Conclusion These results show that inactivation via DNA methylation of the promoter CpG islands of SDH and FH is unlikely to play a major role in sporadic breast carcinomas.

Glioma CpG island methylator phenotype (G-CIMP): biological and clinical implications.

Science.gov (United States)

Malta, Tathiane M; de Souza, Camila F; Sabedot, Thais S; Silva, Tiago C; Mosella, Maritza S; Kalkanis, Steven N; Snyder, James; Castro, Ana Valeria B; Noushmehr, Houtan

2018-04-09

Gliomas are a heterogeneous group of brain tumors with distinct biological and clinical properties. Despite advances in surgical techniques and clinical regimens, treatment of high-grade glioma remains challenging and carries dismal rates of therapeutic success and overall survival. Challenges include the molecular complexity of gliomas, as well as inconsistencies in histopathological grading, resulting in an inaccurate prediction of disease progression and failure in the use of standard therapy. The updated 2016 World Health Organization (WHO) classification of tumors of the central nervous system reflects a refinement of tumor diagnostics by integrating the genotypic and phenotypic features, thereby narrowing the defined subgroups. The new classification recommends molecular diagnosis of isocitrate dehydrogenase (IDH) mutational status in gliomas. IDH-mutant gliomas manifest the cytosine-phosphate-guanine (CpG) island methylator phenotype (G-CIMP). Notably, the recent identification of clinically relevant subsets of G-CIMP tumors (G-CIMP-high and G-CIMP-low) provides a further refinement in glioma classification that is independent of grade and histology. This scheme may be useful for predicting patient outcome and may be translated into effective therapeutic strategies tailored to each patient. In this review, we highlight the evolution of our understanding of the G-CIMP subsets and how recent advances in characterizing the genome and epigenome of gliomas may influence future basic and translational research.

To What Extent Does DNA Methylation Affect Phenotypic Variation in Cattle?

Directory of Open Access Journals (Sweden)

Stephanie McKAY

2015-07-01

to previously identified QTL and GWAS regions using Animal QTL database. The approach described here has provided us with evidence that QTL and GWAS regions overlay genomic regions where methylation may regulate transcription.

DNA methylation modifications associated with chronic fatigue syndrome.

Directory of Open Access Journals (Sweden)

Wilfred C de Vega

Full Text Available Chronic Fatigue Syndrome (CFS, also known as myalgic encephalomyelitis, is a complex multifactorial disease that is characterized by the persistent presence of fatigue and other particular symptoms for a minimum of 6 months. Symptoms fail to dissipate after sufficient rest and have major effects on the daily functioning of CFS sufferers. CFS is a multi-system disease with a heterogeneous patient population showing a wide variety of functional disabilities and its biological basis remains poorly understood. Stable alterations in gene function in the immune system have been reported in several studies of CFS. Epigenetic modifications have been implicated in long-term effects on gene function, however, to our knowledge, genome-wide epigenetic modifications associated with CFS have not been explored. We examined the DNA methylome in peripheral blood mononuclear cells isolated from CFS patients and healthy controls using the Illumina HumanMethylation450 BeadChip array, controlling for invariant probes and probes overlapping polymorphic sequences. Gene ontology (GO and network analysis of differentially methylated genes was performed to determine potential biological pathways showing changes in DNA methylation in CFS. We found an increased abundance of differentially methylated genes related to the immune response, cellular metabolism, and kinase activity. Genes associated with immune cell regulation, the largest coordinated enrichment of differentially methylated pathways, showed hypomethylation within promoters and other gene regulatory elements in CFS. These data are consistent with evidence of multisystem dysregulation in CFS and implicate the involvement of DNA modifications in CFS pathology.

DNA Methylation as a Biomarker for Body Fluid Identification

Directory of Open Access Journals (Sweden)

Rania Gomaa

2017-12-01

Full Text Available Currently, available identification techniques for forensic samples are either enzyme or protein based, which can be subjected to degradation, thus limiting its storage potentials. Epigenetic changes arising due to DNA methylation and histone acetylation can be used for body fluid identification. Markers DACT1, USP49, ZC3H12D, FGF7, cg23521140, cg17610929, chromosome 4 (25287119–25287254, chromosome 11 (72085678–72085798, 57171095–57171236, 1493401–1493538, and chromosome 19 (47395505–47395651 are currently being used for semen identification. Markers cg26107890, cg20691722, cg01774894 and cg14991487 are used to differentiate saliva and vaginal secretions from other body fluids. However, such markers show overlapping methylation pattern. This review article aimed to highlight the feasibility of using DNA methylation of certain genetic markers in body fluid identification and its implications for forensic investigations. The reviewed articles have employed molecular genetics techniques such as Bisulfite sequencing PCR (BSP, methylation specific PCR (MSP, Pyrosequencing, Combined Bisulfite Restriction Analysis (COBRA, Methylation-sensitive Single Nucleotide Primer Extension (SNuPE, and Multiplex SNaPshot Microarray. Bioinformatics software such as MATLAB and BiQ Analyzer has been used. Biological fluids have different methylation patterns and thus, this difference can be used to identify the nature of the biological fluid found at the crime scene. Using DNA methylation to identify the body fluids gives accurate results without consumption of the trace evidence and requires a minute amount of DNA for analysis. Recent studies have incorporated next-generation sequencing aiming to find out more reliable markers that can differentiate between different body fluids. Nonetheless, new DNA methylation markers are yet to be discovered to accurately differentiate between saliva and vaginal secretions with high confidence. Epigenetic changes are

The Helicase Activity of Hyperthermophilic Archaeal MCM is Enhanced at High Temperatures by Lysine Methylation.

Science.gov (United States)

Xia, Yisui; Niu, Yanling; Cui, Jiamin; Fu, Yang; Chen, Xiaojiang S; Lou, Huiqiang; Cao, Qinhong

2015-01-01

Lysine methylation and methyltransferases are widespread in the third domain of life, archaea. Nevertheless, the effects of methylation on archaeal proteins wait to be defined. Here, we report that recombinant sisMCM, an archaeal homolog of Mcm2-7 eukaryotic replicative helicase, is methylated by aKMT4 in vitro. Mono-methylation of these lysine residues occurs coincidently in the endogenous sisMCM protein purified from the hyperthermophilic Sulfolobus islandicus cells as indicated by mass spectra. The helicase activity of mini-chromosome maintenance (MCM) is stimulated by methylation, particularly at temperatures over 70°C. The methylated MCM shows optimal DNA unwinding activity after heat-treatment between 76 and 82°C, which correlates well with the typical growth temperatures of hyperthermophilic Sulfolobus. After methylation, the half life of MCM helicase is dramatically extended at 80°C. The methylated sites are located on the accessible protein surface, which might modulate the intra- and inter- molecular interactions through changing the hydrophobicity and surface charge. Furthermore, the methylation-mimic mutants of MCM show heat resistance helicase activity comparable to the methylated MCM. These data provide the biochemical evidence that posttranslational modifications such as methylation may enhance kinetic stability of proteins under the elevated growth temperatures of hyperthermophilic archaea.

