The Genomic Impact of DNA CpG Methylation on Gene Expression; Relationships in Prostate Cancer.

PubMed

Long, Mark D; Smiraglia, Dominic J; Campbell, Moray J

2017-02-14

The process of DNA CpG methylation has been extensively investigated for over 50 years and revealed associations between changing methylation status of CpG islands and gene expression. As a result, DNA CpG methylation is implicated in the control of gene expression in developmental and homeostasis processes, as well as being a cancer-driver mechanism. The development of genome-wide technologies and sophisticated statistical analytical approaches has ushered in an era of widespread analyses, for example in the cancer arena, of the relationships between altered DNA CpG methylation, gene expression, and tumor status. The remarkable increase in the volume of such genomic data, for example, through investigators from the Cancer Genome Atlas (TCGA), has allowed dissection of the relationships between DNA CpG methylation density and distribution, gene expression, and tumor outcome. In this manner, it is now possible to test that the genome-wide correlations are measurable between changes in DNA CpG methylation and gene expression. Perhaps surprisingly is that these associations can only be detected for hundreds, but not thousands, of genes, and the direction of the correlations are both positive and negative. This, perhaps, suggests that CpG methylation events in cancer systems can act as disease drivers but the effects are possibly more restricted than suspected. Additionally, the positive and negative correlations suggest direct and indirect events and an incomplete understanding. Within the prostate cancer TCGA cohort, we examined the relationships between expression of genes that control DNA methylation, known targets of DNA methylation and tumor status. This revealed that genes that control the synthesis of S -adenosyl-l-methionine (SAM) associate with altered expression of DNA methylation targets in a subset of aggressive tumors.

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

PubMed

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.

Optical biosensing strategies for DNA methylation analysis.

PubMed

Nazmul Islam, Md; Yadav, Sharda; Hakimul Haque, Md; Munaz, Ahmed; Islam, Farhadul; Al Hossain, Md Shahriar; Gopalan, Vinod; Lam, Alfred K; Nguyen, Nam-Trung; Shiddiky, Muhammad J A

2017-06-15

DNA methylation is an epigenetic modification of DNA, where a methyl group is added at the fifth carbon of the cytosine base to form 5 methyl cytosine (5mC) without altering the DNA sequences. It plays important roles in regulating many cellular processes by modulating key genes expression. Alteration in DNA methylation patterns becomes particularly important in the aetiology of different diseases including cancers. Abnormal methylation pattern could contribute to the pathogenesis of cancer either by silencing key tumor suppressor genes or by activating oncogenes. Thus, DNA methylation biosensing can help in the better understanding of cancer prognosis and diagnosis and aid the development of therapies. Over the last few decades, a plethora of optical detection techniques have been developed for analyzing DNA methylation using fluorescence, Raman spectroscopy, surface plasmon resonance (SPR), electrochemiluminescence and colorimetric readouts. This paper aims to comprehensively review the optical strategies for DNA methylation detection. We also present an overview of the remaining challenges of optical strategies that still need to be focused along with the lesson learnt while working with these techniques. Copyright © 2016 Elsevier B.V. All rights reserved.

High-Throughput Analysis of Global DNA Methylation Using Methyl-Sensitive Digestion.

PubMed

Shiratori, Hiromi; Feinweber, Carmen; Knothe, Claudia; Lötsch, Jörn; Thomas, Dominique; Geisslinger, Gerd; Parnham, Michael J; Resch, Eduard

2016-01-01

DNA methylation is a major regulatory process of gene transcription, and aberrant DNA methylation is associated with various diseases including cancer. Many compounds have been reported to modify DNA methylation states. Despite increasing interest in the clinical application of drugs with epigenetic effects, and the use of diagnostic markers for genome-wide hypomethylation in cancer, large-scale screening systems to measure the effects of drugs on DNA methylation are limited. In this study, we improved the previously established fluorescence polarization-based global DNA methylation assay so that it is more suitable for application to human genomic DNA. Our methyl-sensitive fluorescence polarization (MSFP) assay was highly repeatable (inter-assay coefficient of variation = 1.5%) and accurate (r2 = 0.99). According to signal linearity, only 50-80 ng human genomic DNA per reaction was necessary for the 384-well format. MSFP is a simple, rapid approach as all biochemical reactions and final detection can be performed in one well in a 384-well plate without purification steps in less than 3.5 hours. Furthermore, we demonstrated a significant correlation between MSFP and the LINE-1 pyrosequencing assay, a widely used global DNA methylation assay. MSFP can be applied for the pre-screening of compounds that influence global DNA methylation states and also for the diagnosis of certain types of cancer.

DNA methylation as a dynamic regulator of development and disease processes: spotlight on the prostate.

PubMed

Keil, Kimberly P; Vezina, Chad M

2015-01-01

Prostate development, benign hyperplasia and cancer involve androgen and growth factor signaling as well as stromal-epithelial interactions. We review how DNA methylation influences these and related processes in other organ systems such as how proliferation is restricted to specific cell populations during defined temporal windows, how androgens elicit their actions and how cells establish, maintain and remodel DNA methylation in a time and cell specific fashion. We also discuss mechanisms by which hormones and endocrine disrupting chemicals reprogram DNA methylation in the prostate and elsewhere and examine evidence for a reawakening of developmental epigenetic pathways as drivers of prostate cancer and benign prostate hyperplasia.

DNA methylation as a dynamic regulator of development and disease processes: spotlight on the prostate

PubMed Central

Keil, Kimberly P; Vezina, Chad M

2015-01-01

Prostate development, benign hyperplasia and cancer involve androgen and growth factor signaling as well as stromal–epithelial interactions. We review how DNA methylation influences these and related processes in other organ systems such as how proliferation is restricted to specific cell populations during defined temporal windows, how androgens elicit their actions and how cells establish, maintain and remodel DNA methylation in a time and cell specific fashion. We also discuss mechanisms by which hormones and endocrine disrupting chemicals reprogram DNA methylation in the prostate and elsewhere and examine evidence for a reawakening of developmental epigenetic pathways as drivers of prostate cancer and benign prostate hyperplasia. PMID:26077429

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

PubMed

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 DNA digestion.

Genome-wide analysis of salinity-stress induced DNA methylation alterations in cotton (Gossypium hirsutum L.) using the Me-DIP sequencing technology.

PubMed

Lu, X K; Shu, N; Wang, J J; Chen, X G; Wang, D L; Wang, S; Fan, W L; Guo, X N; Guo, L X; Ye, W W

2017-06-29

Cytosine DNA methylation is a significant form of DNA modification closely associated with gene expression in eukaryotes, fungi, animals, and plants. Although the reference genomes of cotton (Gossypium hirsutum L.) have been publically available, the salinity-stress-induced DNA methylome alterations in cotton are not well understood. Here, we constructed a map of genome-wide DNA methylation characteristics of cotton leaves under salt stress using the methylated DNA immunoprecipitation sequencing method. The results showed that the methylation reads on chromosome 9 were most comparable with those on the other chromosomes, but the greatest changes occurred on chromosome 8 under salt stress. The DNA methylation pattern analysis indicated that a relatively higher methylation density was found in the upstream2k and downstream2k elements of the CDS region and CG-islands. Almost 94% of the reads belonged to LTR-gspsy and LTR-copia, and the number of methylation reads in LTR-gypsy was four times greater than that in LTR-copia in both control and stressed samples. The analysis of differentially methylated regions (DMRs) showed that the gene elements upstream2k, intron, and downstream2k were hypomethylated, but the CDS regions were hypermethylated. The GO (Gene Ontology) analysis suggested that the methylated genes were most enriched in cellular processes, metabolic processes, cell parts and catalytic activities, which might be closely correlated with response to NaCl stress. In this study, we completed a genomic DNA methylation profile and conducted a DMR analysis under salt stress, which provided valuable information for the better understanding of epigenetics in response to salt stress in cotton.

DNA methylome of the 20-gigabase Norway spruce genome

PubMed Central

Ausin, Israel; Feng, Suhua; Yu, Chaowei; Liu, Wanlu; Kuo, Hsuan Yu; Jacobsen, Elise L.; Zhai, Jixian; Gallego-Bartolome, Javier; Wang, Lin; Egertsdotter, Ulrika; Street, Nathaniel R.; Jacobsen, Steven E.; Wang, Haifeng

2016-01-01

DNA methylation plays important roles in many biological processes, such as silencing of transposable elements, imprinting, and regulating gene expression. Many studies of DNA methylation have shown its essential roles in angiosperms (flowering plants). However, few studies have examined the roles and patterns of DNA methylation in gymnosperms. Here, we present genome-wide high coverage single-base resolution methylation maps of Norway spruce (Picea abies) from both needles and somatic embryogenesis culture cells via whole genome bisulfite sequencing. On average, DNA methylation levels of CG and CHG of Norway spruce were higher than most other plants studied. CHH methylation was found at a relatively low level; however, at least one copy of most of the RNA-directed DNA methylation pathway genes was found in Norway spruce, and CHH methylation was correlated with levels of siRNAs. In comparison with needles, somatic embryogenesis culture cells that are used for clonally propagating spruce trees showed lower levels of CG and CHG methylation but higher level of CHH methylation, suggesting that like in other species, these culture cells show abnormal methylation patterns. PMID:27911846

Towards understanding the breast cancer epigenome: a comparison of genome-wide DNA methylation and gene expression data

PubMed Central

Michiels, Stefan; Metzger-Filho, Otto; Saini, Kamal S.

2016-01-01

Until recently, an elevated disease risk has been ascribed to a genetic predisposition, however, exciting progress over the past years has discovered alternate elements of inheritance that involve epigenetic regulation. Epigenetic changes are heritably stable alterations that include DNA methylation, histone modifications and RNA-mediated silencing. Aberrant DNA methylation is a common molecular basis for a number of important human diseases, including breast cancer. Changes in DNA methylation profoundly affect global gene expression patterns. What is emerging is a more dynamic and complex association between DNA methylation and gene expression than previously believed. Although many tools have already been developed for analyzing genome-wide gene expression data, tools for analyzing genome-wide DNA methylation have not yet reached the same level of refinement. Here we provide an in-depth analysis of DNA methylation in parallel with gene expression data characteristics and describe the particularities of low-level and high-level analyses of DNA methylation data. Low-level analysis refers to pre-processing of methylation data (i.e. normalization, transformation and filtering), whereas high-level analysis is focused on illustrating the application of the widely used class comparison, class prediction and class discovery methods to DNA methylation data. Furthermore, we investigate the influence of DNA methylation on gene expression by measuring the correlation between the degree of CpG methylation and the level of expression and to explore the pattern of methylation as a function of the promoter region. PMID:26657508

Towards understanding the breast cancer epigenome: a comparison of genome-wide DNA methylation and gene expression data.

PubMed

Singhal, Sandeep K; Usmani, Nawaid; Michiels, Stefan; Metzger-Filho, Otto; Saini, Kamal S; Kovalchuk, Olga; Parliament, Matthew

2016-01-19

Until recently, an elevated disease risk has been ascribed to a genetic predisposition, however, exciting progress over the past years has discovered alternate elements of inheritance that involve epigenetic regulation. Epigenetic changes are heritably stable alterations that include DNA methylation, histone modifications and RNA-mediated silencing. Aberrant DNA methylation is a common molecular basis for a number of important human diseases, including breast cancer. Changes in DNA methylation profoundly affect global gene expression patterns. What is emerging is a more dynamic and complex association between DNA methylation and gene expression than previously believed. Although many tools have already been developed for analyzing genome-wide gene expression data, tools for analyzing genome-wide DNA methylation have not yet reached the same level of refinement. Here we provide an in-depth analysis of DNA methylation in parallel with gene expression data characteristics and describe the particularities of low-level and high-level analyses of DNA methylation data. Low-level analysis refers to pre-processing of methylation data (i.e. normalization, transformation and filtering), whereas high-level analysis is focused on illustrating the application of the widely used class comparison, class prediction and class discovery methods to DNA methylation data. Furthermore, we investigate the influence of DNA methylation on gene expression by measuring the correlation between the degree of CpG methylation and the level of expression and to explore the pattern of methylation as a function of the promoter region.

5-azacytidine promotes microspore embryogenesis initiation by decreasing global DNA methylation, but prevents subsequent embryo development in rapeseed and barley

PubMed Central

Solís, María-Teresa; El-Tantawy, Ahmed-Abdalla; Cano, Vanesa; Risueño, María C.; Testillano, Pilar S.

2015-01-01

Microspores are reprogrammed by stress in vitro toward embryogenesis. This process is an important tool in breeding to obtain double-haploid plants. DNA methylation is a major epigenetic modification that changes in differentiation and proliferation. We have shown changes in global DNA methylation during microspore reprogramming. 5-Azacytidine (AzaC) cannot be methylated and leads to DNA hypomethylation. AzaC is a useful demethylating agent to study DNA dynamics, with a potential application in microspore embryogenesis. This work analyzes the effects of short and long AzaC treatments on microspore embryogenesis initiation and progression in two species, the dicot Brassica napus and the monocot Hordeum vulgare. This involved the quantitative analyses of proembryo and embryo production, the quantification of DNA methylation, 5-methyl-deoxy-cytidine (5mdC) immunofluorescence and confocal microscopy, and the analysis of chromatin organization (condensation/decondensation) by light and electron microscopy. Four days of AzaC treatments (2.5 μM) increased embryo induction, response associated with a decrease of DNA methylation, modified 5mdC, and heterochromatin patterns compared to untreated embryos. By contrast, longer AzaC treatments diminished embryo production. Similar effects were found in both species, indicating that DNA demethylation promotes microspore reprogramming, totipotency acquisition, and embryogenesis initiation, while embryo differentiation requires de novo DNA methylation and is prevented by AzaC. This suggests a role for DNA methylation in the repression of microspore reprogramming and possibly totipotency acquisition. Results provide new insights into the role of epigenetic modifications in microspore embryogenesis and suggest a potential benefit of inhibitors, such as AzaC, to improve the process efficiency in biotechnology and breeding programs. PMID:26161085

Cocoa Consumption Alters the Global DNA Methylation of Peripheral Leukocytes in Humans with Cardiovascular Disease Risk Factors: A Randomized Controlled Trial

PubMed Central

Crescenti, Anna; Solà, Rosa; Valls, Rosa M.; Caimari, Antoni; del Bas, Josep M.; Anguera, Anna; Anglés, Neus; Arola, Lluís

2013-01-01

DNA methylation regulates gene expression and can be modified by different bioactive compounds in foods, such as polyphenols. Cocoa is a rich source of polyphenols, but its role in DNA methylation is still unknown. The objective was to assess the effect of cocoa consumption on DNA methylation and to determine whether the enzymes involved in the DNA methylation process participate in the mechanisms by which cocoa exerts these effects in humans. The global DNA methylation levels in the peripheral blood were evaluated in 214 volunteers who were pre-hypertensive, stage-1 hypertensive or hypercholesterolemic. The volunteers were divided into two groups: 110 subjects who consumed cocoa (6 g/d) for two weeks and 104 control subjects. In addition, the peripheral blood mononuclear cells (PBMCs) from six subjects were treated with a cocoa extract to analyze the mRNA levels of the DNA methyltransferases (DNMTs), methylenetetrahydrofolate reductase (MTHFR), and methionine synthase reductase (MTRR) genes. Cocoa consumption significantly reduced the DNA methylation levels (2.991±0.366 vs. 3.909±0.380, p<0.001). Additionally, we found an association between the cocoa effects on DNA methylation and three polymorphisms located in the MTHFR, MTRR, and DNMT3B genes. Furthermore, in PBMCs, the cocoa extract significantly lowered the mRNA levels of the DNMTs, MTHFR, and MTRR. Our study demonstrates for the first time that the consumption of cocoa decreases the global DNA methylation of peripheral leukocytes in humans with cardiovascular risk factors. In vitro experiments with PBMCs suggest that cocoa may exert this effect partially via the down-regulation of DNMTs, MTHFR and MTRR, which are key genes involved in this epigenetic process. Trial Registration Clinicaltrials.gov NCT00511420 and NCT00502047 PMID:23840361

Cocoa Consumption Alters the Global DNA Methylation of Peripheral Leukocytes in Humans with Cardiovascular Disease Risk Factors: A Randomized Controlled Trial.

PubMed

Crescenti, Anna; Solà, Rosa; Valls, Rosa M; Caimari, Antoni; Del Bas, Josep M; Anguera, Anna; Anglés, Neus; Arola, Lluís

2013-01-01

DNA methylation regulates gene expression and can be modified by different bioactive compounds in foods, such as polyphenols. Cocoa is a rich source of polyphenols, but its role in DNA methylation is still unknown. The objective was to assess the effect of cocoa consumption on DNA methylation and to determine whether the enzymes involved in the DNA methylation process participate in the mechanisms by which cocoa exerts these effects in humans. The global DNA methylation levels in the peripheral blood were evaluated in 214 volunteers who were pre-hypertensive, stage-1 hypertensive or hypercholesterolemic. The volunteers were divided into two groups: 110 subjects who consumed cocoa (6 g/d) for two weeks and 104 control subjects. In addition, the peripheral blood mononuclear cells (PBMCs) from six subjects were treated with a cocoa extract to analyze the mRNA levels of the DNA methyltransferases (DNMTs), methylenetetrahydrofolate reductase (MTHFR), and methionine synthase reductase (MTRR) genes. Cocoa consumption significantly reduced the DNA methylation levels (2.991±0.366 vs. 3.909±0.380, p<0.001). Additionally, we found an association between the cocoa effects on DNA methylation and three polymorphisms located in the MTHFR, MTRR, and DNMT3B genes. Furthermore, in PBMCs, the cocoa extract significantly lowered the mRNA levels of the DNMTs, MTHFR, and MTRR. Our study demonstrates for the first time that the consumption of cocoa decreases the global DNA methylation of peripheral leukocytes in humans with cardiovascular risk factors. In vitro experiments with PBMCs suggest that cocoa may exert this effect partially via the down-regulation of DNMTs, MTHFR and MTRR, which are key genes involved in this epigenetic process. Clinicaltrials.govNCT00511420 and NCT00502047.

[Definition of the specificity of DNA-methyltransferase M.Bsc4I in cell lysate by blocking of restriction endonucleases and computer modeling].

PubMed

Dedkov, V S

2009-01-01

The specificity of DNA-methyltransferase M.Bsc4I was defined in cellular lysate of Bacillus schlegelii 4. For this purpose, we used methylation sensitivity of restriction endonucleases, and also modeling of methylation. The modeling consisted in editing sequences of DNA using replacements of methylated bases and their complementary bases. The substratum DNA processed by M.Bsc4I also were used for studying sensitivity of some restriction endonucleases to methylation. Thus, it was shown that M.Bsc4I methylated 5'-Cm4CNNNNNNNGG-3' and the overlapped dcm-methylation blocked its activity. The offered approach can appear universal enough and simple for definition of specificity of DNA-methyltransferases.

Transient and permanent changes in DNA methylation patterns in inorganic arsenic-mediated epithelial-to-mesenchymal transition.

PubMed

Eckstein, Meredith; Rea, Matthew; Fondufe-Mittendorf, Yvonne N

2017-09-15

Chronic low dose inorganic arsenic exposure causes cells to take on an epithelial-to-mesenchymal phenotype, which is a crucial process in carcinogenesis. Inorganic arsenic is not a mutagen and thus epigenetic alterations have been implicated in this process. Indeed, during the epithelial-to-mesenchymal transition, morphologic changes to cells correlate with changes in chromatin structure and gene expression, ultimately driving this process. However, studies on the effects of inorganic arsenic exposure/withdrawal on the epithelial-to-mesenchymal transition and the impact of epigenetic alterations in this process are limited. In this study we used high-resolution microarray analysis to measure the changes in DNA methylation in cells undergoing inorganic arsenic-induced epithelial-to-mesenchymal transition, and on the reversal of this process, after removal of the inorganic arsenic exposure. We found that cells exposed to chronic, low-dose inorganic arsenic exposure showed 30,530 sites were differentially methylated, and with inorganic arsenic withdrawal several differential methylated sites were reversed, albeit not completely. Furthermore, these changes in DNA methylation mainly correlated with changes in gene expression at most sites tested but not at all. This study suggests that DNA methylation changes on gene expression are not clear-cut and provide a platform to begin to uncover the relationship between DNA methylation and gene expression, specifically within the context of inorganic arsenic treatment. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Global analysis of DNA methylation in young (J1) and senescent (J2) Gossypium hirsutum L. cotyledons by MeDIP-Seq

PubMed Central

Dou, Lingling; Jia, Xiaoyun; Wei, Hengling; Fan, Shuli; Wang, Hantao; Guo, Yaning; Duan, Shan; Pang, Chaoyou; Yu, Shuxun

2017-01-01

DNA methylation is an important epigenetic modification regulating gene expression, genomic imprinting, transposon silencing and chromatin structure in plants and plays an important role in leaf senescence. However, the DNA methylation pattern during Gossypium hirsutum L. cotyledon senescence is poorly understood. In this study, global DNA methylation patterns were compared between two cotyledon development stages, young (J1) and senescence (J2), using methylated DNA immunoprecipitation (MeDIP-Seq). Methylated cytosine occurred mostly in repeat elements, especially LTR/Gypsy in both J1 and J2. When comparing J1 against J2, there were 1222 down-methylated genes and 623 up-methylated genes. Methylated genes were significantly enriched in carbohydrate metabolism, biosynthesis of other secondary metabolites and amino acid metabolism pathways. The global DNA methylation level decreased from J1 to J2, especially in gene promoters, transcriptional termination regions and regions around CpG islands. We further investigated the expression patterns of 9 DNA methyltransferase-associated genes and 2 DNA demethyltransferase-associated genes from young to senescent cotyledons, which were down-regulated during cotyledon development. In this paper, we first reported that senescent cotton cotyledons exhibited lower DNA methylation levels, primarily due to decreased DNA methyltransferase activity and which also play important role in regulating secondary metabolite process. PMID:28715427

Current trends in electrochemical sensing and biosensing of DNA methylation.

PubMed

Krejcova, Ludmila; Richtera, Lukas; Hynek, David; Labuda, Jan; Adam, Vojtech

2017-11-15

DNA methylation plays an important role in physiological and pathological processes. Several genetic diseases and most malignancies tend to be associated with aberrant DNA methylation. Among other analytical methods, electrochemical approaches have been successfully employed for characterisation of DNA methylation patterns that are essential for the diagnosis and treatment of particular diseases. This article discusses current trends in the electrochemical sensing and biosensing of DNA methylation. Particularly, it provides an overview of applied electrode materials, electrode modifications and biorecognition elements applications with an emphasis on strategies that form the core DNA methylation detection approaches. The three main strategies as (i) bisulfite treatment, (ii) cleavage by restriction endonucleases, and (iii) immuno/affinity reaction were described in greater detail. Additionally, the availability of the reviewed platforms for early cancer diagnosis and the approval of methylation inhibitors for anticancer therapy were discussed. Copyright © 2017 Elsevier B.V. All rights reserved.

The effects of cytosine methylation on general transcription factors

NASA Astrophysics Data System (ADS)

Jin, Jianshi; Lian, Tengfei; Gu, Chan; Yu, Kai; Gao, Yi Qin; Su, Xiao-Dong

2016-07-01

DNA methylation on CpG sites is the most common epigenetic modification. Recently, methylation in a non-CpG context was found to occur widely on genomic DNA. Moreover, methylation of non-CpG sites is a highly controlled process, and its level may vary during cellular development. To study non-CpG methylation effects on DNA/protein interactions, we have chosen three human transcription factors (TFs): glucocorticoid receptor (GR), brain and muscle ARNT-like 1 (BMAL1) - circadian locomotor output cycles kaput (CLOCK) and estrogen receptor (ER) with methylated or unmethylated DNA binding sequences, using single-molecule and isothermal titration calorimetry assays. The results demonstrated that these TFs interact with methylated DNA with different effects compared with their cognate DNA sequences. The effects of non-CpG methylation on transcriptional regulation were validated by cell-based luciferase assay at protein level. The mechanisms of non-CpG methylation influencing DNA-protein interactions were investigated by crystallographic analyses and molecular dynamics simulation. With BisChIP-seq assays in HEK-293T cells, we found that GR can recognize highly methylated sites within chromatin in cells. Therefore, we conclude that non-CpG methylation of DNA can provide a mechanism for regulating gene expression through directly affecting the binding of TFs.

DNA methylation-based age prediction from various tissues and body fluids

PubMed Central

Jung, Sang-Eun; Shin, Kyoung-Jin; Lee, Hwan Young

2017-01-01

Aging is a natural and gradual process in human life. It is influenced by heredity, environment, lifestyle, and disease. DNA methylation varies with age, and the ability to predict the age of donor using DNA from evidence materials at a crime scene is of considerable value in forensic investigations. Recently, many studies have reported age prediction models based on DNA methylation from various tissues and body fluids. Those models seem to be very promising because of their high prediction accuracies. In this review, the changes of age-associated DNA methylation and the age prediction models for various tissues and body fluids were examined, and then the applicability of the DNA methylation-based age prediction method to the forensic investigations was discussed. This will improve the understandings about DNA methylation markers and their potential to be used as biomarkers in the forensic field, as well as the clinical field. PMID:28946940

The Influence of Hydroxylation on Maintaining CpG Methylation Patterns: A Hidden Markov Model Approach.

PubMed

Giehr, Pascal; Kyriakopoulos, Charalampos; Ficz, Gabriella; Wolf, Verena; Walter, Jörn

2016-05-01

DNA methylation and demethylation are opposing processes that when in balance create stable patterns of epigenetic memory. The control of DNA methylation pattern formation by replication dependent and independent demethylation processes has been suggested to be influenced by Tet mediated oxidation of 5mC. Several alternative mechanisms have been proposed suggesting that 5hmC influences either replication dependent maintenance of DNA methylation or replication independent processes of active demethylation. Using high resolution hairpin oxidative bisulfite sequencing data, we precisely determine the amount of 5mC and 5hmC and model the contribution of 5hmC to processes of demethylation in mouse ESCs. We develop an extended hidden Markov model capable of accurately describing the regional contribution of 5hmC to demethylation dynamics. Our analysis shows that 5hmC has a strong impact on replication dependent demethylation, mainly by impairing methylation maintenance.

Enhanced GSH synthesis by Bisphenol A exposure promoted DNA methylation process in the testes of adult rare minnow Gobiocypris rarus.

PubMed

Yuan, Cong; Zhang, Yingying; Liu, Yan; Zhang, Ting; Wang, Zaizhao

2016-09-01

DNA methylation is a commonly studied epigenetic modification. The mechanism of BPA on DNA methylation is poorly understood. The present study aims to explore whether GSH synthesis affects DNA methylation in the testes of adult male rare minnow Gobiocypris rarus in response to Bisphenol A (BPA). Male G. rarus was exposed to 1, 15 and 225μgL(-1) BPA for 7 days. The levels of global DNA methylation, hydrogen peroxide (H2O2) and glutathione (GSH) in the testes were analyzed. Meanwhile, the levels of enzymes involved in DNA methylation and de novo GSH synthesis, and the substrate contents for GSH production were measured. Furthermore, gene expression profiles of the corresponding genes of all studied enzymes were analyzed. Results indicated that BPA at 15 and 225μgL(-1) caused hypermethylation of global DNA in the testes. The 15μgL(-1) BPA resulted in significant decrease of ten-eleven translocation proteins (TETs) while 225μgL(-1) BPA caused significant increase of DNA methyltransferase proteins (DNMTs). Moreover, 225μgL(-1) BPA caused significant increase of H2O2 and GSH levels, and the de novo GSH synthesis was enhanced. These results indicated that the significant decrease of the level of TETs may be sufficient to cause the DNA hypermethylation by 15μgL(-1) BPA. However, the significantly increased of DNMTs contributed to the significant increase of DNA methylation levels by 225μgL(-1) BPA. Moreover, the elevated de novo GSH synthesis may promote the DNA methylation process. Copyright © 2016 Elsevier B.V. All rights reserved.

Associations between genetic variation in one-carbon metabolism and LINE-1 DNA methylation in histologically normal breast tissues

USDA-ARS?s Scientific Manuscript database

Genome-wide DNA hypomethylation is an early event in the carcinogenic process. Percent methylation of long interspersed nucleotide element-1 (LINE-1) is a biomarker of genome-wide methylation and is a potential biomarker for breast cancer. Understanding factors associated with percent LINE-1 DNA met...

DNA Methylation and Demethylation in Plant Immunity.

PubMed

Deleris, A; Halter, T; Navarro, L

2016-08-04

Detection of plant and animal pathogens triggers a massive transcriptional reprogramming, which is directed by chromatin-based processes, and ultimately results in antimicrobial immunity. Although the implication of histone modifications in orchestrating biotic stress-induced transcriptional reprogramming has been well characterized, very little was known, until recently, about the role of DNA methylation and demethylation in this process. In this review, we summarize recent findings on the dynamics and biological relevance of DNA methylation and demethylation in plant immunity against nonviral pathogens. In particular, we report the implications of these epigenetic regulatory processes in the transcriptional and co-transcriptional control of immune-responsive genes and discuss their relevance in fine-tuning antimicrobial immune responses. Finally, we discuss the possible yet elusive role of DNA methylation and demethylation in systemic immune responses, transgenerational immune priming, and de novo epiallelism, which could be adaptive.

DNA methylation mediated control of gene expression is critical for development of crown gall tumors.

PubMed

Gohlke, Jochen; Scholz, Claus-Juergen; Kneitz, Susanne; Weber, Dana; Fuchs, Joerg; Hedrich, Rainer; Deeken, Rosalia

2013-01-01

Crown gall tumors develop after integration of the T-DNA of virulent Agrobacterium tumefaciens strains into the plant genome. Expression of the T-DNA-encoded oncogenes triggers proliferation and differentiation of transformed plant cells. Crown gall development is known to be accompanied by global changes in transcription, metabolite levels, and physiological processes. High levels of abscisic acid (ABA) in crown galls regulate expression of drought stress responsive genes and mediate drought stress acclimation, which is essential for wild-type-like tumor growth. An impact of epigenetic processes such as DNA methylation on crown gall development has been suggested; however, it has not yet been investigated comprehensively. In this study, the methylation pattern of Arabidopsis thaliana crown galls was analyzed on a genome-wide scale as well as at the single gene level. Bisulfite sequencing analysis revealed that the oncogenes Ipt, IaaH, and IaaM were unmethylated in crown galls. Nevertheless, the oncogenes were susceptible to siRNA-mediated methylation, which inhibited their expression and subsequently crown gall growth. Genome arrays, hybridized with methylated DNA obtained by immunoprecipitation, revealed a globally hypermethylated crown gall genome, while promoters were rather hypomethylated. Mutants with reduced non-CG methylation developed larger tumors than the wild-type controls, indicating that hypermethylation inhibits plant tumor growth. The differential methylation pattern of crown galls and the stem tissue from which they originate correlated with transcriptional changes. Genes known to be transcriptionally inhibited by ABA and methylated in crown galls became promoter methylated upon treatment of A. thaliana with ABA. This suggests that the high ABA levels in crown galls may mediate DNA methylation and regulate expression of genes involved in drought stress protection. In summary, our studies provide evidence that epigenetic processes regulate gene expression, physiological processes, and the development of crown gall tumors.

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

PubMed

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.

Oxidative stress and alterations in DNA methylation: two sides of the same coin in reproduction.

PubMed

Menezo, Yves J R; Silvestris, Erica; Dale, Brian; Elder, Kay

2016-12-01

The negative effect of oxidative stress on the human reproductive process is no longer a matter for debate. Oxidative stress affects female and male gametes and the developmental capacity of embryos. Its effect can continue through late stages of pregnancy. Metabolic disorders and psychiatric problems can also be caued by DNA methylation and epigenetic errors. Age has a negative effect on oxidative stress and DNA methylation, and recent observations suggest that older men are at risk of transmitting epigenetic disorders to their offspring. Environmental endocrine disruptors can also increase oxidative stress and methylation errors. Oxidative stress and DNA methylation feature a common denominator: the one carbon cycle. This important metabolic pathway stimulates glutathione synthesis and recycles homocysteine, a molecule that interferes with the process of methylation. Glutathione plays a pivotal role during oocyte activation, protecting against reactive oxygen species. Assisted reproductive techniques may exacerbate defects in methylation and epigenesis. Antioxidant supplements are proposed to reduce the risk of potentially harmful effects, but their use has failed to prevent problems and may sometimes be detrimental. New concepts reveal a significant correlation between oxidative stress, methylation processes and epigenesis, and have led to changes in media composition with positive preliminary clinical consequences. Copyright © 2016 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

DNA demethylation activates genes in seed maternal integument development in rice (Oryza sativa L.).

PubMed

Wang, Yifeng; Lin, Haiyan; Tong, Xiaohong; Hou, Yuxuan; Chang, Yuxiao; Zhang, Jian

2017-11-01

DNA methylation is an important epigenetic modification that regulates various plant developmental processes. Rice seed integument determines the seed size. However, the role of DNA methylation in its development remains largely unknown. Here, we report the first dynamic DNA methylomic profiling of rice maternal integument before and after pollination by using a whole-genome bisulfite deep sequencing approach. Analysis of DNA methylation patterns identified 4238 differentially methylated regions underpin 4112 differentially methylated genes, including GW2, DEP1, RGB1 and numerous other regulators participated in maternal integument development. Bisulfite sanger sequencing and qRT-PCR of six differentially methylated genes revealed extensive occurrence of DNA hypomethylation triggered by double fertilization at IAP compared with IBP, suggesting that DNA demethylation might be a key mechanism to activate numerous maternal controlling genes. These results presented here not only greatly expanded the rice methylome dataset, but also shed novel insight into the regulatory roles of DNA methylation in rice seed maternal integument development. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

A novel restriction endonuclease GlaI for rapid and highly sensitive detection of DNA methylation coupled with isothermal exponential amplification reaction.

PubMed

Sun, Yueying; Sun, Yuanyuan; Tian, Weimin; Liu, Chenghui; Gao, Kejian; Li, Zhengping

2018-02-07

Sensitive and accurate detection of site-specific DNA methylation is of critical significance for early diagnosis of human diseases, especially cancers. Herein, for the first time we employ a novel methylation-dependent restriction endonuclease GlaI to detect site-specific DNA methylation in a highly specific and sensitive way by coupling with isothermal exponential amplification reaction (EXPAR). GlaI can only cut the methylated target site with excellent selectivity but leave the unmethylated DNA intact. Then the newly exposed end fragments of methylated DNA can trigger EXPAR for highly efficient signal amplification while the intact unmethylated DNA will not initiate EXPAR at all. As such, only the methylated DNA is quantitatively and faithfully reflected by the real-time fluorescence signal of the GlaI-EXPAR system, and the potential false positive interference from unmethylated DNA can be effectively eliminated. Therefore, by integrating the unique features of GlaI for highly specific methylation discrimination and EXPAR for rapid and powerful signal amplification, the elegant GlaI-EXPAR assay allows the direct quantification of methylated DNA with ultrahigh sensitivity and accuracy. The detection limit of methylated DNA target has been pushed down to the aM level and the whole detection process of GlaI-EXPAR can be accomplished within a short time of 2 h. More importantly, ultrahigh specificity is achieved and as low as 0.01% methylated DNA can be clearly identified in the presence of a large excess of unmethylated DNA. This GlaI-EXPAR is also demonstrated to be capable of determining site-specific DNA methylations in real genomic DNA samples. Sharing the distinct advantages of ultrahigh sensitivity, outstanding specificity and facile operation, this new GlaI-EXPAR strategy may provide a robust and reliable platform for the detection of site-specific DNA methylations with low abundances.

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

PubMed Central

Su, Chang; Wang, Chao; He, Lin; Yang, Chuanping; Wang, Yucheng

2014-01-01

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. PMID:25514241

Cooperative DNA binding and protein/DNA fiber formation increases the activity of the Dnmt3a DNA methyltransferase.