How environmental and genetic factors combine to cause autism: A redox/methylation hypothesis.

Science.gov (United States)

Deth, Richard; Muratore, Christina; Benzecry, Jorge; Power-Charnitsky, Verna-Ann; Waly, Mostafa

2008-01-01

Recently higher rates of autism diagnosis suggest involvement of environmental factors in causing this developmental disorder, in concert with genetic risk factors. Autistic children exhibit evidence of oxidative stress and impaired methylation, which may reflect effects of toxic exposure on sulfur metabolism. We review the metabolic relationship between oxidative stress and methylation, with particular emphasis on adaptive responses that limit activity of cobalamin and folate-dependent methionine synthase. Methionine synthase activity is required for dopamine-stimulated phospholipid methylation, a unique membrane-delimited signaling process mediated by the D4 dopamine receptor that promotes neuronal synchronization and attention, and synchrony is impaired in autism. Genetic polymorphisms adversely affecting sulfur metabolism, methylation, detoxification, dopamine signaling and the formation of neuronal networks occur more frequently in autistic subjects. On the basis of these observations, a "redox/methylation hypothesis of autism" is described, in which oxidative stress, initiated by environment factors in genetically vulnerable individuals, leads to impaired methylation and neurological deficits secondary to reductions in the capacity for synchronizing neural networks.

Targeted cytosine deaminase-uracil phosphoribosyl transferase suicide gene therapy induces small cell lung cancer-specific cytotoxicity and tumor growth delay

DEFF Research Database (Denmark)

Christensen, Camilla L; Gjetting, Torben; Poulsen, Thomas Tuxen

2010-01-01

Small cell lung cancer (SCLC) is a highly malignant cancer for which there is no curable treatment. Novel therapies are therefore in great demand. In the present study we investigated the therapeutic effect of transcriptionally targeted suicide gene therapy for SCLC based on the yeast cytosine...... deaminase (YCD) gene alone or fused with the yeast uracil phosphoribosyl transferase (YUPRT) gene followed by administration of 5-fluorocytosine (5-FC) prodrug. Experimental design: The YCD gene or the YCD-YUPRT gene was placed under regulation of the SCLC-specific promoter insulinoma-associated 1 (INSM1...

Differential DNA methylation profiles of coding and non-coding genes define hippocampal sclerosis in human temporal lobe epilepsy

Science.gov (United States)

Miller-Delaney, Suzanne F.C.; Bryan, Kenneth; Das, Sudipto; McKiernan, Ross C.; Bray, Isabella M.; Reynolds, James P.; Gwinn, Ryder; Stallings, Raymond L.

2015-01-01

Temporal lobe epilepsy is associated with large-scale, wide-ranging changes in gene expression in the hippocampus. Epigenetic changes to DNA are attractive mechanisms to explain the sustained hyperexcitability of chronic epilepsy. Here, through methylation analysis of all annotated C-phosphate-G islands and promoter regions in the human genome, we report a pilot study of the methylation profiles of temporal lobe epilepsy with or without hippocampal sclerosis. Furthermore, by comparative analysis of expression and promoter methylation, we identify methylation sensitive non-coding RNA in human temporal lobe epilepsy. A total of 146 protein-coding genes exhibited altered DNA methylation in temporal lobe epilepsy hippocampus (n = 9) when compared to control (n = 5), with 81.5% of the promoters of these genes displaying hypermethylation. Unique methylation profiles were evident in temporal lobe epilepsy with or without hippocampal sclerosis, in addition to a common methylation profile regardless of pathology grade. Gene ontology terms associated with development, neuron remodelling and neuron maturation were over-represented in the methylation profile of Watson Grade 1 samples (mild hippocampal sclerosis). In addition to genes associated with neuronal, neurotransmitter/synaptic transmission and cell death functions, differential hypermethylation of genes associated with transcriptional regulation was evident in temporal lobe epilepsy, but overall few genes previously associated with epilepsy were among the differentially methylated. Finally, a panel of 13, methylation-sensitive microRNA were identified in temporal lobe epilepsy including MIR27A, miR-193a-5p (MIR193A) and miR-876-3p (MIR876), and the differential methylation of long non-coding RNA documented for the first time. The present study therefore reports select, genome-wide DNA methylation changes in human temporal lobe epilepsy that may contribute to the molecular architecture of the epileptic brain. PMID

Advances in DNA methylation: 5-hydroxymethylcytosine revisited

DEFF Research Database (Denmark)

Dahl, Christina; Grønbæk, Kirsten; Guldberg, Per

2011-01-01

modification involved in gene regulation, X-chromosome inactivation, genomic imprinting, long-term silencing of transposons and cancer development is well described. 5hmC, on the other hand, has only recently entered center stage when it was shown that the Ten-Eleven-Translocation (TET) family of oxygenases...... in cancer development, and developing sequencing methodologies that can accurately distinguish among cytosine, 5mC and 5hmC at single-base-pair resolution....

Chromatin domains and function

NARCIS (Netherlands)

Fransz, P.; Meier, I.

2009-01-01

The inheritance of biological traits involves not only the transfer of genetic information in the form of DNA, but also epigenetic information. The latter is encrypted in a DNA component, methylation of cytosine residues, and in non-DNA components such as histone modifications, non-histone proteins,

Automated sequence- and stereo-specific assignment of methyl-labeled proteins by paramagnetic relaxation and methyl-methyl nuclear overhauser enhancement spectroscopy

Energy Technology Data Exchange (ETDEWEB)

Venditti, Vincenzo; Fawzi, Nicolas L.; Clore, G. Marius, E-mail: mariusc@mail.nih.gov [National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Laboratory of Chemical Physics (United States)

2011-11-15

Methyl-transverse relaxation optimized spectroscopy is rapidly becoming the preferred NMR technique for probing structure and dynamics of very large proteins up to {approx}1 MDa in molecular size. Data interpretation, however, necessitates assignment of methyl groups which still presents a very challenging and time-consuming process. Here we demonstrate that, in combination with a known 3D structure, paramagnetic relaxation enhancement (PRE), induced by nitroxide spin-labels incorporated at only a few surface-exposed engineered cysteines, provides fast, straightforward and robust access to methyl group resonance assignments, including stereoassignments for the methyl groups of leucine and valine. Neither prior assignments, including backbone assignments, for the protein, nor experiments that transfer magnetization between methyl groups and the protein backbone, are required. PRE-derived assignments are refined by 4D methyl-methyl nuclear Overhauser enhancement data, eliminating ambiguities and errors that may arise due to the high sensitivity of PREs to the potential presence of sparsely-populated transient states.