PubMed

Emperle, Max; Rajavelu, Arumugam; Reinhardt, Richard; Jurkowska, Renata Z; Jeltsch, Albert

2014-10-24

The Dnmt3a DNA methyltransferase has been shown to bind cooperatively to DNA and to form large multimeric protein/DNA fibers. However, it has also been reported to methylate DNA in a processive manner, a property that is incompatible with protein/DNA fiber formation. We show here that the DNA methylation rate of Dnmt3a increases more than linearly with increasing enzyme concentration on a long DNA substrate, but not on a short 30-mer oligonucleotide substrate. We also show that addition of a catalytically inactive Dnmt3a mutant, which carries an amino acid exchange in the catalytic center, increases the DNA methylation rate by wild type Dnmt3a on the long substrate but not on the short one. In agreement with this finding, preincubation experiments indicate that stable protein/DNA fibers are formed on the long, but not on the short substrate. In addition, methylation experiments with substrates containing one or two CpG sites did not provide evidence for a processive mechanism over a wide range of enzyme concentrations. These data clearly indicate that Dnmt3a binds to DNA in a cooperative reaction and that the formation of stable protein/DNA fibers increases the DNA methylation rate. Fiber formation occurs at low μm concentrations of Dnmt3a, which are in the range of Dnmt3a concentrations in the nucleus of embryonic stem cells. Understanding the mechanism of Dnmt3a is of vital importance because Dnmt3a is a hotspot of somatic cancer mutations one of which has been implicated in changing Dnmt3a processivity. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

S-adenosylmethionine levels regulate the Schwann cell DNA methylome

PubMed Central

Varela-Rey, Marta; Iruarrizaga-Lejarreta, Marta; Lozano, Juan José; Aransay, Ana María; Fernandez, Agustín F.; Lavin, José Luis; Mósen-Ansorena, David; Berdasco, María; Turmaine, Marc; Luka, Zigmund; Wagner, Conrad; Lu, Shelly C.; Esteller, Manel; Mirsky, Rhona; Jessen, Kristján R.; Fraga, Mario F.; Martínez-Chantar, María L.; Mato, José M.; Woodhoo, Ashwin

2014-01-01

SUMMARY Axonal myelination is essential for rapid saltatory impulse conduction in the nervous system, and malformation or destruction of myelin sheaths leads to motor and sensory disabilities. DNA methylation is an essential epigenetic modification during mammalian development, yet its role in myelination remains obscure. Here, using high-resolution methylome maps, we show that DNA methylation could play a key gene regulatory role in peripheral nerve myelination and that S-adenosylmethionine (SAMe), the principal methyl donor in cytosine methylation, regulates the methylome dynamics during this process. Our studies also point to a possible role of SAMe in establishing the aberrant DNA methylation patterns in a mouse model of diabetic neuropathy, implicating SAMe in the pathogenesis of this disease. These critical observations establish a link between SAMe and DNA methylation status in a defined biological system, and provides a novel mechanism that could direct methylation changes during cellular differentiation and in diverse pathological situations. PMID:24607226

Microplate-based platform for combined chromatin and DNA methylation immunoprecipitation assays

PubMed Central

2011-01-01

Background The processes that compose expression of a given gene are far more complex than previously thought presenting unprecedented conceptual and mechanistic challenges that require development of new tools. Chromatin structure, which is regulated by DNA methylation and histone modification, is at the center of gene regulation. Immunoprecipitations of chromatin (ChIP) and methylated DNA (MeDIP) represent a major achievement in this area that allow researchers to probe chromatin modifications as well as specific protein-DNA interactions in vivo and to estimate the density of proteins at specific sites genome-wide. Although a critical component of chromatin structure, DNA methylation has often been studied independently of other chromatin events and transcription. Results To allow simultaneous measurements of DNA methylation with other genomic processes, we developed and validated a simple and easy-to-use high throughput microplate-based platform for analysis of DNA methylation. Compared to the traditional beads-based MeDIP the microplate MeDIP was more sensitive and had lower non-specific binding. We integrated the MeDIP method with a microplate ChIP assay which allows measurements of both DNA methylation and histone marks at the same time, Matrix ChIP-MeDIP platform. We illustrated several applications of this platform to relate DNA methylation, with chromatin and transcription events at selected genes in cultured cells, human cancer and in a model of diabetic kidney disease. Conclusion The high throughput capacity of Matrix ChIP-MeDIP to profile tens and potentially hundreds of different genomic events at the same time as DNA methylation represents a powerful platform to explore complex genomic mechanism at selected genes in cultured cells and in whole tissues. In this regard, Matrix ChIP-MeDIP should be useful to complement genome-wide studies where the rich chromatin and transcription database resources provide fruitful foundation to pursue mechanistic, functional and diagnostic information at genes of interest in health and disease. PMID:22098709

Cytosine methylation of sperm DNA in horse semen after cryopreservation.

PubMed

Aurich, Christine; Schreiner, Bettina; Ille, Natascha; Alvarenga, Marco; Scarlet, Dragos

2016-09-15

Semen processing may contribute to epigenetic changes in spermatozoa. We have therefore addressed changes in sperm DNA cytosine methylation induced by cryopreservation of stallion semen. The relative amount of 5-methylcytosine relative to the genomic cytosine content of sperm DNA was analyzed by ELISA. In experiment 1, raw semen (n = 6 stallions, one ejaculate each) was shock-frozen. Postthaw semen motility and membrane integrity were completely absent, whereas DNA methylation was similar in raw (0.4 ± 0.2%) and shock-frozen (0.3 ± 0.1%) semen (not significant). In experiment 2, three ejaculates per stallion (n = 6) were included. Semen quality and DNA methylation was assessed before addition of the freezing extender and after freezing-thawing with either Ghent (G) or BotuCrio (BC) extender. Semen motility, morphology, and membrane integrity were significantly reduced by cryopreservation but not influenced by the extender (e.g., total motility: G 69.5 ± 2.0, BC 68.4 ± 2.2%; P < 0.001 vs. centrifugation). Cryopreservation significantly (P < 0.01) increased the level of DNA methylation (before freezing 0.6 ± 0.1%, postthaw G 6.4 ± 3.7, BC 4.4 ± 1.5%; P < 0.01), but no differences between the freezing extenders were seen. The level of DNA methylation was not correlated to semen motility, morphology, or membrane integrity. The results demonstrate that semen processing for cryopreservation increases the DNA methylation level in stallion semen. We conclude that assessment of sperm DNA methylation allows for evaluation of an additional parameter characterizing semen quality. The lower fertility rates of mares after insemination with frozen-thawed semen may at least in part be explained by cytosine methylation of sperm-DNA induced by the cryopreservation procedure. Copyright © 2016 Elsevier Inc. All rights reserved.

Structural and Functional Coordination of DNA and Histone Methylation

PubMed Central

Cheng, Xiaodong

2014-01-01

One of the most fundamental questions in the control of gene expression in mammals is how epigenetic methylation patterns of DNA and histones are established, erased, and recognized. This central process in controlling gene expression includes coordinated covalent modifications of DNA and its associated histones. This article focuses on structural aspects of enzymatic activities of histone (arginine and lysine) methylation and demethylation and functional links between the methylation status of the DNA and histones. An interconnected network of methyltransferases, demethylases, and accessory proteins is responsible for changing or maintaining the modification status of specific regions of chromatin. PMID:25085914

Loss of ATRX, associated with DNA methylation pattern of chromosome end, impacted biological behaviors of astrocytic tumors

PubMed Central

Zhang, Wei; Yang, Pei; Zhang, Chuanbao; Li, Mingyang; Yao, Kun; Wang, Hongjun; Li, Qingbin; Jiang, Chuanlu; Jiang, Tao

2015-01-01

Loss of ATRX leads to epigenetic alterations, including abnormal levels of DNA methylation at repetitive elements such as telomeres in murine cells. We conducted an extensive DNA methylation and mRNA expression profile study on a cohort of 82 patients with astrocytic tumors to study whether ATRX expression was associated with DNA methylation level in astrocytic tumors and in which cellular functions it participated. We observed that astrocytic tumors with lower ATRX expression harbored higher DNA methylation level at chromatin end and astrocytic tumors with ATRX-low had distinct gene expression profile and DNA methylation profile compared with ATRX-high tumors. Then, we uncovered that several ATRX associated biological functions in the DNA methylation and mRNA expression profile (GEP), including apoptotic process, DNA-dependent positive regulation of transcription, chromatin modification, and observed that ATRX expression was companied by MGMT methylation and expression. We also found that loss of ATRX caused by siRNA induced apoptotic cells increasing, reduced tumor cell proliferation and repressed the cell migration in glioma cells. Our results showed ATRX-related regulatory functions of the combined profiles from DNA methylation and mRNA expression in astrocytic tumors, and delineated that loss of ATRX impacted biological behaviors of astrocytic tumor cells, providing important resources for future dissection of ATRX role in glioma. PMID:25971279

Loss of ATRX, associated with DNA methylation pattern of chromosome end, impacted biological behaviors of astrocytic tumors.

PubMed

Cai, Jinquan; Chen, Jing; Zhang, Wei; Yang, Pei; Zhang, Chuanbao; Li, Mingyang; Yao, Kun; Wang, Hongjun; Li, Qingbin; Jiang, Chuanlu; Jiang, Tao

2015-07-20

Loss of ATRX leads to epigenetic alterations, including abnormal levels of DNA methylation at repetitive elements such as telomeres in murine cells. We conducted an extensive DNA methylation and mRNA expression profile study on a cohort of 82 patients with astrocytic tumors to study whether ATRX expression was associated with DNA methylation level in astrocytic tumors and in which cellular functions it participated. We observed that astrocytic tumors with lower ATRX expression harbored higher DNA methylation level at chromatin end and astrocytic tumors with ATRX-low had distinct gene expression profile and DNA methylation profile compared with ATRX-high tumors. Then, we uncovered that several ATRX associated biological functions in the DNA methylation and mRNA expression profile (GEP), including apoptotic process, DNA-dependent positive regulation of transcription, chromatin modification, and observed that ATRX expression was companied by MGMT methylation and expression. We also found that loss of ATRX caused by siRNA induced apoptotic cells increasing, reduced tumor cell proliferation and repressed the cell migration in glioma cells. Our results showed ATRX-related regulatory functions of the combined profiles from DNA methylation and mRNA expression in astrocytic tumors, and delineated that loss of ATRX impacted biological behaviors of astrocytic tumor cells, providing important resources for future dissection of ATRX role in glioma.

Extensive genetic and DNA methylation variation contribute to heterosis in triploid loquat hybrids.

PubMed

Liu, Chao; Wang, Mingbo; Wang, Lingli; Guo, Qigao; Liang, Guolu

2018-04-24

We aim to overcome the unclear origin of the loquat and elucidate the heterosis mechanism of the triploid loquat. Here we investigated the genetic and epigenetic variations between the triploid plant and its parental lines using amplified fragment length polymorphism (AFLP) and methylation-sensitive amplified fragment length polymorphism (MSAP) analyses. We show that in addition to genetic variations, extensive DNA methylation variation occurred during the formation process of triploid loquat, with the triploid hybrid having increased DNA methylation compared to the parents. Furthermore, a correlation existed between genetic variation and DNA methylation remodeling, suggesting that genome instability may lead to DNA methylation variation or vice versa. Sequence analysis of the MSAP bands revealed that over 53% of them overlap with protein-coding genes, which may indicate a functional role of the differential DNA methylation in gene regulation and hence heterosis phenotypes. Consistent with this, the genetic and epigenetic alterations were associated closely to the heterosis phenotypes of triploid loquat, and this association varied for different traits. Our results suggested that the formation of triploid is accompanied by extensive genetic and DNA methylation variation, and these changes contribute to the heterosis phenotypes of the triploid loquats from the two cross lines.

Genome-wide DNA methylation reprogramming in response to inorganic arsenic links inhibition of CTCF binding, DNMT expression and cellular transformation

NASA Astrophysics Data System (ADS)

Rea, Matthew; Eckstein, Meredith; Eleazer, Rebekah; Smith, Caroline; Fondufe-Mittendorf, Yvonne N.

2017-02-01

Chronic low dose inorganic arsenic (iAs) exposure leads to changes in gene expression and epithelial-to-mesenchymal transformation. During this transformation, cells adopt a fibroblast-like phenotype accompanied by profound gene expression changes. While many mechanisms have been implicated in this transformation, studies that focus on the role of epigenetic alterations in this process are just emerging. DNA methylation controls gene expression in physiologic and pathologic states. Several studies show alterations in DNA methylation patterns in iAs-mediated pathogenesis, but these studies focused on single genes. We present a comprehensive genome-wide DNA methylation analysis using methyl-sequencing to measure changes between normal and iAs-transformed cells. Additionally, these differential methylation changes correlated positively with changes in gene expression and alternative splicing. Interestingly, most of these differentially methylated genes function in cell adhesion and communication pathways. To gain insight into how genomic DNA methylation patterns are regulated during iAs-mediated carcinogenesis, we show that iAs probably targets CTCF binding at the promoter of DNA methyltransferases, regulating their expression. These findings reveal how CTCF binding regulates DNA methyltransferase to reprogram the methylome in response to an environmental toxin.

Genome-wide DNA methylation reprogramming in response to inorganic arsenic links inhibition of CTCF binding, DNMT expression and cellular transformation

PubMed Central

Rea, Matthew; Eckstein, Meredith; Eleazer, Rebekah; Smith, Caroline; Fondufe-Mittendorf , Yvonne N.

2017-01-01

Chronic low dose inorganic arsenic (iAs) exposure leads to changes in gene expression and epithelial-to-mesenchymal transformation. During this transformation, cells adopt a fibroblast-like phenotype accompanied by profound gene expression changes. While many mechanisms have been implicated in this transformation, studies that focus on the role of epigenetic alterations in this process are just emerging. DNA methylation controls gene expression in physiologic and pathologic states. Several studies show alterations in DNA methylation patterns in iAs-mediated pathogenesis, but these studies focused on single genes. We present a comprehensive genome-wide DNA methylation analysis using methyl-sequencing to measure changes between normal and iAs-transformed cells. Additionally, these differential methylation changes correlated positively with changes in gene expression and alternative splicing. Interestingly, most of these differentially methylated genes function in cell adhesion and communication pathways. To gain insight into how genomic DNA methylation patterns are regulated during iAs-mediated carcinogenesis, we show that iAs probably targets CTCF binding at the promoter of DNA methyltransferases, regulating their expression. These findings reveal how CTCF binding regulates DNA methyltransferase to reprogram the methylome in response to an environmental toxin. PMID:28150704

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.

PubMed

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 Mediated Control of Gene Expression Is Critical for Development of Crown Gall Tumors

PubMed Central

Kneitz, Susanne; Weber, Dana; Fuchs, Joerg; Hedrich, Rainer; Deeken, Rosalia

2013-01-01

Crown gall tumors develop after integration of the T-DNA of virulent Agrobacterium tumefaciens strains into the plant genome. Expression of the T-DNA–encoded oncogenes triggers proliferation and differentiation of transformed plant cells. Crown gall development is known to be accompanied by global changes in transcription, metabolite levels, and physiological processes. High levels of abscisic acid (ABA) in crown galls regulate expression of drought stress responsive genes and mediate drought stress acclimation, which is essential for wild-type-like tumor growth. An impact of epigenetic processes such as DNA methylation on crown gall development has been suggested; however, it has not yet been investigated comprehensively. In this study, the methylation pattern of Arabidopsis thaliana crown galls was analyzed on a genome-wide scale as well as at the single gene level. Bisulfite sequencing analysis revealed that the oncogenes Ipt, IaaH, and IaaM were unmethylated in crown galls. Nevertheless, the oncogenes were susceptible to siRNA–mediated methylation, which inhibited their expression and subsequently crown gall growth. Genome arrays, hybridized with methylated DNA obtained by immunoprecipitation, revealed a globally hypermethylated crown gall genome, while promoters were rather hypomethylated. Mutants with reduced non-CG methylation developed larger tumors than the wild-type controls, indicating that hypermethylation inhibits plant tumor growth. The differential methylation pattern of crown galls and the stem tissue from which they originate correlated with transcriptional changes. Genes known to be transcriptionally inhibited by ABA and methylated in crown galls became promoter methylated upon treatment of A. thaliana with ABA. This suggests that the high ABA levels in crown galls may mediate DNA methylation and regulate expression of genes involved in drought stress protection. In summary, our studies provide evidence that epigenetic processes regulate gene expression, physiological processes, and the development of crown gall tumors. PMID:23408907

DNA methylation Landscape of body size variation in sheep.

PubMed

Cao, Jiaxue; Wei, Caihong; Liu, Dongming; Wang, Huihua; Wu, Mingming; Xie, Zhiyuan; Capellini, Terence D; Zhang, Li; Zhao, Fuping; Li, Li; Zhong, Tao; Wang, Linjie; Lu, Jian; Liu, Ruizao; Zhang, Shifang; Du, Yongfei; Zhang, Hongping; Du, Lixin

2015-10-16

Sub-populations of Chinese Mongolian sheep exhibit significant variance in body mass. In the present study, we sequenced the whole genome DNA methylation in these breeds to detect whether DNA methylation plays a role in determining the body mass of sheep by Methylated DNA immunoprecipitation - sequencing method. A high quality methylation map of Chinese Mongolian sheep was obtained in this study. We identified 399 different methylated regions located in 93 human orthologs, which were previously reported as body size related genes in human genome-wide association studies. We tested three regions in LTBP1, and DNA methylation of two CpG sites showed significant correlation with its RNA expression. Additionally, a particular set of differentially methylated windows enriched in the "development process" (GO: 0032502) was identified as potential candidates for association with body mass variation. Next, we validated small part of these windows in 5 genes; DNA methylation of SMAD1, TSC1 and AKT1 showed significant difference across breeds, and six CpG were significantly correlated with RNA expression. Interestingly, two CpG sites showed significant correlation with TSC1 protein expression. This study provides a thorough understanding of body size variation in sheep from an epigenetic perspective.

Genomewide analysis indicates that queen larvae have lower methylation levels in the honey bee ( Apis mellifera)

NASA Astrophysics Data System (ADS)

Shi, Yuan Yuan; Yan, Wei Yu; Huang, Zachary Y.; Wang, Zi Long; Wu, Xiao Bo; Zeng, Zhi Jiang

2013-02-01

The honey bee is a social insect characterized by caste differentiation, by which a young larva can develop into either a queen or a worker. Despite possessing the same genome, queen and workers display marked differences in reproductive capacity, physiology, and behavior. Recent studies have shown that DNA methylation plays important roles in caste differentiation. To further explore the roles of DNA methylation in this process, we analyzed DNA methylome profiles of both queen larvae (QL) and worker larvae (WL) of different ages (2, 4, and 6 day old), by using methylated DNA immunoprecipitation-sequencing (meDIP-seq) technique. The global DNA methylation levels varied between the larvae of two castes. DNA methylation increased from 2-day- to 4-day-old QL and then decreased in 6-day-old larvae. In WL, methylation levels increased with age. The methylcytosines in both larvae were enriched in introns, followed by coding sequence (CDS) regions, CpG islands, 2 kbp downstream and upstream of genes, and 5' and 3' untranslated regions (UTRs). The number of differentially methylated genes (DMGs) in 2-, 4-, and 6-day-old QL and WL was 725, 3,013, and 5,049, respectively. Compared to 4- and 6-day-old WL, a large number of genes in QL were downmethylated, which were involved in many processes including development, reproduction, and metabolic regulation. In addition, some DMGs were concerned with caste differentiation.

Smoke-related DNA methylation changes in the etiology of human disease.

PubMed

Besingi, Welisane; Johansson, Asa

2014-05-01

Exposure to environmental and lifestyle factors, such as cigarette smoking, affect the epigenome and might mediate risk for diseases and cancers. We have performed a genome-wide DNA methylation study to determine the effect of smoke and snuff (smokeless tobacco) on DNA methylation. A total of 95 sites were differentially methylated [false discovery rate (FDR) q-values < 0.05] in smokers and a subset of the differentially methylated loci were also differentially expressed in smokers. We found no sites, neither any biological functions nor molecular processes enriched for smoke-less tobacco-related differential DNA methylation. This suggests that methylation changes are not caused by the basic components of the tobacco but from its burnt products. Instead, we see a clear enrichment (FDR q-value < 0.05) for genes, including CPOX, CDKN1A and PTK2, involved in response to arsenic-containing substance, which agrees with smoke containing small amounts of arsenic. A large number of biological functions and molecular processes with links to disease conditions are also enriched (FDR q-value < 0.05) for smoke-related DNA methylation changes. These include 'insulin receptor binding', and 'negative regulation of glucose import' which are associated with diabetes, 'positive regulation of interleukin-6-mediated signaling pathway', 'regulation of T-helper 2 cell differentiation', 'positive regulation of interleukin-13 production' which are associated with the immune system and 'sertoli cell fate commitment' which is important for male fertility. Since type 2 diabetes, repressed immune system and infertility have previously been associated with smoking, our results suggest that this might be mediated by DNA methylation changes.

DNA methylation in complex disease: applications in nursing research, practice, and policy.

PubMed

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

2013-01-01

DNA methylation is an epigenomic modification that is essential to normal human development and biological processes. DNA methylation patterns are heritable and dynamic throughout the life span. Environmental exposures can alter DNA methylation patterns, contributing to the development of complex disease. Identification and modulation of environmental factors influencing disease susceptibility through alterations in DNA methylation are amenable to nursing intervention and form the basis for individualized patient care. Here we describe the evidence supporting the translation of DNA methylation analyses as a tool for screening, diagnosis, and treatment of complex disease in nursing research and practice. The ethical, legal, social, and economic considerations of advances in genomics are considered as a model for epigenomic policy. We conclude that contemporary and informed nurse scientists and clinicians are uniquely poised to apply innovations in epigenomic research to clinical populations and develop appropriate policies that guide equitable and ethical use of new strategies to improve patient care. Copyright © 2013 Elsevier Inc. All rights reserved.

A Feature Selection Algorithm to Compute Gene Centric Methylation from Probe Level Methylation Data.

PubMed

Baur, Brittany; Bozdag, Serdar

2016-01-01

DNA methylation is an important epigenetic event that effects gene expression during development and various diseases such as cancer. Understanding the mechanism of action of DNA methylation is important for downstream analysis. In the Illumina Infinium HumanMethylation 450K array, there are tens of probes associated with each gene. Given methylation intensities of all these probes, it is necessary to compute which of these probes are most representative of the gene centric methylation level. In this study, we developed a feature selection algorithm based on sequential forward selection that utilized different classification methods to compute gene centric DNA methylation using probe level DNA methylation data. We compared our algorithm to other feature selection algorithms such as support vector machines with recursive feature elimination, genetic algorithms and ReliefF. We evaluated all methods based on the predictive power of selected probes on their mRNA expression levels and found that a K-Nearest Neighbors classification using the sequential forward selection algorithm performed better than other algorithms based on all metrics. We also observed that transcriptional activities of certain genes were more sensitive to DNA methylation changes than transcriptional activities of other genes. Our algorithm was able to predict the expression of those genes with high accuracy using only DNA methylation data. Our results also showed that those DNA methylation-sensitive genes were enriched in Gene Ontology terms related to the regulation of various biological processes.

Comparative Analyses of DNA Methylation and Sequence Evolution Using Nasonia Genomes

PubMed Central

Park, Jungsun; Peng, Zuogang; Zeng, Jia; Elango, Navin; Park, Taesung; Wheeler, Dave; Werren, John H.; Yi, Soojin V.

2011-01-01

The functional and evolutionary significance of DNA methylation in insect genomes remains to be resolved. Nasonia is well situated for comparative analyses of DNA methylation and genome evolution, since the genomes of a moderately distant outgroup species as well as closely related sibling species are available. Using direct sequencing of bisulfite-converted DNA, we uncovered a substantial level of DNA methylation in 17 of 18 Nasonia vitripennis genes and a strong correlation between methylation level and CpG depletion. Notably, in the sex-determining locus transformer, the exon that is alternatively spliced between the sexes is heavily methylated in both males and females, whereas other exons are only sparsely methylated. Orthologous genes of the honeybee and Nasonia show highly similar relative levels of CpG depletion, despite ∼190 My divergence. Densely and sparsely methylated genes in these species also exhibit similar functional enrichments. We found that the degree of CpG depletion is negatively correlated with substitution rates between closely related Nasonia species for synonymous, nonsynonymous, and intron sites. This suggests that mutation rates increase with decreasing levels of germ line methylation. Thus, DNA methylation is prevalent in the Nasonia genome, may participate in regulatory processes such as sex determination and alternative splicing, and is correlated with several aspects of genome and sequence evolution. PMID:21693438

Exploring the roles of DNA methylation in the metal-reducing bacterium Shewanella oneidensis MR-1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bendall, Matthew L.; Luong, Khai; Wetmore, Kelly M.

2013-08-30

We performed whole genome analyses of DNA methylation in Shewanella 17 oneidensis MR-1 to examine its possible role in regulating gene expression and 18 other cellular processes. Single-Molecule Real Time (SMRT) sequencing 19 revealed extensive methylation of adenine (N6mA) throughout the 20 genome. These methylated bases were located in five sequence motifs, 21 including three novel targets for Type I restriction/modification enzymes. The 22 sequence motifs targeted by putative methyltranferases were determined via 23 SMRT sequencing of gene knockout mutants. In addition, we found S. 24 oneidensis MR-1 cultures grown under various culture conditions displayed 25 different DNA methylation patterns.more » However, the small number of differentially 26 methylated sites could not be directly linked to the much larger number of 27 differentially expressed genes in these conditions, suggesting DNA methylation is 28 not a major regulator of gene expression in S. oneidensis MR-1. The enrichment 29 of methylated GATC motifs in the origin of replication indicate DNA methylation 30 may regulate genome replication in a manner similar to that seen in Escherichia 31 coli. Furthermore, comparative analyses suggest that many 32 Gammaproteobacteria, including all members of the Shewanellaceae family, may 33 also utilize DNA methylation to regulate genome replication.« less

Caulobacter crescentus Cell Cycle-Regulated DNA Methyltransferase Uses a Novel Mechanism for Substrate Recognition.

PubMed

Woodcock, Clayton B; Yakubov, Aziz B; Reich, Norbert O

2017-08-01

Caulobacter crescentus relies on DNA methylation by the cell cycle-regulated methyltransferase (CcrM) in addition to key transcription factors to control the cell cycle and direct cellular differentiation. CcrM is shown here to efficiently methylate its cognate recognition site 5'-GANTC-3' in single-stranded and hemimethylated double-stranded DNA. We report the K m , k cat , k methylation , and K d for single-stranded and hemimethylated substrates, revealing discrimination of 10 7 -fold for noncognate sequences. The enzyme also shows a similar discrimination against single-stranded RNA. Two independent assays clearly show that CcrM is highly processive with single-stranded and hemimethylated DNA. Collectively, the data provide evidence that CcrM and other DNA-modifying enzymes may use a new mechanism to recognize DNA in a key epigenetic process.

Dynamic changes of genome-wide DNA methylation during soybean seed development

USDA-ARS?s Scientific Manuscript database

Seed development is programmed by expression of many genes in plants. Seed maturation is an important developmental process to soybean seed quality and yield. DNA methylation is a major epigenetic modification regulating gene expression. However, little is known about the dynamic nature of DNA me...

Neonatal exposure to diethylstilbestrol alters expression of DNA methyltransferases and methylation of genomic DNA in the mouse uterus.

PubMed

Sato, Koji; Fukata, Hideki; Kogo, Yasushi; Ohgane, Jun; Shiota, Kunio; Mori, Chisato

2009-01-01

Perinatal exposure to diethylstilbestrol (DES) can have numerous adverse effects on the reproductive organs later in life, such as vaginal clear-cell adenocarcinoma. Epigenetic processes including DNA methylation may be involved in the mechanisms. We subcutaneously injected DES to neonatal C57BL/6 mice. At days 5, 14, and 30, expressions of DNA methyltransferases (Dnmts) Dnmt1, Dnmt3a, and Dnmt3b, and transcription factors Sp1 and Sp3 were examined. We also performed restriction landmark genomic scanning (RLGS) to detect aberrant DNA methylation. Real-time RT-PCR revealed that expressions of Dnmt1, Dnmt3b, and Sp3 were decreased at day 5 in DES-treated mice, and that those of Dnmt1, Dnmt3a, and Sp1 were also decreased at day 14. RLGS analysis revealed that 5 genomic loci were demethylated, and 5 other loci were methylated by DES treatment. Two loci were cloned, and differential DNA methylation was quantified. Our results indicated that DES altered the expression levels of Dnmts and DNA methylation.

Comparative Analysis of DNA Methylation Reveals Specific Regulations on Ethylene Pathway in Tomato Fruit

PubMed Central

Zuo, Jinhua; Wang, Yunxiang; Zhu, Benzhong; Luo, Yunbo; Wang, Qing; Gao, Lipu

2018-01-01

DNA methylation is an essential feature of epigenetic regulation and plays a role in various physiological and biochemical processes at CG, CHG, and CHH sites in plants. LeERF1 is an ethylene response factor (ERF) found in tomatoes which plays an important role in ethylene signal transduction. To explore the characteristics of DNA methylation in the ethylene pathway, sense-/antisense-LeERF1 transgenic tomato fruit were chosen for deep sequencing and bioinformatics parsing. The methylation type with the greatest distribution was CG, (71.60–72.80%) and CHH was found least frequently (10.70–12.50%). The level of DNA methylation was different among different tomato genomic regions. The differentially methylated regions (DMRs) and the differentially expressed genes (DEGs) were conjointly analyzed and 3030 different expressed genes were found, of which several are involved in ethylene synthesis and signaling transduction (such as ACS, ACO, MADS-Box, ERFs, and F-box). Furthermore, the relationships between DNA methylation and microRNAs (miRNAs) were also deciphered, providing basic information for the further study of DNA methylation and small RNAs involved in the ethylene pathway. PMID:29883429

Patterns of DNA Methylation Across the Leptin Core Promoter in Four Diverse Asian and North American Populations.

PubMed

Mosher, M J; Melton, P E; Stapleton, P; Schanfield, M S; Crawford, M H

2016-04-01

DNA methylation is the most widely studied of epigenetic mechanisms, with environmental effects recorded through patterned attachments of methyl groups along the DNA that are capable of modifying gene expression without altering the DNA sequencing. The degree to which these patterns of DNA methylation are heritable, the expected range of normality across populations, and the phenotypic relevance of pattern variation remain unclear. Genes regulating metabolic pathways appear to be vulnerable to ongoing nutritional programming over the life course, as dietary nutrients are significant environmental determinants of DNA methylation, supplying both the methyl groups and energy to generate the methylation process. Here we examine methylation patterns along a region of the metabolic gene leptin (LEP). LEP's putative functions include regulation of energy homeostasis, with its signals affecting energy intake and expenditure, adipogenesis and energy storage, lipid and glucose metabolism, bone metabolism, and reproductive endocrine function. A pattern of differential methylation across CpG sites of the LEP core promoter has been previously identified; however, any consistency of pattern or its phenotypic significance is not fully elucidated among populations. Using DNA extracted from unfractionated white blood cells of peripheral blood samples, our pilot study, divided into two parts, examined the significance of variation in DNA methylation patterns along the leptin core promoter in four populations (phase 1) and used biomarkers reflecting leptin's functional process in two of those populations, western Buryat of Siberia and the Mennonite of central Kansas, to investigate the relevance of the ethnic variation identified in the DNA methylation (phase 2). LEP's core promoter region contains both the binding site for C/EBPα (CCAAT/enhancer binding protein alpha), which tempers the final step in adipocyte maturity and capacity to synthesize leptin, and the TATA motif controlling leptin synthesis. Previous studies report that increased methylation in this region is correlated to decreased gene expression, suggesting tissue-specific methylation variation at this region ( Melzner et al. 2002 ). We hypothesized that evidence of nutritional epigenetic programming would be identified through variation in patterns of DNA methylation and that functional relevance of that variation among populations would be identified through biomarkers that reflect leptin's metabolic signals: serum leptin levels, lipoproteins of the lipid transport system, and anthropometric measures. In phase 1, our combined analyses of 313 individuals documented a distinct and consistent overall pattern of differential DNA methylation across seven CpG sites of LEP core promoter in all ethnicities and both sexes. This pattern replicates those identified in previous studies, suggesting a conserved core promoter region across populations. Phase 2 analyses of two of the four populations (n = 239), correlating methylation at the C/EBPα transcription binding site (TBS) with metabolic and anthropometric biomarkers reflecting LEP roles, showed that stature, which reflects bone growth and remodeling, was significantly and inversely correlated with the percentage of DNA methylation at this site in both sexes. We suggest that variation in DNA methylation along the LEP core promoter plays a substantial role in energy signals affecting both adipogenesis and bone metabolism.

DNA Methylation Biomarkers: Cancer and Beyond

PubMed Central

Mikeska, Thomas; Craig, Jeffrey M.

2014-01-01

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. PMID:25229548

Characterization of DNA methyltransferase and demethylase genes in Fragaria vesca.

PubMed

Gu, Tingting; Ren, Shuai; Wang, Yuanhua; Han, Yuhui; Li, Yi

2016-06-01

DNA methylation is an epigenetic modification essential for gene regulations in plants, but understanding on how it is involved in fruit development, especially in non-climacteric fleshy fruit, is limited. The diploid woodland strawberry (Fragaria vesca) is an important model for non-climacteric fruit crops. In this study, we identified DNA methyltransferase genes and demethylase genes in Fragaria vesca and other angiosperm species. In accordance with previous studies, our phylogenetic analyses of those DNA methylation modifiers support the clustering of those genes into several classes. Our data indicate that whole-genome duplications and tandem duplications contributed to the expansion of those DNA methylation modifiers in angiosperms. We have further demonstrated that some DNA methylase and demethylase genes reach their highest expression levels in strawberry fleshy fruits when turning from white to red, suggesting that DNA methylation might undergo a dramatic change at the onset of fleshy fruit-ripening process. In addition, we have observed that expression of some DNA demethylase genes increases in response to various abiotic stresses including heat, cold, drought and salinity. Collectively, our study indicates a regulatory role of DNA methylation in the turning stage of non-climacteric fleshy fruit and responses to environment stimuli, and would facilitate functional studies of DNA methylation in the growth and development of non-climacteric fruits.

Dynamics of DNA methylomes underlie oyster development

PubMed Central

Sourdaine, Pascal; Guo, Ximing; Favrel, Pascal

2017-01-01

DNA methylation is a critical epigenetic regulator of development in mammals and social insects, but its significance in development outside these groups is not understood. Here we investigated the genome-wide dynamics of DNA methylation in a mollusc model, the oyster Crassostrea gigas, from the egg to the completion of organogenesis. Large-scale methylation maps reveal that the oyster genome displays a succession of methylated and non methylated regions, which persist throughout development. Differentially methylated regions (DMRs) are strongly regulated during cleavage and metamorphosis. The distribution and levels of methylated DNA within genomic features (exons, introns, promoters, repeats and transposons) show different developmental lansdscapes marked by a strong increase in the methylation of exons against introns after metamorphosis. Kinetics of methylation in gene-bodies correlate to their transcription regulation and to distinct functional gene clusters, and DMRs at cleavage and metamorphosis bear the genes functionally related to these steps, respectively. This study shows that DNA methylome dynamics underlie development through transcription regulation in the oyster, a lophotrochozoan species. To our knowledge, this is the first demonstration of such epigenetic regulation outside vertebrates and ecdysozoan models, bringing new insights into the evolution and the epigenetic regulation of developmental processes. PMID:28594821

Dynamics of DNA methylomes underlie oyster development.

PubMed

Riviere, Guillaume; He, Yan; Tecchio, Samuele; Crowell, Elizabeth; Gras, Michaël; Sourdaine, Pascal; Guo, Ximing; Favrel, Pascal

2017-06-01

DNA methylation is a critical epigenetic regulator of development in mammals and social insects, but its significance in development outside these groups is not understood. Here we investigated the genome-wide dynamics of DNA methylation in a mollusc model, the oyster Crassostrea gigas, from the egg to the completion of organogenesis. Large-scale methylation maps reveal that the oyster genome displays a succession of methylated and non methylated regions, which persist throughout development. Differentially methylated regions (DMRs) are strongly regulated during cleavage and metamorphosis. The distribution and levels of methylated DNA within genomic features (exons, introns, promoters, repeats and transposons) show different developmental lansdscapes marked by a strong increase in the methylation of exons against introns after metamorphosis. Kinetics of methylation in gene-bodies correlate to their transcription regulation and to distinct functional gene clusters, and DMRs at cleavage and metamorphosis bear the genes functionally related to these steps, respectively. This study shows that DNA methylome dynamics underlie development through transcription regulation in the oyster, a lophotrochozoan species. To our knowledge, this is the first demonstration of such epigenetic regulation outside vertebrates and ecdysozoan models, bringing new insights into the evolution and the epigenetic regulation of developmental processes.

Physiological differences and changes in global DNA methylation levels in Agave angustifolia Haw. albino variant somaclones during the micropropagation process.

PubMed

Duarte-Aké, Fátima; Castillo-Castro, Eduardo; Pool, Felipe Barredo; Espadas, Francisco; Santamaría, Jorge M; Robert, Manuel L; De-la-Peña, Clelia

2016-12-01

Global DNA methylation changes caused by in vitro conditions are associated with the subculturing and phenotypic variation in Agave angustifolia Haw. While the relationship between the development of albinism and in vitro culture is well documented, the role of epigenetic processes in this development leaves some important questions unanswered. During the micropropagation of Agave angustifolia Haw., we found three different phenotypes, green (G), variegated (V) and albino (A). To understand the physiological and epigenetic differences among the somaclones, we analyzed several morphophysiological parameters and changes in the DNA methylation patterns in the three phenotypes during their in vitro development. We found that under in vitro conditions, the V plantlets maintained their CAM photosynthetic capacity, while the A variant showed no pigments and lost its CAM photosynthetic ability. Epigenetic analysis revealed that global DNA methylation increased in the G phenotype during the first two subcultures. However, after that time, DNA methylation levels declined. This hypomethylation correlated with the appearance of V shoots in the G plantlets. A similar correlation occurred in the V phenotype, where an increase of 2 % in the global DNA methylation levels was correlated with the generation of A shoots in the V plantlets. This suggests that an "epigenetic stress memory" during in vitro conditions causes a chromatin shift that favors the generation of variegated and albino shoots.

Quantitative analysis of DNA methylation in the promoter region of the methylguanine-O(6) -DNA-methyltransferase gene by COBRA and subsequent native capillary gel electrophoresis.

PubMed

Goedecke, Simon; Mühlisch, Jörg; Hempel, Georg; Frühwald, Michael C; Wünsch, Bernhard

2015-12-01

Along with histone modifications, RNA interference and delayed replication timing, DNA methylation belongs to the key processes in epigenetic regulation of gene expression. Therefore, reliable information about the methylation level of particular DNA fragments is of major interest. Herein the methylation level at two positions of the promoter region of the gene methylguanine-O(6) -DNA-Methyltransferase (MGMT) was investigated. Previously, it was demonstrated that the epigenetic status of this DNA region correlates with response to alkylating anticancer agents. An automated CGE method with LIF detection was established to separate the six DNA fragments resulting from combined bisulfite restriction analysis of the methylated and non-methylated MGMT promoter. In COBRA, the DNA was treated with bisulfite converting cytosine into uracil. During PCR uracil pairs with adenine, which changes the original recognition site of the restriction enzyme Taql. Artificial probes generated by mixing appropriate amounts of DNA after bisulfite treatment and PCR amplification were used for validation of the method. The methylation levels of these samples could be determined with high accuracy and precision. DNA samples prepared by mixing the corresponding clones first and then performing PCR amplification led to non-linear correlation between the corrected peak areas and the methylation levels. This effect is explained by slightly different PCR amplification of DNA with different sequences present in the mixture. The superiority of CGE over PAGE was clearly demonstrated. Finally, the established method was used to analyze the methylation levels of human brain tumor tissue samples. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

DNA Methylation and Methylation Polymorphism in Genetically Stable In vitro Regenerates of Jatropha curcas L. Using Methylation-Sensitive AFLP Markers.

PubMed

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.

Insufficient DNA methylation affects healthy aging and promotes age-related health problems.

PubMed

Liu, Liang; van Groen, Thomas; Kadish, Inga; Li, Yuanyuan; Wang, Deli; James, Smitha R; Karpf, Adam R; Tollefsbol, Trygve O

2011-08-01

DNA methylation plays an integral role in development and aging through epigenetic regulation of genome function. DNA methyltransferase 1 (Dnmt1) is the most prevalent DNA methyltransferase that maintains genomic methylation stability. To further elucidate the function of Dnmt1 in aging and age-related diseases, we exploited the Dnmt1+/- mouse model to investigate how Dnmt1 haploinsufficiency impacts the aging process by assessing the changes of several major aging phenotypes. We confirmed that Dnmt1 haploinsufficiency indeed decreases DNA methylation as a result of reduced Dnmt1 expression. To assess the effect of Dnmt1 haploinsufficiency on general body composition, we performed dual-energy X-ray absorptiometry analysis and showed that reduced Dnmt1 activity decreased bone mineral density and body weight, but with no significant impact on mortality or body fat content. Using behavioral tests, we demonstrated that Dnmt1 haploinsufficiency impairs learning and memory functions in an age-dependent manner. Taken together, our findings point to the interesting likelihood that reduced genomic methylation activity adversely affects the healthy aging process without altering survival and mortality. Our studies demonstrated that cognitive functions of the central nervous system are modulated by Dnmt1 activity and genomic methylation, highlighting the significance of the original epigenetic hypothesis underlying memory coding and function.

Nutrients and DNA Methylation

USDA-ARS?s Scientific Manuscript database

Epigenetics is a new mechanism responsible for development, aging, and disease process such as cancer development. One major epigenetic phenomenon is DNA methylation, which attributes to gene expression and integrity. Deepening the knowledge on one-carbon metabolism is very important to understandin...

Stepwise DNA Methylation Changes Are Linked to Escape from Defined Proliferation Barriers and Mammary Epithelial Cell Immortalization

DOE Office of Scientific and Technical Information (OSTI.GOV)

Novak, Petr; Jensen, Taylor J.; Garbe, James C.

The timing and progression of DNA methylation changes during carcinogenesis are not completely understood. To develop a timeline of aberrant DNA methylation events during malignant transformation, we analyzed genome-wide DNA methylation patterns in an isogenic human mammary epithelial cell (HMEC) culture model of transformation. To acquire immortality and malignancy, the cultured finite lifespan HMEC must overcome two distinct proliferation barriers. The first barrier, stasis, is mediated by the retinoblastoma protein and can be overcome by loss of p16(INK4A) expression. HMEC that escape stasis and continue to proliferate become genomically unstable before encountering a second more stringent proliferation barrier, telomere dysfunctionmore » due to telomere attrition. Rare cells that acquire telomerase expression may escape this barrier, become immortal, and develop further malignant properties. Our analysis of HMEC transitioning from finite lifespan to malignantly transformed showed that aberrant DNA methylation changes occur in a stepwise fashion early in the transformation process. The first aberrant DNA methylation step coincides with overcoming stasis, and results in few to hundreds of changes, depending on how stasis was overcome. A second step coincides with immortalization and results in hundreds of additional DNA methylation changes regardless of the immortalization pathway. A majority of these DNA methylation changes are also found in malignant breast cancer cells. These results show that large-scale epigenetic remodeling occurs in the earliest steps of mammary carcinogenesis, temporally links DNA methylation changes and overcoming cellular proliferation barriers, and provides a bank of potential epigenetic biomarkers that mayprove useful in breast cancer risk assessment.« less

Establishment and functions of DNA methylation in the germline

PubMed Central

Stewart, Kathleen R; Veselovska, Lenka; Kelsey, Gavin

2016-01-01

Epigenetic modifications established during gametogenesis regulate transcription and other nuclear processes in gametes, but also have influences in the zygote, embryo and postnatal life. This is best understood for DNA methylation which, established at discrete regions of the oocyte and sperm genomes, governs genomic imprinting. In this review, we describe how imprinting has informed our understanding of de novo DNA methylation mechanisms, highlight how recent genome-wide profiling studies have provided unprecedented insights into establishment of the sperm and oocyte methylomes and consider the fate and function of gametic methylation and other epigenetic modifications after fertilization. PMID:27659720

Association of in vitro fertilization with global and IGF2/H19 methylation variation in newborn twins.

PubMed

Loke, Y J; Galati, J C; Saffery, R; Craig, J M

2015-04-01

In vitro fertilization (IVF) and its subset intracytoplasmic sperm injection (ICSI), are widely used medical treatments for conception. There has been controversy over whether IVF is associated with adverse short- and long-term health outcomes of offspring. As with other prenatal factors, epigenetic change is thought to be a molecular mediator of any in utero programming effects. Most studies focused on DNA methylation at gene-specific and genomic level, with only a few on associations between DNA methylation and IVF. Using buccal epithelium from 208 twin pairs from the Peri/Postnatal Epigenetic Twin Study (PETS), we investigated associations between IVF and DNA methylation on a global level, using the proxies of Alu and LINE-1 interspersed repeats in addition to two locus-specific regulatory regions within IGF2/H19, controlling for 13 potentially confounding factors. Using multiple correction testing, we found strong evidence that IVF-conceived twins have lower DNA methylation in Alu, and weak evidence of lower methylation in one of the two IGF2/H19 regulatory regions and LINE-1, compared with naturally conceived twins. Weak evidence of a relationship between ICSI and DNA methylation within IGF2/H19 regulatory region was found, suggesting that one or more of the processes associated with IVF/ICSI may contribute to these methylation differences. Lower within- and between-pair DNA methylation variation was also found in IVF-conceived twins for LINE-1, Alu and one IGF2/H19 regulatory region. Although larger sample sizes are needed, our results provide additional insight to the possible influence of IVF and ICSI on DNA methylation. To our knowledge, this is the largest study to date investigating the association of IVF and DNA methylation.

DNA methylation of the serotonin transporter gene (SLC6A4) is associated with brain function involved in processing emotional stimuli.

PubMed

Frodl, Thomas; Szyf, Moshe; Carballedo, Angela; Ly, Victoria; Dymov, Sergiy; Vaisheva, Farida; Morris, Derek; Fahey, Ciara; Meaney, James; Gill, Michael; Booij, Linda

2015-09-01

The aim of the present study was to investigate the association of fMRI blood oxygen-level dependent (BOLD) reactivity with the level of epigenetic methylation of SLC6A4 in blood DNA from a sample of healthy participants and patients with major depressive disorder (MDD). We investigated patients with MDD and healthy controls using fMRI and an emotional attention-shifting task. We assessed site-specific DNA methylation of a previously characterized SLC6A4 region in peripheral blood DNA using pyrosequencing. Our study involved 25 patients with MDD and 35 healthy controls. Activation in the anterior insula elicited by negative emotional content was significantly positively associated with the degree of SLC6A4 methylation. Significantly negative associations were observed between activation in the posterior insula and the degree of SLC6A4 methylation when judging the geometry of pictures after seeing negative in contrast to positive emotional stimuli. Healthy controls with a high degree of SLC6A4 methylation depicted significantly more activity elicited by positive stimuli in limbic regions and more activity elicited by negative stimuli in limbic as well as cognitive control regions than those with a low degree of SLC6A4 methylation. It is impossible to measure methylation directly in the brain and thus we assessed peripheral methylation of SLC6A4. Since the association was cross-sectional, no conclusion about cause and effect can be drawn. Our study provides further support to the hypothesis that particular DNA methylation states that are associated with brain function during emotion processing are detectable in the periphery.

Grandmaternal stress during pregnancy and DNA methylation of the third generation: an epigenome-wide association study

PubMed Central

Serpeloni, F; Radtke, K; de Assis, S G; Henning, F; Nätt, D; Elbert, T

2017-01-01

Stress during pregnancy may impact subsequent generations, which is demonstrated by an increased susceptibility to childhood and adulthood health problems in the children and grandchildren. Although the importance of the prenatal environment is well reported with regards to future physical and emotional outcomes, little is known about the molecular mechanisms that mediate the long-term consequences of early stress across generations. Recent studies have identified DNA methylation as a possible mediator of the impact of prenatal stress in the offspring. Whether psychosocial stress during pregnancy also affects DNA methylation of the grandchildren is still not known. In the present study we examined the multigenerational hypothesis, that is, grandmaternal exposure to psychosocial stress during pregnancy affecting DNA methylation of the grandchildren. We determined the genome-wide DNA methylation profile in 121 children (65 females and 56 males) and tested for associations with exposure to grandmaternal interpersonal violence during pregnancy. We observed methylation variations of five CpG sites significantly (FDR<0.05) associated with the grandmother’s report of exposure to violence while pregnant with the mothers of the children. The results revealed differential methylation of genes previously shown to be involved in circulatory system processes (FDR<0.05). This study provides support for DNA methylation as a biological mechanism involved in the transmission of stress across generations and motivates further investigations to examine prenatal-dependent DNA methylation as a potential biomarker for health problems. PMID:28809857

Grandmaternal stress during pregnancy and DNA methylation of the third generation: an epigenome-wide association study.

PubMed

Serpeloni, F; Radtke, K; de Assis, S G; Henning, F; Nätt, D; Elbert, T

2017-08-15

Stress during pregnancy may impact subsequent generations, which is demonstrated by an increased susceptibility to childhood and adulthood health problems in the children and grandchildren. Although the importance of the prenatal environment is well reported with regards to future physical and emotional outcomes, little is known about the molecular mechanisms that mediate the long-term consequences of early stress across generations. Recent studies have identified DNA methylation as a possible mediator of the impact of prenatal stress in the offspring. Whether psychosocial stress during pregnancy also affects DNA methylation of the grandchildren is still not known. In the present study we examined the multigenerational hypothesis, that is, grandmaternal exposure to psychosocial stress during pregnancy affecting DNA methylation of the grandchildren. We determined the genome-wide DNA methylation profile in 121 children (65 females and 56 males) and tested for associations with exposure to grandmaternal interpersonal violence during pregnancy. We observed methylation variations of five CpG sites significantly (FDR<0.05) associated with the grandmother's report of exposure to violence while pregnant with the mothers of the children. The results revealed differential methylation of genes previously shown to be involved in circulatory system processes (FDR<0.05). This study provides support for DNA methylation as a biological mechanism involved in the transmission of stress across generations and motivates further investigations to examine prenatal-dependent DNA methylation as a potential biomarker for health problems.

Expression of exogenous DNA methyltransferases: application in molecular and cell biology.

PubMed

Dyachenko, O V; Tarlachkov, S V; Marinitch, D V; Shevchuk, T V; Buryanov, Y I

2014-02-01

DNA methyltransferases might be used as powerful tools for studies in molecular and cell biology due to their ability to recognize and modify nitrogen bases in specific sequences of the genome. Methylation of the eukaryotic genome using exogenous DNA methyltransferases appears to be a promising approach for studies on chromatin structure. Currently, the development of new methods for targeted methylation of specific genetic loci using DNA methyltransferases fused with DNA-binding proteins is especially interesting. In the present review, expression of exogenous DNA methyltransferase for purposes of in vivo analysis of the functional chromatin structure along with investigation of the functional role of DNA methylation in cell processes are discussed, as well as future prospects for application of DNA methyltransferases in epigenetic therapy and in plant selection.

Global DNA methylation as a possible biomarker for diabetic retinopathy.

PubMed

Maghbooli, Zhila; Hossein-nezhad, Arash; Larijani, Bagher; Amini, Manochehr; Keshtkar, Abbasali

2015-02-01

We evaluated whether global levels of DNA methylation status were associated with retinopathy as well as providing a predictive role of DNA methylation in developing retinopathy in a case-control study of 168 patients with type 2 diabetes. The 5-methylcytosine content was assessed by reversed-phase high-pressure liquid chromatography of peripheral blood leukocytes to determine an individual's global DNA methylation status in the two groups, either with or without retinopathy. The global DNA methylation levels were significantly higher in diabetic retinopathy patients compared with those in non-retinopathy patients (4.90 ± 0.12 vs. 4.22 ± 0.13, respectively; p = 0.001). There was a significant increasing trend in global DNA methylation levels in terms of progressing retinopathy (without retinopathy, 4.22 ± 0.13; non-proliferative diabetic retinopathy, 4.62 ± 0.17; proliferative diabetic retinopathy, 5.07 ± 0.21) (p = 0.006). Additionally, global DNA methylation independent of retinopathy risk factors, which include dyslipidaemia, hypertension, hyperglycaemia and duration of diabetes, was a predictive factor for retinopathy (OR = 1.53, p = 0.015). Global DNA methylation is modulated during or possibly before the primary stage of diabetes. This observation verifies the metabolic memory effect of hyperglycaemia in early stage of an aetiological process that leads to type 2 diabetes and its associated complications. Copyright © 2014 John Wiley & Sons, Ltd.

DNA methylation and healthy human aging.

PubMed

Jones, Meaghan J; Goodman, Sarah J; Kobor, Michael S

2015-12-01

The process of aging results in a host of changes at the cellular and molecular levels, which include senescence, telomere shortening, and changes in gene expression. Epigenetic patterns also change over the lifespan, suggesting that epigenetic changes may constitute an important component of the aging process. The epigenetic mark that has been most highly studied is DNA methylation, the presence of methyl groups at CpG dinucleotides. These dinucleotides are often located near gene promoters and associate with gene expression levels. Early studies indicated that global levels of DNA methylation increase over the first few years of life and then decrease beginning in late adulthood. Recently, with the advent of microarray and next-generation sequencing technologies, increases in variability of DNA methylation with age have been observed, and a number of site-specific patterns have been identified. It has also been shown that certain CpG sites are highly associated with age, to the extent that prediction models using a small number of these sites can accurately predict the chronological age of the donor. Together, these observations point to the existence of two phenomena that both contribute to age-related DNA methylation changes: epigenetic drift and the epigenetic clock. In this review, we focus on healthy human aging throughout the lifetime and discuss the dynamics of DNA methylation as well as how interactions between the genome, environment, and the epigenome influence aging rates. We also discuss the impact of determining 'epigenetic age' for human health and outline some important caveats to existing and future studies. © 2015 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

DNA and histone methylation in gastric carcinogenesis

PubMed Central

Calcagno, Danielle Queiroz; Gigek, Carolina Oliveira; Chen, Elizabeth Suchi; Burbano, Rommel Rodriguez; Smith, Marília de Arruda Cardoso

2013-01-01

Epigenetic alterations contribute significantly to the development and progression of gastric cancer, one of the leading causes of cancer death worldwide. Epigenetics refers to the number of modifications of the chromatin structure that affect gene expression without altering the primary sequence of DNA, and these changes lead to transcriptional activation or silencing of the gene. Over the years, the study of epigenetic processes has increased, and novel therapeutic approaches that target DNA methylation and histone modifications have emerged. A greater understanding of epigenetics and the therapeutic potential of manipulating these processes is necessary for gastric cancer treatment. Here, we review recent research on the effects of aberrant DNA and histone methylation on the onset and progression of gastric tumors and the development of compounds that target enzymes that regulate the epigenome. PMID:23482412

Exercise-associated DNA methylation change in skeletal muscle and the importance of imprinted genes: a bioinformatics meta-analysis.

PubMed

Brown, William M

2015-12-01

Epigenetics is the study of processes--beyond DNA sequence alteration--producing heritable characteristics. For example, DNA methylation modifies gene expression without altering the nucleotide sequence. A well-studied DNA methylation-based phenomenon is genomic imprinting (ie, genotype-independent parent-of-origin effects). We aimed to elucidate: (1) the effect of exercise on DNA methylation and (2) the role of imprinted genes in skeletal muscle gene networks (ie, gene group functional profiling analyses). Gene ontology (ie, gene product elucidation)/meta-analysis. 26 skeletal muscle and 86 imprinted genes were subjected to g:Profiler ontology analysis. Meta-analysis assessed exercise-associated DNA methylation change. g:Profiler found four muscle gene networks with imprinted loci. Meta-analysis identified 16 articles (387 genes/1580 individuals) associated with exercise. Age, method, sample size, sex and tissue variation could elevate effect size bias. Only skeletal muscle gene networks including imprinted genes were reported. Exercise-associated effect sizes were calculated by gene. Age, method, sample size, sex and tissue variation were moderators. Six imprinted loci (RB1, MEG3, UBE3A, PLAGL1, SGCE, INS) were important for muscle gene networks, while meta-analysis uncovered five exercise-associated imprinted loci (KCNQ1, MEG3, GRB10, L3MBTL1, PLAGL1). DNA methylation decreased with exercise (60% of loci). Exercise-associated DNA methylation change was stronger among older people (ie, age accounted for 30% of the variation). Among older people, genes exhibiting DNA methylation decreases were part of a microRNA-regulated gene network functioning to suppress cancer. Imprinted genes were identified in skeletal muscle gene networks and exercise-associated DNA methylation change. Exercise-associated DNA methylation modification could rewind the 'epigenetic clock' as we age. CRD42014009800. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/

Rapid response to changing environments during biological invasions: DNA methylation perspectives.

PubMed

Huang, Xuena; Li, Shiguo; Ni, Ping; Gao, Yangchun; Jiang, Bei; Zhou, Zunchun; Zhan, Aibin

2017-12-01

Dissecting complex interactions between species and their environments has long been a research hot spot in the fields of ecology and evolutionary biology. The well-recognized Darwinian evolution has well-explained long-term adaptation scenarios; however, "rapid" processes of biological responses to environmental changes remain largely unexplored, particularly molecular mechanisms such as DNA methylation that have recently been proposed to play crucial roles in rapid environmental adaptation. Invasive species, which have capacities to successfully survive rapidly changing environments during biological invasions, provide great opportunities to study molecular mechanisms of rapid environmental adaptation. Here, we used the methylation-sensitive amplified polymorphism (MSAP) technique in an invasive model ascidian, Ciona savignyi, to investigate how species interact with rapidly changing environments at the whole-genome level. We detected quite rapid DNA methylation response: significant changes of DNA methylation frequency and epigenetic differentiation between treatment and control groups occurred only after 1 hr of high-temperature exposure or after 3 hr of low-salinity challenge. In addition, we detected time-dependent hemimethylation changes and increased intragroup epigenetic divergence induced by environmental stresses. Interestingly, we found evidence of DNA methylation resilience, as most stress-induced DNA methylation variation maintained shortly (~48 hr) and quickly returned back to the control levels. Our findings clearly showed that invasive species could rapidly respond to acute environmental changes through DNA methylation modifications, and rapid environmental changes left significant epigenetic signatures at the whole-genome level. All these results provide fundamental background to deeply investigate the contribution of DNA methylation mechanisms to rapid contemporary environmental adaptation. © 2017 John Wiley & Sons Ltd.

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

PubMed Central

2012-01-01

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. PMID:22251412

[Epigenetics of prostate cancer].

PubMed

Yi, Xiao-Ming; Zhou, Wen-Quan

2010-07-01

Prostate cancer is one of the most common malignant tumors in males, and its etiology and pathogenesis remain unclear. Epigenesis is involved in prostate cancer at all stages of the process, and closely related with its growth and metastasis. DNA methylation and histone modification are the most important manifestations of epigenetics in prostate cancer. The mechanisms of carcinogenesis of DNA methylation include whole-genome hypomethylation, aberrant local hypermethylation of promoters and genomic instability. DNA methylation is closely related to the process of prostate cancer, as in DNA damage repair, hormone response, tumor cell invasion/metastasis, cell cycle regulation, and so on. Histone modification causes corresponding changes in chromosome structure and the level of gene transcription, and it may affect the cycle, differentiation and apoptosis of cells, resulting in prostate cancer. Some therapies have been developed targeting the epigenetic changes in prostate cancer, including DNA methyltransferases and histone deacetylase inhibitors, and have achieved certain desirable results.

Epigenetic profiling of ADHD symptoms trajectories: a prospective, methylome-wide study.

PubMed

Walton, E; Pingault, J-B; Cecil, C A M; Gaunt, T R; Relton, C L; Mill, J; Barker, E D

2017-02-01

Attention-deficit/hyperactivity disorder (ADHD) is a prevalent developmental disorder, associated with a range of long-term impairments. Variation in DNA methylation, an epigenetic mechanism, is implicated in both neurobiological functioning and psychiatric health. However, the potential role of DNA methylation in ADHD symptoms is currently unclear. In this study, we examined data from the Avon Longitudinal Study of Parents and Children (ALSPAC)-specifically the subsample forming the Accessible Resource for Integrated Epigenomics Studies (ARIES)-that includes (1) peripheral measures of DNA methylation (Illumina 450k) at birth (n=817, 49% male) and age 7 (n=892, 50% male) and (2) trajectories of ADHD symptoms (7-15 years). We first employed a genome-wide analysis to test whether DNA methylation at birth associates with later ADHD trajectories; and then followed up at age 7 to investigate the stability of associations across early childhood. We found that DNA methylation at birth differentiated ADHD trajectories across multiple genomic locations, including probes annotated to SKI (involved in neural tube development), ZNF544 (previously implicated in ADHD), ST3GAL3 (linked to intellectual disability) and PEX2 (related to perixosomal processes). None of these probes maintained an association with ADHD trajectories at age 7. Findings lend novel insights into the epigenetic landscape of ADHD symptoms, highlighting the potential importance of DNA methylation variation in genes related to neurodevelopmental and peroxisomal processes that play a key role in the maturation and stability of cortical circuits.

P-Hint-Hunt: a deep parallelized whole genome DNA methylation detection tool.

PubMed

Peng, Shaoliang; Yang, Shunyun; Gao, Ming; Liao, Xiangke; Liu, Jie; Yang, Canqun; Wu, Chengkun; Yu, Wenqiang

2017-03-14

The increasing studies have been conducted using whole genome DNA methylation detection as one of the most important part of epigenetics research to find the significant relationships among DNA methylation and several typical diseases, such as cancers and diabetes. In many of those studies, mapping the bisulfite treated sequence to the whole genome has been the main method to study DNA cytosine methylation. However, today's relative tools almost suffer from inaccuracies and time-consuming problems. In our study, we designed a new DNA methylation prediction tool ("Hint-Hunt") to solve the problem. By having an optimal complex alignment computation and Smith-Waterman matrix dynamic programming, Hint-Hunt could analyze and predict the DNA methylation status. But when Hint-Hunt tried to predict DNA methylation status with large-scale dataset, there are still slow speed and low temporal-spatial efficiency problems. In order to solve the problems of Smith-Waterman dynamic programming and low temporal-spatial efficiency, we further design a deep parallelized whole genome DNA methylation detection tool ("P-Hint-Hunt") on Tianhe-2 (TH-2) supercomputer. To the best of our knowledge, P-Hint-Hunt is the first parallel DNA methylation detection tool with a high speed-up to process large-scale dataset, and could run both on CPU and Intel Xeon Phi coprocessors. Moreover, we deploy and evaluate Hint-Hunt and P-Hint-Hunt on TH-2 supercomputer in different scales. The experimental results illuminate our tools eliminate the deviation caused by bisulfite treatment in mapping procedure and the multi-level parallel program yields a 48 times speed-up with 64 threads. P-Hint-Hunt gain a deep acceleration on CPU and Intel Xeon Phi heterogeneous platform, which gives full play of the advantages of multi-cores (CPU) and many-cores (Phi).

Neuronal DNA Methyltransferases: Epigenetic Mediators between Synaptic Activity and Gene Expression?

PubMed Central

Bayraktar, Gonca; Kreutz, Michael R.

2017-01-01

DNMT3A and 3B are the main de novo DNA methyltransferases (DNMTs) in the brain that introduce new methylation marks to non-methylated DNA in postmitotic neurons. DNA methylation is a key epigenetic mark that is known to regulate important cellular processes in neuronal development and brain plasticity. Accumulating evidence disclosed rapid and dynamic changes in DNA methylation of plasticity-relevant genes that are important for learning and memory formation. To understand how DNMTs contribute to brain function and how they are regulated by neuronal activity is a prerequisite for a deeper appreciation of activity-dependent gene expression in health and disease. This review discusses the functional role of de novo methyltransferases and in particular DNMT3A1 in the adult brain with special emphasis on synaptic plasticity, memory formation, and brain disorders. PMID:28513272

[Epigenetic heredity (deoxyribonucleic acid methylation): Clinical context in neurodegenerative disorders and ATXN2 gene].

PubMed

Laffita-Mesa, José Miguel; Bauer, Peter

2014-10-21

Epigenetics is the group of changes in the phenotype which are related with the process independently of the primary DNA sequence. These changes are intimately related with changes in the gene expression level and its profile across the body. These are mediated by histone tail modifications, DNA methylation, micro-RNAs, with chromatin remodeling remaining as the foundation of epigenetic changes. DNA methylation involves the covalent addition of methyl group to cytosine of the DNA, which is mediated by methyltransferases enzymes. DNA methylation regulates gene expression by repressing transcription, while de-methylation activates gene transcription. Several human diseases are related with the epigenetic process: cancer, Alzheimer disease, stroke, Parkinson disease, and diabetes. We present here the basis of epigenetic inheritance and show the pathogenic mechanisms relating epigenetics in human diseases, specifically with regard to neurodegeneration. We discuss current concepts aimed at understanding the contribution of epigenetics to human neurodegenerative diseases. We also discuss recent findings obtained in our and other centers regarding the ATXN2 gene that causes spinocerebellar ataxia 2 and amyotrophic lateral sclerosis. Epigenetics play a pivotal role in the pathogenesis of human diseases and in several neurodegenerative disorders, and this knowledge will illuminate the pathways in the diagnostic and therapeutic field, which ultimately will be translated into the clinic context of neurodegenerative diseases. Copyright © 2013 Elsevier España, S.L.U. All rights reserved.

Cytosine DNA Methylation Is Found in Drosophila melanogaster but Absent in Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Other Yeast Species

PubMed Central

2014-01-01

The methylation of cytosine to 5-methylcytosine (5-meC) is an important epigenetic DNA modification in many bacteria, plants, and mammals, but its relevance for important model organisms, including Caenorhabditis elegans and Drosophila melanogaster, is still equivocal. By reporting the presence of 5-meC in a broad variety of wild, laboratory, and industrial yeasts, a recent study also challenged the dogma about the absence of DNA methylation in yeast species. We would like to bring to attention that the protocol used for gas chromatography/mass spectrometry involved hydrolysis of the DNA preparations. As this process separates cytosine and 5-meC from the sugar phosphate backbone, this method is unable to distinguish DNA- from RNA-derived 5-meC. We employed an alternative LC–MS/MS protocol where by targeting 5-methyldeoxycytidine moieties after enzymatic digestion, only 5-meC specifically derived from DNA is quantified. This technique unambiguously identified cytosine DNA methylation in Arabidopsis thaliana (14.0% of cytosines methylated), Mus musculus (7.6%), and Escherichia coli (2.3%). Despite achieving a detection limit at 250 attomoles (corresponding to <0.00002 methylated cytosines per nonmethylated cytosine), we could not confirm any cytosine DNA methylation in laboratory and industrial strains of Saccharomyces cerevisiae, Schizosaccharomyces pombe, Saccharomyces boulardii, Saccharomyces paradoxus, or Pichia pastoris. The protocol however unequivocally confirmed DNA methylation in adult Drosophila melanogaster at a value (0.034%) that is up to 2 orders of magnitude below the detection limit of bisulphite sequencing. Thus, 5-meC is a rare DNA modification in drosophila but absent in yeast. PMID:24640988

Mapping of Variable DNA Methylation Across Multiple Cell Types Defines a Dynamic Regulatory Landscape of the Human Genome.

PubMed

Gu, Junchen; Stevens, Michael; Xing, Xiaoyun; Li, Daofeng; Zhang, Bo; Payton, Jacqueline E; Oltz, Eugene M; Jarvis, James N; Jiang, Kaiyu; Cicero, Theodore; Costello, Joseph F; Wang, Ting

2016-04-07

DNA methylation is an important epigenetic modification involved in many biological processes and diseases. Many studies have mapped DNA methylation changes associated with embryogenesis, cell differentiation, and cancer at a genome-wide scale. Our understanding of genome-wide DNA methylation changes in a developmental or disease-related context has been steadily growing. However, the investigation of which CpGs are variably methylated in different normal cell or tissue types is still limited. Here, we present an in-depth analysis of 54 single-CpG-resolution DNA methylomes of normal human cell types by integrating high-throughput sequencing-based methylation data. We found that the ratio of methylated to unmethylated CpGs is relatively constant regardless of cell type. However, which CpGs made up the unmethylated complement was cell-type specific. We categorized the 26,000,000 human autosomal CpGs based on their methylation levels across multiple cell types to identify variably methylated CpGs and found that 22.6% exhibited variable DNA methylation. These variably methylated CpGs formed 660,000 variably methylated regions (VMRs), encompassing 11% of the genome. By integrating a multitude of genomic data, we found that VMRs enrich for histone modifications indicative of enhancers, suggesting their role as regulatory elements marking cell type specificity. VMRs enriched for transcription factor binding sites in a tissue-dependent manner. Importantly, they enriched for GWAS variants, suggesting that VMRs could potentially be implicated in disease and complex traits. Taken together, our results highlight the link between CpG methylation variation, genetic variation, and disease risk for many human cell types. Copyright © 2016 Gu et al.

Genome-wide DNA methylation patterns in LSH mutant reveals de-repression of repeat elements and redundant epigenetic silencing pathways

PubMed Central

Yu, Weishi; McIntosh, Carl; Lister, Ryan; Zhu, Iris; Han, Yixing; Ren, Jianke; Landsman, David; Lee, Eunice; Briones, Victorino; Terashima, Minoru; Leighty, Robert; Ecker, Joseph R.

2014-01-01

Cytosine methylation is critical in mammalian development and plays a role in diverse biologic processes such as genomic imprinting, X chromosome inactivation, and silencing of repeat elements. Several factors regulate DNA methylation in early embryogenesis, but their precise role in the establishment of DNA methylation at a given site remains unclear. We have generated a comprehensive methylation map in fibroblasts derived from the murine DNA methylation mutant Hells−/− (helicase, lymphoid specific, also known as LSH). It has been previously shown that HELLS can influence de novo methylation of retroviral sequences and endogenous genes. Here, we describe that HELLS controls cytosine methylation in a nuclear compartment that is in part defined by lamin B1 attachment regions. Despite widespread loss of cytosine methylation at regulatory sequences, including promoter regions of protein-coding genes and noncoding RNA genes, overall relative transcript abundance levels in the absence of HELLS are similar to those in wild-type cells. A subset of promoter regions shows increases of the histone modification H3K27me3, suggesting redundancy of epigenetic silencing mechanisms. Furthermore, HELLS modulates CG methylation at all classes of repeat elements and is critical for repression of a subset of repeat elements. Overall, we provide a detailed analysis of gene expression changes in relation to DNA methylation alterations, which contributes to our understanding of the biological role of cytosine methylation. PMID:25170028

Identification of methylation haplotype blocks aids in deconvolution of heterogeneous tissue samples and tumor tissue-of-origin mapping from plasma DNA.

PubMed

Guo, Shicheng; Diep, Dinh; Plongthongkum, Nongluk; Fung, Ho-Lim; Zhang, Kang; Zhang, Kun

2017-04-01

Adjacent CpG sites in mammalian genomes can be co-methylated owing to the processivity of methyltransferases or demethylases, yet discordant methylation patterns have also been observed, which are related to stochastic or uncoordinated molecular processes. We focused on a systematic search and investigation of regions in the full human genome that show highly coordinated methylation. We defined 147,888 blocks of tightly coupled CpG sites, called methylation haplotype blocks, after analysis of 61 whole-genome bisulfite sequencing data sets and validation with 101 reduced-representation bisulfite sequencing data sets and 637 methylation array data sets. Using a metric called methylation haplotype load, we performed tissue-specific methylation analysis at the block level. Subsets of informative blocks were further identified for deconvolution of heterogeneous samples. Finally, using methylation haplotypes we demonstrated quantitative estimation of tumor load and tissue-of-origin mapping in the circulating cell-free DNA of 59 patients with lung or colorectal cancer.

Epigenetic response to environmental change: DNA methylation varies with invasion status.

PubMed

Schrey, Aaron W; Robbins, Travis R; Lee, Jacob; Dukes, David W; Ragsdale, Alexandria K; Thawley, Christopher J; Langkilde, Tracy

2016-04-01

Epigenetic mechanisms may be important for a native species' response to rapid environmental change. Red Imported Fire Ants ( Solenopsis invicta Santschi, 1916) were recently introduced to areas occupied by the Eastern Fence Lizard ( Sceloporus undulatus Bosc & Daudin, 1801). Behavioral, morphological and physiological phenotypes of the Eastern Fence Lizard have changed following invasion, creating a natural biological system to investigate environmentally induced epigenetic changes. We tested for variation in DNA methylation patterns in Eastern Fence Lizard populations associated with different histories of invasion by Red Imported Fire Ants. At methylation sensitive amplified fragment length polymorphism loci, we detected a higher diversity of methylation in Eastern Fence Lizard populations from Fire Ant uninvaded versus invaded sites, and uninvaded sites had higher methylation. Our results suggest that invasive species may alter methylation frequencies and the pattern of methylation among native individuals. While our data indicate a high level of intrinsic variability in DNA methylation, DNA methylation at some genomic loci may underlie observed phenotypic changes in Eastern Fence Lizard populations in response to invasion of Red Imported Fire Ants. This process may be important in facilitating adaptation of native species to novel pressures imposed by a rapidly changing environment.

Improved results of LINE-1 methylation analysis in formalin-fixed, paraffin-embedded tissues with the application of a heating step during the DNA extraction process.

PubMed

Wen, Xianyu; Jeong, Seorin; Kim, Younghoon; Bae, Jeong Mo; Cho, Nam Yun; Kim, Jung Ho; Kang, Gyeong Hoon

2017-01-01

Formalin-fixed, paraffin-embedded (FFPE) tissues are important resources for profiling DNA methylation changes and for studying a variety of diseases. However, formalin fixation introduces inter-strand crosslinking, which might cause incomplete bisulfite conversion of unmethylated cytosines, which might lead to falsely elevated measurements of methylation levels in pyrosequencing assays. Long interspersed nucleotide element-1 (LINE-1) is a major constituent of repetitive transposable DNA elements, and its methylation is referred to correlates with global DNA methylation. To identify whether formalin fixation might impact the measured values of methylation in LINE-1 repetitive elements and whether prolonged heat-induced denaturation of DNA might reduce the artificial increases in measured values caused by formalin fixation, we analyzed paired fresh-frozen (FF) and FFPE xenograft tissue samples for their methylation levels in LINE-1 using a pyrosequencing assay. To further confirm the effect of a heating step in the measurement of LINE-1 or single gene methylation levels, we analyzed FFPE tissue samples of gastric cancer and colorectal cancer for their methylation status in LINE-1 and eight single genes, respectively. Formalin fixation led to an increase in the measured values of LINE-1 methylation regardless of the duration of fixation. Prolonged heating of the DNA at 95 °C for 30 min before bisulfite conversion was found (1) to decrease the discrepancy in the measured values between the paired FF and FFPE tissue samples, (2) to decrease the standard deviation of the measured value of LINE-1 methylation levels in FFPE tissue samples of gastric cancer, and (3) to improve the performance in the measurement of single gene methylation levels in FFPE tissue samples of colorectal cancer. Formalin fixation leads to artificial increases in the measured values of LINE-1 methylation, and the application of prolonged heating of DNA samples decreases the discrepancy in the measured values of LINE-1 methylation between paired FF and FFPE tissue samples. The application of prolonged heating of DNA samples improves bisulfite conversion-based measurement of LINE-1 or single gene methylation levels in FFPE tissue samples.

Krebs cycle intermediates regulate DNA and histone methylation: epigenetic impact on the aging process.

PubMed

Salminen, Antero; Kauppinen, Anu; Hiltunen, Mikko; Kaarniranta, Kai

2014-07-01

Many aging theories have proposed that mitochondria and energy metabolism have a major role in the aging process. There are recent studies indicating that Krebs cycle intermediates can shape the epigenetic landscape of chromatin by regulating DNA and histone methylation. A growing evidence indicates that epigenetics plays an important role in the regulation of healthspan but also is involved in the aging process. 2-Oxoglutarate (α-ketoglutarate) is a key metabolite in the Krebs cycle but it is also an obligatory substrate for 2-oxoglutarate-dependent dioxygenases (2-OGDO). The 2-OGDO enzyme family includes the major enzymes of DNA and histone demethylation, i.e. Ten-Eleven Translocation (TETs) and Jumonji C domain containing (JmjC) demethylases. In addition, 2-OGDO members can regulate collagen synthesis and hypoxic responses in a non-epigenetical manner. Interestingly, succinate and fumarate, also Krebs cycle intermediates, are potent inhibitors of 2-OGDO enzymes, i.e. the balance of Krebs cycle reactions can affect the level of DNA and histone methylation and thus control gene expression. We will review the epigenetic mechanisms through which Krebs cycle intermediates control the DNA and histone methylation. We propose that age-related disturbances in the Krebs cycle function induce stochastic epigenetic changes in chromatin structures which in turn promote the aging process. Copyright © 2014 Elsevier B.V. All rights reserved.