Epigenetic Pathways in Human Disease: The Impact of DNA Methylation on Stress-Related Pathogenesis and Current Challenges in Biomarker Development

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M. Austin Argentieri

2017-04-01

Full Text Available HPA axis genes implicated in glucocorticoid regulation play an important role in regulating the physiological impact of social and environmental stress, and have become a focal point for investigating the role of glucocorticoid regulation in the etiology of disease. We conducted a systematic review to critically assess the full range of clinical associations that have been reported in relation to DNA methylation of CRH, CRH-R1/2, CRH-BP, AVP, POMC, ACTH, ACTH-R, NR3C1, FKBP5, and HSD11β1/2 genes in adults. A total of 32 studies were identified. There is prospective evidence for an association between HSD11β2 methylation and hypertension, and functional evidence of an association between NR3C1 methylation and both small cell lung cancer (SCLC and breast cancer. Strong associations have been reported between FKBP5 and NR3C1 methylation and PTSD, and biologically-plausible associations have been reported between FKBP5 methylation and Alzheimer's Disease. Mixed associations between NR3C1 methylation and mental health outcomes have been reported according to different social and environmental exposures, and according to varying gene regions investigated. We conclude by highlighting key challenges and future research directions that will need to be addressed in order to develop both clinically meaningful prognostic biomarkers and an evidence base that can inform public policy practice.

Structural and Kinetic Evidence for an Extended Hydrogen-Bonding Network in Catalysis of Methyl Group Transfer

International Nuclear Information System (INIS)

Doukov, T.; Hemmi, H.; Drennan, C.; Ragsdale, S.

2007-01-01

The methyltetrahydrofolate (CH 3 -H 4 folate) corrinoid-ironsulfur protein (CFeSP) methyltransferase (MeTr) catalyzes transfer of the methyl group of CH3-H4folate to cob(I)amide. This key step in anaerobic CO and CO 2 fixation is similar to the first half-reaction in the mechanisms of other cobalamin-dependent methyltransferases. Methyl transfer requires electrophilic activation of the methyl group of CH 3 -H 4 folate, which includes proton transfer to the N5 group of the pterin ring and poises the methyl group for reaction with the Co(I) nucleophile. The structure of the binary CH 3 -H 4 folate/MeTr complex (revealed here) lacks any obvious proton donor near the N5 group. Instead, an Asn residue and water molecules are found within H-bonding distance of N5. Structural and kinetic experiments described here are consistent with the involvement of an extended H-bonding network in proton transfer to N5 of the folate that includes an Asn (Asn-199 in MeTr), a conserved Asp (Asp-160), and a water molecule. This situation is reminiscent of purine nucleoside phosphorylase, which involves protonation of the purine N7 in the transition state and is accomplished by an extended H-bond network that includes water molecules, a Glu residue, and an Asn residue (Kicska, G. A., Tyler, P. C., Evans, G. B., Furneaux, R. H., Shi, W., Fedorov, A., Lewandowicz, A., Cahill, S. M., Almo, S. C., and Schramm, V. L. (2002) Biochemistry 41, 14489-14498). In MeTr, the Asn residue swings from a distant position to within H-bonding distance of the N5 atom upon CH 3 -H 4 folate binding. An N199A variant exhibits only ∼20-fold weakened affinity for CH 3 -H 4 folate but a much more marked 20,000-40,000-fold effect on catalysis, suggesting that Asn-199 plays an important role in stabilizing a transition state or high energy intermediate for methyl transfer

Methylation diet and methyl group genetics in risk for adenomatous polyp occurrence

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Mark Lucock

2015-06-01

Conclusion: A methylation diet influences methyl group synthesis in the regulation of blood homocysteine level, and is modulated by genetic interactions. Methylation-related nutrients also interact with key genes to modify risk of AP, a precursor of colorectal cancer. Independent of diet, two methylation-related genes (A2756G-MS and A66G-MSR were directly associated with AP occurrence.

Reactions of guanine with methyl chloride and methyl bromide: O6-methylation versus charge transfer complex formation

Science.gov (United States)

Shukla, P. K.; Mishra, P. C.; Suhai, S.

Density functional theory (DFT) at the B3LYP/6-31+G* and B3LYP/AUG-cc-pVDZ levels was employed to study O6-methylation of guanine due to its reactions with methyl chloride and methyl bromide and to obtain explanation as to why the methyl halides cause genotoxicity and possess mutagenic and carcinogenic properties. Geometries of the various isolated species involved in the reactions, reactant complexes (RCs), and product complexes (PCs) were optimized in gas phase. Transition states connecting the reactant complexes with the product complexes were also optimized in gas phase at the same levels of theory. The reactant complexes, product complexes, and transition states were solvated in aqueous media using the polarizable continuum model (PCM) of the self-consistent reaction field theory. Zero-point energy (ZPE) correction to total energy and the corresponding thermal energy correction to enthalpy were made in each case. The reactant complexes of the keto form of guanine with methyl chloride and methyl bromide in water are appreciably more stable than the corresponding complexes involving the enol form of guanine. The nature of binding in the product complexes was found to be of the charge transfer type (O6mG+ · X-, X dbond Cl, Br). Binding of HCl, HBr, and H2O molecules to the PCs obtained with the keto form of guanine did not alter the positions of the halide anions in the PCs, and the charge transfer character of the PCs was also not modified due to this binding. Further, the complexes obtained due to the binding of HCl, HBr, and H2O molecules to the PCs had greater stability than the isolated PCs. The reaction barriers involved in the formation of PCs were found to be quite high (?50 kcal/mol). Mechanisms of genotoxicity, mutagenesis and carcinogenesis caused by the methyl halides appear to involve charge transfer-type complex formation. Thus the mechanisms of these processes involving the methyl halides appear to be quite different from those that involve the

Trans-methylation reactions in plants: focus on the activated methyl cycle.