The identification of age-associated cancer markers by an integrative analysis of dynamic DNA methylation changes.

PubMed

Wang, Yihan; Zhang, Jingyu; Xiao, Xingjun; Liu, Hongbo; Wang, Fang; Li, Song; Wen, Yanhua; Wei, Yanjun; Su, Jianzhong; Zhang, Yunming; Zhang, Yan

2016-03-07

As one of the most widely studied epigenetic modifications, DNA methylation has an important influence on human traits and cancers. Dynamic variations in DNA methylation have been reported in malignant neoplasm and aging; however, the mechanisms remain poorly understood. By constructing an age-associated and cancer-related weighted network (ACWN) based on the correlation of the methylation level and the protein-protein interaction, we found that DNA methylation changes associated with age were closely related to the occurrence of cancer. Additional analysis of 102 module genes mined from the ACWN revealed discrimination based on two main patterns. One pattern involved methylation levels that increased with aging and were higher in cancer patients compared with normal controls (HH pattern). The other pattern involved methylation levels that decreased with aging and were lower in cancer compared with normal (LL pattern). Upon incorporation with gene expression levels, 25 genes were filtered based on negative regulation by DNA methylation. These genes were regarded as potential cancer risk markers that were influenced by age in the process of carcinogenesis. Our results will facilitate further studies regarding the impact of the epigenetic effects of aging on diseases and will aid in the development of tailored cancer preventive strategies.

The identification of age-associated cancer markers by an integrative analysis of dynamic DNA methylation changes

PubMed Central

Wang, Yihan; Zhang, Jingyu; Xiao, Xingjun; Liu, Hongbo; Wang, Fang; Li, Song; Wen, Yanhua; Wei, Yanjun; Su, Jianzhong; Zhang, Yunming; Zhang, Yan

2016-01-01

As one of the most widely studied epigenetic modifications, DNA methylation has an important influence on human traits and cancers. Dynamic variations in DNA methylation have been reported in malignant neoplasm and aging; however, the mechanisms remain poorly understood. By constructing an age-associated and cancer-related weighted network (ACWN) based on the correlation of the methylation level and the protein-protein interaction, we found that DNA methylation changes associated with age were closely related to the occurrence of cancer. Additional analysis of 102 module genes mined from the ACWN revealed discrimination based on two main patterns. One pattern involved methylation levels that increased with aging and were higher in cancer patients compared with normal controls (HH pattern). The other pattern involved methylation levels that decreased with aging and were lower in cancer compared with normal (LL pattern). Upon incorporation with gene expression levels, 25 genes were filtered based on negative regulation by DNA methylation. These genes were regarded as potential cancer risk markers that were influenced by age in the process of carcinogenesis. Our results will facilitate further studies regarding the impact of the epigenetic effects of aging on diseases and will aid in the development of tailored cancer preventive strategies. PMID:26949191

The role of cytosine methylation on charge transport through a DNA strand

DOE Office of Scientific and Technical Information (OSTI.GOV)

Qi, Jianqing, E-mail: jqqi@uw.edu; Anantram, M. P., E-mail: anantmp@uw.edu; Govind, Niranjan, E-mail: niri.govind@pnnl.gov

Cytosine methylation has been found to play a crucial role in various biological processes, including a number of human diseases. The detection of this small modification remains challenging. In this work, we computationally explore the possibility of detecting methylated DNA strands through direct electrical conductance measurements. Using density functional theory and the Landauer-Büttiker method, we study the electronic properties and charge transport through an eight base-pair methylated DNA strand and its native counterpart. We first analyze the effect of cytosine methylation on the tight-binding parameters of two DNA strands and then model the transmission of the electrons and conductance throughmore » the strands both with and without decoherence. We find that the main difference of the tight-binding parameters between the native DNA and the methylated DNA lies in the on-site energies of (methylated) cytosine bases. The intra- and inter-strand hopping integrals between two nearest neighboring guanine base and (methylated) cytosine base also change with the addition of the methyl groups. Our calculations show that in the phase-coherent limit, the transmission of the methylated strand is close to the native strand when the energy is nearby the highest occupied molecular orbital level and larger than the native strand by 5 times in the bandgap. The trend in transmission also holds in the presence of the decoherence with the same rate. The lower conductance for the methylated strand in the experiment is suggested to be caused by the more stable structure due to the introduction of the methyl groups. We also study the role of the exchange-correlation functional and the effect of contact coupling by choosing coupling strengths ranging from weak to strong coupling limit.« less

DNA methylation signature of human fetal alcohol spectrum disorder.

PubMed

Portales-Casamar, Elodie; Lussier, Alexandre A; Jones, Meaghan J; MacIsaac, Julia L; Edgar, Rachel D; Mah, Sarah M; Barhdadi, Amina; Provost, Sylvie; Lemieux-Perreault, Louis-Philippe; Cynader, Max S; Chudley, Albert E; Dubé, Marie-Pierre; Reynolds, James N; Pavlidis, Paul; Kobor, Michael S

2016-01-01

Prenatal alcohol exposure is the leading preventable cause of behavioral and cognitive deficits, which may affect between 2 and 5 % of children in North America. While the underlying mechanisms of alcohol's effects on development remain relatively unknown, emerging evidence implicates epigenetic mechanisms in mediating the range of symptoms observed in children with fetal alcohol spectrum disorder (FASD). Thus, we investigated the effects of prenatal alcohol exposure on genome-wide DNA methylation in the NeuroDevNet FASD cohort, the largest cohort of human FASD samples to date. Genome-wide DNA methylation patterns of buccal epithelial cells (BECs) were analyzed using the Illumina HumanMethylation450 array in a Canadian cohort of 206 children (110 FASD and 96 controls). Genotyping was performed in parallel using the Infinium HumanOmni2.5-Quad v1.0 BeadChip. After correcting for the effects of genetic background, we found 658 significantly differentially methylated sites between FASD cases and controls, with 41 displaying differences in percent methylation change >5 %. Furthermore, 101 differentially methylated regions containing two or more CpGs were also identified, overlapping with 95 different genes. The majority of differentially methylated genes were highly expressed at the level of mRNA in brain samples from the Allen Brain Atlas, and independent DNA methylation data from cortical brain samples showed high correlations with BEC DNA methylation patterns. Finally, overrepresentation analysis of genes with up-methylated CpGs revealed a significant enrichment for neurodevelopmental processes and diseases, such as anxiety, epilepsy, and autism spectrum disorders. These findings suggested that prenatal alcohol exposure is associated with distinct DNA methylation patterns in children and adolescents, raising the possibility of an epigenetic biomarker of FASD.

The role of cytosine methylation on charge transport through a DNA strand

NASA Astrophysics Data System (ADS)

Qi, Jianqing; Govind, Niranjan; Anantram, M. P.

2015-09-01

Cytosine methylation has been found to play a crucial role in various biological processes, including a number of human diseases. The detection of this small modification remains challenging. In this work, we computationally explore the possibility of detecting methylated DNA strands through direct electrical conductance measurements. Using density functional theory and the Landauer-Büttiker method, we study the electronic properties and charge transport through an eight base-pair methylated DNA strand and its native counterpart. We first analyze the effect of cytosine methylation on the tight-binding parameters of two DNA strands and then model the transmission of the electrons and conductance through the strands both with and without decoherence. We find that the main difference of the tight-binding parameters between the native DNA and the methylated DNA lies in the on-site energies of (methylated) cytosine bases. The intra- and inter-strand hopping integrals between two nearest neighboring guanine base and (methylated) cytosine base also change with the addition of the methyl groups. Our calculations show that in the phase-coherent limit, the transmission of the methylated strand is close to the native strand when the energy is nearby the highest occupied molecular orbital level and larger than the native strand by 5 times in the bandgap. The trend in transmission also holds in the presence of the decoherence with the same rate. The lower conductance for the methylated strand in the experiment is suggested to be caused by the more stable structure due to the introduction of the methyl groups. We also study the role of the exchange-correlation functional and the effect of contact coupling by choosing coupling strengths ranging from weak to strong coupling limit.

Targeting epigenetic regulations in cancer

PubMed Central

Ning, Bo; Li, Wenyuan; Zhao, Wei; Wang, Rongfu

2016-01-01

Epigenetic regulation of gene expression is a dynamic and reversible process with DNA methylation, histone modifications, and chromatin remodeling. Recently, groundbreaking studies have demonstrated the importance of DNA and chromatin regulatory proteins from different aspects, including stem cell, development, and tumor genesis. Abnormal epigenetic regulation is frequently associated with diseases and drugs targeting DNA methylation and histone acetylation have been approved for cancer therapy. Although the network of epigenetic regulation is more complex than people expect, new potential druggable chromatin-associated proteins are being discovered and tested for clinical application. Here we review the key proteins that mediate epigenetic regulations through DNA methylation, the acetylation and methylation of histones, and the reader proteins that bind to modified histones. We also discuss cancer associations and recent progress of pharmacological development of these proteins. PMID:26508480

Epigenomics of Development in Populus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Strauss, Steve; Freitag, Michael; Mockler, Todd

2013-01-10

We conducted research to determine the role of epigenetic modifications during tree development using poplar (Populus trichocarpa), a model woody feedstock species. Using methylated DNA immunoprecipitation (MeDIP) or chromatin immunoprecipitation (ChIP), followed by high-throughput sequencing, we are analyzed DNA and histone methylation patterns in the P. trichocarpa genome in relation to four biological processes: bud dormancy and release, mature organ maintenance, in vitro organogenesis, and methylation suppression. Our project is now completed. We have 1) produced 22 transgenic events for a gene involved in DNA methylation suppression and studied its phenotypic consequences; 2) completed sequencing of methylated DNA from elevenmore » target tissues in wildtype P. trichocarpa; 3) updated our customized poplar genome browser using the open-source software tools (2.13) and (V2.2) of the P. trichocarpa genome; 4) produced summary data for genome methylation in P. trichocarpa, including distribution of methylation across chromosomes and in and around genes; 5) employed bioinformatic and statistical methods to analyze differences in methylation patterns among tissue types; and 6) used bisulfite sequencing of selected target genes to confirm bioinformatics and sequencing results, and gain a higher-resolution view of methylation at selected genes 7) compared methylation patterns to expression using available microarray data. Our main findings of biological significance are the identification of extensive regions of the genome that display developmental variation in DNA methylation; highly distinctive gene-associated methylation profiles in reproductive tissues, particularly male catkins; a strong whole genome/all tissue inverse association of methylation at gene bodies and promoters with gene expression; a lack of evidence that tissue specificity of gene expression is associated with gene methylation; and evidence that genome methylation is a significant impediment to tissue dedifferentiation and redifferentiation in vitro.« less

VEZF1 Elements Mediate Protection from DNA Methylation

PubMed Central

Strogantsev, Ruslan; Gaszner, Miklos; Hair, Alan; Felsenfeld, Gary; West, Adam G.

2010-01-01

There is growing consensus that genome organization and long-range gene regulation involves partitioning of the genome into domains of distinct epigenetic chromatin states. Chromatin insulator or barrier elements are key components of these processes as they can establish boundaries between chromatin states. The ability of elements such as the paradigm β-globin HS4 insulator to block the range of enhancers or the spread of repressive histone modifications is well established. Here we have addressed the hypothesis that a barrier element in vertebrates should be capable of defending a gene from silencing by DNA methylation. Using an established stable reporter gene system, we find that HS4 acts specifically to protect a gene promoter from de novo DNA methylation. Notably, protection from methylation can occur in the absence of histone acetylation or transcription. There is a division of labor at HS4; the sequences that mediate protection from methylation are separable from those that mediate CTCF-dependent enhancer blocking and USF-dependent histone modification recruitment. The zinc finger protein VEZF1 was purified as the factor that specifically interacts with the methylation protection elements. VEZF1 is a candidate CpG island protection factor as the G-rich sequences bound by VEZF1 are frequently found at CpG island promoters. Indeed, we show that VEZF1 elements are sufficient to mediate demethylation and protection of the APRT CpG island promoter from DNA methylation. We propose that many barrier elements in vertebrates will prevent DNA methylation in addition to blocking the propagation of repressive histone modifications, as either process is sufficient to direct the establishment of an epigenetically stable silent chromatin state. PMID:20062523

Modeling DNA methylation by analyzing the individual configurations of single molecules

PubMed Central

Affinito, Ornella; Scala, Giovanni; Palumbo, Domenico; Florio, Ermanno; Monticelli, Antonella; Miele, Gennaro; Avvedimento, Vittorio Enrico; Usiello, Alessandro; Chiariotti, Lorenzo; Cocozza, Sergio

2016-01-01

ABSTRACT DNA methylation is often analyzed by reporting the average methylation degree of each cytosine. In this study, we used a single molecule methylation analysis in order to look at the methylation conformation of individual molecules. Using D-aspartate oxidase as a model gene, we performed an in-depth methylation analysis through the developmental stages of 3 different mouse tissues (brain, lung, and gut), where this gene undergoes opposite methylation destiny. This approach allowed us to track both methylation and demethylation processes at high resolution. The complexity of these dynamics was markedly simplified by introducing the concept of methylation classes (MCs), defined as the number of methylated cytosines per molecule, irrespective of their position. The MC concept smooths the stochasticity of the system, allowing a more deterministic description. In this framework, we also propose a mathematical model based on the Markov chain. This model aims to identify the transition probability of a molecule from one MC to another during methylation and demethylation processes. The results of our model suggest that: 1) both processes are ruled by a dominant class of phenomena, namely, the gain or loss of one methyl group at a time; and 2) the probability of a single CpG site becoming methylated or demethylated depends on the methylation status of the whole molecule at that time. PMID:27748645

BioVLAB-mCpG-SNP-EXPRESS: A system for multi-level and multi-perspective analysis and exploration of DNA methylation, sequence variation (SNPs), and gene expression from multi-omics data.

PubMed

Chae, Heejoon; Lee, Sangseon; Seo, Seokjun; Jung, Daekyoung; Chang, Hyeonsook; Nephew, Kenneth P; Kim, Sun

2016-12-01

Measuring gene expression, DNA sequence variation, and DNA methylation status is routinely done using high throughput sequencing technologies. To analyze such multi-omics data and explore relationships, reliable bioinformatics systems are much needed. Existing systems are either for exploring curated data or for processing omics data in the form of a library such as R. Thus scientists have much difficulty in investigating relationships among gene expression, DNA sequence variation, and DNA methylation using multi-omics data. In this study, we report a system called BioVLAB-mCpG-SNP-EXPRESS for the integrated analysis of DNA methylation, sequence variation (SNPs), and gene expression for distinguishing cellular phenotypes at the pairwise and multiple phenotype levels. The system can be deployed on either the Amazon cloud or a publicly available high-performance computing node, and the data analysis and exploration of the analysis result can be conveniently done using a web-based interface. In order to alleviate analysis complexity, all the process are fully automated, and graphical workflow system is integrated to represent real-time analysis progression. The BioVLAB-mCpG-SNP-EXPRESS system works in three stages. First, it processes and analyzes multi-omics data as input in the form of the raw data, i.e., FastQ files. Second, various integrated analyses such as methylation vs. gene expression and mutation vs. methylation are performed. Finally, the analysis result can be explored in a number of ways through a web interface for the multi-level, multi-perspective exploration. Multi-level interpretation can be done by either gene, gene set, pathway or network level and multi-perspective exploration can be explored from either gene expression, DNA methylation, sequence variation, or their relationship perspective. The utility of the system is demonstrated by performing analysis of phenotypically distinct 30 breast cancer cell line data set. BioVLAB-mCpG-SNP-EXPRESS is available at http://biohealth.snu.ac.kr/software/biovlab_mcpg_snp_express/. Copyright © 2016 Elsevier Inc. All rights reserved.

Sperm DNA fragmentation affects epigenetic feature in human male pronucleus.

PubMed

Rajabi, H; Mohseni-Kouchesfehani, H; Eslami-Arshaghi, T; Salehi, M

2018-02-01

To evaluate whether the sperm DNA fragmentation affects male pronucleus epigenetic factors, semen analysis was performed and DNA fragmentation was assessed by the method of sperm chromatin structure assay (SCSA). Human-mouse interspecies fertilisation was used to create human male pronucleus. Male pronucleus DNA methylation and H4K12 acetylation were evaluated by immunostaining. Results showed a significant positive correlation between the level of sperm DNA fragmentation and DNA methylation in male pronuclei. In other words, an increase in DNA damage caused an upsurge in DNA methylation. In the case of H4K12 acetylation, no correlation was detected between DNA damage and the level of histone acetylation in the normal group, but results for the group in which male pronuclei were derived from sperm cells with DNA fragmentation, increased DNA damage led to a decreased acetylation level. Sperm DNA fragmentation interferes with the active demethylation process and disrupts the insertion of histones into the male chromatin in the male pronucleus, following fertilisation. © 2017 Blackwell Verlag GmbH.

DNA methylation in adult diffuse gliomas.

PubMed

LeBlanc, Veronique G; Marra, Marco A

2016-11-01

Adult diffuse gliomas account for the majority of primary malignant brain tumours, and are in most cases lethal. Current therapies are often only marginally effective, and improved options will almost certainly benefit from further insight into the various processes contributing to gliomagenesis and pathology. While molecular characterization of these tumours classifies them on the basis of genetic alterations and chromosomal abnormalities, DNA methylation patterns are increasingly understood to play a role in glioma pathogenesis. Indeed, a subset of gliomas associated with improved survival is characterized by the glioma CpG island methylator phenotype (G-CIMP), which can be induced by the expression of mutant isocitrate dehydrogenase (IDH1/2). Aberrant methylation of particular genes or regulatory elements, within the context of G-CIMP-positive and/or negative tumours, has also been shown to be associated with differential survival. In this review, we provide an overview of the current knowledge regarding the role of DNA methylation in adult diffuse gliomas. In particular, we discuss IDH mutations and G-CIMP, MGMT promoter methylation, DNA methylation-mediated microRNA regulation and aberrant methylation of specific genes or groups of genes. © The Author 2016. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Modulation of DNA methylation by human papillomavirus E6 and E7 oncoproteins in cervical cancer

PubMed Central

Sen, Prakriti; Ganguly, Pooja; Ganguly, Niladri

2018-01-01

Human papillomaviruses (HPVs) are double stranded circular DNA viruses that infect cutaneous and mucosal epithelial cells. Almost 99% of cervical cancer has a HPV infection. The early oncoproteins E6 and E7 are important in this cellular transformation process. Epigenetic mechanisms have long been known to result in decisive alterations in DNA, leading to alterations in DNA-protein interactions, alterations in chromatin structure and compaction and significant alterations in gene expression. The enzymes responsible for these epigenetic modifications are DNA methyl transferases (DNMTs), histone acetylases and deacetylases. Epigenetics has an important role in cancer development by modifying the cellular micro environment. In this review, the authors discuss the role of HPV oncoproteins E6 and E7 in modulating the epigenetic mechanisms inside the host cell. The oncoproteins induce the expression of DNMTs which lead to aberrant DNA methylations and disruption of the normal epigenetic processes. The E7 oncoprotein may additionally directly bind and induce methyl transferase activity of the enzyme. These modulations lead to altered gene expression levels, particularly the genes involved in apoptosis, cell cycle and cell adhesion. In addition, the present review discusses how epigenetic mechanisms may be targeted for possible therapeutic interventions for HPV mediated cervical cancer. PMID:29285184

Analysis of DNA Methylation in Young People: Limited Evidence for an Association Between Victimization Stress and Epigenetic Variation in Blood.

PubMed

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

2018-06-01

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.

Inter-Species Grafting Caused Extensive and Heritable Alterations of DNA Methylation in Solanaceae Plants

PubMed Central

Lin, Yan; Ma, Yiqiao; Liu, Gang; Yu, Xiaoming; Zhong, Silin; Liu, Bao

2013-01-01

Background Grafting has been extensively used to enhance the performance of horticultural crops. Since Charles Darwin coined the term “graft hybrid” meaning that asexual combination of different plant species may generate products that are genetically distinct, highly discrepant opinions exist supporting or against the concept. Recent studies have documented that grafting enables exchanges of both RNA and DNA molecules between the grafting partners, thus providing a molecular basis for grafting-induced genetic variation. DNA methylation is known as prone to alterations as a result of perturbation of internal and external conditions. Given characteristics of grafting, it is interesting to test whether the process may cause an alteration of this epigenetic marker in the grafted organismal products. Methodology/Principal Findings We analyzed relative global DNA methylation levels and locus-specific methylation patterns by the MSAP marker and locus-specific bisulfite-sequencing in the seed plants (wild-type controls), self- and hetero-grafted scions/rootstocks, selfed progenies of scions and their seed-plant controls, involving three Solanaceae species. We quantified expression of putative genes involved in establishing and/or maintaining DNA methylation by q-(RT)-PCR. We found that (1) hetero-grafting caused extensive alteration of DNA methylation patterns in a locus-specific manner, especially in scions, although relative methylation levels remain largely unaltered; (2) the altered methylation patterns in the hetero-grafting-derived scions could be inherited to sexual progenies with some sites showing further alterations or revisions; (3) hetero-grafting caused dynamic changes in steady-state transcript abundance of genes encoding for a set of enzymes functionally relevant to DNA methylation. Conclusions/Significance Our results demonstrate that inter-species grafting in plants could produce extensive and heritable alterations in DNA methylation. We suggest that these readily altered, yet heritable, epigenetic modifications due to interspecies hetero-grafting may shed one facet of insight into the molecular underpinnings for the still contentious concept of graft hybrid. PMID:23614002

Regulation of DNA methylation on EEF1D and RPL8 expression in cattle.

PubMed

Liu, Xuan; Yang, Jie; Zhang, Qin; Jiang, Li

2017-10-01

Dynamic changes to the epigenome play a critical role in a variety of biology processes and complex traits. Many important candidate genes have been identified through our previous genome wide association study (GWAS) on milk production traits in dairy cattle. However, the underlying mechanism of candidate genes have not yet been clearly understood. In this study, we analyzed the methylation variation of the candidate genes, EEF1D and RPL8, which were identified to be strongly associated with milk production traits in dairy cattle in our previous studies, and its effect on protein and mRNA expression. We compared DNA methylation profiles and gene expression levels of EEF1D and RPL8 in five different tissues (heart, liver, mammary gland, ovary and muscle) of three cows. Both genes showed the highest expression level in mammary gland. For RPL8, there was no difference in the DNA methylation pattern in the five tissues, suggesting no effect of DNA methylation on gene expression. For EEF1D, the DNA methylation levels of its first CpG island differed in the five tissues and were negatively correlated with the gene expression levels. To further investigate the function of DNA methylation on the expression of EEF1D, we collected blood samples of three cows at early stage of lactation and in dry period and analyzed its expression and the methylation status of the first CpG island in blood. As a result, the mRNA expression of EEF1D in the dry period was higher than that at the early stage of lactation, while the DNA methylation level in the dry period was lower than that at the early stage of lactation. Our result suggests that the DNA methylation of EEF1D plays an important role in the spatial and temporal regulation of its expression and possibly have an effect on the milk production traits.

DNA methylation mediates neural processing after odor learning in the honeybee

PubMed Central

Biergans, Stephanie D.; Claudianos, Charles; Reinhard, Judith; Galizia, C. Giovanni

2017-01-01

DNA methyltransferases (Dnmts) - epigenetic writers catalyzing the transfer of methyl-groups to cytosine (DNA methylation) – regulate different aspects of memory formation in many animal species. In honeybees, Dnmt activity is required to adjust the specificity of olfactory reward memories and bees’ relearning capability. The physiological relevance of Dnmt-mediated DNA methylation in neural networks, however, remains unknown. Here, we investigated how Dnmt activity impacts neuroplasticity in the bees’ primary olfactory center, the antennal lobe (AL) an equivalent of the vertebrate olfactory bulb. The AL is crucial for odor discrimination, an indispensable process in forming specific odor memories. Using pharmacological inhibition, we demonstrate that Dnmt activity influences neural network properties during memory formation in vivo. We show that Dnmt activity promotes fast odor pattern separation in trained bees. Furthermore, Dnmt activity during memory formation increases both the number of responding glomeruli and the response magnitude to a novel odor. These data suggest that Dnmt activity is necessary for a form of homoeostatic network control which might involve inhibitory interneurons in the AL network. PMID:28240742

Aberrant DNA methylation patterns of spermatozoa in men with unexplained infertility.

PubMed

Urdinguio, Rocío G; Bayón, Gustavo F; Dmitrijeva, Marija; Toraño, Estela G; Bravo, Cristina; Fraga, Mario F; Bassas, Lluís; Larriba, Sara; Fernández, Agustín F

2015-05-01

Are there DNA methylation alterations in sperm that could explain the reduced biological fertility of male partners from couples with unexplained infertility? DNA methylation patterns, not only at specific loci but also at Alu Yb8 repetitive sequences, are altered in infertile individuals compared with fertile controls. Aberrant DNA methylation of sperm has been associated with human male infertility in patients demonstrating either deficiencies in the process of spermatogenesis or low semen quality. Case and control prospective study. This study compares 46 sperm samples obtained from 17 normospermic fertile men and 29 normospermic infertile patients. Illumina Infinium HD Human Methylation 450K arrays were used to identify genomic regions showing differences in sperm DNA methylation patterns between five fertile and seven infertile individuals. Additionally, global DNA methylation of sperm was measured using the Methylamp Global DNA Methylation Quantification Ultra kit (Epigentek) in 14 samples, and DNA methylation at several repetitive sequences (LINE-1, Alu Yb8, NBL2, D4Z4) measured by bisulfite pyrosequencing in 44 sperm samples. A sperm-specific DNA methylation pattern was obtained by comparing the sperm methylomes with the DNA methylomes of differentiated somatic cells using data obtained from methylation arrays (Illumina 450 K) of blood, neural and glial cells deposited in public databases. In this study we conduct, for the first time, a genome-wide study to identify alterations of sperm DNA methylation in individuals with unexplained infertility that may account for the differences in their biological fertility compared with fertile individuals. We have identified 2752 CpGs showing aberrant DNA methylation patterns, and more importantly, these differentially methylated CpGs were significantly associated with CpG sites which are specifically methylated in sperm when compared with somatic cells. We also found statistically significant (P < 0.001) associations between DNA hypomethylation and regions corresponding to those which, in somatic cells, are enriched in the repressive histone mark H3K9me3, and between DNA hypermethylation and regions enriched in H3K4me1 and CTCF, suggesting that the relationship between chromatin context and aberrant DNA methylation of sperm in infertile men could be locus-dependent. Finally, we also show that DNA methylation patterns, not only at specific loci but also at several repetitive sequences (LINE-1, Alu Yb8, NBL2, D4Z4), were lower in sperm than in somatic cells. Interestingly, sperm samples at Alu Yb8 repetitive sequences of infertile patients showed significantly lower DNA methylation levels than controls. Our results are descriptive and further studies would be needed to elucidate the functional effects of aberrant DNA methylation on male fertility. Overall, our data suggest that aberrant sperm DNA methylation might contribute to fertility impairment in couples with unexplained infertility and they provide a promising basis for future research. This work has been financially supported by Fundación Cientifica de la AECC (to R.G.U.); IUOPA (to G.F.B.); FICYT (to E.G.T.); the Spanish National Research Council (CSIC; 200820I172 to M.F.F.); Fundación Ramón Areces (to M.F.F); the Plan Nacional de I+D+I 2008-2011/2013-2016/FEDER (PI11/01728 to AF.F., PI12/01080 to M.F.F. and PI12/00361 to S.L.); the PN de I+D+I 2008-20011 and the Generalitat de Catalunya (2009SGR01490). A.F.F. is sponsored by ISCIII-Subdirección General de Evaluación y Fomento de la Investigación (CP11/00131). S.L. is sponsored by the Researchers Stabilization Program from the Spanish National Health System (CES09/020). The IUOPA is supported by the Obra Social Cajastur, Spain. © The Author 2015. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

DNA methylation alterations in response to pesticide exposure in vitro

PubMed Central

Zhang, Xiao; Wallace, Andrew D.; Du, Pan; Kibbe, Warren A.; Jafari, Nadereh; Xie, Hehuang; Lin, Simon; Baccarelli, Andrea; Soares, Marcelo Bento; Hou, Lifang

2013-01-01

Although pesticides are subject to extensive carcinogenicity testing before regulatory approval, pesticide exposure has repeatedly been associated with various cancers. This suggests that pesticides may cause cancer via non-mutagenicity mechanisms. The present study provides evidence to support the hypothesis that pesticide-induced cancer may be mediated in part by epigenetic mechanisms. We examined whether exposure to 7 commonly used pesticides (i.e., fonofos, parathion, terbufos, chlorpyrifos, diazinon, malathion, and phorate) induces DNA methylation alterations in vitro. We conducted genome-wide DNA methylation analyses on DNA samples obtained from the human hematopoietic K562 cell line exposed to ethanol (control) and several OPs using the Illumina Infinium HumanMethylation27 BeadChip. Bayesian-adjusted t-tests were used to identify differentially methylated gene promoter CpG sites. In this report, we present our results on three pesticides (fonofos, parathion, and terbufos) that clustered together based on principle component analysis and hierarchical clustering. These three pesticides induced similar methylation changes in the promoter regions of 712 genes, while also exhibiting their own OP-specific methylation alterations. Functional analysis of methylation changes specific to each OP, or common to all three OPs, revealed that differential methylation was associated with numerous genes that are involved in carcinogenesis-related processes. Our results provide experimental evidence that pesticides may modify gene promoter DNA methylation levels, suggesting that epigenetic mechanisms may contribute to pesticide-induced carcinogenesis. Further studies in other cell types and human samples are required, as well as determining the impact of these methylation changes on gene expression. PMID:22847954

Circulating DNA and its methylation level in inflammatory bowel disease and related colon cancer.

PubMed

Bai, Xuming; Zhu, Yaqun; Pu, Wangyang; Xiao, Li; Li, Kai; Xing, Chungen; Jin, Yong

2015-01-01

Both of chronic inflammation and abnormal immune in inflammatory bowel disease can induce colon cancer. Previous research showed that cell apoptosis and necrosis become the main source of circulating DNA in the peripheral blood during tumorigenesis that reduced along with methylation degree. However, its role in the process of colitis transforming to colon cancer is not clarified. Drinking 3% DSS was used to establish colitis model, while 3% dextran sodium sulfate (DSS) combined with azo oxidation methane (AOM) intraperitoneal injection was applied to establish colitis related colon cancer model. Circulating DNA and its methylation level in peripheral blood were tested. Morphology observation, HE staining, and p53 and β-catenin expression detection confirmed that drinking 3% DSS and 3% DSS combined with AOM intraperitoneal injection can successfully establish colitis and colitis associated colorectal cancer models. Circulating DNA level in colitis and colon cancer mice increased by gradient compared with control, while significant difference was observed between each other. Circulating DNA methylation level decreased obviously in colitis and colon cancer, and significant difference was observed between each other. Abnormal protein expression, circulating DNA and its methylation level in ulcerative colitis associated colorectal tissues change in gradient, suggesting that circulating DNA and its methylation level can be treated as new markers for colitis cancer transformation that has certain significance to explore the mechanism of human ulcerative colitis canceration.

Inhibition of DNA methyltransferases regulates cocaine self-administration by rats: a genome-wide DNA methylation study.

PubMed

Fonteneau, M; Filliol, D; Anglard, P; Befort, K; Romieu, P; Zwiller, J

2017-03-01

DNA methylation is a major epigenetic process which regulates the accessibility of genes to the transcriptional machinery. In the present study, we investigated whether modifying the global DNA methylation pattern in the brain would alter cocaine intake by rats, using the cocaine self-administration test. The data indicate that treatment of rats with the DNA methyltransferase inhibitors 5-aza-2'-deoxycytidine (dAZA) and zebularine enhanced the reinforcing properties of cocaine. To obtain some insights about the underlying neurobiological mechanisms, a genome-wide methylation analysis was undertaken in the prefrontal cortex of rats self-administering cocaine and treated with or without dAZA. The study identified nearly 189 000 differentially methylated regions (DMRs), about half of them were located inside gene bodies, while only 9% of DMRs were found in the promoter regions of genes. About 99% of methylation changes occurred outside CpG islands. Gene expression studies confirmed the inverse correlation usually observed between increased methylation and transcriptional activation when methylation occurs in the gene promoter. This inverse correlation was not observed when methylation took place inside gene bodies. Using the literature-based Ingenuity Pathway Analysis, we explored how the differentially methylated genes were related. The analysis showed that increase in cocaine intake by rats in response to DNA methyltransferase inhibitors underlies plasticity mechanisms which mainly concern axonal growth and synaptogenesis as well as spine remodeling. Together with the Akt/PI3K pathway, the Rho-GTPase family was found to be involved in the plasticity underlying the effect of dAZA on the observed behavioral changes. © 2016 John Wiley & Sons Ltd and International Behavioural and Neural Genetics Society.

MethVisual - visualization and exploratory statistical analysis of DNA methylation profiles from bisulfite sequencing.

PubMed

Zackay, Arie; Steinhoff, Christine

2010-12-15

Exploration of DNA methylation and its impact on various regulatory mechanisms has become a very active field of research. Simultaneously there is an arising need for tools to process and analyse the data together with statistical investigation and visualisation. MethVisual is a new application that enables exploratory analysis and intuitive visualization of DNA methylation data as is typically generated by bisulfite sequencing. The package allows the import of DNA methylation sequences, aligns them and performs quality control comparison. It comprises basic analysis steps as lollipop visualization, co-occurrence display of methylation of neighbouring and distant CpG sites, summary statistics on methylation status, clustering and correspondence analysis. The package has been developed for methylation data but can be also used for other data types for which binary coding can be inferred. The application of the package, as well as a comparison to existing DNA methylation analysis tools and its workflow based on two datasets is presented in this paper. The R package MethVisual offers various analysis procedures for data that can be binarized, in particular for bisulfite sequenced methylation data. R/Bioconductor has become one of the most important environments for statistical analysis of various types of biological and medical data. Therefore, any data analysis within R that allows the integration of various data types as provided from different technological platforms is convenient. It is the first and so far the only specific package for DNA methylation analysis, in particular for bisulfite sequenced data available in R/Bioconductor enviroment. The package is available for free at http://methvisual.molgen.mpg.de/ and from the Bioconductor Consortium http://www.bioconductor.org.

MethVisual - visualization and exploratory statistical analysis of DNA methylation profiles from bisulfite sequencing

PubMed Central

2010-01-01

Background Exploration of DNA methylation and its impact on various regulatory mechanisms has become a very active field of research. Simultaneously there is an arising need for tools to process and analyse the data together with statistical investigation and visualisation. Findings MethVisual is a new application that enables exploratory analysis and intuitive visualization of DNA methylation data as is typically generated by bisulfite sequencing. The package allows the import of DNA methylation sequences, aligns them and performs quality control comparison. It comprises basic analysis steps as lollipop visualization, co-occurrence display of methylation of neighbouring and distant CpG sites, summary statistics on methylation status, clustering and correspondence analysis. The package has been developed for methylation data but can be also used for other data types for which binary coding can be inferred. The application of the package, as well as a comparison to existing DNA methylation analysis tools and its workflow based on two datasets is presented in this paper. Conclusions The R package MethVisual offers various analysis procedures for data that can be binarized, in particular for bisulfite sequenced methylation data. R/Bioconductor has become one of the most important environments for statistical analysis of various types of biological and medical data. Therefore, any data analysis within R that allows the integration of various data types as provided from different technological platforms is convenient. It is the first and so far the only specific package for DNA methylation analysis, in particular for bisulfite sequenced data available in R/Bioconductor enviroment. The package is available for free at http://methvisual.molgen.mpg.de/ and from the Bioconductor Consortium http://www.bioconductor.org. PMID:21159174

Characterization of the Methylation Status of Pax7 and Myogenic Regulator Factors in Cell Myogenic Differentiation.

PubMed

Chao, Zhe; Zheng, Xin-Li; Sun, Rui-Ping; Liu, Hai-Long; Huang, Li-Li; Cao, Zong-Xi; Deng, Chang-Yan; Wang, Feng

2016-07-01

Epigenetic processes in the development of skeletal muscle have been appreciated for over a decade. DNA methylation is a major epigenetic modification important for regulating gene expression and suppressing spurious transcription. Up to now, the importance of epigenetic marks in the regulation of Pax7 and myogenic regulatory factors (MRFs) expression is far less explored. In the present study, semi-quantitative the real-time polymerase chain reaction (RT-PCR) analyses showed MyoD and Myf5 were expressed in activated and quiescent C2C12 cells. MyoG was expressed in a later stage of myogenesis. Pax7 was weakly expressed in differentiated C2C12 cells. To further understand the regulation of expression of these genes, the DNA methylation status of Pax7, MyoD, and Myf5 was determined by bisulfite sequencing PCR. During the C2C12 myoblasts fusion process, the changes of promoter and exon 1 methylation of Pax7, MyoD, and Myf5 genes were observed. In addition, an inverse relationship of low methylation and high expression was found. These results suggest that DNA methylation may be an important mechanism regulating Pax7 and MRFs transcription in cell myogenic differentiation.

Tumor purity and differential methylation in cancer epigenomics.