Science.gov (United States)

Rahikainen, Moona; Alegre, Sara; Trotta, Andrea; Pascual, Jesús; Kangasjärvi, Saijaliisa

2018-02-01

Trans-methylation reactions are vital in basic metabolism, epigenetic regulation, RNA metabolism, and posttranslational control of protein function and therefore fundamental in determining the physiological processes in all living organisms. The plant kingdom is additionally characterized by the production of secondary metabolites that undergo specific hydroxylation, oxidation and methylation reactions to obtain a wide array of different chemical structures. Increasing research efforts have started to reveal the enzymatic pathways underlying the biosynthesis of complex metabolites in plants. Further engineering of these enzymatic machineries offers significant possibilities in the development of bio-based technologies, but necessitates deep understanding of their potential metabolic and regulatory interactions. Trans-methylation reactions are tightly coupled with the so-called activated methyl cycle (AMC), an essential metabolic circuit that maintains the trans-methylation capacity in all living cells. Tight regulation of the AMC is crucial in ensuring accurate trans-methylation reactions in different subcellular compartments, cell types, developmental stages and environmental conditions. This review addresses the organization and posttranslational regulation of the AMC and elaborates its critical role in determining metabolic regulation through modulation of methyl utilization in stress-exposed plants. © 2017 Scandinavian Plant Physiology Society.

Cytosine hypomethylation at CHG and CHH sites in the pleiotropic ...

Indian Academy of Sciences (India)

2013-12-16

Dec 16, 2013 ... The abasic gap left behind by the demethylase action is filled by any of the DNA ...... Inactivation of a DNA methylation pathway in maize reproduc- tive organs ... of diversity for morphological and yield related traits among.

21 CFR 177.2000 - Vinylidene chloride/methyl acrylate/methyl methacrylate polymers.

Science.gov (United States)

2010-04-01

... methacrylate polymers. 177.2000 Section 177.2000 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF...: POLYMERS Substances for Use as Basic Components of Single and Repeated Use Food Contact Surfaces § 177.2000 Vinylidene chloride/methyl acrylate/methyl methacrylate polymers. The vinylidene chloride/methyl acrylate...

Induction of cytosine arabinoside-resistant human myeloid leukemia cell death through autophagy regulation by hydroxychloroquine.

Science.gov (United States)

Kim, Yundeok; Eom, Ju-In; Jeung, Hoi-Kyung; Jang, Ji Eun; Kim, Jin Seok; Cheong, June-Won; Kim, Young Sam; Min, Yoo Hong

2015-07-01

We investigated the effects of the autophagy inhibitor hydroxychloroquine (HCQ) on cell death of cytosine arabinoside (Ara-C)-resistant human acute myeloid leukemia (AML) cells. Ara-C-sensitive (U937, AML-2) and Ara-C-resistant (U937/AR, AML-2/AR) human AML cell lines were used to evaluate HCQ-regulated cytotoxicity, autophagy, and apoptosis as well as effects on cell death-related signaling pathways. We found that HCQ-induced dose- and time-dependent cell death in Ara-C-resistant cells compared to Ara-C-sensitive cell lines. The extent of cell death and features of HCQ-induced autophagic markers including increase in microtubule-associated protein light chain 3 (LC3) I conversion to LC3-II, beclin-1, ATG5, as well as green fluorescent protein-LC3 positive puncta and autophagosome were remarkably greater in U937/AR cells. Also, p62/SQSTM1 was increased in response to HCQ. p62/SQSTM1 protein interacts with both LC3-II and ubiquitin protein and is degraded in autophagosomes. Therefore, a reduction of p62/SQSTM1 indicates increased autophagic degradation, whereas an increase of p62/SQSTM1 by HCQ indicates inhibited autophagic degradation. Knock down of p62/SQSTM1 using siRNA were prevented the HCQ-induced LC3-II protein level as well as significantly reduced the HCQ-induced cell death in U937/AR cells. Also, apoptotic cell death and caspase activation in U937/AR cells were increased by HCQ, provided evidence that HCQ-induced autophagy blockade. Taken together, our data show that HCQ-induced apoptotic cell death in Ara-C-resistant AML cells through autophagy regulation. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

Whole-genome methylation caller designed for methyl- DNA ...

African Journals Online (AJOL)

etchie

2013-02-20

Feb 20, 2013 ... Key words: Methyl-DNA immunoprecipitation, next-generation sequencing, Hidden ... its response to environmental cues. .... have a great potential to become the most cost-effective ... hg18 reference genome (set to 0 if not present in retrieved reads). ..... DNA methylation patterns and epigenetic memory.

Targeted inhibition of osteosarcoma tumor growth by bone marrow-derived mesenchymal stem cells expressing cytosine deaminase/5-fluorocytosine in tumor-bearing mice.

Science.gov (United States)

NguyenThai, Quynh-Anh; Sharma, Neelesh; Luong, Do Huynh; Sodhi, Simrinder Singh; Kim, Jeong-Hyun; Kim, Nameun; Oh, Sung-Jong; Jeong, Dong Kee

2015-01-01

Mesenchymal stem cells (MSCs) are considered as an attractive approach for gene or drug delivery in cancer therapy. In the present study, the ability of human bone marrow-derived MSCs expressing the cytosine deaminase/5-fluorocytosine prodrug (CD/5-FC MSCs) to target the human osteosarcoma cell line Cal72 was evaluated. The stable CD/5-FC MSC cell line was established by transfection of pEGFP containing the cytosine deaminase gene into MSCs with G418 selection. The anti-tumor effect was verified by a bystander effect assay in vitro and co-injection of Cal72 and CD/5-FC MSCs in cancer-bearing mice. The therapeutic CD/5-FC MSCs retained the characteristics of multipotent cells, such as differentiation into adipocytes/osteocytes and expression of mesenchymal markers (CD90 and CD44), and showed migration toward Cal72 cells to a greater extent than the native MSCs. The bystander effect assay showed that the CD/5-FC MSCs significantly augmented Cal72 cytotoxicity in direct co-culture and in the presence of 5-FC through the application of conditioned medium. In osteosarcoma-bearing mice, the CD/5-FC MSCs inhibited tumor growth compared to control mice subcutaneously injected with only Cal72 cells. Taken together, these findings suggest that CD/5-FC MSCs may be suitable for targeting human osteosarcoma. Copyright © 2015 John Wiley & Sons, Ltd.

Effects of methyl mercury exposure on pancreatic beta cell development and function.

Science.gov (United States)

Schumacher, Lauren; Abbott, Louise C

2017-01-01

Methyl mercury is an environmental contaminant of worldwide concern. Since the discovery of methyl mercury exposure due to eating contaminated fish as the underlying cause of the Minamata disaster, the scientific community has known about the sensitivity of the developing central nervous system to mercury toxicity. Warnings are given to pregnant women and young children to limit consumption of foods containing methyl mercury to protect the embryonic, fetal and postnatally developing central nervous system. However, evidence also suggests that exposure to methyl mercury or various forms of inorganic mercury may also affect development and function of other organs. Numerous reports indicate a worldwide increase in diabetes, particularly type 2 diabetes. Quite recently, methyl mercury has been shown to have adverse effects on pancreatic beta (β) cell development and function, resulting in insulin resistance and hyperglycemia and may even lead to the development of diabetes. This review discusses possible mechanisms by which methyl mercury exposure may adversely affect pancreatic β cell development and function, and the role that methyl mercury exposure may have in the reported worldwide increase in diabetes, particularly type 2 diabetes. While additional information is needed regarding associations between mercury exposure and specific mechanisms of the pathogenesis of diabetes in the human population, methyl mercury's adverse effects on the body's natural sources of antioxidants suggest that one possible therapeutic strategy could involve supplementation with antioxidants. Thus, it is important that additional investigation be undertaken into the role of methyl mercury exposure and reduced pancreatic β cell function. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Tissue culture-induced alteration in cytosine methylation in new rice ...