PubMed

Wang, Fayou; Zhang, Naiqian; Wang, Jun; Wu, Hao; Zheng, Xiaoqi

2016-11-01

DNA methylation is an epigenetic modification of DNA molecule that plays a vital role in gene expression regulation. It is not only involved in many basic biological processes, but also considered an important factor for tumorigenesis and other human diseases. Study of DNA methylation has been an active field in cancer epigenomics research. With the advances of high-throughput technologies and the accumulation of enormous amount of data, method development for analyzing these data has gained tremendous interests in the fields of computational biology and bioinformatics. In this review, we systematically summarize the recent developments of computational methods and software tools in high-throughput methylation data analysis with focus on two aspects: differential methylation analysis and tumor purity estimation in cancer studies. © The Author 2016. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Salt Stress Induced Variation in DNA Methylation Pattern and Its Influence on Gene Expression in Contrasting Rice Genotypes

PubMed Central

Karan, Ratna; DeLeon, Teresa; Biradar, Hanamareddy; Subudhi, Prasanta K.

2012-01-01

Background Salinity is a major environmental factor limiting productivity of crop plants including rice in which wide range of natural variability exists. Although recent evidences implicate epigenetic mechanisms for modulating the gene expression in plants under environmental stresses, epigenetic changes and their functional consequences under salinity stress in rice are underexplored. DNA methylation is one of the epigenetic mechanisms regulating gene expression in plant’s responses to environmental stresses. Better understanding of epigenetic regulation of plant growth and response to environmental stresses may create novel heritable variation for crop improvement. Methodology/Principal Findings Methylation sensitive amplification polymorphism (MSAP) technique was used to assess the effect of salt stress on extent and patterns of DNA methylation in four genotypes of rice differing in the degree of salinity tolerance. Overall, the amount of DNA methylation was more in shoot compared to root and the contribution of fully methylated loci was always more than hemi-methylated loci. Sequencing of ten randomly selected MSAP fragments indicated gene-body specific DNA methylation of retrotransposons, stress responsive genes, and chromatin modification genes, distributed on different rice chromosomes. Bisulphite sequencing and quantitative RT-PCR analysis of selected MSAP loci showed that cytosine methylation changes under salinity as well as gene expression varied with genotypes and tissue types irrespective of the level of salinity tolerance of rice genotypes. Conclusions/Significance The gene body methylation may have an important role in regulating gene expression in organ and genotype specific manner under salinity stress. Association between salt tolerance and methylation changes observed in some cases suggested that many methylation changes are not “directed”. The natural genetic variation for salt tolerance observed in rice germplasm may be independent of the extent and pattern of DNA methylation which may have been induced by abiotic stress followed by accumulation through the natural selection process. PMID:22761959

Genome-wide DNA methylation patterns in wild samples of two morphotypes of threespine stickleback (Gasterosteus aculeatus).

PubMed

Smith, Gilbert; Smith, Carl; Kenny, John G; Chaudhuri, Roy R; Ritchie, Michael G

2015-04-01

Epigenetic marks such as DNA methylation play important biological roles in gene expression regulation and cellular differentiation during development. To examine whether DNA methylation patterns are potentially associated with naturally occurring phenotypic differences, we examined genome-wide DNA methylation within Gasterosteus aculeatus, using reduced representation bisulfite sequencing. First, we identified highly methylated regions of the stickleback genome, finding such regions to be located predominantly within genes, and associated with genes functioning in metabolism and biosynthetic processes, cell adhesion, signaling pathways, and blood vessel development. Next, we identified putative differentially methylated regions (DMRs) of the genome between complete and low lateral plate morphs of G. aculeatus. We detected 77 DMRs that were mainly located in intergenic regions. Annotations of genes associated with these DMRs revealed potential functions in a number of known divergent adaptive phenotypes between G. aculeatus ecotypes, including cardiovascular development, growth, and neuromuscular development. © The Author 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

DNA modifications in models of alcohol use disorders

PubMed Central

Tulisiak, Christopher T.; Harris, R. Adron; Ponomarev, Igor

2016-01-01

Chronic alcohol use and abuse result in widespread changes to gene expression, some of which contribute to the development of alcohol use disorders (AUD). Gene expression is, in part, controlled by a group of regulatory systems often referred to as epigenetic factors, which includes, among other mechanisms, chemical marks made on the histone proteins around which genomic DNA is wound to form chromatin, and on nucleotides of the DNA itself. In particular, alcohol has been shown to perturb the epigenetic machinery, leading to changes in gene expression and cellular functions characteristic of AUD and, ultimately, to altered behavior. DNA modifications in particular are seeing increasing research in the context of alcohol use and abuse. To date, studies of DNA modifications in AUD have primarily looked at global methylation profiles in human brain and blood, gene-specific methylation profiles in animal models, methylation changes associated with prenatal ethanol exposure, and the potential therapeutic abilities of DNA methyltransferase inhibitors. Future studies may be aimed at identifying changes to more recently discovered DNA modifications, utilizing new methods to discriminate methylation profiles between cell types and clarifying how alcohol influences the methylomes of cell type populations and how this may affect downstream processes. These studies and more in-depth probing of DNA methylation will be key to determining whether DNA-level epigenetic regulation plays a causative role in AUD and can thus be targeted for treatment of the disorder. PMID:27865607

Variations in DNA methylation, acetylated histone H4, and methylated histone H3 during Pinus radiata needle maturation in relation to the loss of in vitro organogenic capability.

PubMed

Valledor, Luis; Meijón, Mónica; Hasbún, Rodrigo; Jesús Cañal, Maria; Rodríguez, Roberto

2010-03-15

Needle differentiation is a very complex process associated with the formation of a mature photosynthetic organ. From meristem differentiation to leaf maturation, gene control must play an important role switching required genes on and off to define tissue functions, with the epigenetic code being one of the main regulation mechanisms. In this work, we examined the connections between the variation in the levels of some epigenetic players (DNA methylation, acetylated histone H4 and histone H3 methylation at Lys 4 and Lys 9) at work during needle maturation. Our results indicate that needle maturation, which is associated with a decrease in organogenic capability, is related to an increase in heterochromatin-related epigenetic markers (high DNA methylation and low acetylated histone H4 levels, and the presence of histone H3 methylated at lys 9). Immunohistochemical analyses also showed that the DNA methylation of palisade parenchyma cell layers during the transition from immature to mature scions is associated with the loss of the capacity to induce adventitious organs. Copyright 2009 Elsevier GmbH. All rights reserved.

Kidney Dysfunction in Adult Offspring Exposed In Utero to Type 1 Diabetes Is Associated with Alterations in Genome-Wide DNA Methylation

PubMed Central

Gautier, Jean-François; Porcher, Raphaël; Abi Khalil, Charbel; Bellili-Munoz, Naima; Fetita, Lila Sabrina; Travert, Florence; Choukem, Simeon-Pierre; Riveline, Jean-Pierre; Hadjadj, Samy; Larger, Etienne; Boudou, Philippe; Blondeau, Bertrand; Roussel, Ronan; Ferré, Pascal; Ravussin, Eric; Rouzet, François; Marre, Michel

2015-01-01

Background Fetal exposure to hyperglycemia impacts negatively kidney development and function. Objective Our objective was to determine whether fetal exposure to moderate hyperglycemia is associated with epigenetic alterations in DNA methylation in peripheral blood cells and whether those alterations are related to impaired kidney function in adult offspring. Design Twenty nine adult, non-diabetic offspring of mothers with type 1 diabetes (T1D) (case group) were matched with 28 offspring of T1D fathers (control group) for the study of their leukocyte genome-wide DNA methylation profile (27,578 CpG sites, Human Methylation 27 BeadChip, Illumina Infinium). In a subset of 19 cases and 18 controls, we assessed renal vascular development by measuring Glomerular Filtration Rate (GFR) and Effective Renal Plasma Flow (ERPF) at baseline and during vasodilatation produced by amino acid infusion. Results Globally, DNA was under-methylated in cases vs. controls. Among the 87 CpG sites differently methylated, 74 sites were less methylated and 13 sites more methylated in cases vs. controls. None of these CpG sites were located on a gene known to be directly involved in kidney development and/or function. However, the gene encoding DNA methyltransferase 1 (DNMT1)—a key enzyme involved in gene expression during early development–was under-methylated in cases. The average methylation of the 74 under-methylated sites differently correlated with GFR in cases and controls. Conclusion Alterations in methylation profile imprinted by the hyperglycemic milieu of T1D mothers during fetal development may impact kidney function in adult offspring. The involved pathways seem to be a nonspecific imprinting process rather than specific to kidney development or function. PMID:26258530

Kidney Dysfunction in Adult Offspring Exposed In Utero to Type 1 Diabetes Is Associated with Alterations in Genome-Wide DNA Methylation.

PubMed

Gautier, Jean-François; Porcher, Raphaël; Abi Khalil, Charbel; Bellili-Munoz, Naima; Fetita, Lila Sabrina; Travert, Florence; Choukem, Simeon-Pierre; Riveline, Jean-Pierre; Hadjadj, Samy; Larger, Etienne; Boudou, Philippe; Blondeau, Bertrand; Roussel, Ronan; Ferré, Pascal; Ravussin, Eric; Rouzet, François; Marre, Michel

2015-01-01

Fetal exposure to hyperglycemia impacts negatively kidney development and function. Our objective was to determine whether fetal exposure to moderate hyperglycemia is associated with epigenetic alterations in DNA methylation in peripheral blood cells and whether those alterations are related to impaired kidney function in adult offspring. Twenty nine adult, non-diabetic offspring of mothers with type 1 diabetes (T1D) (case group) were matched with 28 offspring of T1D fathers (control group) for the study of their leukocyte genome-wide DNA methylation profile (27,578 CpG sites, Human Methylation 27 BeadChip, Illumina Infinium). In a subset of 19 cases and 18 controls, we assessed renal vascular development by measuring Glomerular Filtration Rate (GFR) and Effective Renal Plasma Flow (ERPF) at baseline and during vasodilatation produced by amino acid infusion. Globally, DNA was under-methylated in cases vs. controls. Among the 87 CpG sites differently methylated, 74 sites were less methylated and 13 sites more methylated in cases vs. controls. None of these CpG sites were located on a gene known to be directly involved in kidney development and/or function. However, the gene encoding DNA methyltransferase 1 (DNMT1)--a key enzyme involved in gene expression during early development--was under-methylated in cases. The average methylation of the 74 under-methylated sites differently correlated with GFR in cases and controls. Alterations in methylation profile imprinted by the hyperglycemic milieu of T1D mothers during fetal development may impact kidney function in adult offspring. The involved pathways seem to be a nonspecific imprinting process rather than specific to kidney development or function.

DNA Demethylation Rescues the Impaired Osteogenic Differentiation Ability of Human Periodontal Ligament Stem Cells in High Glucose

PubMed Central

Liu, Zhi; Chen, Tian; Sun, Wenhua; Yuan, Zongyi; Yu, Mei; Chen, Guoqing; Guo, Weihua; Xiao, Jingang; Tian, Weidong

2016-01-01

Diabetes mellitus, characterized by abnormally high blood glucose levels, gives rise to impaired bone remodeling. In response to high glucose (HG), the attenuated osteogenic differentiation capacity of human periodontal ligament stem cells (hPDLSCs) is associated with the loss of alveolar bone. Recently, DNA methylation was reported to affect osteogenic differentiation of stem cells in pathological states. However, the intrinsic mechanism linking DNA methylation to osteogenic differentiation ability in the presence of HG is still unclear. In this study, we found that diabetic rats with increased DNA methylation levels in periodontal ligaments exhibited reduced bone mass and density. In vitro application of 5-aza-2′-deoxycytidine (5-aza-dC), a DNA methyltransferase inhibitor, to decrease DNA methylation levels in hPDLSCs, rescued the osteogenic differentiation capacity of hPDLSCs under HG conditions. Moreover, we demonstrated that the canonical Wnt signaling pathway was activated during this process and, under HG circumstances, the 5-aza-dC-rescued osteogenic differentiation capacity was blocked by Dickkopf-1, an effective antagonist of the canonical Wnt signaling pathway. Taken together, these results demonstrate for the first time that suppression of DNA methylation is able to facilitate the osteogenic differentiation capacity of hPDLSCs exposed to HG, through activation of the canonical Wnt signaling pathway. PMID:27273319

First evidence of DNA methylation in insect Tribolium castaneum: environmental regulation of DNA methylation within heterochromatin.

PubMed

Feliciello, Isidoro; Parazajder, Josip; Akrap, Ivana; Ugarković, Durđica

2013-05-01

DNA methylation has been studied in many eukaryotic organisms, in particular vertebrates, and was implicated in developmental and phenotypic variations. Little is known about the role of DNA methylation in invertebrates, although insects are considered as excellent models for studying the evolution of DNA methylation. In the red flour beetle, Tribolium castaneum (Tenebrionidae, Coleoptera), no evidence of DNA methylation has been found till now. In this paper, a cytosine methylation in Tribolium castaneum embryos was detected by methylation sensitive restriction endonucleases and immuno-dot blot assay. DNA methylation in embryos is followed by a global demethylation in larvae, pupae and adults. DNA demethylation seems to proceed actively through 5-hydroxymethylcytosine, most probably by the action of TET enzyme. Bisulfite sequencing of a highly abundant satellite DNA located in pericentromeric heterochromatin revealed similar profile of cytosine methylation in adults and embryos. Cytosine methylation was not only restricted to CpG sites but was found at CpA, CpT and CpC sites. In addition, complete cytosine demethylation of heterochromatic satellite DNA was induced by heat stress. The results reveal existence of DNA methylation cycling in T. castaneum ranging from strong overall cytosine methylation in embryos to a weak DNA methylation in other developmental stages. Nevertheless, DNA methylation is preserved within heterochromatin during development, indicating its role in heterochromatin formation and maintenance. It is, however, strongly affected by heat stress, suggesting a role for DNA methylation in heterochromatin structure modulation during heat stress response.

Gestational Alcohol Exposure Altered DNA Methylation Status in the Developing Fetus

PubMed Central

Mandal, Chanchal; Halder, Debasish; Jung, Kyoung Hwa; Chai, Young Gyu

2017-01-01

Ethanol is well known as a teratogenic factor that is capable of inducing a wide range of developmental abnormalities if the developing fetus is exposed to it. Duration and dose are the critical parameters of exposure that affect teratogenic variation to the developing fetus. It is suggested that ethanol interferes with epigenetic processes especially DNA methylation. We aimed to organize all of the available information on the alteration of DNA methylation by ethanol in utero. Thus, we have summarized all published information regarding alcohol-mediated alterations in DNA methylation during gestation. We tried to arrange information in a way that anyone can easily find the alcohol exposure time, doses, sampling time, and major changes in genomic level. Manuscript texts will also represent the correlation between ethanol metabolites and subsequent changes in methylome patterns. We hope that this review will help future researchers to further examine the issues associated with ethanol exposure. PMID:28657590

Overlapping DNA Methylation Dynamics in Mouse Intestinal Cell Differentiation and Early Stages of Malignant Progression

PubMed Central

Forn, Marta; Díez-Villanueva, Anna; Merlos-Suárez, Anna; Muñoz, Mar; Lois, Sergi; Carriò, Elvira; Jordà, Mireia; Bigas, Anna; Batlle, Eduard; Peinado, Miguel A.

2015-01-01

Mouse models of intestinal crypt cell differentiation and tumorigenesis have been used to characterize the molecular mechanisms underlying both processes. DNA methylation is a key epigenetic mark and plays an important role in cell identity and differentiation programs and cancer. To get insights into the dynamics of cell differentiation and malignant transformation we have compared the DNA methylation profiles along the mouse small intestine crypt and early stages of tumorigenesis. Genome-scale analysis of DNA methylation together with microarray gene expression have been applied to compare intestinal crypt stem cells (EphB2high), differentiated cells (EphB2negative), ApcMin/+ adenomas and the corresponding non-tumor adjacent tissue, together with small and large intestine samples and the colon cancer cell line CT26. Compared with late stages, small intestine crypt differentiation and early stages of tumorigenesis display few and relatively small changes in DNA methylation. Hypermethylated loci are largely shared by the two processes and affect the proximities of promoter and enhancer regions, with enrichment in genes associated with the intestinal stem cell signature and the PRC2 complex. The hypermethylation is progressive, with minute levels in differentiated cells, as compared with intestinal stem cells, and reaching full methylation in advanced stages. Hypomethylation shows different signatures in differentiation and cancer and is already present in the non-tumor tissue adjacent to the adenomas in ApcMin/+ mice, but at lower levels than advanced cancers. This study provides a reference framework to decipher the mechanisms driving mouse intestinal tumorigenesis and also the human counterpart. PMID:25933092

The Role of Cytosine Methylation on Charge Transport through a DNA Strand

DOE Office of Scientific and Technical Information (OSTI.GOV)

Qi, Jianqing; Govind, Niranjan; Anantram, M. P.

Cytosine methylation has been found to play a crucial role in various biological processes, including a number of human diseases. The detection of this small modifi-cation remains challenging. In this work, we computationally explore the possibility of detecting methylated DNA strands through direct electrical conductance measurements. Using density functional theory and the Landauer-Buttiker method, we study the electronic properties and charge transport through an eight base-pair methylated DNA strand and its native counterpart. Specifically, we compare the results generated with the widely used B3LYP exchange-correlation (XC) functional and CAM-B3LYP based tuned range-separated hybrid density functional. We first analyze the effectmore » of cytosine methylation on the tight-binding parameters of two DNA strands and then model the transmission of the electrons and conductance through the strands both with and without decoherence. We find that with both functionals, the main difference of the tight-binding parameters between the native DNA and the methylated DNA lies in the on-site energies of (methylated) cytosine bases. The intra- and interstrand hopping integrals between two nearest neighboring guanine base and (methylated) cytosine base also change with the addition of the methyl groups. Our calculations show that in the phase-coherent limit, the transmission of the methylated strand is close to the native strand when the energy is nearby the highest occupied molecular orbital (HOMO) level and larger than the native strand by 5 times in the bandgap. The trend in transmission also holds in the presence of the decoherence with both functionals. We also study the effect of contact coupling by choosing coupling strengths ranging from weak to strong coupling limit. Our results suggest that the effect of the two different functionals is to alter the on-site energies of the DNA bases at the HOMO level, while the transport properties don't depend much on the two functionals.« less

Preterm Birth and Its Long-Term Effects: Methylation to Mechanisms

PubMed Central

Parets, Sasha E.; Bedient, Carrie E.; Menon, Ramkumar; Smith, Alicia K.

2014-01-01

The epigenetic patterns established during development may influence gene expression over a lifetime and increase susceptibility to chronic disease. Being born preterm (<37 weeks of gestation) is associated with increased risk mortality and morbidity from birth until adulthood. This brief review explores the potential role of DNA methylation in preterm birth (PTB) and its possible long-term consequences and provides an overview of the physiological processes central to PTB and recent DNA methylation studies of PTB. PMID:25256426

Association of Brain DNA methylation in SORL1, ABCA7, HLA-DRB5, SLC24A4, and BIN1 with pathological diagnosis of Alzheimer disease.

PubMed

Yu, Lei; Chibnik, Lori B; Srivastava, Gyan P; Pochet, Nathalie; Yang, Jingyun; Xu, Jishu; Kozubek, James; Obholzer, Nikolaus; Leurgans, Sue E; Schneider, Julie A; Meissner, Alexander; De Jager, Philip L; Bennett, David A

2015-01-01

Recent large-scale genome-wide association studies have discovered several genetic variants associated with Alzheimer disease (AD); however, the extent to which DNA methylation in these AD loci contributes to the disease susceptibility remains unknown. To examine the association of brain DNA methylation in 28 reported AD loci with AD pathologies. Ongoing community-based clinical pathological cohort studies of aging and dementia (the Religious Orders Study and the Rush Memory and Aging Project) among 740 autopsied participants 66.0 to 108.3 years old. DNA methylation levels at individual CpG sites generated from dorsolateral prefrontal cortex tissue using a bead assay. Pathological diagnosis of AD by National Institute on Aging-Reagan criteria following a standard postmortem examination. Overall, 447 participants (60.4%) met the criteria for pathological diagnosis of AD. Brain DNA methylation in SORL1, ABCA7, HLA-DRB5, SLC24A4, and BIN1 was associated with pathological AD. The association was robustly retained after replacing the binary trait of pathological AD with 2 quantitative and molecular specific hallmarks of AD, namely, Aβ load and paired helical filament tau tangle density. Furthermore, RNA expression of transcripts of SORL1 and ABCA7 was associated with paired helical filament tau tangle density, and the expression of BIN1 was associated with Aβ load. Brain DNA methylation in multiple AD loci is associated with AD pathologies. The results provide further evidence that disruption of DNA methylation is involved in the pathological process of AD.

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

PubMed

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.

Evolution of DNA Methylation across Insects

PubMed Central

Vogel, Kevin J.; Moore, Allen J.; Schmitz, Robert J.

2017-01-01

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. PMID:28025279

Dynamics and biological relevance of DNA demethylation in Arabidopsis antibacterial defense.

PubMed

Yu, Agnès; Lepère, Gersende; Jay, Florence; Wang, Jingyu; Bapaume, Laure; Wang, Yu; Abraham, Anne-Laure; Penterman, Jon; Fischer, Robert L; Voinnet, Olivier; Navarro, Lionel

2013-02-05

DNA methylation is an epigenetic mark that silences transposable elements (TEs) and repeats. Whereas the establishment and maintenance of DNA methylation are relatively well understood, little is known about their dynamics and biological relevance in plant and animal innate immunity. Here, we show that some TEs are demethylated and transcriptionally reactivated during antibacterial defense in Arabidopsis. This effect is correlated with the down-regulation of key transcriptional gene silencing factors and is partly dependent on an active demethylation process. DNA demethylation restricts multiplication and vascular propagation of the bacterial pathogen Pseudomonas syringae in leaves and, accordingly, some immune-response genes, containing repeats in their promoter regions, are negatively regulated by DNA methylation. This study provides evidence that DNA demethylation is part of a plant-induced immune response, potentially acting to prime transcriptional activation of some defense genes linked to TEs/repeats.

GPU-BSM: A GPU-Based Tool to Map Bisulfite-Treated Reads

PubMed Central

Manconi, Andrea; Orro, Alessandro; Manca, Emanuele; Armano, Giuliano; Milanesi, Luciano

2014-01-01

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. PMID:24842718

Association of Tissue-Specific DNA Methylation Alterations with α-Thalassemia Southeast Asian Deletion

PubMed Central

Pangeson, Tanapat; Sanguansermsri, Phanchana; Sanguansermsri, Torpong; Seeratanachot, Teerapat; Suwanakhon, Narutchala; Srikummool, Metawee; Kaewkong, Worasak; Mahingsa, Khwanruedee

2017-01-01

In the wild-type allele, DNA methylation levels of 10 consecutive CpG sites adjacent to the upstream 5′-breakpoint of α-thalassemia Southeast Asian (SEA) deletion are not different between placenta and leukocytes. However, no previous study has reported the map of DNA methylation in the SEA allele. This report aims to show that the SEA mutation is associated with DNA methylation changes, resulting in differential methylation between placenta and leukocytes. Methylation-sensitive high-resolution analysis was used to compare DNA methylation among placenta, leukocytes, and unmethylated control DNA. The result indicates that the DNA methylation between placenta and leukocyte DNA is different and shows that the CpG status of both is not fully unmethylated. Mapping of individual CpG sites was performed by targeted bisulfite sequencing. The DNA methylation level of the 10 consecutive CpG sites was different between placenta and leukocyte DNA. When the 10th CpG of the mutation allele was considered as a hallmark for comparing DNA methylation level, it was totally different from the unmethylated 10th CpG of the wild-type allele. Finally, the distinct DNA methylation patterns between both DNA were extracted. In total, 24 patterns were found in leukocyte samples and 9 patterns were found in placenta samples. This report shows that the large deletion is associated with DNA methylation change. In further studies for clinical application, the distinct DNA methylation pattern might be a potential marker for detecting cell-free fetal DNA. PMID:29162979

DNA methylation in lung tissues of mouse offspring exposed in utero to polycyclic aromatic hydrocarbons.

PubMed

Fish, Trevor J; Benninghoff, Abby D

2017-11-01

Polycyclic aromatic hydrocarbons (PAHs) comprise an important class of environmental pollutants that are known to cause lung cancer in animals and are suspected lung carcinogens in humans. Moreover, evidence from cell-based studies points to PAHs as modulators of the epigenome. The objective of this work was to assess patterns of genome-wide DNA methylation in lung tissues of adult offspring initiated in utero with the transplacental PAH carcinogens dibenzo [def,p]chrysene (DBC) or benzo [a]pyrene (BaP). Genome-wide methylation patterns for normal (not exposed), normal adjacent and lung tumor tissues obtained from adult offspring were determined using methylated DNA immunoprecipitation (MeDIP) with the NimbleGen mouse DNA methylation CpG island array. Lung tumor incidence in 45-week old mice initiated with BaP was 32%, much lower than that of the DBC-exposed offspring at 96%. Also, male offspring appeared more susceptible to BaP as compared to females. Distinct patterns of DNA methylation were associated with non-exposed, normal adjacent and adenocarcinoma lung tissues, as determined by principal components, hierarchical clustering and gene ontology analyses. From these methylation profiles, a set of genes of interest was identified that includes potential important targets for epigenetic modification during the process of lung tumorigenesis in animals exposed to environmental PAHs. Copyright © 2017 Elsevier Ltd. All rights reserved.

MeDReaders: a database for transcription factors that bind to methylated DNA.

PubMed

Wang, Guohua; Luo, Ximei; Wang, Jianan; Wan, Jun; Xia, Shuli; Zhu, Heng; Qian, Jiang; Wang, Yadong

2018-01-04

Understanding the molecular principles governing interactions between transcription factors (TFs) and DNA targets is one of the main subjects for transcriptional regulation. Recently, emerging evidence demonstrated that some TFs could bind to DNA motifs containing highly methylated CpGs both in vitro and in vivo. Identification of such TFs and elucidation of their physiological roles now become an important stepping-stone toward understanding the mechanisms underlying the methylation-mediated biological processes, which have crucial implications for human disease and disease development. Hence, we constructed a database, named as MeDReaders, to collect information about methylated DNA binding activities. A total of 731 TFs, which could bind to methylated DNA sequences, were manually curated in human and mouse studies reported in the literature. In silico approaches were applied to predict methylated and unmethylated motifs of 292 TFs by integrating whole genome bisulfite sequencing (WGBS) and ChIP-Seq datasets in six human cell lines and one mouse cell line extracted from ENCODE and GEO database. MeDReaders database will provide a comprehensive resource for further studies and aid related experiment designs. The database implemented unified access for users to most TFs involved in such methylation-associated binding actives. The website is available at http://medreader.org/. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

SET oncoprotein accumulation regulates transcription through DNA demethylation and histone hypoacetylation.

PubMed

Almeida, Luciana O; Neto, Marinaldo P C; Sousa, Lucas O; Tannous, Maryna A; Curti, Carlos; Leopoldino, Andreia M

2017-04-18

Epigenetic modifications are essential in the control of normal cellular processes and cancer development. DNA methylation and histone acetylation are major epigenetic modifications involved in gene transcription and abnormal events driving the oncogenic process. SET protein accumulates in many cancer types, including head and neck squamous cell carcinoma (HNSCC); SET is a member of the INHAT complex that inhibits gene transcription associating with histones and preventing their acetylation. We explored how SET protein accumulation impacts on the regulation of gene expression, focusing on DNA methylation and histone acetylation. DNA methylation profile of 24 tumour suppressors evidenced that SET accumulation decreased DNA methylation in association with loss of 5-methylcytidine, formation of 5-hydroxymethylcytosine and increased TET1 levels, indicating an active DNA demethylation mechanism. However, the expression of some suppressor genes was lowered in cells with high SET levels, suggesting that loss of methylation is not the main mechanism modulating gene expression. SET accumulation also downregulated the expression of 32 genes of a panel of 84 transcription factors, and SET directly interacted with chromatin at the promoter of the downregulated genes, decreasing histone acetylation. Gene expression analysis after cell treatment with 5-aza-2'-deoxycytidine (5-AZA) and Trichostatin A (TSA) revealed that histone acetylation reversed transcription repression promoted by SET. These results suggest a new function for SET in the regulation of chromatin dynamics. In addition, TSA diminished both SET protein levels and SET capability to bind to gene promoter, suggesting that administration of epigenetic modifier agents could be efficient to reverse SET phenotype in cancer.

Methylated site display (MSD)-AFLP, a sensitive and affordable method for analysis of CpG methylation profiles.

PubMed

Aiba, Toshiki; Saito, Toshiyuki; Hayashi, Akiko; Sato, Shinji; Yunokawa, Harunobu; Maruyama, Toru; Fujibuchi, Wataru; Kurita, Hisaka; Tohyama, Chiharu; Ohsako, Seiichiroh

2017-03-09

It has been pointed out that environmental factors or chemicals can cause diseases that are developmental in origin. To detect abnormal epigenetic alterations in DNA methylation, convenient and cost-effective methods are required for such research, in which multiple samples are processed simultaneously. We here present methylated site display (MSD), a unique technique for the preparation of DNA libraries. By combining it with amplified fragment length polymorphism (AFLP) analysis, we developed a new method, MSD-AFLP. Methylated site display libraries consist of only DNAs derived from DNA fragments that are CpG methylated at the 5' end in the original genomic DNA sample. To test the effectiveness of this method, CpG methylation levels in liver, kidney, and hippocampal tissues of mice were compared to examine if MSD-AFLP can detect subtle differences in the levels of tissue-specific differentially methylated CpGs. As a result, many CpG sites suspected to be tissue-specific differentially methylated were detected. Nucleotide sequences adjacent to these methyl-CpG sites were identified and we determined the methylation level by methylation-sensitive restriction endonuclease (MSRE)-PCR analysis to confirm the accuracy of AFLP analysis. The differences of the methylation level among tissues were almost identical among these methods. By MSD-AFLP analysis, we detected many CpGs showing less than 5% statistically significant tissue-specific difference and less than 10% degree of variability. Additionally, MSD-AFLP analysis could be used to identify CpG methylation sites in other organisms including humans. MSD-AFLP analysis can potentially be used to measure slight changes in CpG methylation level. Regarding the remarkable precision, sensitivity, and throughput of MSD-AFLP analysis studies, this method will be advantageous in a variety of epigenetics-based research.

Minimal evidence for consistent changes in maize DNA methylation patterns following environmental stress.

PubMed

Eichten, Steven R; Springer, Nathan M

2015-01-01

DNA methylation is a chromatin modification that is sometimes associated with epigenetic regulation of gene expression. As DNA methylation can be reversible at some loci, it is possible that methylation patterns may change within an organism that is subjected to environmental stress. In order to assess the effects of abiotic stress on DNA methylation patterns in maize (Zea mays), seeding plants were subjected to heat, cold, and UV stress treatments. Tissue was later collected from individual adult plants that had been subjected to stress or control treatments and used to perform DNA methylation profiling to determine whether there were consistent changes in DNA methylation triggered by specific stress treatments. DNA methylation profiling was performed by immunoprecipitation of methylated DNA followed by microarray hybridization to allow for quantitative estimates of DNA methylation abundance throughout the low-copy portion of the maize genome. By comparing the DNA methylation profiles of each individual plant to the average of the control plants it was possible to identify regions of the genome with variable DNA methylation. However, we did not find evidence of consistent DNA methylation changes resulting from the stress treatments used in this study. Instead, the data suggest that there is a low-rate of stochastic variation that is present in both control and stressed plants.

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

PubMed

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.

DNA methylation in insects: on the brink of the epigenomic era.

PubMed

Glastad, K M; Hunt, Brendan G; Yi, S V; Goodisman, M A D

2011-10-01

DNA methylation plays an important role in gene regulation in animals. However, the evolution and function of DNA methylation has only recently emerged as the subject of widespread study in insects. In this review we profile the known distribution of DNA methylation systems across insect taxa and synthesize functional inferences from studies of DNA methylation in insects and vertebrates. Unlike vertebrate genomes, which tend to be globally methylated, DNA methylation is primarily targeted to genes in insects. Nevertheless, mounting evidence suggests that a specialized role exists for genic methylation in the regulation of transcription, and possibly mRNA splicing, in both insects and mammals. Investigations in several insect taxa further reveal that DNA methylation is preferentially targeted to ubiquitously expressed genes and may play a key role in the regulation of phenotypic plasticity. We suggest that insects are particularly amenable to advancing our understanding of the biological functions of DNA methylation, because insects are evolutionarily diverse, display several lineage-specific losses of DNA methylation and possess tractable patterns of DNA methylation in moderately sized genomes. © 2011 The Authors. Insect Molecular Biology © 2011 The Royal Entomological Society.

A polycomb-mediated epigenetic field defect precedes invasive cervical carcinoma

PubMed Central

Wijetunga, Neil Ari; Ben-Dayan, Miriam; Tozour, Jessica; Burk, Robert D.; Schlecht, Nicolas F.; Einstein, Mark H.; Greally, John M.

2016-01-01

Human papillomavirus (HPV)-associated cervical carcinoma is preceded by stages of cervical intra-epithelial neoplasia (CIN) that can variably progress to malignancy. Understanding the different molecular processes involved in the progression of pre-malignant CIN is critical to the development of improved predictive and interventional capabilities. We tested the role of regulators of transcription in both the development and the progression of HPV-associated CIN, performing the most comprehensive genomic survey to date of DNA methylation in HPV-associated cervical neoplasia, testing ~2 million loci throughout the human genome in biopsies from 78 HPV+ women, identifying changes starting in early CIN and maintained through carcinogenesis. We identified loci at which DNA methylation is consistently altered, beginning early in the course of neoplastic disease and progressing with disease advancement. While the loss of DNA methylation occurs mostly at intergenic regions, acquisition of DNA methylation is at sites involved in transcriptional regulation, with strong enrichment for targets of polycomb repression. Using an independent cohort from The Cancer Genome Atlas, we validated the loci with increased DNA methylation and found that these regulatory changes were associated with locally decreased gene expression. Secondary validation using immunohistochemistry showed that the progression of neoplasia was associated with increasing polycomb protein expression specifically in the cervical epithelium. We find that perturbations of genomic regulatory processes occur early and persist in cervical carcinoma. The results indicate a polycomb-mediated epigenetic field defect in cervical neoplasia that may represent a target for early, topical interventions using polycomb inhibitors. PMID:27557505

A polycomb-mediated epigenetic field defect precedes invasive cervical carcinoma.

PubMed

Wijetunga, Neil Ari; Ben-Dayan, Miriam; Tozour, Jessica; Burk, Robert D; Schlecht, Nicolas F; Einstein, Mark H; Greally, John M

2016-09-20

Human papillomavirus (HPV)-associated cervical carcinoma is preceded by stages of cervical intra-epithelial neoplasia (CIN) that can variably progress to malignancy. Understanding the different molecular processes involved in the progression of pre-malignant CIN is critical to the development of improved predictive and interventional capabilities. We tested the role of regulators of transcription in both the development and the progression of HPV-associated CIN, performing the most comprehensive genomic survey to date of DNA methylation in HPV-associated cervical neoplasia, testing ~2 million loci throughout the human genome in biopsies from 78 HPV+ women, identifying changes starting in early CIN and maintained through carcinogenesis. We identified loci at which DNA methylation is consistently altered, beginning early in the course of neoplastic disease and progressing with disease advancement. While the loss of DNA methylation occurs mostly at intergenic regions, acquisition of DNA methylation is at sites involved in transcriptional regulation, with strong enrichment for targets of polycomb repression. Using an independent cohort from The Cancer Genome Atlas, we validated the loci with increased DNA methylation and found that these regulatory changes were associated with locally decreased gene expression. Secondary validation using immunohistochemistry showed that the progression of neoplasia was associated with increasing polycomb protein expression specifically in the cervical epithelium. We find that perturbations of genomic regulatory processes occur early and persist in cervical carcinoma. The results indicate a polycomb-mediated epigenetic field defect in cervical neoplasia that may represent a target for early, topical interventions using polycomb inhibitors.

Assessment of global and gene-specific DNA methylation in rat liver and kidney in response to non-genotoxic carcinogen exposure

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ozden, Sibel, E-mail: stopuz@istanbul.edu.tr; Turgut Kara, Neslihan; Sezerman, Osman Ugur

Altered expression of tumor suppressor genes and oncogenes, which is regulated in part at the level of DNA methylation, is an important event involved in non-genotoxic carcinogenesis. This may serve as a marker for early detection of non-genotoxic carcinogens. Therefore, we evaluated the effects of non-genotoxic hepatocarcinogens, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), hexachlorobenzene (HCB), methapyrilene (MPY) and male rat kidney carcinogens, d-limonene, p-dichlorobenzene (DCB), chloroform and ochratoxin A (OTA) on global and CpG island promoter methylation in their respective target tissues in rats. No significant dose-related effects on global DNA hypomethylation were observed in tissues of rats compared to vehicle controls using LC–MS/MSmore » in response to short-term non-genotoxic carcinogen exposure. Initial experiments investigating gene-specific methylation using methylation-specific PCR and bisulfite sequencing, revealed partial methylation of p16 in the liver of rats treated with HCB and TCDD. However, no treatment related effects on the methylation status of Cx32, e-cadherin, VHL, c-myc, Igfbp2, and p15 were observed. We therefore applied genome-wide DNA methylation analysis using methylated DNA immunoprecipitation combined with microarrays to identify alterations in gene-specific methylation. Under the conditions of our study, some genes were differentially methylated in response to MPY and TCDD, whereas d-limonene, DCB and chloroform did not induce any methylation changes. 90-day OTA treatment revealed enrichment of several categories of genes important in protein kinase activity and mTOR cell signaling process which are related to OTA nephrocarcinogenicity. - Highlights: • Studied non-genotoxic carcinogens caused no change on global DNA hypomethylation. • d-Limonene, DCB and chloroform did not show any genome-wide methylation changes. • Some genes were differentially methylated in response to MPY, TCDD and OTA. • Protein kinase activity and mTOR cell signaling are involved in OTA carcinogenicity. • Our data highlight Cpne4 may be a potential biomarker for non-genotoxic carcinogens.« less

Evolution of DNA Methylation across Insects.