African Journals Online (AJOL)

Zizania DNA introgression could induce a large number of genetic and epigenetic changes of the new rice recombinant inbred lines genome. In this present study, we employed inter-simple sequence repeat (ISSR) to further study the genetic and epigenetic changes that are induced by tissue culture. Changes induced by ...

DNA Methylation and Methylation Polymorphism in Genetically Stable In vitro Regenerates of Jatropha curcas L. Using Methylation-Sensitive AFLP Markers.

Science.gov (United States)

Rathore, Mangal S; Jha, Bhavanath

2016-03-01

The present investigation aimed to evaluate the degree and pattern of DNA methylation using methylation-sensitive AFLP (MS-AFLP) markers in genetically stable in vitro regenerates of Jatropha curcas L.. The genetically stable in vitro regenerates were raised through direct organogenesis via enhanced axillary shoot bud proliferation (Protocol-1) and in vitro-derived leaf regeneration (Protocol-2). Ten selective combinations of MS-AFLP primers produced 462 and 477 MS-AFLP bands in Protocol-1 (P-1) and Protocol-2 (P-2) regenerates, respectively. In P-1 regenerates, 15.8-31.17 % DNA was found methylated with an average of 25.24 %. In P-2 regenerates, 15.93-32.7 % DNA was found methylated with an average of 24.11 %. Using MS-AFLP in P-1 and P-2 regenerates, 11.52-25.53 % and 13.33-25.47 % polymorphism in methylated DNA was reported, respectively. Compared to the mother plant, P-1 regenerates showed hyper-methylation while P-2 showed hypo-methylation. The results clearly indicated alternation in degree and pattern of DNA methylation; hence, epigenetic instability in the genetically stable in vitro regenerates of J. curcas, developed so far using two different regeneration systems and explants of two different origins. The homologous nucleotide fragments in genomes of P-1 and P-2 regenerates showing methylation re-patterning might be involved in immediate adaptive responses and developmental processes through differential regulation of transcriptome under in vitro conditions.

Analysis of DNA Hydroxymethylation Using Colorimetric Assay.

Science.gov (United States)

Golubov, Andrey; Kovalchuk, Igor

2017-01-01

Hydroxymethylcytosine (hmC or 5-hmC) is a nitrogen base occurring as a result of cytosine methylation followed by replacing a methyl group with a hydroxyl group through active oxidation. 5-hmC is considered to be one of the forms of epigenetic modification and is suggested as an intermediate step in a semi-active loss of DNA methylation mark. 5-hmC plays an important role in the epigenetic regulation of gene expression in animals, although its role in plants remains controversial. Here, we present a colorimetric method of quantification of 5-hmC using Brassica rapa DNA.

Traumatic stress and accelerated DNA methylation age: A meta-analysis.

Science.gov (United States)

Wolf, Erika J; Maniates, Hannah; Nugent, Nicole; Maihofer, Adam X; Armstrong, Don; Ratanatharathorn, Andrew; Ashley-Koch, Allison E; Garrett, Melanie; Kimbrel, Nathan A; Lori, Adriana; Va Mid-Atlantic Mirecc Workgroup; Aiello, Allison E; Baker, Dewleen G; Beckham, Jean C; Boks, Marco P; Galea, Sandro; Geuze, Elbert; Hauser, Michael A; Kessler, Ronald C; Koenen, Karestan C; Miller, Mark W; Ressler, Kerry J; Risbrough, Victoria; Rutten, Bart P F; Stein, Murray B; Ursano, Robert J; Vermetten, Eric; Vinkers, Christiaan H; Uddin, Monica; Smith, Alicia K; Nievergelt, Caroline M; Logue, Mark W

2018-06-01

Recent studies examining the association between posttraumatic stress disorder (PTSD) and accelerated aging, as defined by DNA methylation-based estimates of cellular age that exceed chronological age, have yielded mixed results. We conducted a meta-analysis of trauma exposure and PTSD diagnosis and symptom severity in association with accelerated DNA methylation age using data from 9 cohorts contributing to the Psychiatric Genomics Consortium PTSD Epigenetics Workgroup (combined N = 2186). Associations between demographic and cellular variables and accelerated DNA methylation age were also examined, as was the moderating influence of demographic variables. Meta-analysis of regression coefficients from contributing cohorts revealed that childhood trauma exposure (when measured with the Childhood Trauma Questionnaire) and lifetime PTSD severity evidenced significant, albeit small, meta-analytic associations with accelerated DNA methylation age (ps = 0.028 and 0.016, respectively). Sex, CD4T cell proportions, and natural killer cell proportions were also significantly associated with accelerated DNA methylation age (all ps age. There was no evidence of moderation of the trauma or PTSD variables by demographic factors. Results suggest that traumatic stress is associated with advanced epigenetic age and raise the possibility that cells integral to immune system maintenance and responsivity play a role in this. This study highlights the need for additional research into the biological mechanisms linking traumatic stress to accelerated DNA methylation age and the importance of furthering our understanding of the neurobiological and health consequences of PTSD. Published by Elsevier Ltd.

MECP2 Is a Frequently Amplified Oncogene with a Novel Epigenetic Mechanism That Mimics the Role of Activated RAS in Malignancy

DEFF Research Database (Denmark)

Neupane, Manish; Clark, Allison P.; Landini, Serena

2016-01-01

An unbiased genome-scale screen for unmutated genes that drive cancer growth when overexpressed identified methyl cytosine-guanine dinucleotide (CpG) binding protein 2 (MECP2) as a novel oncogene. MECP2 resides in a region of the X-chromosome that is significantly amplified across 18% of cancers,...

The global DNA methylation surrogate LINE-1 methylation is correlated with MGMT promoter methylation and is a better prognostic factor for glioma.