PubMed

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.

DNA modifications in models of alcohol use disorders.

PubMed

Tulisiak, Christopher T; Harris, R Adron; Ponomarev, Igor

2017-05-01

Chronic alcohol use and abuse result in widespread changes to gene expression, some of which contribute to the development of alcohol-use disorders (AUD). Gene expression is controlled, in part, by a group of regulatory systems often referred to as epigenetic factors, which includes, among other mechanisms, chemical marks made on the histone proteins around which genomic DNA is wound to form chromatin, and on nucleotides of the DNA itself. In particular, alcohol has been shown to perturb the epigenetic machinery, leading to changes in gene expression and cellular functions characteristic of AUD and, ultimately, to altered behavior. DNA modifications in particular are seeing increasing research in the context of alcohol use and abuse. To date, studies of DNA modifications in AUD have primarily looked at global methylation profiles in human brain and blood, gene-specific methylation profiles in animal models, methylation changes associated with prenatal ethanol exposure, and the potential therapeutic abilities of DNA methyltransferase inhibitors. Future studies may be aimed at identifying changes to more recently discovered DNA modifications, utilizing new methods to discriminate methylation profiles between cell types, thus clarifying how alcohol influences the methylomes of cell-type populations and how this may affect downstream processes. These studies and more in-depth probing of DNA methylation will be key to determining whether DNA-level epigenetic regulation plays a causative role in AUD and can thus be targeted for treatment of the disorder. Copyright © 2016 Elsevier Inc. All rights reserved.

Dimorphic DNA methylation during temperature-dependent sex determination in the sea turtle Lepidochelys olivacea.

PubMed

Venegas, Daniela; Marmolejo-Valencia, Alejandro; Valdes-Quezada, Christian; Govenzensky, Tzipe; Recillas-Targa, Félix; Merchant-Larios, Horacio

2016-09-15

Sex determination in vertebrates depends on the expression of a conserved network of genes. Sea turtles such as Lepidochelys olivacea have temperature-dependent sex determination. The present work analyses some of the epigenetic processes involved in this. We describe sexual dimorphism in global DNA methylation patterns between ovaries and testes of L. olivacea and show that the differences may arise from a combination of DNA methylation and demethylation events that occur during sex determination. Irrespective of incubation temperature, 5-hydroxymethylcytosine was abundant in the bipotential gonad; however, following sex determination, this modification was no longer found in pre-Sertoli cells in the testes. These changes correlate with the establishment of the sexually dimorphic DNA methylation patterns, down regulation of Sox9 gene expression in ovaries and irreversible gonadal commitment towards a male or female differentiation pathway. Thus, DNA methylation changes may be necessary for the stabilization of the gene expression networks that drive the differentiation of the bipotential gonad to form either an ovary or a testis in L. olivacea and probably among other species that manifest temperature-dependent sex determination. Copyright © 2016 Elsevier Inc. All rights reserved.

Epigenomics of Total Acute Sleep Deprivation in Relation to Genome-Wide DNA Methylation Profiles and RNA Expression.

PubMed

Nilsson, Emil K; Boström, Adrian E; Mwinyi, Jessica; Schiöth, Helgi B

2016-06-01

Despite an established link between sleep deprivation and epigenetic processes in humans, it remains unclear to what extent sleep deprivation modulates DNA methylation. We performed a within-subject randomized blinded study with 16 healthy subjects to examine the effect of one night of total sleep deprivation (TSD) on the genome-wide methylation profile in blood compared with that in normal sleep. Genome-wide differences in methylation between both conditions were assessed by applying a paired regression model that corrected for monocyte subpopulations. In addition, the correlations between the methylation of genes detected to be modulated by TSD and gene expression were examined in a separate, publicly available cohort of 10 healthy male donors (E-GEOD-49065). Sleep deprivation significantly affected the DNA methylation profile both independently and in dependency of shifts in monocyte composition. Our study detected differential methylation of 269 probes. Notably, one CpG site was located 69 bp upstream of ING5, which has been shown to be differentially expressed after sleep deprivation. Gene set enrichment analysis detected the Notch and Wnt signaling pathways to be enriched among the differentially methylated genes. These results provide evidence that total acute sleep deprivation alters the methylation profile in healthy human subjects. This is, to our knowledge, the first study that systematically investigated the impact of total acute sleep deprivation on genome-wide DNA methylation profiles in blood and related the epigenomic findings to the expression data.

Looking beyond the DNA sequence: the relevance of DNA methylation processes for the stress-diathesis model of depression.

PubMed

Booij, Linda; Wang, Dongsha; Lévesque, Mélissa L; Tremblay, Richard E; Szyf, Moshe

2013-01-01

The functioning of the hypothalamic-pituitary-adrenal (HPA) axis and serotonergic (5-HT) system are known to be intertwined with mood. Alterations in these systems are often associated with depression. However, neither are sufficient to cause depression in and of themselves. It is now becoming increasingly clear that the environment plays a crucial role, particularly, the perinatal environment. In this review, we posit that early environmental stress triggers a series of epigenetic mechanisms that adapt the genome and programme the HPA axis and 5-HT system for survival in a harsh environment. We focus on DNA methylation as it is the most stable epigenetic mark. Given that DNA methylation patterns are in large part set within the perinatal period, long-term gene expression programming by DNA methylation is especially vulnerable to environmental insults during this period. We discuss specific examples of genes in the 5-HT system (serotonin transporter) and HPA axis (glucocorticoid receptor and arginine vasopressin enhancer) whose DNA methylation state is associated with early life experience and may potentially lead to depression vulnerability. We conclude with a discussion on the relevance of studying epigenetic mechanisms in peripheral tissue as a proxy for those occurring in the human brain and suggest avenues for future research.

Looking beyond the DNA sequence: the relevance of DNA methylation processes for the stress–diathesis model of depression

PubMed Central

Booij, Linda; Wang, Dongsha; Lévesque, Mélissa L.; Tremblay, Richard E.; Szyf, Moshe

2013-01-01

The functioning of the hypothalamic–pituitary–adrenal (HPA) axis and serotonergic (5-HT) system are known to be intertwined with mood. Alterations in these systems are often associated with depression. However, neither are sufficient to cause depression in and of themselves. It is now becoming increasingly clear that the environment plays a crucial role, particularly, the perinatal environment. In this review, we posit that early environmental stress triggers a series of epigenetic mechanisms that adapt the genome and programme the HPA axis and 5-HT system for survival in a harsh environment. We focus on DNA methylation as it is the most stable epigenetic mark. Given that DNA methylation patterns are in large part set within the perinatal period, long-term gene expression programming by DNA methylation is especially vulnerable to environmental insults during this period. We discuss specific examples of genes in the 5-HT system (serotonin transporter) and HPA axis (glucocorticoid receptor and arginine vasopressin enhancer) whose DNA methylation state is associated with early life experience and may potentially lead to depression vulnerability. We conclude with a discussion on the relevance of studying epigenetic mechanisms in peripheral tissue as a proxy for those occurring in the human brain and suggest avenues for future research. PMID:23440465

ARSENITE EXPOSURE CAUSES BOTH HYPOMETHYLATION AND HYPERMETHYLATION IN HUMAN CELL LINES IN CULTURE AT LOW CONCENTRATIONS

EPA Science Inventory

We and others have hypothesized that a mechanism of arsenic carcinogenesis could involve alteration of DNA methylation since this process utilizes a methyltransferase and consumes S-adenosylmethionine (SAM) as the methyl donor. We analyzed differentially methylated regions of ge...

Association between fetal exposure to phthalate endocrine disruptor and genome-wide DNA methylation at birth.

PubMed

Chen, Chung-Hsing; Jiang, Shih Sheng; Chang, I-Shou; Wen, Hui-Ju; Sun, Chien-Wen; Wang, Shu-Li

2018-04-01

Phthalic acid esters are ubiquitous and antiandrogenic, and may cause systemic effects in humans, particularly with in utero exposure. Epigenetic modification, such as DNA methylation, has been hypothesized to be an important mechanism that mediates certain biological processes and pathogenic effects of in utero phthalate exposure. The aim of this study was to examine the association between genome-wide DNA methylation at birth and prenatal exposure to phthalate. We studied 64 infant-mother pairs included in TMICS (Taiwan Maternal and Infant Cohort Study), a long-term follow-up birth cohort from the general population. DNA methylation levels at more than 450,000 CpG sites were measured in cord blood samples using Illumina Infinium HumanMethylation450 BeadChips. The concentrations of three metabolites of di-(2-ethylhexyl) phthalate (DEHP) were measured using liquid chromatography tandem-mass spectrometry (LC-MS/MS) in urine samples collected from the pregnant women during 28-36 weeks gestation. We identified 25 CpG sites whose methylation levels in cord blood were significantly correlated with prenatal DEHP exposure using a false discovery rate (FDR) of 5% (q-value < 0.05). Via gene-set enrichment analysis (GSEA), we also found that there was significant enrichment of genes involved in the androgen response, estrogen response, and spermatogenesis within those genes showing DNA methylation changes in response to exposure. Specifically, PA2G4, HMGCR, and XRCC6 genes were involved in genes in response to androgen. Phthalate exposure in utero may cause significant alterations in the DNA methylation in cord blood. These changes in DNA methylation might serve as biomarkers of maternal exposure to phthalate in infancy and potential candidates for studying mechanisms via which phthalate may impact on health in later life. Future investigations are warranted. Copyright © 2018 Elsevier Inc. All rights reserved.

Genetic and DNA Methylation Changes in Cotton (Gossypium) Genotypes and Tissues

PubMed Central

Osabe, Kenji; Clement, Jenny D.; Bedon, Frank; Pettolino, Filomena A.; Ziolkowski, Lisa; Llewellyn, Danny J.; Finnegan, E. Jean; Wilson, Iain W.

2014-01-01

In plants, epigenetic regulation is important in normal development and in modulating some agronomic traits. The potential contribution of DNA methylation mediated gene regulation to phenotypic diversity and development in cotton was investigated between cotton genotypes and various tissues. DNA methylation diversity, genetic diversity, and changes in methylation context were investigated using methylation-sensitive amplified polymorphism (MSAP) assays including a methylation insensitive enzyme (BsiSI), and the total DNA methylation level was measured by high-performance liquid chromatography (HPLC). DNA methylation diversity was greater than the genetic diversity in the selected cotton genotypes and significantly different levels of DNA methylation were identified between tissues, including fibre. The higher DNA methylation diversity (CHG methylation being more diverse than CG methylation) in cotton genotypes suggest epigenetic regulation may be important for cotton, and the change in DNA methylation between fibre and other tissues hints that some genes may be epigenetically regulated for fibre development. The novel approach using BsiSI allowed direct comparison between genetic and epigenetic diversity, and also measured CC methylation level that cannot be detected by conventional MSAP. PMID:24465864

Genetic and DNA methylation changes in cotton (Gossypium) genotypes and tissues.

PubMed

Osabe, Kenji; Clement, Jenny D; Bedon, Frank; Pettolino, Filomena A; Ziolkowski, Lisa; Llewellyn, Danny J; Finnegan, E Jean; Wilson, Iain W

2014-01-01

In plants, epigenetic regulation is important in normal development and in modulating some agronomic traits. The potential contribution of DNA methylation mediated gene regulation to phenotypic diversity and development in cotton was investigated between cotton genotypes and various tissues. DNA methylation diversity, genetic diversity, and changes in methylation context were investigated using methylation-sensitive amplified polymorphism (MSAP) assays including a methylation insensitive enzyme (BsiSI), and the total DNA methylation level was measured by high-performance liquid chromatography (HPLC). DNA methylation diversity was greater than the genetic diversity in the selected cotton genotypes and significantly different levels of DNA methylation were identified between tissues, including fibre. The higher DNA methylation diversity (CHG methylation being more diverse than CG methylation) in cotton genotypes suggest epigenetic regulation may be important for cotton, and the change in DNA methylation between fibre and other tissues hints that some genes may be epigenetically regulated for fibre development. The novel approach using BsiSI allowed direct comparison between genetic and epigenetic diversity, and also measured CC methylation level that cannot be detected by conventional MSAP.

A simple modification to the luminometric methylation assay to control for the effects of DNA fragmentation.

PubMed

Duman, Elif Aysimi; Kriaucionis, Skirmantas; Dunn, John J; Hatchwell, Eli

2015-05-01

Variations in DNA methylation have been implicated in a number of disorders. Changes in global DNA methylation levels have long been associated with various types of cancer. One of the recently described methods for determining global DNA methylation levels is the LUminometric Methylation Assay (LUMA), which utilizes methylation sensitive and insensitive restriction endonucleases and pyrosequencing technology for quantification. Here we provide evidence suggesting that the global methylation level reported by LUMA is affected by the integrity of the DNA being analyzed. The less intact the DNA, the lower the global methylation levels reported by LUMA. In order to overcome this problem, we propose the use of undigested DNA alongside digested samples. Finally, we demonstrate that this results in a more accurate assessment of global DNA methylation levels.

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

PubMed

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.

Methylation Status of the Follistatin Gene at Different Development Stages of Japanese Flounder (Paralichthys olivaceus)

NASA Astrophysics Data System (ADS)

Huang, Yajuan; Hu, Nan; Si, Yufeng; Li, Siping; Wu, Shuxian; Zhang, Meizhao; Wen, Haishen; Li, Jifang; Li, Yun; He, Feng

2018-06-01

Follistatin (Fst) is a hyperplasia factor that plays a crucial role in muscle development. DNA methylation, a significant process, regulates gene expression. The aim of our study is to examine the DNA methylation and expression patterns of Fst gene at five different development stages of Japanese flounder (stage A, 7 dph; stage B, 90 dph; stage C, about 180 dph; stage D, about 24 months; stage E, about 36 months). The muscle tissue of Japanese flounder was obtained at different development stages in this experiment. DNA methylation levels in the promoter and exon 2 of Fst were determined by bisulfite sequencing, and the relative expression of the Fst gene at the five stages was measured by quantitative PCR. The results showed that the lowest methylation level was at stage A and the highest methylation level was at stage B. Moreover, the highest expression level of the Fst gene was observed at stage A. The mRNA abundance was negatively correlated with DNA methylation level. Three CpG islands in the promoter region and three CpG islands in exon 2 of Fst were found in the binding sequence of the putative transcription factor. These results offered a theoretical basis for the mechanism of Fst gene regulation to muscle development at different development stages.

Epigenetics and colorectal cancer pathogenesis.

PubMed

Bardhan, Kankana; Liu, Kebin

2013-06-05

Colorectal cancer (CRC) develops through a multistage process that results from the progressive accumulation of genetic mutations, and frequently as a result of mutations in the Wnt signaling pathway. However, it has become evident over the past two decades that epigenetic alterations of the chromatin, particularly the chromatin components in the promoter regions of tumor suppressors and oncogenes, play key roles in CRC pathogenesis. Epigenetic regulation is organized at multiple levels, involving primarily DNA methylation and selective histone modifications in cancer cells. Assessment of the CRC epigenome has revealed that virtually all CRCs have aberrantly methylated genes and that the average CRC methylome has thousands of abnormally methylated genes. Although relatively less is known about the patterns of specific histone modifications in CRC, selective histone modifications and resultant chromatin conformation have been shown to act, in concert with DNA methylation, to regulate gene expression to mediate CRC pathogenesis. Moreover, it is now clear that not only DNA methylation but also histone modifications are reversible processes. The increased understanding of epigenetic regulation of gene expression in the context of CRC pathogenesis has led to development of epigenetic biomarkers for CRC diagnosis and epigenetic drugs for CRC therapy.

Epigenetics and Colorectal Cancer Pathogenesis

PubMed Central

Bardhan, Kankana; Liu, Kebin

2013-01-01

Colorectal cancer (CRC) develops through a multistage process that results from the progressive accumulation of genetic mutations, and frequently as a result of mutations in the Wnt signaling pathway. However, it has become evident over the past two decades that epigenetic alterations of the chromatin, particularly the chromatin components in the promoter regions of tumor suppressors and oncogenes, play key roles in CRC pathogenesis. Epigenetic regulation is organized at multiple levels, involving primarily DNA methylation and selective histone modifications in cancer cells. Assessment of the CRC epigenome has revealed that virtually all CRCs have aberrantly methylated genes and that the average CRC methylome has thousands of abnormally methylated genes. Although relatively less is known about the patterns of specific histone modifications in CRC, selective histone modifications and resultant chromatin conformation have been shown to act, in concert with DNA methylation, to regulate gene expression to mediate CRC pathogenesis. Moreover, it is now clear that not only DNA methylation but also histone modifications are reversible processes. The increased understanding of epigenetic regulation of gene expression in the context of CRC pathogenesis has led to development of epigenetic biomarkers for CRC diagnosis and epigenetic drugs for CRC therapy. PMID:24216997

The methylation of nuclear and mitochondrial DNA in ageing phenotypes and longevity.

PubMed

Bacalini, Maria Giulia; D'Aquila, Patrizia; Marasco, Elena; Nardini, Christine; Montesanto, Alberto; Franceschi, Claudio; Passarino, Giuseppe; Garagnani, Paolo; Bellizzi, Dina

2017-07-01

An increasing body of data is progressively indicating that the comprehension of the epigenetic landscape, actively integrated with the genetic elements, is crucial to delineate the molecular basis of the inter-individual complexity of ageing process. Indeed, it has emerged that DNA methylation changes occur during ageing, consisting mainly in a progressive process of genome demethylation, in a hypermethylation of gene-specific CpG dinucleotides, as well as in an inter-individual divergence of the epigenome due to stochastic events and environmental exposures throughout life, namely as epigenetic drift. Additionally, it has also come to light an implication of the mitochondrial genome in the regulation of the intracellular epigenetic landscape, as demonstrated by the being itself object of epigenetic modifications. An overview of DNA methylation changes occurring during ageing process at both nuclear and mitochondrial level will be described in this review, also taking into account the recent and promising data available on the 5-hydroxymethylcytosine. Copyright © 2017 Elsevier B.V. All rights reserved.

Regional differences in mitochondrial DNA methylation in human post-mortem brain tissue.

PubMed

Devall, Matthew; Smith, Rebecca G; Jeffries, Aaron; Hannon, Eilis; Davies, Matthew N; Schalkwyk, Leonard; Mill, Jonathan; Weedon, Michael; Lunnon, Katie

2017-01-01

DNA methylation is an important epigenetic mechanism involved in gene regulation, with alterations in DNA methylation in the nuclear genome being linked to numerous complex diseases. Mitochondrial DNA methylation is a phenomenon that is receiving ever-increasing interest, particularly in diseases characterized by mitochondrial dysfunction; however, most studies have been limited to the investigation of specific target regions. Analyses spanning the entire mitochondrial genome have been limited, potentially due to the amount of input DNA required. Further, mitochondrial genetic studies have been previously confounded by nuclear-mitochondrial pseudogenes. Methylated DNA Immunoprecipitation Sequencing is a technique widely used to profile DNA methylation across the nuclear genome; however, reads mapped to mitochondrial DNA are often discarded. Here, we have developed an approach to control for nuclear-mitochondrial pseudogenes within Methylated DNA Immunoprecipitation Sequencing data. We highlight the utility of this approach in identifying differences in mitochondrial DNA methylation across regions of the human brain and pre-mortem blood. We were able to correlate mitochondrial DNA methylation patterns between the cortex, cerebellum and blood. We identified 74 nominally significant differentially methylated regions ( p  < 0.05) in the mitochondrial genome, between anatomically separate cortical regions and the cerebellum in matched samples ( N  = 3 matched donors). Further analysis identified eight significant differentially methylated regions between the total cortex and cerebellum after correcting for multiple testing. Using unsupervised hierarchical clustering analysis of the mitochondrial DNA methylome, we were able to identify tissue-specific patterns of mitochondrial DNA methylation between blood, cerebellum and cortex. Our study represents a comprehensive analysis of the mitochondrial methylome using pre-existing Methylated DNA Immunoprecipitation Sequencing data to identify brain region-specific patterns of mitochondrial DNA methylation.

Blood as a surrogate marker for tissue-specific DNA methylation and changes due to folate depletion in post-partum female mice.

PubMed

McKay, Jill A; Xie, Long; Harris, Sarah; Wong, Yi K; Ford, Dianne; Mathers, John C

2011-07-01

DNA methylation patterns are tissue specific and may influence tissue-specific gene regulation. Human studies investigating DNA methylation in relation to environmental factors primarily use blood-derived DNA as a surrogate for DNA from target tissues. It is therefore important to know if DNA methylation changes in blood in response to environmental changes reflect those in target tissues. Folate intake can influence DNA methylation, via altered methyl donor supply. Previously, manipulations of maternal folate intake during pregnancy altered the patterns of DNA methylation in offspring but, to our knowledge, the consequences for maternal DNA methylation are unknown. Given the increased requirement for folate during pregnancy, mothers may be susceptible to aberrant DNA methylation due to folate depletion. Female mice were fed folate-adequate (2 mg folic acid/kg diet) or folate-deplete (0.4 mg folic acid/kg diet) diets prior to mating and during pregnancy and lactation. Following weaning, dams were killed and DNA methylation was assessed by pyrosequencing® in blood, liver, and kidney at the Esr1, Igf2 differentially methylated region (DMR)1, Igf2 DMR2, Slc39a4CGI1, and Slc39a4CGI2 loci. We observed tissue-specific differences in methylation at all loci. Folate depletion reduced Igf2 DMR1 and Slc39a4CGI1 methylation across all tissues and altered Igf2 DMR2 methylation in a tissue-specific manner (p<0.05). Blood-derived DNA methylation measurements may not always reflect methylation within other tissues. Further measurements of blood-derived and tissue-specific methylation patterns are warranted to understand the complexity of tissue-specific responses to altered nutritional exposure. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Prenatal maternal immune activation causes epigenetic differences in adolescent mouse brain

PubMed Central

Basil, P; Li, Q; Dempster, E L; Mill, J; Sham, P-C; Wong, C C Y; McAlonan, G M

2014-01-01

Epigenetic processes such as DNA methylation have been implicated in the pathophysiology of neurodevelopmental disorders including schizophrenia and autism. Epigenetic changes can be induced by environmental exposures such as inflammation. Here we tested the hypothesis that prenatal inflammation, a recognized risk factor for schizophrenia and related neurodevelopmental conditions, alters DNA methylation in key brain regions linked to schizophrenia, namely the dopamine rich striatum and endocrine regulatory centre, the hypothalamus. DNA methylation across highly repetitive elements (long interspersed element 1 (LINE1) and intracisternal A-particles (IAPs)) were used to proxy global DNA methylation. We also investigated the Mecp2 gene because it regulates transcription of LINE1 and has a known association with neurodevelopmental disorders. Brain tissue was harvested from 6 week old offspring of mice exposed to the viral analog PolyI:C or saline on gestation day 9. We used Sequenom EpiTYPER assay to quantitatively analyze differences in DNA methylation at IAPs, LINE1 elements and the promoter region of Mecp2. In the hypothalamus, prenatal exposure to PolyI:C caused significant global DNA hypomethylation (t=2.44, P=0.019, PolyI:C mean 69.67%, saline mean 70.19%), especially in females, and significant hypomethylation of the promoter region of Mecp2, (t=3.32, P=0.002; PolyI:C mean 26.57%, saline mean 34.63%). IAP methylation was unaltered. DNA methylation in the striatum was not significantly altered. This study provides the first experimental evidence that exposure to inflammation during prenatal life is associated with epigenetic changes, including Mecp2 promoter hypomethylation. This suggests that environmental and genetic risk factors associated with neurodevelopmental disorders may act upon similar pathways. This is important because epigenetic changes are potentially modifiable and their investigation may open new avenues for treatment. PMID:25180573

A Novel Computational Method for Detecting DNA Methylation Sites with DNA Sequence Information and Physicochemical Properties.

PubMed

Pan, Gaofeng; Jiang, Limin; Tang, Jijun; Guo, Fei

2018-02-08

DNA methylation is an important biochemical process, and it has a close connection with many types of cancer. Research about DNA methylation can help us to understand the regulation mechanism and epigenetic reprogramming. Therefore, it becomes very important to recognize the methylation sites in the DNA sequence. In the past several decades, many computational methods-especially machine learning methods-have been developed since the high-throughout sequencing technology became widely used in research and industry. In order to accurately identify whether or not a nucleotide residue is methylated under the specific DNA sequence context, we propose a novel method that overcomes the shortcomings of previous methods for predicting methylation sites. We use k -gram, multivariate mutual information, discrete wavelet transform, and pseudo amino acid composition to extract features, and train a sparse Bayesian learning model to do DNA methylation prediction. Five criteria-area under the receiver operating characteristic curve (AUC), Matthew's correlation coefficient (MCC), accuracy (ACC), sensitivity (SN), and specificity-are used to evaluate the prediction results of our method. On the benchmark dataset, we could reach 0.8632 on AUC, 0.8017 on ACC, 0.5558 on MCC, and 0.7268 on SN. Additionally, the best results on two scBS-seq profiled mouse embryonic stem cells datasets were 0.8896 and 0.9511 by AUC, respectively. When compared with other outstanding methods, our method surpassed them on the accuracy of prediction. The improvement of AUC by our method compared to other methods was at least 0.0399 . For the convenience of other researchers, our code has been uploaded to a file hosting service, and can be downloaded from: https://figshare.com/s/0697b692d802861282d3.

DNA methylation in amphioxus: from ancestral functions to new roles in vertebrates.

PubMed

Albalat, Ricard; Martí-Solans, Josep; Cañestro, Cristian

2012-03-01

In vertebrates, DNA methylation is an epigenetic mechanism that modulates gene transcription, and plays crucial roles during development, cell fate maintenance, germ cell pluripotency and inheritable genome imprinting. DNA methylation might also play a role as a genome defense mechanism against the mutational activity derived from transposon mobility. In contrast to the heavily methylated genomes in vertebrates, most genomes in invertebrates are poorly or just moderately methylated, and the function of DNA methylation remains unclear. Here, we review the DNA methylation system in the cephalochordate amphioxus, which belongs to the most basally divergent group of our own phylum, the chordates. First, surveys of the amphioxus genome database reveal the presence of the DNA methylation machinery, DNA methyltransferases and methyl-CpG-binding domain proteins. Second, comparative genomics and analyses of conserved synteny between amphioxus and vertebrates provide robust evidence that the DNA methylation machinery of amphioxus represents the ancestral toolkit of chordates, and that its expansion in vertebrates was originated by the two rounds of whole-genome duplication that occurred in stem vertebrates. Third, in silico analysis of CpGo/e ratios throughout the amphioxus genome suggests a bimodal distribution of DNA methylation, consistent with a mosaic pattern comprising domains of methylated DNA interspersed with domains of unmethylated DNA, similar to the situation described in ascidians, but radically different to the globally methylated vertebrate genomes. Finally, we discuss potential roles of the DNA methylation system in amphioxus in the context of chordate genome evolution and the origin of vertebrates.

Corruption of the intra-gene DNA methylation architecture is a hallmark of cancer.

PubMed

Bartlett, Thomas E; Zaikin, Alexey; Olhede, Sofia C; West, James; Teschendorff, Andrew E; Widschwendter, Martin

2013-01-01

Epigenetic processes--including DNA methylation--are increasingly seen as having a fundamental role in chronic diseases like cancer. It is well known that methylation levels at particular genes or loci differ between normal and diseased tissue. Here we investigate whether the intra-gene methylation architecture is corrupted in cancer and whether the variability of levels of methylation of individual CpGs within a defined gene is able to discriminate cancerous from normal tissue, and is associated with heterogeneous tumour phenotype, as defined by gene expression. We analysed 270985 CpGs annotated to 18272 genes, in 3284 cancerous and 681 normal samples, corresponding to 14 different cancer types. In doing so, we found novel differences in intra-gene methylation pattern across phenotypes, particularly in those genes which are crucial for stem cell biology; our measures of intra-gene methylation architecture are a better determinant of phenotype than measures based on mean methylation level alone (K-S test [Formula: see text] in all 14 diseases tested). These per-gene methylation measures also represent a considerable reduction in complexity, compared to conventional per-CpG beta-values. Our findings strongly support the view that intra-gene methylation architecture has great clinical potential for the development of DNA-based cancer biomarkers.

Neurospora Importin α Is Required for Normal Heterochromatic Formation and DNA Methylation

PubMed Central

Klocko, Andrew D.; Rountree, Michael R.; Grisafi, Paula L.; Hays, Shan M.; Adhvaryu, Keyur K.; Selker, Eric U.

2015-01-01

Heterochromatin and associated gene silencing processes play roles in development, genome defense, and chromosome function. In many species, constitutive heterochromatin is decorated with histone H3 tri-methylated at lysine 9 (H3K9me3) and cytosine methylation. In Neurospora crassa, a five-protein complex, DCDC, catalyzes H3K9 methylation, which then directs DNA methylation. Here, we identify and characterize a gene important for DCDC function, dim-3 (defective in methylation-3), which encodes the nuclear import chaperone NUP-6 (Importin α). The critical mutation in dim-3 results in a substitution in an ARM repeat of NUP-6 and causes a substantial loss of H3K9me3 and DNA methylation. Surprisingly, nuclear transport of all known proteins involved in histone and DNA methylation, as well as a canonical transport substrate, appear normal in dim-3 strains. Interactions between DCDC members also appear normal, but the nup-6dim-3 allele causes the DCDC members DIM-5 and DIM-7 to mislocalize from heterochromatin and NUP-6dim-3 itself is mislocalized from the nuclear envelope, at least in conidia. GCN-5, a member of the SAGA histone acetyltransferase complex, also shows altered localization in dim-3, raising the possibility that NUP-6 is necessary to localize multiple chromatin complexes following nucleocytoplasmic transport. PMID:25793375

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

PubMed

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 < 0.05). The top pathways enriched among the DMCpGs included developmental processes, immune system regulation, regulation of cell signaling, and small GTPase-mediated signal transduction. The associations between the methylation of selected DMCpGs with childhood 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 < 0.01), while 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.

Current and Emerging Technologies for the Analysis of the Genome-Wide and Locus-Specific DNA Methylation Patterns.

PubMed

Tost, Jörg

2016-01-01

DNA methylation is the most studied epigenetic modification, and altered DNA methylation patterns have been identified in cancer and more recently also in many other complex diseases. Furthermore, DNA methylation is influenced by a variety of environmental factors, and the analysis of DNA methylation patterns might allow deciphering previous exposure. Although a large number of techniques to study DNA methylation either genome-wide or at specific loci have been devised, they all are based on a limited number of principles for differentiating the methylation state, viz., methylation-specific/methylation-dependent restriction enzymes, antibodies or methyl-binding proteins, chemical-based enrichment, or bisulfite conversion. Second-generation sequencing has largely replaced microarrays as readout platform and is also becoming more popular for locus-specific DNA methylation analysis. In this chapter, the currently used methods for both genome-wide and locus-specific analysis of 5-methylcytosine and as its oxidative derivatives, such as 5-hydroxymethylcytosine, are reviewed in detail, and the advantages and limitations of each approach are discussed. Furthermore, emerging technologies avoiding PCR amplification and allowing a direct readout of DNA methylation are summarized, together with novel applications, such as the detection of DNA methylation in single cells or in circulating cell-free DNA.

Regulatory link between DNA methylation and active demethylation in Arabidopsis

PubMed Central

Lei, Mingguang; Zhang, Huiming; Julian, Russell; Tang, Kai; Xie, Shaojun; Zhu, Jian-Kang

2015-01-01

De novo DNA methylation through the RNA-directed DNA methylation (RdDM) pathway and active DNA demethylation play important roles in controlling genome-wide DNA methylation patterns in plants. Little is known about how cells manage the balance between DNA methylation and active demethylation activities. Here, we report the identification of a unique RdDM target sequence, where DNA methylation is required for maintaining proper active DNA demethylation of the Arabidopsis genome. In a genetic screen for cellular antisilencing factors, we isolated several REPRESSOR OF SILENCING 1 (ros1) mutant alleles, as well as many RdDM mutants, which showed drastically reduced ROS1 gene expression and, consequently, transcriptional silencing of two reporter genes. A helitron transposon element (TE) in the ROS1 gene promoter negatively controls ROS1 expression, whereas DNA methylation of an RdDM target sequence between ROS1 5′ UTR and the promoter TE region antagonizes this helitron TE in regulating ROS1 expression. This RdDM target sequence is also targeted by ROS1, and defective DNA demethylation in loss-of-function ros1 mutant alleles causes DNA hypermethylation of this sequence and concomitantly causes increased ROS1 expression. Our results suggest that this sequence in the ROS1 promoter region serves as a DNA methylation monitoring sequence (MEMS) that senses DNA methylation and active DNA demethylation activities. Therefore, the ROS1 promoter functions like a thermostat (i.e., methylstat) to sense DNA methylation levels and regulates DNA methylation by controlling ROS1 expression. PMID:25733903

Profile analysis and prediction of tissue-specific CpG island methylation classes

PubMed Central

2009-01-01

Background The computational prediction of DNA methylation has become an important topic in the recent years due to its role in the epigenetic control of normal and cancer-related processes. While previous prediction approaches focused merely on differences between methylated and unmethylated DNA sequences, recent experimental results have shown the presence of much more complex patterns of methylation across tissues and time in the human genome. These patterns are only partially described by a binary model of DNA methylation. In this work we propose a novel approach, based on profile analysis of tissue-specific methylation that uncovers significant differences in the sequences of CpG islands (CGIs) that predispose them to a tissue- specific methylation pattern. Results We defined CGI methylation profiles that separate not only between constitutively methylated and unmethylated CGIs, but also identify CGIs showing a differential degree of methylation across tissues and cell-types or a lack of methylation exclusively in sperm. These profiles are clearly distinguished by a number of CGI attributes including their evolutionary conservation, their significance, as well as the evolutionary evidence of prior methylation. Additionally, we assess profile functionality with respect to the different compartments of protein coding genes and their possible use in the prediction of DNA methylation. Conclusion Our approach provides new insights into the biological features that determine if a CGI has a functional role in the epigenetic control of gene expression and the features associated with CGI methylation susceptibility. Moreover, we show that the ability to predict CGI methylation is based primarily on the quality of the biological information used and the relationships uncovered between different sources of knowledge. The strategy presented here is able to predict, besides the constitutively methylated and unmethylated classes, two more tissue specific methylation classes conserving the accuracy provided by leading binary methylation classification methods. PMID:19383127

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

PubMed Central

Nwaobi, Sinifunanya E.; Olsen, Michelle L.

2015-01-01

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. PMID:26436772

Global Proteomic Analysis in Trypanosomes Reveals Unique Proteins and Conserved Cellular Processes Impacted by Arginine Methylation

PubMed Central

Lott, Kaylen; Li, Jun; Fisk, John C.; Wang, Hao; Aletta, John M.; Qu, Jun; Read, Laurie K.

2013-01-01

Arginine methylation is a common posttranslational modification with reported functions in transcription, RNA processing and translation, and DNA repair. Trypanosomes encode five protein arginine methyltransferases, suggesting that arginine methylation exerts widespread impacts on the biology of these organisms. Here, we performed a global proteomic analysis of T. brucei to identify arginine methylated proteins and their sites of modification. Using an approach entailing two-dimensional chromatographic separation, and alternating electron transfer dissociation and collision induced dissociation, we identified 1332 methylarginines in 676 proteins. The resulting data set represents the largest compilation of arginine methylated proteins in any organism to date. Functional classification revealed numerous arginine methylated proteins involved in flagellar function, RNA metabolism, DNA replication and repair, and intracellular protein trafficking. Thus, arginine methylation has the potential to impact aspects of T. brucei gene expression, cell biology, and pathogenesis. Interestingly, pathways with known methylated proteins in higher eukaryotes were identified in this study, but often different components of the pathway were methylated in trypanosomes. Methylarginines were often identified in glycine rich contexts, although exceptions to this rule were detected. Collectively, these data inform on a multitude of aspects of trypanosome biology and serve as a guide for the identification of homologous arginine methylated proteins in higher eukaryotes. PMID:23872088

Two-step epigenetic Mendelian randomization: a strategy for establishing the causal role of epigenetic processes in pathways to disease

PubMed Central

Relton, Caroline L; Davey Smith, George

2012-01-01

The burgeoning interest in the field of epigenetics has precipitated the need to develop approaches to strengthen causal inference when considering the role of epigenetic mediators of environmental exposures on disease risk. Epigenetic markers, like any other molecular biomarker, are vulnerable to confounding and reverse causation. Here, we present a strategy, based on the well-established framework of Mendelian randomization, to interrogate the causal relationships between exposure, DNA methylation and outcome. The two-step approach first uses a genetic proxy for the exposure of interest to assess the causal relationship between exposure and methylation. A second step then utilizes a genetic proxy for DNA methylation to interrogate the causal relationship between DNA methylation and outcome. The rationale, origins, methodology, advantages and limitations of this novel strategy are presented. PMID:22422451

Direct detection of methylation in genomic DNA

PubMed Central

Bart, A.; van Passel, M. W. J.; van Amsterdam, K.; van der Ende, A.

2005-01-01

The identification of methylated sites on bacterial genomic DNA would be a useful tool to study the major roles of DNA methylation in prokaryotes: distinction of self and nonself DNA, direction of post-replicative mismatch repair, control of DNA replication and cell cycle, and regulation of gene expression. Three types of methylated nucleobases are known: N6-methyladenine, 5-methylcytosine and N4-methylcytosine. The aim of this study was to develop a method to detect all three types of DNA methylation in complete genomic DNA. It was previously shown that N6-methyladenine and 5-methylcytosine in plasmid and viral DNA can be detected by intersequence trace comparison of methylated and unmethylated DNA. We extended this method to include N4-methylcytosine detection in both in vitro and in vivo methylated DNA. Furthermore, application of intersequence trace comparison was extended to bacterial genomic DNA. Finally, we present evidence that intrasequence comparison suffices to detect methylated sites in genomic DNA. In conclusion, we present a method to detect all three natural types of DNA methylation in bacterial genomic DNA. This provides the possibility to define the complete methylome of any prokaryote. PMID:16091626

DNA methylation: the future of crime scene investigation?

PubMed

Gršković, Branka; Zrnec, Dario; Vicković, Sanja; Popović, Maja; Mršić, Gordan

2013-07-01

Proper detection and subsequent analysis of biological evidence is crucial for crime scene reconstruction. The number of different criminal acts is increasing rapidly. Therefore, forensic geneticists are constantly on the battlefield, trying hard to find solutions how to solve them. One of the essential defensive lines in the fight against the invasion of crime is relying on DNA methylation. In this review, the role of DNA methylation in body fluid identification and other DNA methylation applications are discussed. Among other applications of DNA methylation, age determination of the donor of biological evidence, analysis of the parent-of-origin specific DNA methylation markers at imprinted loci for parentage testing and personal identification, differentiation between monozygotic twins due to their different DNA methylation patterns, artificial DNA detection and analyses of DNA methylation patterns in the promoter regions of circadian clock genes are the most important ones. Nevertheless, there are still a lot of open chapters in DNA methylation research that need to be closed before its final implementation in routine forensic casework.

Mass Spectrometry Based Ultrasensitive DNA Methylation Profiling Using Target Fragmentation Assay.

PubMed

Lin, Xiang-Cheng; Zhang, Ting; Liu, Lan; Tang, Hao; Yu, Ru-Qin; Jiang, Jian-Hui

2016-01-19

Efficient tools for profiling DNA methylation in specific genes are essential for epigenetics and clinical diagnostics. Current DNA methylation profiling techniques have been limited by inconvenient implementation, requirements of specific reagents, and inferior accuracy in quantifying methylation degree. We develop a novel mass spectrometry method, target fragmentation assay (TFA), which enable to profile methylation in specific sequences. This method combines selective capture of DNA target from restricted cleavage of genomic DNA using magnetic separation with MS detection of the nonenzymatic hydrolysates of target DNA. This method is shown to be highly sensitive with a detection limit as low as 0.056 amol, allowing direct profiling of methylation using genome DNA without preamplification. Moreover, this method offers a unique advantage in accurately determining DNA methylation level. The clinical applicability was demonstrated by DNA methylation analysis using prostate tissue samples, implying the potential of this method as a useful tool for DNA methylation profiling in early detection of related diseases.

Investigating the epigenetic effects of a prototype smoke-derived carcinogen in human cells.

PubMed

Tommasi, Stella; Kim, Sang-in; Zhong, Xueyan; Wu, Xiwei; Pfeifer, Gerd P; Besaratinia, Ahmad

2010-05-12

Global loss of DNA methylation and locus/gene-specific gain of DNA methylation are two distinct hallmarks of carcinogenesis. Aberrant DNA methylation is implicated in smoking-related lung cancer. In this study, we have comprehensively investigated the modulation of DNA methylation consequent to chronic exposure to a prototype smoke-derived carcinogen, benzo[a]pyrene diol epoxide (B[a]PDE), in genomic regions of significance in lung cancer, in normal human cells. We have used a pulldown assay for enrichment of the CpG methylated fraction of cellular DNA combined with microarray platforms, followed by extensive validation through conventional bisulfite-based analysis. Here, we demonstrate strikingly similar patterns of DNA methylation in non-transformed B[a]PDE-treated cells vs control using high-throughput microarray-based DNA methylation profiling confirmed by conventional bisulfite-based DNA methylation analysis. The absence of aberrant DNA methylation in our model system within a timeframe that precedes cellular transformation suggests that following carcinogen exposure, other as yet unknown factors (secondary to carcinogen treatment) may help initiate global loss of DNA methylation and region-specific gain of DNA methylation, which can, in turn, contribute to lung cancer development. Unveiling the initiating events that cause aberrant DNA methylation in lung cancer has tremendous public health relevance, as it can help define future strategies for early detection and prevention of this highly lethal disease.

Investigating the Epigenetic Effects of a Prototype Smoke-Derived Carcinogen in Human Cells

PubMed Central

Tommasi, Stella; Kim, Sang-in; Zhong, Xueyan; Wu, Xiwei; Pfeifer, Gerd P.; Besaratinia, Ahmad

2010-01-01

Global loss of DNA methylation and locus/gene-specific gain of DNA methylation are two distinct hallmarks of carcinogenesis. Aberrant DNA methylation is implicated in smoking-related lung cancer. In this study, we have comprehensively investigated the modulation of DNA methylation consequent to chronic exposure to a prototype smoke-derived carcinogen, benzo[a]pyrene diol epoxide (B[a]PDE), in genomic regions of significance in lung cancer, in normal human cells. We have used a pulldown assay for enrichment of the CpG methylated fraction of cellular DNA combined with microarray platforms, followed by extensive validation through conventional bisulfite-based analysis. Here, we demonstrate strikingly similar patterns of DNA methylation in non-transformed B[a]PDE-treated cells vs control using high-throughput microarray-based DNA methylation profiling confirmed by conventional bisulfite-based DNA methylation analysis. The absence of aberrant DNA methylation in our model system within a timeframe that precedes cellular transformation suggests that following carcinogen exposure, other as yet unknown factors (secondary to carcinogen treatment) may help initiate global loss of DNA methylation and region-specific gain of DNA methylation, which can, in turn, contribute to lung cancer development. Unveiling the initiating events that cause aberrant DNA methylation in lung cancer has tremendous public health relevance, as it can help define future strategies for early detection and prevention of this highly lethal disease. PMID:20485678

Inheritance and Variation of Genomic DNA Methylation in Diploid and Triploid Pacific Oyster (Crassostrea gigas).

PubMed

Jiang, Qun; Li, Qi; Yu, Hong; Kong, Lingfeng

2016-02-01

DNA methylation is an important epigenetic mechanism that could be responsive to environmental changes indicating a potential role in natural selection and adaption. In order to evaluate an evolutionary role of DNA methylation, it is essential to first gain a better insight into inheritability. To address this question, this study investigated DNA methylation variation from parents to offspring in the Pacific oyster Crassostrea gigas using fluorescent-labeled methylation-sensitive amplified polymorphism (F-MSAP) analysis. Most of parental methylated loci were stably transmitted to offspring segregating following Medelian expectation. However, methylated loci deviated more often than non-methylated loci and offspring showed a few de novo methylated loci indicating DNA methylation changes from parents to offspring. Interestingly, some male-specific methylated loci were found in this study which might help to explore sex determination in oyster. Despite environmental stimuli, genomic stresses such as polyploidization also can induce methylation changes. This study also compared global DNA methylation level and individual methylated loci between diploid and triploid oysters. Results showed no difference in global methylation state but a few ploidy-specific loci were detected. DNA methylation variation during polyploidization was less than autonomous methylation variation from parents to offspring.

Epigenetic and physiological effects of gibberellin inhibitors and chemical pruners on the floral transition of azalea.

PubMed

Meijón, Mónica; Cañal, María Jesús; Valledor, Luis; Rodríguez, Roberto; Feito, Isabel

2011-03-01

The ability to control the timing of flowering is a key strategy in planning the production of ornamental species such as azaleas; however, it requires a thorough understanding of floral transition. DNA methylation is involved in controlling the functional state of chromatin and gene expression during floral induction pathways in response to environmental and developmental signals. Plant hormone signalling is also known to regulate suites of morphogenic processes in plants and its role in flowering-time control is starting to emerge as a key controlling step. This work investigates if the gibberellin (GA) inhibitors and chemical pinching applied in improvement of azalea flowering alter the dynamics of DNA methylation or the levels of polyamines (PAs), GAs and cytokinins (CKs) during floral transition, and whether these changes could be related to the effects observed on flowering ability. DNA methylation during floral transition and endogenous content of PAs, GAs and CKs were analysed after the application of GA synthesis inhibitors (daminozide, paclobutrazol and chlormequat chloride) and a chemical pruner (fatty acids). The application of GA biosynthesis inhibitors caused alterations in levels of PAs, GAs and CKs and in global DNA methylation levels during floral transition; also, these changes in plant growth regulators and DNA methylation were correlated with flower development. DNA methylation, PA, GA and CK levels can be used as predictive markers of plant floral capacity in azalea. Copyright © Physiologia Plantarum 2010.

Detection of regional DNA methylation using DNA-graphene affinity interactions.

PubMed

Haque, Md Hakimul; Gopalan, Vinod; Yadav, Sharda; Islam, Md Nazmul; Eftekhari, Ehsan; Li, Qin; Carrascosa, Laura G; Nguyen, Nam-Trung; Lam, Alfred K; Shiddiky, Muhammad J A

2017-01-15

We report a new method for the detection of regional DNA methylation using base-dependent affinity interaction (i.e., adsorption) of DNA with graphene. Due to the strongest adsorption affinity of guanine bases towards graphene, bisulfite-treated guanine-enriched methylated DNA leads to a larger amount of the adsorbed DNA on the graphene-modified electrodes in comparison to the adenine-enriched unmethylated DNA. The level of the methylation is quantified by monitoring the differential pulse voltammetric current as a function of the adsorbed DNA. The assay is sensitive to distinguish methylated and unmethylated DNA sequences at single CpG resolution by differentiating changes in DNA methylation as low as 5%. Furthermore, this method has been used to detect methylation levels in a collection of DNA samples taken from oesophageal cancer tissues. Copyright © 2016 Elsevier B.V. All rights reserved.

Ontogeny-Driven rDNA Rearrangement, Methylation, and Transcription, and Paternal Influence

PubMed Central

Shiao, Yih-Horng; Leighty, Robert M.; Wang, Cuiju; Ge, Xin; Crawford, Erik B.; Spurrier, Joshua M.; McCann, Sean D.; Fields, Janet R.; Fornwald, Laura; Riffle, Lisa; Driver, Craig; Quiñones, Octavio A.; Wilson, Ralph E.; Kasprzak, Kazimierz S.; Travlos, Gregory S.; Alvord, W. Gregory; Anderson, Lucy M.

2011-01-01

Gene rearrangement occurs during development in some cell types and this genome dynamics is modulated by intrinsic and extrinsic factors, including growth stimulants and nutrients. This raises a possibility that such structural change in the genome and its subsequent epigenetic modifications may also take place during mammalian ontogeny, a process undergoing finely orchestrated cell division and differentiation. We tested this hypothesis by comparing single nucleotide polymorphism-defined haplotype frequencies and DNA methylation of the rDNA multicopy gene between two mouse ontogenic stages and among three adult tissues of individual mice. Possible influences to the genetic and epigenetic dynamics by paternal exposures were also examined for Cr(III) and acid saline extrinsic factors. Variables derived from litters, individuals, and duplicate assays in large mouse populations were examined using linear mixed-effects model. We report here that active rDNA rearrangement, represented by changes of haplotype frequencies, arises during ontogenic progression from day 8 embryos to 6-week adult mice as well as in different tissue lineages and is modifiable by paternal exposures. The rDNA methylation levels were also altered in concordance with this ontogenic progression and were associated with rDNA haplotypes. Sperm showed highest level of methylation, followed by lungs and livers, and preferentially selected haplotypes that are positively associated with methylation. Livers, maintaining lower levels of rDNA methylation compared with lungs, expressed more rRNA transcript. In vitro transcription demonstrated haplotype-dependent rRNA expression. Thus, the genome is also dynamic during mammalian ontogeny and its rearrangement may trigger epigenetic changes and subsequent transcriptional controls, that are further influenced by paternal exposures. PMID:21765958

A comprehensive resource of genomic, epigenomic and transcriptomic sequencing data for the black truffle Tuber melanosporum

PubMed Central

2014-01-01

Background Tuber melanosporum, also known in the gastronomic community as “truffle”, features one of the largest fungal genomes (125 Mb) with an exceptionally high transposable element (TE) and repetitive DNA content (>58%). The main purpose of DNA methylation in fungi is TE silencing. As obligate outcrossing organisms, truffles are bound to a sexual mode of propagation, which together with TEs is thought to represent a major force driving the evolution of DNA methylation. Thus, it was of interest to examine if and how T. melanosporum exploits DNA methylation to maintain genome integrity. Findings We performed whole-genome DNA bisulfite sequencing and mRNA sequencing on different developmental stages of T. melanosporum; namely, fruitbody (“truffle”), free-living mycelium and ectomycorrhiza. The data revealed a high rate of cytosine methylation (>44%), selectively targeting TEs rather than genes with a strong preference for CpG sites. Whole genome DNA sequencing uncovered multiple TE-enriched, copy number variant regions bearing a significant fraction of hypomethylated and expressed TEs, almost exclusively in free-living mycelium propagated in vitro. Treatment of mycelia with 5-azacytidine partially reduced DNA methylation and increased TE transcription. Our transcriptome assembly also resulted in the identification of a set of novel transcripts from 614 genes. Conclusions The datasets presented here provide valuable and comprehensive (epi)genomic information that can be of interest for evolutionary genomics studies of multicellular (filamentous) fungi, in particular Ascomycetes belonging to the subphylum, Pezizomycotina. Evidence derived from comparative methylome and transcriptome analyses indicates that a non-exhaustive and partly reversible methylation process operates in truffles. PMID:25392735

A comprehensive resource of genomic, epigenomic and transcriptomic sequencing data for the black truffle Tuber melanosporum.

PubMed

Chen, Pao-Yang; Montanini, Barbara; Liao, Wen-Wei; Morselli, Marco; Jaroszewicz, Artur; Lopez, David; Ottonello, Simone; Pellegrini, Matteo

2014-01-01

Tuber melanosporum, also known in the gastronomic community as "truffle", features one of the largest fungal genomes (125 Mb) with an exceptionally high transposable element (TE) and repetitive DNA content (>58%). The main purpose of DNA methylation in fungi is TE silencing. As obligate outcrossing organisms, truffles are bound to a sexual mode of propagation, which together with TEs is thought to represent a major force driving the evolution of DNA methylation. Thus, it was of interest to examine if and how T. melanosporum exploits DNA methylation to maintain genome integrity. We performed whole-genome DNA bisulfite sequencing and mRNA sequencing on different developmental stages of T. melanosporum; namely, fruitbody ("truffle"), free-living mycelium and ectomycorrhiza. The data revealed a high rate of cytosine methylation (>44%), selectively targeting TEs rather than genes with a strong preference for CpG sites. Whole genome DNA sequencing uncovered multiple TE-enriched, copy number variant regions bearing a significant fraction of hypomethylated and expressed TEs, almost exclusively in free-living mycelium propagated in vitro. Treatment of mycelia with 5-azacytidine partially reduced DNA methylation and increased TE transcription. Our transcriptome assembly also resulted in the identification of a set of novel transcripts from 614 genes. The datasets presented here provide valuable and comprehensive (epi)genomic information that can be of interest for evolutionary genomics studies of multicellular (filamentous) fungi, in particular Ascomycetes belonging to the subphylum, Pezizomycotina. Evidence derived from comparative methylome and transcriptome analyses indicates that a non-exhaustive and partly reversible methylation process operates in truffles.

Dynamics of DNA methylation and Histone H4 acetylation during floral bud differentiation in azalea

PubMed Central

2010-01-01

Background The ability to control the timing of flowering is a key strategy for planning production in ornamental species such as azalea, however it requires a thorough understanding of floral transition. Floral transition is achieved through a complex genetic network and regulated by multiple environmental and endogenous cues. Dynamic changes between chromatin states facilitating or inhibiting DNA transcription regulate the expression of floral induction pathways in response to environmental and developmental signals. DNA methylation and histone modifications are involved in controlling the functional state of chromatin and gene expression. Results The results of this work indicate that epigenetic mechanisms such as DNA methylation and histone H4 acetylation have opposite and particular dynamics during the transition from vegetative to reproductive development in the apical shoots of azalea. Global levels of DNA methylation and histone H4 acetylation as well as immunodetection of 5-mdC and acetylated H4, in addition to a morphological study have permitted the delimitation of four basic phases in the development of the azalea bud and allowed the identification of a stage of epigenetic reprogramming which showed a sharp decrease of whole DNA methylation similar to that is defined in other developmental processes in plants and in mammals. Conclusion The epigenetic control and reorganization of chromatin seem to be decisive for coordinating floral development in azalea. DNA methylation and H4 deacetylation act simultaneously and co-ordinately, restructuring the chromatin and regulating the gene expression during soot apical meristem development and floral differentiation. PMID:20067625

Genome-Wide Analysis of DNA Methylation before-and after Exercise in the Thoroughbred Horse with MeDIP-Seq

PubMed Central

Gim, Jeong-An; Hong, Chang Pyo; Kim, Dae-Soo; Moon, Jae-Woo; Choi, Yuri; Eo, Jungwoo; Kwon, Yun-Jeong; Lee, Ja-Rang; Jung, Yi-Deun; Bae, Jin-Han; Choi, Bong-Hwan; Ko, Junsu; Song, Sanghoon; Ahn, Kung; Ha, Hong-Seok; Yang, Young Mok; Lee, Hak-Kyo; Park, Kyung-Do; Do, Kyoung-Tag; Han, Kyudong; Yi, Joo Mi; Cha, Hee-Jae; Ayarpadikannan, Selvam; Cho, Byung-Wook; Bhak, Jong; Kim, Heui-Soo

2015-01-01

Athletic performance is an important criteria used for the selection of superior horses. However, little is known about exercise-related epigenetic processes in the horse. DNA methylation is a key mechanism for regulating gene expression in response to environmental changes. We carried out comparative genomic analysis of genome-wide DNA methylation profiles in the blood samples of two different thoroughbred horses before and after exercise by methylated-DNA immunoprecipitation sequencing (MeDIP-Seq). Differentially methylated regions (DMRs) in the pre-and post-exercise blood samples of superior and inferior horses were identified. Exercise altered the methylation patterns. After 30 min of exercise, 596 genes were hypomethylated and 715 genes were hypermethylated in the superior horse, whereas in the inferior horse, 868 genes were hypomethylated and 794 genes were hypermethylated. These genes were analyzed based on gene ontology (GO) annotations and the exercise-related pathway patterns in the two horses were compared. After exercise, gene regions related to cell division and adhesion were hypermethylated in the superior horse, whereas regions related to cell signaling and transport were hypermethylated in the inferior horse. Analysis of the distribution of methylated CpG islands confirmed the hypomethylation in the gene-body methylation regions after exercise. The methylation patterns of transposable elements also changed after exercise. Long interspersed nuclear elements (LINEs) showed abundance of DMRs. Collectively, our results serve as a basis to study exercise-based reprogramming of epigenetic traits. PMID:25666347

Genome-wide methylation and gene expression changes in newborn rats following maternal protein restriction and reversal by folic acid.

PubMed

Altobelli, Gioia; Bogdarina, Irina G; Stupka, Elia; Clark, Adrian J L; Langley-Evans, Simon

2013-01-01

A large body of evidence from human and animal studies demonstrates that the maternal diet during pregnancy can programme physiological and metabolic functions in the developing fetus, effectively determining susceptibility to later disease. The mechanistic basis of such programming is unclear but may involve resetting of epigenetic marks and fetal gene expression. The aim of this study was to evaluate genome-wide DNA methylation and gene expression in the livers of newborn rats exposed to maternal protein restriction. On day one postnatally, there were 618 differentially expressed genes and 1183 differentially methylated regions (FDR 5%). The functional analysis of differentially expressed genes indicated a significant effect on DNA repair/cycle/maintenance functions and of lipid, amino acid metabolism and circadian functions. Enrichment for known biological functions was found to be associated with differentially methylated regions. Moreover, these epigenetically altered regions overlapped genetic loci associated with metabolic and cardiovascular diseases. Both expression changes and DNA methylation changes were largely reversed by supplementing the protein restricted diet with folic acid. Although the epigenetic and gene expression signatures appeared to underpin largely different biological processes, the gene expression profile of DNA methyl transferases was altered, providing a potential link between the two molecular signatures. The data showed that maternal protein restriction is associated with widespread differential gene expression and DNA methylation across the genome, and that folic acid is able to reset both molecular signatures.

Epigenome-wide association study of smoking and DNA methylation in non-small cell lung neoplasms.

PubMed

Freeman, Joshua R; Chu, Su; Hsu, Thomas; Huang, Yen-Tsung

2016-10-25

Tobacco smoke is a well-established lung cancer carcinogen. We hypothesize that epigenetic processes underlie carcinogenesis. The objective of this study is to examine the effects of smoke exposure on DNA methylation to search for novel susceptibility loci. We obtained epigenome-wide DNA methylation data from lung adenocarcinoma (LUAD) and lung squamous cell (LUSC) tissues in The Cancer Genome Atlas (TCGA). We performed a two-stage discovery (n = 326) and validation (n = 185) analysis to investigate the association of epigenetic DNA methylation level with cigarette smoking pack-years. We also externally validated our findings in an independent dataset. Linear model with least square estimator and spline regression were performed to examine the association between DNA methylation and smoking. We identified five CpG sites highly associated with pack-years of cigarette smoking. Smoking was negatively associated with methylation levels in cg25771041 (WWTR1, p = 3.6 × 10-9), cg16200496 (NFIX, p = 3.4 × 10-12), cg22515201 (PLA2G6, p = 1.0 × 10-9) and cg24823993 (NHP2L1, p = 5.1 × 10-8) and positively associated with the methylation level in cg11875268 (SMUG1, p = 4.3 × 10-8). The CpG-smoking association was stronger in LUSC than LUAD. Of the five loci, smoking explained the most variation in cg16200496 (R2 = 0.098 [both types] and 0.144 [LUSC]). We identified 5 novel CpG candidates that demonstrate differential methylation patterns associated with smoke exposure in lung neoplasms.

The MBD7 complex promotes expression of methylated transgenes without significantly altering their methylation status

PubMed Central

Li, Dongming; Palanca, Ana Marie S; Won, So Youn; Gao, Lei; Feng, Ying; Vashisht, Ajay A; Liu, Li; Zhao, Yuanyuan; Liu, Xigang; Wu, Xiuyun; Li, Shaofang; Le, Brandon; Kim, Yun Ju; Yang, Guodong; Li, Shengben; Liu, Jinyuan; Wohlschlegel, James A; Guo, Hongwei; Mo, Beixin; Chen, Xuemei; Law, Julie A

2017-01-01

DNA methylation is associated with gene silencing in eukaryotic organisms. Although pathways controlling the establishment, maintenance and removal of DNA methylation are known, relatively little is understood about how DNA methylation influences gene expression. Here we identified a METHYL-CpG-BINDING DOMAIN 7 (MBD7) complex in Arabidopsis thaliana that suppresses the transcriptional silencing of two LUCIFERASE (LUC) reporters via a mechanism that is largely downstream of DNA methylation. Although mutations in components of the MBD7 complex resulted in modest increases in DNA methylation concomitant with decreased LUC expression, we found that these hyper-methylation and gene expression phenotypes can be genetically uncoupled. This finding, along with genome-wide profiling experiments showing minimal changes in DNA methylation upon disruption of the MBD7 complex, places the MBD7 complex amongst a small number of factors acting downstream of DNA methylation. This complex, however, is unique as it functions to suppress, rather than enforce, DNA methylation-mediated gene silencing. DOI: http://dx.doi.org/10.7554/eLife.19893.001 PMID:28452714

Intratumor DNA methylation heterogeneity reflects clonal evolution in aggressive prostate cancer.

PubMed

Brocks, David; Assenov, Yassen; Minner, Sarah; Bogatyrova, Olga; Simon, Ronald; Koop, Christina; Oakes, Christopher; Zucknick, Manuela; Lipka, Daniel Bernhard; Weischenfeldt, Joachim; Feuerbach, Lars; Cowper-Sal Lari, Richard; Lupien, Mathieu; Brors, Benedikt; Korbel, Jan; Schlomm, Thorsten; Tanay, Amos; Sauter, Guido; Gerhäuser, Clarissa; Plass, Christoph

2014-08-07

Despite much evidence on epigenetic abnormalities in cancer, it is currently unclear to what extent epigenetic alterations can be associated with tumors' clonal genetic origins. Here, we show that the prostate intratumor heterogeneity in DNA methylation and copy-number patterns can be explained by a unified evolutionary process. By assaying multiple topographically distinct tumor sites, premalignant lesions, and lymph node metastases within five cases of prostate cancer, we demonstrate that both DNA methylation and copy-number heterogeneity consistently reflect the life history of the tumors. Furthermore, we show cases of genetic or epigenetic convergent evolution and highlight the diversity in the evolutionary origins and aberration spectrum between tumor and metastatic subclones. Importantly, DNA methylation can complement genetic data by serving as a proxy for activity at regulatory domains, as we show through identification of high epigenetic heterogeneity at androgen-receptor-bound enhancers. Epigenome variation thereby expands on the current genome-centric view on tumor heterogeneity. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

DOT1L and H3K79 Methylation in Transcription and Genomic Stability.

PubMed

Wood, Katherine; Tellier, Michael; Murphy, Shona

2018-02-27

The organization of eukaryotic genomes into chromatin provides challenges for the cell to accomplish basic cellular functions, such as transcription, DNA replication and repair of DNA damage. Accordingly, a range of proteins modify and/or read chromatin states to regulate access to chromosomal DNA. Yeast Dot1 and the mammalian homologue DOT1L are methyltransferases that can add up to three methyl groups to histone H3 lysine 79 (H3K79). H3K79 methylation is implicated in several processes, including transcription elongation by RNA polymerase II, the DNA damage response and cell cycle checkpoint activation. DOT1L is also an important drug target for treatment of mixed lineage leukemia (MLL)-rearranged leukemia where aberrant transcriptional activation is promoted by DOT1L mislocalisation. This review summarizes what is currently known about the role of Dot1/DOT1L and H3K79 methylation in transcription and genomic stability.

Impact of DNA methylation on trophoblast function

PubMed Central

2011-01-01

The influence of epigenetics is evident in many fields of medicine today. This is also true in placentology, where versatile epigenetic mechanisms that regulate expression of genes have shown to have important influence on trophoblast implantation and placentation. Such gene regulation can be established in different ways and on different molecular levels, the most common being the DNA methylation. DNA methylation has been shown today as an important predictive component in assessing clinical prognosis of certain malignant tumors; in addition, it opens up new possibilities for non-invasive prenatal diagnosis utilizing cell-free fetal DNA methods. By using a well known demethylating agent 5-azacytidine in pregnant rat model, we have been able to change gene expression and, consequently, the processes of trophoblast differentiation and placental development. In this review, we describe how changes in gene methylation effect trophoblast development and placentation and offer our perspective on use of trophoblast epigenetic research for better understanding of not only placenta development but cancer cell growth and invasion as well. PMID:22414254

Inferring genome-wide interplay landscape between DNA methylation and transcriptional regulation.

PubMed

Tang, Binhua; Wang, Xin

2015-01-01

DNA methylation and transcriptional regulation play important roles in cancer cell development and differentiation processes. Based on the currently available cell line profiling information from the ENCODE Consortium, we propose a Bayesian inference model to infer and construct genome-wide interaction landscape between DNA methylation and transcriptional regulation, which sheds light on the underlying complex functional mechanisms important within the human cancer and disease context. For the first time, we select all the currently available cell lines (>=20) and transcription factors (>=80) profiling information from the ENCODE Consortium portal. Through the integration of those genome-wide profiling sources, our genome-wide analysis detects multiple functional loci of interest, and indicates that DNA methylation is cell- and region-specific, due to the interplay mechanisms with transcription regulatory activities. We validate our analysis results with the corresponding RNA-sequencing technique for those detected genomic loci. Our results provide novel and meaningful insights for the interplay mechanisms of transcriptional regulation and gene expression for the human cancer and disease studies.

Genome-wide DNA methylation patterns of bovine blastocysts derived from in vivo embryos subjected to in vitro culture before, during or after embryonic genome activation.

PubMed

Salilew-Wondim, Dessie; Saeed-Zidane, Mohammed; Hoelker, Michael; Gebremedhn, Samuel; Poirier, Mikhaël; Pandey, Hari Om; Tholen, Ernst; Neuhoff, Christiane; Held, Eva; Besenfelder, Urban; Havlicek, Vita; Rings, Franca; Fournier, Eric; Gagné, Dominic; Sirard, Marc-André; Robert, Claude; Gad, Ahmed; Schellander, Karl; Tesfaye, Dawit

2018-06-01

Aberrant DNA methylation patterns of genes required for development are common in in vitro produced embryos. In this regard, we previously identified altered DNA methylation patterns of in vivo developed blastocysts from embryos which spent different stages of development in vitro, indicating carryover effects of suboptimal culture conditions on epigenetic signatures of preimplantation embryos. However, epigenetic responses of in vivo originated embryos to suboptimal culture conditions are not fully understood. Therefore, here we investigated DNA methylation patterns of in vivo derived bovine embryos subjected to in vitro culture condition before, during or after major embryonic genome activation (EGA). For this, in vivo produced 2-, 8- and 16-cell stage embryos were cultured in vitro until the blastocyst stage and blastocysts were used for genome-wide DNA methylation analysis. The 2- and 8-cell flushed embryo groups showed lower blastocyst rates compared to the 16-cell flush group. This was further accompanied by increased numbers of differentially methylated genomic regions (DMRs) in blastocysts of the 2- and 8-cell flush groups compared to the complete in vivo control ones. Moreover, 1623 genomic loci including imprinted genes were hypermethylated in blastocyst of 2-, 8- and 16-cell flushed groups, indicating the presence of genomic regions which are sensitive to the in vitro culture at any stage of embryonic development. Furthermore, hypermethylated genomic loci outnumbered hypomethylated ones in blastocysts of 2- and 16-cell flushed embryo groups, but the opposite occurred in the 8-cell group. Moreover, DMRs which were unique to blastocysts of the 2-cell flushed group and inversely correlated with corresponding mRNA expression levels were involved in plasma membrane lactate transport, amino acid transport and phosphorus metabolic processes, whereas DMRs which were specific to the 8-cell group and inversely correlated with corresponding mRNA expression levels were involved in several biological processes including regulation of fatty acids and steroid biosynthesis processes. In vivo embryos subjected to in vitro culture before and during major embryonic genome activation (EGA) are prone to changes in DNA methylation marks and exposure of in vivo embryos to in vitro culture during the time of EGA increased hypomethylated genomic loci in blastocysts.

DNA Methylation and Cancer Diagnosis

PubMed Central

Delpu, Yannick; Cordelier, Pierre; Cho, William C.; Torrisani, Jérôme

2013-01-01

DNA methylation is a major epigenetic modification that is strongly involved in the physiological control of genome expression. DNA methylation patterns are largely modified in cancer cells and can therefore be used to distinguish cancer cells from normal tissues. This review describes the main technologies available for the detection and the discovery of aberrantly methylated DNA patterns. It also presents the different sources of biological samples suitable for DNA methylation studies. We discuss the interest and perspectives on the use of DNA methylation measurements for cancer diagnosis through examples of methylated genes commonly documented in the literature. The discussion leads to our consideration for why DNA methylation is not commonly used in clinical practice through an examination of the main requirements that constitute a reliable biomarker. Finally, we describe the main DNA methylation inhibitors currently used in clinical trials and those that exhibit promising results. PMID:23873296

Sulfur mustard-induced epigenetic modifications over time - a pilot study.

PubMed

Simons, Thilo; Steinritz, Dirk; Bölck, Birgit; Schmidt, Annette; Popp, Tanja; Thiermann, Horst; Gudermann, Thomas; Bloch, Wilhelm; Kehe, Kai

2018-09-01

The chemical warfare agent sulfur mustard (SM) can cause long-term health effects that may occur even years after a single exposure. The underlying pathophysiology is unknown, but epigenetic mechanisms are discussed as feasible explanation. "Epigenetics" depicts regulation of gene function without affecting the DNA sequence itself. DNA-methylation and covalent histone modifications (methylation or acetylation) are regarded as important processes. In the present in vitro study using early endothelial cells (EEC), we analyzed SM-induced DNA methylation over time and compared results to an in vivo skin sample that was obtained approx. one year after an accidental SM exposure. EEC were exposed to low SM concentrations (0.5 and 1.0μM). DNA methylation and histone acetylation (H3-K9, H3-K27, H4-K8) or histone di-methylation (H3-K9, H3-K27, H3-K36) were investigated 24h after exposure, and after 2 or 4 additional cell passages. The human skin sample was assessed in parallel. SM had only some minor effects on histone modifications. However, a significant and pronounced increase of DNA methylation was detected in the late cell passages as well as in the skin sample. Our results indicate that SM does indeed cause epigenetic modifications that appear to persist over time. Copyright © 2017 Elsevier B.V. All rights reserved.

Salicylic acid and nitric oxide alleviate high temperature induced oxidative damage in Lablab purpureus L plants by regulating bio-physical processes and DNA methylation.

PubMed

Rai, Krishna Kumar; Rai, Nagendra; Rai, Shashi Pandey

2018-07-01

Salicylic acid (SA) and sodium nitroprusside (SNP, NO donor) modulates plant growth and development processes and recent findings have also revealed their involvement in the regulation of epigenetic factors under stress condition. In the present study, some of these factors were comparatively studied in hyacinth bean plants subjected to high temperature (HT) environment (40-42 °C) with and without exogenous application of SA and SNP under field condition. Exogenous application of SA and SNP substantially modulated the growth and biophysical process of hyacinth bean plants under HT environment. Exogenous application of SA and SNP also remarkably regulated the activities of antioxidant enzymes, modulated mRNA level of certain enzymes, improves plant water relation, enhance photosynthesis and thereby increasing plant defence under HT. Coupled restriction enzyme digestion-random amplification (CRED-RA) technique revealed that many methylation changes were "dose dependent" and HT significantly increased DNA damages as evidenced by both increase and decrease in bands profiles, methylation and de-methylation pattern. Thus, the result of the present study clearly shows that exogenous SA and SNP regulates DNA methylation pattern, modulates stress-responsive genes and can impart transient HT tolerance by synchronizing growth and physiological acclimatization of plants, thus narrowing the gaps between physio-biochemical and molecular events in addressing HT tolerance. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

ASCIZ regulates lesion-specific Rad51 focus formation and apoptosis after methylating DNA damage

PubMed Central

McNees, Carolyn J; Conlan, Lindus A; Tenis, Nora; Heierhorst, Jörg

2005-01-01

Nuclear Rad51 focus formation is required for homology-directed repair of DNA double-strand breaks (DSBs), but its regulation in response to non-DSB lesions is poorly understood. Here we report a novel human SQ/TQ cluster domain-containing protein termed ASCIZ that forms Rad51-containing foci in response to base-modifying DNA methylating agents but not in response to DSB-inducing agents. ASCIZ foci seem to form prior to Rad51 recruitment, and an ASCIZ core domain can concentrate Rad51 in focus-like structures independently of DNA damage. ASCIZ depletion dramatically increases apoptosis after methylating DNA damage and impairs Rad51 focus formation in response to methylating agents but not after ionizing radiation. ASCIZ focus formation and increased apoptosis in ASCIZ-depleted cells depend on the mismatch repair protein MLH1. Interestingly, ASCIZ foci form efficiently during G1 phase, when sister chromatids are unavailable as recombination templates. We propose that ASCIZ acts as a lesion-specific focus scaffold in a Rad51-dependent pathway that resolves cytotoxic repair intermediates, most likely single-stranded DNA gaps, resulting from MLH1-dependent processing of base lesions. PMID:15933716

ASCIZ regulates lesion-specific Rad51 focus formation and apoptosis after methylating DNA damage.

PubMed

McNees, Carolyn J; Conlan, Lindus A; Tenis, Nora; Heierhorst, Jörg

2005-07-06

Nuclear Rad51 focus formation is required for homology-directed repair of DNA double-strand breaks (DSBs), but its regulation in response to non-DSB lesions is poorly understood. Here we report a novel human SQ/TQ cluster domain-containing protein termed ASCIZ that forms Rad51-containing foci in response to base-modifying DNA methylating agents but not in response to DSB-inducing agents. ASCIZ foci seem to form prior to Rad51 recruitment, and an ASCIZ core domain can concentrate Rad51 in focus-like structures independently of DNA damage. ASCIZ depletion dramatically increases apoptosis after methylating DNA damage and impairs Rad51 focus formation in response to methylating agents but not after ionizing radiation. ASCIZ focus formation and increased apoptosis in ASCIZ-depleted cells depend on the mismatch repair protein MLH1. Interestingly, ASCIZ foci form efficiently during G1 phase, when sister chromatids are unavailable as recombination templates. We propose that ASCIZ acts as a lesion-specific focus scaffold in a Rad51-dependent pathway that resolves cytotoxic repair intermediates, most likely single-stranded DNA gaps, resulting from MLH1-dependent processing of base lesions.

Alterations in DNA methylation corresponding with lung inflammation and as a biomarker for disease development after MWCNT exposure.