Directory of Open Access Journals (Sweden)

Fumiharu Ohka

Full Text Available Gliomas are the most frequently occurring primary brain tumor in the central nervous system of adults. Glioblastoma multiformes (GBMs, WHO grade 4 have a dismal prognosis despite the use of the alkylating agent, temozolomide (TMZ, and even low grade gliomas (LGGs, WHO grade 2 eventually transform to malignant secondary GBMs. Although GBM patients benefit from promoter hypermethylation of the O(6-methylguanine-DNA methyltransferase (MGMT that is the main determinant of resistance to TMZ, recent studies suggested that MGMT promoter methylation is of prognostic as well as predictive significance for the efficacy of TMZ. Glioma-CpG island methylator phenotype (G-CIMP in the global genome was shown to be a significant predictor of improved survival in patients with GBM. Collectively, we hypothesized that MGMT promoter methylation might reflect global DNA methylation. Additionally in LGGs, the significance of MGMT promoter methylation is still undetermined. In the current study, we aimed to determine the correlation between clinical, genetic, and epigenetic profiles including LINE-1 and different cancer-related genes and the clinical outcome in newly diagnosed 57 LGG and 54 GBM patients. Here, we demonstrated that (1 IDH1/2 mutation is closely correlated with MGMT promoter methylation and 1p/19q codeletion in LGGs, (2 LINE-1 methylation levels in primary and secondary GBMs are lower than those in LGGs and normal brain tissues, (3 LINE-1 methylation is proportional to MGMT promoter methylation in gliomas, and (4 higher LINE-1 methylation is a favorable prognostic factor in primary GBMs, even compared to MGMT promoter methylation. As a global DNA methylation marker, LINE-1 may be a promising marker in gliomas.

Recognition of methylated DNA through methyl-CpG binding domain proteins

DEFF Research Database (Denmark)

Zou, Xueqing; Ma, Wen; Solov'yov, Ilia

2012-01-01

DNA methylation is a key regulatory control route in epigenetics, involving gene silencing and chromosome inactivation. It has been recognized that methyl-CpG binding domain (MBD) proteins play an important role in interpreting the genetic information encoded by methylated DNA (mDNA). Although...... the function of MBD proteins has attracted considerable attention and is well characterized, the mechanism underlying mDNA recognition by MBD proteins is still poorly understood. In this article, we demonstrate that the methyl-CpG dinucleotides are recognized at the MBD-mDNA interface by two MBD arginines...

Induction of epigenetic variation in Arabidopsis by over-expression of DNA METHYLTRANSFERASE1 (MET1.

Directory of Open Access Journals (Sweden)

Samuel Brocklehurst

Full Text Available Epigenetic marks such as DNA methylation and histone modification can vary among plant accessions creating epi-alleles with different levels of expression competence. Mutations in epigenetic pathway functions are powerful tools to induce epigenetic variation. As an alternative approach, we investigated the potential of over-expressing an epigenetic function, using DNA METHYLTRANSFERASE1 (MET1 for proof-of-concept. In Arabidopsis thaliana, MET1 controls maintenance of cytosine methylation at symmetrical CG positions. At some loci, which contain dense DNA methylation in CG- and non-CG context, loss of MET1 causes joint loss of all cytosines methylation marks. We find that over-expression of both catalytically active and inactive versions of MET1 stochastically generates new epi-alleles at loci encoding transposable elements, non-coding RNAs and proteins, which results for most loci in an increase in expression. Individual transformants share some common phenotypes and genes with altered gene expression. Altered expression states can be transmitted to the next generation, which does not require the continuous presence of the MET1 transgene. Long-term stability and epigenetic features differ for individual loci. Our data show that over-expression of MET1, and potentially of other genes encoding epigenetic factors, offers an alternative strategy to identify epigenetic target genes and to create novel epi-alleles.

Green chemistry perspectives of methane conversion via oxidative methylation of aromatics over zeolite catalysts

Energy Technology Data Exchange (ETDEWEB)

Adebajo, M.O. [University of Queensland, St Lucia, Qld. (Australia)

2007-06-15

This paper provides a general overview of the recent work that we and other researchers have done on the utilisation of methane for catalytic methylation of aromatic compounds and for direct coal liquefaction for the production of liquid hydrocarbons. In particular, the paper presents a detailed description of more recent substantial experimental evidence that we have provided for the requirement of oxygen as a stoichiometry reactant for benzene methylation with methane over moderately acidic zeolite catalysts. The reaction, which has been termed 'oxidative methylation', was thus postulated to involve a two-step mechanism involving intermediate methanol formation by methane partial oxidation, followed by benzene methylation with methanol in the second step. However, strongly acidic zeolites can cause cracking of benzene to yield methylated products in the absence of oxygen. The participation of methane and oxygen, and the effective use of zeolite catalysts in this methylation reaction definitely have some positive green chemistry implications. Thus, the results of these previous studies are also discussed in this review in light of the principles and tools of green chemistry. Various metrics were used to evaluate the greenness, cost-effectiveness, and material and energy efficiency of the oxidative methylation reaction.

Evasion of short interfering RNA-directed antiviral silencing in Musa acuminata persistently infected with six distinct banana streak pararetroviruses.

Science.gov (United States)

Rajeswaran, Rajendran; Seguin, Jonathan; Chabannes, Matthieu; Duroy, Pierre-Olivier; Laboureau, Nathalie; Farinelli, Laurent; Iskra-Caruana, Marie-Line; Pooggin, Mikhail M

2014-10-01

Vegetatively propagated crop plants often suffer from infections with persistent RNA and DNA viruses. Such viruses appear to evade the plant defenses that normally restrict viral replication and spread. The major antiviral defense mechanism is based on RNA silencing generating viral short interfering RNAs (siRNAs) that can potentially repress viral genes posttranscriptionally through RNA cleavage and transcriptionally through DNA cytosine methylation. Here we examined the RNA silencing machinery of banana plants persistently infected with six pararetroviruses after many years of vegetative propagation. Using deep sequencing, we reconstructed consensus master genomes of the viruses and characterized virus-derived and endogenous small RNAs. Consistent with the presence of endogenous siRNAs that can potentially establish and maintain DNA methylation, the banana genomic DNA was extensively methylated in both healthy and virus-infected plants. A novel class of abundant 20-nucleotide (nt) endogenous small RNAs with 5'-terminal guanosine was identified. In all virus-infected plants, 21- to 24-nt viral siRNAs accumulated at relatively high levels (up to 22% of the total small RNA population) and covered the entire circular viral DNA genomes in both orientations. The hotspots of 21-nt and 22-nt siRNAs occurred within open reading frame (ORF) I and II and the 5' portion of ORF III, while 24-nt siRNAs were more evenly distributed along the viral genome. Despite the presence of abundant viral siRNAs of different size classes, the viral DNA was largely free of cytosine methylation. Thus, the virus is able to evade siRNA-directed DNA methylation and thereby avoid transcriptional silencing. This evasion of silencing likely contributes to the persistence of pararetroviruses in banana plants. We report that DNA pararetroviruses in Musa acuminata banana plants are able to evade DNA cytosine methylation and transcriptional gene silencing, despite being targeted by the host silencing

Analysis of DNA Methylation in Young People: Limited Evidence for an Association Between Victimization Stress and Epigenetic Variation in Blood.

Science.gov (United States)

Marzi, Sarah J; Sugden, Karen; Arseneault, Louise; Belsky, Daniel W; Burrage, Joe; Corcoran, David L; Danese, Andrea; Fisher, Helen L; Hannon, Eilis; Moffitt, Terrie E; Odgers, Candice L; Pariante, Carmine; Poulton, Richie; Williams, Benjamin S; Wong, Chloe C Y; Mill, Jonathan; Caspi, Avshalom

2018-01-12

DNA methylation has been proposed as an epigenetic mechanism by which early-life experiences become "embedded" in the genome and alter transcriptional processes to compromise health. The authors sought to investigate whether early-life victimization stress is associated with genome-wide DNA methylation. The authors tested the hypothesis that victimization is associated with DNA methylation in the Environmental Risk (E-Risk) Longitudinal Study, a nationally representative 1994-1995 birth cohort of 2,232 twins born in England and Wales and assessed at ages 5, 7, 10, 12, and 18 years. Multiple forms of victimization were ascertained in childhood and adolescence (including physical, sexual, and emotional abuse; neglect; exposure to intimate-partner violence; bullying; cyber-victimization; and crime). Epigenome-wide analyses of polyvictimization across childhood and adolescence revealed few significant associations with DNA methylation in peripheral blood at age 18, but these analyses were confounded by tobacco smoking and/or did not survive co-twin control tests. Secondary analyses of specific forms of victimization revealed sparse associations with DNA methylation that did not replicate across different operationalizations of the same putative victimization experience. Hypothesis-driven analyses of six candidate genes in the stress response (NR3C1, FKBP5, BDNF, AVP, CRHR1, SLC6A4) did not reveal predicted associations with DNA methylation in probes annotated to these genes. Findings from this epidemiological analysis of the epigenetic effects of early-life stress do not support the hypothesis of robust changes in DNA methylation in victimized young people. We need to come to terms with the possibility that epigenetic epidemiology is not yet well matched to experimental, nonhuman models in uncovering the biological embedding of stress.

Molecular characterization of a rice mutator-phenotype derived from an incompatible cross-pollination reveals transgenerational mobilization of multiple transposable elements and extensive epigenetic instability

Directory of Open Access Journals (Sweden)

Xu Chunming

2009-05-01

Full Text Available Abstract Background Inter-specific hybridization occurs frequently in plants, which may induce genetic and epigenetic instabilities in the resultant hybrids, allopolyploids and introgressants. It remains unclear however whether pollination by alien pollens of an incompatible species may impose a "biological stress" even in the absence of genome-merger or genetic introgression, whereby genetic and/or epigenetic instability of the maternal recipient genome might be provoked. Results We report here the identification of a rice mutator-phenotype from a set of rice plants derived from a crossing experiment involving two remote and apparently incompatible species, Oryza sativa L. and Oenothera biennis L. The mutator-phenotype (named Tong211-LP showed distinct alteration in several traits, with the most striking being substantially enlarged panicles. Expectably, gel-blotting by total genomic DNA of the pollen-donor showed no evidence for introgression. Characterization of Tong211-LP (S0 and its selfed progenies (S1 ruled out contamination (via seed or pollen or polyploidy as a cause for its dramatic phenotypic changes, but revealed transgenerational mobilization of several previously characterized transposable elements (TEs, including a MITE (mPing, and three LTR retrotransposons (Osr7, Osr23 and Tos17. AFLP and MSAP fingerprinting revealed extensive, transgenerational alterations in cytosine methylation and to a less extent also genetic variation in Tong211-LP and its immediate progenies. mPing mobility was found to correlate with cytosine methylation alteration detected by MSAP but not with genetic variation detected by AFLP. Assay by q-RT-PCR of the steady-state transcript abundance of a set of genes encoding for the various putative DNA methyltransferases, 5-methylcytosine DNA glycosylases, and small interference RNA (siRNA pathway-related proteins showed that, relative to the rice parental line, heritable perturbation in expression of 12 out of

Titanium dioxide nanoparticles enhance inorganic arsenic bioavailability and methylation in two freshwater algae species.

Science.gov (United States)

Luo, Zhuanxi; Wang, Zhenhong; Yan, Yameng; Li, Jinli; Yan, Changzhou; Xing, Baoshan

2018-07-01

The effect of titanium dioxide nanoparticles (nano-TiO 2 ) on the bioaccumulation and biotransformation of arsenic (As) remains largely unknown. In this study, we exposed two freshwater algae (Microcystis aeruginosa and Scenedesmus obliquus) to inorganic As (arsenite and arsenate) with the aim of increasing our understanding on As bioaccumulation and methylation in the presence of nano-TiO 2 . Direct evidence from transmission electron microscope (TEM) images show that nano-TiO 2 (anatase) entered exposed algae. Thus, nano-TiO 2 as carriers boosted As accumulation and methylation in these two algae species, which varied between inorganic As speciation and algae species. Specifically, nano-TiO 2 could markedly enhance arsenate (As(V)) accumulation in M. aeruginosa and arsenite (As(III)) accumulation in S. obliquus. Similarly, we found evidence of higher As methylation activity in the M. aeruginosa of As(III) 2 mg L -1 nano-TiO 2 treatment. Although this was also true for the S. obliquus (As(V)) treatment, this species exhibited higher As methylation compared to M. aeruginosa, being more sensitive to As associated with nano-TiO 2 compared to M. aeruginosa. Due to changes in pH levels inside these exposed algae, As dissociation from nano-TiO 2 inside algal cells enhanced As methylation. Accordingly, the potential influence of nanoparticles on the bioaccumulation and biotransformation of their co-contaminants deserves more attention. Copyright © 2018 Elsevier Ltd. All rights reserved.

Effects of nickel, chromate, and arsenite on histone 3 lysine methylation

International Nuclear Information System (INIS)

Zhou Xue; Li Qin; Arita, Adriana; Sun Hong; Costa, Max

2009-01-01

Occupational exposure to nickel (Ni), chromium (Cr), and arsenic (As) containing compounds has been associated with lung cancer and other adverse health effects. Their carcinogenic properties may be attributable in part, to activation and/or repression of gene expression induced by changes in the DNA methylation status and histone tail post-translational modifications. Here we show that individual treatment with nickel, chromate, and arsenite all affect the gene activating mark H3K4 methylation. We found that nickel (1 mM), chromate (10 μM), and arsenite (1 μM) significantly increase tri-methyl H3K4 after 24 h exposure in human lung carcinoma A549 cells. Seven days of exposure to lower levels of nickel (50 and 100 μM), chromate (0.5 and 1 μM) or arsenite (0.1, 0.5 and 1 μM) also increased tri-methylated H3K4 in A549 cells. This mark still remained elevated and inherited through cell division 7 days following removal of 1 μM arsenite. We also demonstrate by dual staining immunofluorescence microscopy that both H3K4 tri-methyl and H3K9 di-methyl marks increase globally after 24 h exposure to each metal treatment in A549 cells. However, the tri-methyl H3K4 and di-methyl H3K9 marks localize in different regions in the nucleus of the cell. Thus, our study provides further evidence that a mechanism(s) of carcinogenicity of nickel, chromate, and arsenite metal compounds may involve alterations of various histone tail modifications that may in turn affect the expression of genes that may cause transformation

A genome-wide methylation study on obesity Differential variability and differential methylation

NARCIS (Netherlands)

Xu, Xiaojing; Su, Shaoyong; Barnes, Vernon A.; De Miguel, Carmen; Pollock, Jennifer; Ownby, Dennis; Shi, Huidong; Zhu, Haidong; Snieder, Harold; Wang, Xiaoling

2013-01-01

Besides differential methylation, DNA methylation variation has recently been proposed and demonstrated to be a potential contributing factor to cancer risk. Here we aim to examine whether differential variability in methylation is also an important feature of obesity, a typical non-malignant common

Maternal intake of methyl-group donors affects DNA methylation of metabolic genes in infants.

Science.gov (United States)

Pauwels, Sara; Ghosh, Manosij; Duca, Radu Corneliu; Bekaert, Bram; Freson, Kathleen; Huybrechts, Inge; Langie, Sabine A S; Koppen, Gudrun; Devlieger, Roland; Godderis, Lode

2017-01-01

Maternal nutrition during pregnancy and infant nutrition in the early postnatal period (lactation) are critically involved in the development and health of the newborn infant. The Maternal Nutrition and Offspring's Epigenome (MANOE) study was set up to assess the effect of maternal methyl-group donor intake (choline, betaine, folate, methionine) on infant DNA methylation. Maternal intake of dietary methyl-group donors was assessed using a food-frequency questionnaire (FFQ). Before and during pregnancy, we evaluated maternal methyl-group donor intake through diet and supplementation (folic acid) in relation to gene-specific ( IGF2 DMR, DNMT1 , LEP , RXRA ) buccal epithelial cell DNA methylation in 6 months old infants ( n  = 114) via pyrosequencing. In the early postnatal period, we determined the effect of maternal choline intake during lactation (in mothers who breast-fed for at least 3 months) on gene-specific buccal DNA methylation ( n  = 65). Maternal dietary and supplemental intake of methyl-group donors (folate, betaine, folic acid), only in the periconception period, was associated with buccal cell DNA methylation in genes related to growth ( IGF2 DMR), metabolism ( RXRA ), and appetite control ( LEP ). A negative association was found between maternal folate and folic acid intake before pregnancy and infant LEP (slope = -1.233, 95% CI -2.342; -0.125, p  = 0.0298) and IGF2 DMR methylation (slope = -0.706, 95% CI -1.242; -0.107, p  = 0.0101), respectively. Positive associations were observed for maternal betaine (slope = 0.875, 95% CI 0.118; 1.633, p  = 0.0241) and folate (slope = 0.685, 95% CI 0.245; 1.125, p  = 0.0027) intake before pregnancy and RXRA methylation. Buccal DNMT1 methylation in the infant was negatively associated with maternal methyl-group donor intake in the first and second trimester of pregnancy and negatively in the third trimester. We found no clear association between maternal choline intake

Crystal structure of MboIIA methyltransferase

OpenAIRE

Osipiuk, Jerzy; Walsh, Martin A.; Joachimiak, Andrzej

2003-01-01

DNA methyltransferases (MTases) are sequence-specific enzymes which transfer a methyl group from S-adenosyl-l-methionine (AdoMet) to the amino group of either cytosine or adenine within a recognized DNA sequence. Methylation of a base in a specific DNA sequence protects DNA from nucleolytic cleavage by restriction enzymes recognizing the same DNA sequence. We have determined at 1.74 Å resolution the crystal structure of a β-class DNA MTase MboIIA (M·MboIIA) from the bacterium Moraxella bovis,...

Lung function discordance in monozygotic twins and associated differences in blood DNA methylation

DEFF Research Database (Denmark)

Bolund, Anneli C S; Starnawska, Anna; Miller, Martin R

2017-01-01

Background: Lung function is an important predictor of morbidity and mortality, with accelerated lung function decline reported to have immense consequences for the world's healthcare systems. The lung function decline across individual's lifetime is a consequence of age-related changes in lung...... as TGF-β-receptor-related genes, may be involved in the cross-sectional level and longitudinal change in lung function in middle-aged monozygotic twins....... and genetic factors. DNA methylation plays a crucial role in regulation of gene expression, with increasing evidence linking aberrant DNA methylation levels with a number of common human diseases. In this study, we investigated possible associations between genome-wide DNA methylation levels and lung function...

Aberrantly methylated DNA as a biomarker in breast cancer.

Science.gov (United States)

Kristiansen, Søren; Jørgensen, Lars M; Guldberg, Per; Sölétormos, György

2013-01-01

Aberrant DNA hypermethylation at gene promoters is a frequent event in human breast cancer. Recent genome-wide studies have identified hundreds of genes that exhibit differential methylation between breast cancer cells and normal breast tissue. Due to the tumor-specific nature of DNA hypermethylation events, their use as tumor biomarkers is usually not hampered by analytical signals from normal cells, which is a general problem for existing protein tumor markers used for clinical assessment of breast cancer. There is accumulating evidence that DNA-methylation changes in breast cancer patients occur early during tumorigenesis. This may open up for effective screening, and analysis of blood or nipple aspirate may later help in diagnosing breast cancer. As a more detailed molecular characterization of different types of breast cancer becomes available, the ability to divide patients into subgroups based on DNA biomarkers may improve prognosis. Serial monitoring of DNA-methylation markers in blood during treatment may be useful, particularly when the cancer burden is below the detection level for standard imaging techniques. Overall, aberrant DNA methylation has a great potential as a versatile biomarker tool for screening, diagnosis, prognosis and monitoring of breast cancer. Standardization of methods and biomarker panels will be required to fully exploit this clinical potential.