PubMed

Brown, Traci A; Lee, Joong Won; Holian, Andrij; Porter, Virginia; Fredriksen, Harley; Kim, Minju; Cho, Yoon Hee

2016-01-01

Use of multi-walled carbon nanotubes (MWCNT) is growing which increases occupational exposures to these materials. Their toxic potential makes it important to have an in-depth understanding of the inflammation and disease that develops due to exposure. Epigenetics is one area of interest that has been quickly developing to assess disease processes due to its ability to change gene expression and thus the lung environment after exposure. In this study, promoter methylation of inflammatory genes (IFN-γ and TNF-α) was measured after MWCNT exposure using the pyrosequencing assay and found to correlate with initial cytokine production. In addition, methylation of a gene involved in tissue fibrosis (Thy-1) was also altered in a way that matched collagen deposition. In addition to using epigenetics to better understand disease processes, it has also been used as a biomarker of exposure and disease. In this study, global methylation was determined in the lung to ascertain whether MWCNT alter global methylation at the site of exposure and if those alterations coincide with disease development. Then, global methylation levels were determined in the blood to ascertain whether global methylation could be used as a biomarker of exposure in a more easily accessible tissue. Using the LuUminometric Methylation Assay (LUMA) and 5-Methylcytosine (5-mC) Quantification assay, we found that MWCNT lead to DNA hypomethylation in the lung and blood, which coincided with disease development. This study provides initial data showing that alterations in gene-specific methylation correspond with an inflammatory response to MWCNT exposure. In addition, global DNA methylation in the lung and blood coincides with MWCNT-induced disease development, suggesting its potential as a biomarker of both exposure and disease development.

Signatures of DNA Methylation across Insects Suggest Reduced DNA Methylation Levels in Holometabola

PubMed Central

Provataris, Panagiotis; Meusemann, Karen; Niehuis, Oliver; Grath, Sonja; Misof, Bernhard

2018-01-01

Abstract It has been experimentally shown that DNA methylation is involved in the regulation of gene expression and the silencing of transposable element activity in eukaryotes. The variable levels of DNA methylation among different insect species indicate an evolutionarily flexible role of DNA methylation in insects, which due to a lack of comparative data is not yet well-substantiated. Here, we use computational methods to trace signatures of DNA methylation across insects by analyzing transcriptomic and genomic sequence data from all currently recognized insect orders. We conclude that: 1) a functional methylation system relying exclusively on DNA methyltransferase 1 is widespread across insects. 2) DNA methylation has potentially been lost or extremely reduced in species belonging to springtails (Collembola), flies and relatives (Diptera), and twisted-winged parasites (Strepsiptera). 3) Holometabolous insects display signs of reduced DNA methylation levels in protein-coding sequences compared with hemimetabolous insects. 4) Evolutionarily conserved insect genes associated with housekeeping functions tend to display signs of heavier DNA methylation in comparison to the genomic/transcriptomic background. With this comparative study, we provide the much needed basis for experimental and detailed comparative analyses required to gain a deeper understanding on the evolution and function of DNA methylation in insects. PMID:29697817

A multi-scale analysis of bull sperm methylome revealed both species peculiarities and conserved tissue-specific features.

PubMed

Perrier, Jean-Philippe; Sellem, Eli; Prézelin, Audrey; Gasselin, Maxime; Jouneau, Luc; Piumi, François; Al Adhami, Hala; Weber, Michaël; Fritz, Sébastien; Boichard, Didier; Le Danvic, Chrystelle; Schibler, Laurent; Jammes, Hélène; Kiefer, Hélène

2018-05-29

Spermatozoa have a remarkable epigenome in line with their degree of specialization, their unique nature and different requirements for successful fertilization. Accordingly, perturbations in the establishment of DNA methylation patterns during male germ cell differentiation have been associated with infertility in several species. While bull semen is widely used in artificial insemination, the literature describing DNA methylation in bull spermatozoa is still scarce. The purpose of this study was therefore to characterize the bull sperm methylome relative to both bovine somatic cells and the sperm of other mammals through a multiscale analysis. The quantification of DNA methylation at CCGG sites using luminometric methylation assay (LUMA) highlighted the undermethylation of bull sperm compared to the sperm of rams, stallions, mice, goats and men. Total blood cells displayed a similarly high level of methylation in bulls and rams, suggesting that undermethylation of the bovine genome was specific to sperm. Annotation of CCGG sites in different species revealed no striking bias in the distribution of genome features targeted by LUMA that could explain undermethylation of bull sperm. To map DNA methylation at a genome-wide scale, bull sperm was compared with bovine liver, fibroblasts and monocytes using reduced representation bisulfite sequencing (RRBS) and immunoprecipitation of methylated DNA followed by microarray hybridization (MeDIP-chip). These two methods exhibited differences in terms of genome coverage, and consistently, two independent sets of sequences differentially methylated in sperm and somatic cells were identified for RRBS and MeDIP-chip. Remarkably, in the two sets most of the differentially methylated sequences were hypomethylated in sperm. In agreement with previous studies in other species, the sequences that were specifically hypomethylated in bull sperm targeted processes relevant to the germline differentiation program (piRNA metabolism, meiosis, spermatogenesis) and sperm functions (cell adhesion, fertilization), as well as satellites and rDNA repeats. These results highlight the undermethylation of bull spermatozoa when compared with both bovine somatic cells and the sperm of other mammals, and raise questions regarding the dynamics of DNA methylation in bovine male germline. Whether sperm undermethylation has potential interactions with structural variation in the cattle genome may deserve further attention.

Methylation interactions in Arabidopsis hybrids require RNA-directed DNA methylation and are influenced by genetic variation

PubMed Central

Zhang, Qingzhu; Wang, Dong; Lang, Zhaobo; He, Li; Yang, Lan; Zeng, Liang; Li, Yanqiang; Zhao, Cheng; Huang, Huan; Zhang, Heng; Zhang, Huiming; Zhu, Jian-Kang

2016-01-01

DNA methylation is a conserved epigenetic mark in plants and many animals. How parental alleles interact in progeny to influence the epigenome is poorly understood. We analyzed the DNA methylomes of Arabidopsis Col and C24 ecotypes, and their hybrid progeny. Hybrids displayed nonadditive DNA methylation levels, termed methylation interactions, throughout the genome. Approximately 2,500 methylation interactions occurred at regions where parental DNA methylation levels are similar, whereas almost 1,000 were at differentially methylated regions in parents. Methylation interactions were characterized by an abundance of 24-nt small interfering RNAs. Furthermore, dysfunction of the RNA-directed DNA methylation pathway abolished methylation interactions but did not affect the increased biomass observed in hybrid progeny. Methylation interactions correlated with altered genetic variation within the genome, suggesting that they may play a role in genome evolution. PMID:27382183

Choline nutrition programs brain development via DNA and histone methylation.

PubMed

Blusztajn, Jan Krzysztof; Mellott, Tiffany J

2012-06-01

Choline is an essential nutrient for humans. Metabolically choline is used for the synthesis of membrane phospholipids (e.g. phosphatidylcholine), as a precursor of the neurotransmitter acetylcholine, and, following oxidation to betaine, choline functions as a methyl group donor in a pathway that produces S-adenosylmethionine. As a methyl donor choline influences DNA and histone methylation--two central epigenomic processes that regulate gene expression. Because the fetus and neonate have high demands for choline, its dietary intake during pregnancy and lactation is particularly important for normal development of the offspring. Studies in rodents have shown that high choline intake during gestation improves cognitive function in adulthood and prevents memory decline associated with old age. These behavioral changes are accompanied by electrophysiological, neuroanatomical, and neurochemical changes and by altered patterns of expression of multiple cortical and hippocampal genes including those encoding key proteins that contribute to the biochemical mechanisms of learning and memory. These actions of choline are observed long after the exposure to the nutrient ended (months) and correlate with fetal hepatic and cerebral cortical choline-evoked changes in global- and gene-specific DNA cytosine methylation and with dramatic changes of the methylation pattern of lysine residues 4, 9 and 27 of histone H3. Moreover, gestational choline modulates the expression of DNA (Dnmt1, Dnmt3a) and histone (G9a/Ehmt2/Kmt1c, Suv39h1/Kmt1a) methyltransferases. In addition to the central role of DNA and histone methylation in brain development, these processes are highly dynamic in adult brain, modulate the expression of genes critical for synaptic plasticity, and are involved in mechanisms of learning and memory. A recent study documented that in a cohort of normal elderly people, verbal and visual memory function correlated positively with the amount of dietary choline consumption. It will be important to determine if these actions of choline on human cognition are mediated by epigenomic mechanisms or by its influence on acetylcholine or phospholipid synthesis.

Choline nutrition programs brain development via DNA and histone methylation

PubMed Central

Blusztajn, Jan Krzysztof; Mellott, Tiffany J.

2017-01-01

Choline is an essential nutrient for humans. Metabolically choline is used for the synthesis of membrane phospholipids (e.g. phosphatidylcholine), as a precursor of the neurotransmitter acetylcholine, and, following oxidation to betaine, choline functions as a methyl group donor in a pathway that produces S-adenosylmethionine. As a methyl donor choline influences DNA and histone methylation – two central epigenomic processes that regulate gene expression. Because the fetus and neonate have high demands for choline, its dietary intake during pregnancy and lactation is particularly important for normal development of the offspring. Studies in rodents have shown that high choline intake during gestation improves cognitive function in adulthood and prevents memory decline associated with old age. These behavioral changes are accompanied by electrophysiological, neuroanatomical, and neurochemical changes and by altered patterns of expression of multiple cortical and hippocampal genes including those encoding key proteins that contribute to the biochemical mechanisms of learning and memory. These actions of choline are observed long after the exposure to the nutrient ended (months) and correlate with fetal hepatic and cerebral cortical choline-evoked changes in global- and gene-specific DNA cytosine methylation and with dramatic changes of the methylation pattern of lysine residues 4, 9 and 27 of histone H3. Moreover, gestational choline modulates the expression of DNA (Dnmt1, Dnmt3a) and histone (G9a/Ehmt2/Kmt1c, Suv39h1/Kmt1a) methyltransferases. In addition to the central role of DNA and histone methylation in brain development, these processes are highly dynamic in adult brain, modulate the expression of genes critical for synaptic plasticity, and are involved in mechanisms of learning and memory. A recent study documented that in a cohort of normal elderly people, verbal and visual memory function correlated positively with the amount of dietary choline consumption. It will be important to determine if these actions of choline on human cognition are mediated by epigenomic mechanisms or by its influence on acetylcholine or phospholipid synthesis. PMID:22483275

CpG PatternFinder: a Windows-based utility program for easy and rapid identification of the CpG methylation status of DNA.

PubMed

Xu, Yi-Hua; Manoharan, Herbert T; Pitot, Henry C

2007-09-01

The bisulfite genomic sequencing technique is one of the most widely used techniques to study sequence-specific DNA methylation because of its unambiguous ability to reveal DNA methylation status to the order of a single nucleotide. One characteristic feature of the bisulfite genomic sequencing technique is that a number of sample sequence files will be produced from a single DNA sample. The PCR products of bisulfite-treated DNA samples cannot be sequenced directly because they are heterogeneous in nature; therefore they should be cloned into suitable plasmids and then sequenced. This procedure generates an enormous number of sample DNA sequence files as well as adding extra bases belonging to the plasmids to the sequence, which will cause problems in the final sequence comparison. Finding the methylation status for each CpG in each sample sequence is not an easy job. As a result CpG PatternFinder was developed for this purpose. The main functions of the CpG PatternFinder are: (i) to analyze the reference sequence to obtain CpG and non-CpG-C residue position information. (ii) To tailor sample sequence files (delete insertions and mark deletions from the sample sequence files) based on a configuration of ClustalW multiple alignment. (iii) To align sample sequence files with a reference file to obtain bisulfite conversion efficiency and CpG methylation status. And, (iv) to produce graphics, highlighted aligned sequence text and a summary report which can be easily exported to Microsoft Office suite. CpG PatternFinder is designed to operate cooperatively with BioEdit, a freeware on the internet. It can handle up to 100 files of sample DNA sequences simultaneously, and the total CpG pattern analysis process can be finished in minutes. CpG PatternFinder is an ideal software tool for DNA methylation studies to determine the differential methylation pattern in a large number of individuals in a population. Previously we developed the CpG Analyzer program; CpG PatternFinder is our further effort to create software tools for DNA methylation studies.

DNA Methylation Profiling of Embryonic Stem Cell Differentiation into the Three Germ Layers

PubMed Central

Isagawa, Takayuki; Nagae, Genta; Shiraki, Nobuaki; Fujita, Takanori; Sato, Noriko; Ishikawa, Shumpei; Kume, Shoen; Aburatani, Hiroyuki

2011-01-01

Embryogenesis is tightly regulated by multiple levels of epigenetic regulation such as DNA methylation, histone modification, and chromatin remodeling. DNA methylation patterns are erased in primordial germ cells and in the interval immediately following fertilization. Subsequent developmental reprogramming occurs by de novo methylation and demethylation. Variance in DNA methylation patterns between different cell types is not well understood. Here, using methylated DNA immunoprecipitation and tiling array technology, we have comprehensively analyzed DNA methylation patterns at proximal promoter regions in mouse embryonic stem (ES) cells, ES cell-derived early germ layers (ectoderm, endoderm and mesoderm) and four adult tissues (brain, liver, skeletal muscle and sperm). Most of the methylated regions are methylated across all three germ layers and in the three adult somatic tissues. This commonly methylated gene set is enriched in germ cell-associated genes that are generally transcriptionally inactive in somatic cells. We also compared DNA methylation patterns by global mapping of histone H3 lysine 4/27 trimethylation, and found that gain of DNA methylation correlates with loss of histone H3 lysine 4 trimethylation. Our combined findings indicate that differentiation of ES cells into the three germ layers is accompanied by an increased number of commonly methylated DNA regions and that these tissue-specific alterations in methylation occur for only a small number of genes. DNA methylation at the proximal promoter regions of commonly methylated genes thus appears to be an irreversible mark which functions to fix somatic lineage by repressing the transcription of germ cell-specific genes. PMID:22016810

DNA methylation profiling of embryonic stem cell differentiation into the three germ layers.

PubMed

Isagawa, Takayuki; Nagae, Genta; Shiraki, Nobuaki; Fujita, Takanori; Sato, Noriko; Ishikawa, Shumpei; Kume, Shoen; Aburatani, Hiroyuki

2011-01-01

Embryogenesis is tightly regulated by multiple levels of epigenetic regulation such as DNA methylation, histone modification, and chromatin remodeling. DNA methylation patterns are erased in primordial germ cells and in the interval immediately following fertilization. Subsequent developmental reprogramming occurs by de novo methylation and demethylation. Variance in DNA methylation patterns between different cell types is not well understood. Here, using methylated DNA immunoprecipitation and tiling array technology, we have comprehensively analyzed DNA methylation patterns at proximal promoter regions in mouse embryonic stem (ES) cells, ES cell-derived early germ layers (ectoderm, endoderm and mesoderm) and four adult tissues (brain, liver, skeletal muscle and sperm). Most of the methylated regions are methylated across all three germ layers and in the three adult somatic tissues. This commonly methylated gene set is enriched in germ cell-associated genes that are generally transcriptionally inactive in somatic cells. We also compared DNA methylation patterns by global mapping of histone H3 lysine 4/27 trimethylation, and found that gain of DNA methylation correlates with loss of histone H3 lysine 4 trimethylation. Our combined findings indicate that differentiation of ES cells into the three germ layers is accompanied by an increased number of commonly methylated DNA regions and that these tissue-specific alterations in methylation occur for only a small number of genes. DNA methylation at the proximal promoter regions of commonly methylated genes thus appears to be an irreversible mark which functions to fix somatic lineage by repressing the transcription of germ cell-specific genes.

Microsphere-Based Multiplex Analysis of DNA Methylation in Acute Myeloid Leukemia

PubMed Central

Wertheim, Gerald B.W.; Smith, Catherine; Figueroa, Maria E.; Kalos, Michael; Bagg, Adam; Carroll, Martin; Master, Stephen R.

2015-01-01

Aberrant regulation of DNA methylation is characteristic of cancer cells and clearly influences phenotypes of various malignancies. Despite clear correlations between DNA methylation and patient outcome, tests that directly measure multiple-locus DNA methylation are typically expensive and technically challenging. Previous studies have demonstrated that the prognosis of patients with acute myeloid leukemia can be predicted by the DNA methylation pattern of 18 loci. We have developed a novel strategy, termed microsphere HpaII tiny fragment enrichment by ligation-mediated PCR (MELP), to simultaneously analyze the DNA methylation pattern at these loci using methylation-specific DNA digestion, fluorescently labeled microspheres, and branched DNA hybridization. The method uses techniques that are inexpensive and easily performed in a molecular laboratory. MELP accurately reflects the methylation levels at each locus analyzed and segregates patients with acute myeloid leukemia into prognostic subgroups. Our results demonstrate the usefulness of MELP as a platform for simultaneous evaluation of DNA methylation of multiple loci. PMID:24373919

Collaborations between CpG sites in DNA methylation

NASA Astrophysics Data System (ADS)

Song, You; Ren, Honglei; Lei, Jinzhi

2017-08-01

DNA methylation patterns have profound impacts on genome stability, gene expression and development. The molecular base of DNA methylation patterns has long been focused at single CpG sites level. Here, we construct a kinetic model of DNA methylation with collaborations between CpG sites, from which a correlation function was established based on experimental data. The function consists of three parts that suggest three possible sources of the correlation: movement of enzymes along DNA, collaboration between DNA methylation and nucleosome modification, and global enzyme concentrations within a cell. Moreover, the collaboration strength between DNA methylation and nucleosome modification is universal for mouse early embryo cells. The obtained correlation function provides insightful understanding for the mechanisms of inheritance of DNA methylation patterns.

Genome-Wide Negative Feedback Drives Transgenerational DNA Methylation Dynamics in Arabidopsis

PubMed Central

Kassam, Mohamed; Duvernois-Berthet, Evelyne; Cortijo, Sandra; Takashima, Kazuya; Saze, Hidetoshi; Toyoda, Atsushi; Fujiyama, Asao; Colot, Vincent; Kakutani, Tetsuji

2015-01-01

Epigenetic variations of phenotypes, especially those associated with DNA methylation, are often inherited over multiple generations in plants. The active and inactive chromatin states are heritable and can be maintained or even be amplified by positive feedback in a transgenerational manner. However, mechanisms controlling the transgenerational DNA methylation dynamics are largely unknown. As an approach to understand the transgenerational dynamics, we examined long-term effect of impaired DNA methylation in Arabidopsis mutants of the chromatin remodeler gene DDM1 (Decrease in DNA Methylation 1) through whole genome DNA methylation sequencing. The ddm1 mutation induces a drastic decrease in DNA methylation of transposable elements (TEs) and repeats in the initial generation, while also inducing ectopic DNA methylation at hundreds of loci. Unexpectedly, this ectopic methylation can only be seen after repeated self-pollination. The ectopic cytosine methylation is found primarily in the non-CG context and starts from 3’ regions within transcription units and spreads upstream. Remarkably, when chromosomes with reduced DNA methylation were introduced from a ddm1 mutant into a DDM1 wild-type background, the ddm1-derived chromosomes also induced analogous de novo accumulation of DNA methylation in trans. These results lead us to propose a model to explain the transgenerational DNA methylation redistribution by genome-wide negative feedback. The global negative feedback, together with local positive feedback, would ensure robust and balanced differentiation of chromatin states within the genome. PMID:25902052

DNA methylation of retrotransposons, DNA transposons and genes in sugar beet (Beta vulgaris L.).

PubMed

Zakrzewski, Falk; Schmidt, Martin; Van Lijsebettens, Mieke; Schmidt, Thomas

2017-06-01

The methylation of cytosines shapes the epigenetic landscape of plant genomes, coordinates transgenerational epigenetic inheritance, represses the activity of transposable elements (TEs), affects gene expression and, hence, can influence the phenotype. Sugar beet (Beta vulgaris ssp. vulgaris), an important crop that accounts for 30% of worldwide sugar needs, has a relatively small genome size (758 Mbp) consisting of approximately 485 Mbp repetitive DNA (64%), in particular satellite DNA, retrotransposons and DNA transposons. Genome-wide cytosine methylation in the sugar beet genome was studied in leaves and leaf-derived callus with a focus on repetitive sequences, including retrotransposons and DNA transposons, the major groups of repetitive DNA sequences, and compared with gene methylation. Genes showed a specific methylation pattern for CG, CHG (H = A, C, and T) and CHH sites, whereas the TE pattern differed, depending on the TE class (class 1, retrotransposons and class 2, DNA transposons). Along genes and TEs, CG and CHG methylation was higher than that of adjacent genomic regions. In contrast to the relatively low CHH methylation in retrotransposons and genes, the level of CHH methylation in DNA transposons was strongly increased, pointing to a functional role of asymmetric methylation in DNA transposon silencing. Comparison of genome-wide DNA methylation between sugar beet leaves and callus revealed a differential methylation upon tissue culture. Potential epialleles were hypomethylated (lower methylation) at CG and CHG sites in retrotransposons and genes and hypermethylated (higher methylation) at CHH sites in DNA transposons of callus when compared with leaves. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

Detection of DNA Methylation by Whole-Genome Bisulfite Sequencing.

PubMed

Li, Qing; Hermanson, Peter J; Springer, Nathan M

2018-01-01

DNA methylation plays an important role in the regulation of the expression of transposons and genes. Various methods have been developed to assay DNA methylation levels. Bisulfite sequencing is considered to be the "gold standard" for single-base resolution measurement of DNA methylation levels. Coupled with next-generation sequencing, whole-genome bisulfite sequencing (WGBS) allows DNA methylation to be evaluated at a genome-wide scale. Here, we described a protocol for WGBS in plant species with large genomes. This protocol has been successfully applied to assay genome-wide DNA methylation levels in maize and barley. This protocol has also been successfully coupled with sequence capture technology to assay DNA methylation levels in a targeted set of genomic regions.

Live-Cell Imaging of DNA Methylation Based on Synthetic-Molecule/Protein Hybrid Probe.

PubMed

Kumar, Naresh; Hori, Yuichiro; Kikuchi, Kazuya

2018-06-04

The epigenetic modification of DNA involves the conversion of cytosine to 5-methylcytosine, also known as DNA methylation. DNA methylation is important in modulating gene expression and thus, regulating genome and cellular functions. Recent studies have shown that aberrations in DNA methylation are associated with various epigenetic disorders or diseases including cancer. This stimulates great interest in the development of methods that can detect and visualize DNA methylation. For instance, fluorescent proteins (FPs) in conjugation with methyl-CpG-binding domain (MBD) have been employed for live-cell imaging of DNA methylation. However, the FP-based approach showed fluorescence signals for both the DNA-bound and -unbound states and thus differentiation between these states is difficult. Synthetic-molecule/protein hybrid probes can provide an alternative to overcome this restriction. In this article, we discuss the synthetic-molecule/protein hybrid probe that we developed recently for live-cell imaging of DNA methylation, which exhibited fluorescence enhancement only after binding to methylated DNA. © 2018 The Chemical Society of Japan & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

EHMT2 directs DNA methylation for efficient gene silencing in mouse embryos

PubMed Central

Auclair, Ghislain; Borgel, Julie; Sanz, Lionel A.; Vallet, Judith; Guibert, Sylvain; Dumas, Michael; Cavelier, Patricia; Girardot, Michael; Forné, Thierry; Feil, Robert; Weber, Michael

2016-01-01

The extent to which histone modifying enzymes contribute to DNA methylation in mammals remains unclear. Previous studies suggested a link between the lysine methyltransferase EHMT2 (also known as G9A and KMT1C) and DNA methylation in the mouse. Here, we used a model of knockout mice to explore the role of EHMT2 in DNA methylation during mouse embryogenesis. The Ehmt2 gene is expressed in epiblast cells but is dispensable for global DNA methylation in embryogenesis. In contrast, EHMT2 regulates DNA methylation at specific sequences that include CpG-rich promoters of germline-specific genes. These loci are bound by EHMT2 in embryonic cells, are marked by H3K9 dimethylation, and have strongly reduced DNA methylation in Ehmt2−/− embryos. EHMT2 also plays a role in the maintenance of germline-derived DNA methylation at one imprinted locus, the Slc38a4 gene. Finally, we show that DNA methylation is instrumental for EHMT2-mediated gene silencing in embryogenesis. Our findings identify EHMT2 as a critical factor that facilitates repressive DNA methylation at specific genomic loci during mammalian development. PMID:26576615

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.).

PubMed

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 between chilling and methylation changes was observed, which suggested that chilling acquisition during dormancy in apple is likely to affect the epigenetic regulation through DNA methylation.

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.)

PubMed Central

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 between chilling and methylation changes was observed, which suggested that chilling acquisition during dormancy in apple is likely to affect the epigenetic regulation through DNA methylation. PMID:26901339

Liquid biopsy - Performance of the PAXgene® Blood ccfDNA Tubes for the isolation and characterization of cell-free plasma DNA from tumor patients.

PubMed

Schmidt, Bernd; Reinicke, Dana; Reindl, Iris; Bork, Ines; Wollschläger, Bettina; Lambrecht, Nina; Fleischhacker, Michael

2017-06-01

In most research laboratories the use of EDTA tubes for the isolation of plasma DNA from tumor patients is standard. Unfortunately these tubes do not allow for an extended storage of samples before processing and prevent EDTA tubes from being shipped at ambient temperature. The aim of our study was to compare the quantity and quality of plasma DNA isolated from EDTA and PAXgene® Blood ccfDNA Tubes in different downstream applications. We enrolled 29 patients in our study. Blood samples were drawn into EDTA and PAXgene® Blood ccfDNA Tubes and were processed on day 0 and day 7 after storage at ambient temperature. The plasma DNA from 10 patients was isolated manually. For the DNA isolation from the plasma of 19 additional patients we used the automated QIAsymphony system. The total amount DNA from all samples was measured with a quantitative real-time PCR assay. In addition the amount of methylated mSHOX2 plasma DNA was determined. While the 7day storage lead to an increased amount of total DNA in almost all EDTA tubes, this effect was only seen in very few PAXgene® Blood ccfDNA Tubes. The stabilization solution which prevents the lysis of blood cells had no effect on the method for quantification of methylated sequences in these samples. The quantity and quality of plasma DNA from both types of blood draw tubes are comparable. DNA from PAXgene® Blood ccfDNA was successfully used for PCR-based quantification of total amount of cell-free DNA and for methylation analysis as well. Copyright © 2017 Elsevier B.V. All rights reserved.

Effects of soluble and particulate Cr(VI) on genome-wide DNA methylation in human B lymphoblastoid cells.

PubMed

Lou, Jianlin; Wang, Yu; Chen, Junqiang; Ju, Li; Yu, Min; Jiang, Zhaoqiang; Feng, Lingfang; Jin, Lingzhi; Zhang, Xing

2015-10-01

Several previous studies highlighted the potential epigenetic effects of Cr(VI), especially DNA methylation. However, few studies have compared the effects of Cr(VI) on DNA methylation profiles between soluble and particulate chromate in vitro. Accordingly, Illumina Infinium Human Methylation 450K BeadChip array was used to analyze DNA methylation profiles of human B lymphoblastoid cells exposed to potassium dichromate or lead chromate, and the cell viability was also studied. Array based DNA methylation analysis showed that the impacts of Cr(VI) on DNA methylation were limited, only about 40 differentially methylated CpG sites, with an overlap of 15CpG sites, were induced by both potassium dichromate and lead chromate. The results of mRNA expression showed that after Cr(VI) treatment, mRNA expression changes of four genes (TBL1Y, FZD5, IKZF2, and KIAA1949) were consistent with their DNA methylation alteration, but DNA methylation changes of other six genes did not correlate with mRNA expression. In conclusion, both of soluble and particulate Cr(VI) could induce a small amount of differentially methylated sites in human B lymphoblastoid cells, and the correlations between DNA methylation changes and mRNA expression varied between different genes. Copyright © 2015 Elsevier B.V. All rights reserved.

Epigenetic DNA Methylation Profiling with MSRE: A Quantitative NGS Approach Using a Parkinson's Disease Test Case

PubMed Central

Marsh, Adam G.; Cottrell, Matthew T.; Goldman, Morton F.

2016-01-01

Epigenetics is a rapidly developing field focused on deciphering chemical fingerprints that accumulate on human genomes over time. As the nascent idea of precision medicine expands to encompass epigenetic signatures of diagnostic and prognostic relevance, there is a need for methodologies that provide high-throughput DNA methylation profiling measurements. Here we report a novel quantification methodology for computationally reconstructing site-specific CpG methylation status from next generation sequencing (NGS) data using methyl-sensitive restriction endonucleases (MSRE). An integrated pipeline efficiently incorporates raw NGS metrics into a statistical discrimination platform to identify functional linkages between shifts in epigenetic DNA methylation and disease phenotypes in samples being analyzed. In this pilot proof-of-concept study we quantify and compare DNA methylation in blood serum of individuals with Parkinson's Disease relative to matched healthy blood profiles. Even with a small study of only six samples, a high degree of statistical discrimination was achieved based on CpG methylation profiles between groups, with 1008 statistically different CpG sites (p < 0.0025, after false discovery rate correction). A methylation load calculation was used to assess higher order impacts of methylation shifts on genes and pathways and most notably identified FGF3, FGF8, HTT, KMTA5, MIR8073, and YWHAG as differentially methylated genes with high relevance to Parkinson's Disease and neurodegeneration (based on PubMed literature citations). Of these, KMTA5 is a histone methyl-transferase gene and HTT is Huntington Disease Protein or Huntingtin, for which there are well established neurodegenerative impacts. The future need for precision diagnostics now requires more tools for exploring epigenetic processes that may be linked to cellular dysfunction and subsequent disease progression. PMID:27853465

DNA methylation of intragenic CpG islands depends on their transcriptional activity during differentiation and disease

PubMed Central

Jeziorska, Danuta M.; Murray, Robert J. S.; De Gobbi, Marco; Gaentzsch, Ricarda; Garrick, David; Ayyub, Helena; Chen, Taiping; Li, En; Telenius, Jelena; Lynch, Magnus; Graham, Bryony; Smith, Andrew J. H.; Lund, Jonathan N.; Hughes, Jim R.; Higgs, Douglas R.

2017-01-01

The human genome contains ∼30,000 CpG islands (CGIs). While CGIs associated with promoters nearly always remain unmethylated, many of the ∼9,000 CGIs lying within gene bodies become methylated during development and differentiation. Both promoter and intragenic CGIs may also become abnormally methylated as a result of genome rearrangements and in malignancy. The epigenetic mechanisms by which some CGIs become methylated but others, in the same cell, remain unmethylated in these situations are poorly understood. Analyzing specific loci and using a genome-wide analysis, we show that transcription running across CGIs, associated with specific chromatin modifications, is required for DNA methyltransferase 3B (DNMT3B)-mediated DNA methylation of many naturally occurring intragenic CGIs. Importantly, we also show that a subgroup of intragenic CGIs is not sensitive to this process of transcription-mediated methylation and that this correlates with their individual intrinsic capacity to initiate transcription in vivo. We propose a general model of how transcription could act as a primary determinant of the patterns of CGI methylation in normal development and differentiation, and in human disease. PMID:28827334

Histone modification alteration coordinated with acquisition of promoter DNA methylation during Epstein-Barr virus infection

PubMed Central

Funata, Sayaka; Matsusaka, Keisuke; Yamanaka, Ryota; Yamamoto, Shogo; Okabe, Atsushi; Fukuyo, Masaki; Aburatani, Hiroyuki; Fukayama, Masashi; Kaneda, Atsushi

2017-01-01

Aberrant DNA hypermethylation is a major epigenetic mechanism to inactivate tumor suppressor genes in cancer. Epstein-Barr virus positive gastric cancer is the most frequently hypermethylated tumor among human malignancies. Herein, we performed comprehensive analysis of epigenomic alteration during EBV infection, by Infinium HumanMethylation 450K BeadChip for DNA methylation and ChIP-sequencing for histone modification alteration during EBV infection into gastric cancer cell line MKN7. Among 7,775 genes with increased DNA methylation in promoter regions, roughly half were “DNA methylation-sensitive” genes, which acquired DNA methylation in the whole promoter regions and thus were repressed. These included anti-oncogenic genes, e.g. CDKN2A. The other half were “DNA methylation-resistant” genes, where DNA methylation is acquired in the surrounding of promoter regions, but unmethylated status is protected in the vicinity of transcription start site. These genes thereby retained gene expression, and included DNA repair genes. Histone modification was altered dynamically and coordinately with DNA methylation alteration. DNA methylation-sensitive genes significantly correlated with loss of H3K27me3 pre-marks or decrease of active histone marks, H3K4me3 and H3K27ac. Apoptosis-related genes were significantly enriched in these epigenetically repressed genes. Gain of active histone marks significantly correlated with DNA methylation-resistant genes. Genes related to mitotic cell cycle and DNA repair were significantly enriched in these epigenetically activated genes. Our data show that orchestrated epigenetic alterations are important in gene regulation during EBV infection, and histone modification status in promoter regions significantly associated with acquisition of de novo DNA methylation or protection of unmethylated status at transcription start site. PMID:28903418

Histone modification alteration coordinated with acquisition of promoter DNA methylation during Epstein-Barr virus infection.

PubMed

Funata, Sayaka; Matsusaka, Keisuke; Yamanaka, Ryota; Yamamoto, Shogo; Okabe, Atsushi; Fukuyo, Masaki; Aburatani, Hiroyuki; Fukayama, Masashi; Kaneda, Atsushi

2017-08-15

Aberrant DNA hypermethylation is a major epigenetic mechanism to inactivate tumor suppressor genes in cancer. Epstein-Barr virus positive gastric cancer is the most frequently hypermethylated tumor among human malignancies. Herein, we performed comprehensive analysis of epigenomic alteration during EBV infection, by Infinium HumanMethylation 450K BeadChip for DNA methylation and ChIP-sequencing for histone modification alteration during EBV infection into gastric cancer cell line MKN7. Among 7,775 genes with increased DNA methylation in promoter regions, roughly half were "DNA methylation-sensitive" genes, which acquired DNA methylation in the whole promoter regions and thus were repressed. These included anti-oncogenic genes, e.g. CDKN2A . The other half were "DNA methylation-resistant" genes, where DNA methylation is acquired in the surrounding of promoter regions, but unmethylated status is protected in the vicinity of transcription start site. These genes thereby retained gene expression, and included DNA repair genes. Histone modification was altered dynamically and coordinately with DNA methylation alteration. DNA methylation-sensitive genes significantly correlated with loss of H3K27me3 pre-marks or decrease of active histone marks, H3K4me3 and H3K27ac. Apoptosis-related genes were significantly enriched in these epigenetically repressed genes. Gain of active histone marks significantly correlated with DNA methylation-resistant genes. Genes related to mitotic cell cycle and DNA repair were significantly enriched in these epigenetically activated genes. Our data show that orchestrated epigenetic alterations are important in gene regulation during EBV infection, and histone modification status in promoter regions significantly associated with acquisition of de novo DNA methylation or protection of unmethylated status at transcription start site.

DNA methylation aberrancies as a guide for surveillance and treatment of human cancers

PubMed Central

Liang, Gangning; Weisenberger, Daniel J.

2017-01-01

ABSTRACT DNA methylation aberrancies are hallmarks of human cancers and are characterized by global DNA hypomethylation of repetitive elements and non-CpG rich regions concomitant with locus-specific DNA hypermethylation. DNA methylation changes may result in altered gene expression profiles, most notably the silencing of tumor suppressors, microRNAs, endogenous retorviruses and tumor antigens due to promoter DNA hypermethylation, as well as oncogene upregulation due to gene-body DNA hypermethylation. Here, we review DNA methylation aberrancies in human cancers, their use in cancer surveillance and the interplay between DNA methylation and histone modifications in gene regulation. We also summarize DNA methylation inhibitors and their therapeutic effects in cancer treatment. In this context, we describe the integration of DNA methylation inhibitors with conventional chemotherapies, DNA repair inhibitors and immune-based therapies, to bring the epigenome closer to its normal state and increase sensitivity to other therapeutic agents to improve patient outcome and survival. PMID:28358281

Peripheral DNA methylation, cognitive decline and brain aging: pilot findings from the Whitehall II imaging study.

PubMed

Chouliaras, Leonidas; Pishva, Ehsan; Haapakoski, Rita; Zsoldos, Eniko; Mahmood, Abda; Filippini, Nicola; Burrage, Joe; Mill, Jonathan; Kivimäki, Mika; Lunnon, Katie; Ebmeier, Klaus P

2018-05-01

The present study investigated the link between peripheral DNA methylation (DNAm), cognitive impairment and brain aging. We tested the association between blood genome-wide DNAm profiles using the Illumina 450K arrays, cognitive dysfunction and brain MRI measures in selected participants of the Whitehall II imaging sub-study. Eight differentially methylated regions were associated with cognitive impairment. Accelerated aging based on the Hannum epigenetic clock was associated with mean diffusivity and global fractional anisotropy. We also identified modules of co-methylated loci associated with white matter hyperintensities. These co-methylation modules were enriched among pathways relevant to β-amyloid processing and glutamatergic signaling. Our data support the notion that blood DNAm changes may have utility as a biomarker for cognitive dysfunction and brain aging.

DNA methylation dynamics during in vivo differentiation of blood and skin stem cells

PubMed Central

Bock, Christoph; Beerman, Isabel; Lien, Wen-Hui; Smith, Zachary D.; Gu, Hongcang; Boyle, Patrick; Gnirke, Andreas; Fuchs, Elaine; Rossi, Derrick J.; Meissner, Alexander

2012-01-01

DNA methylation is a mechanism of epigenetic regulation that is common to all vertebrates. Functional studies underscore its relevance for tissue homeostasis, but the global dynamics of DNA methylation during in vivo differentiation remain underexplored. Here we report high-resolution DNA methylation maps of adult stem cell differentiation in mouse, focusing on 19 purified cell populations of the blood and skin lineages. DNA methylation changes were locus-specific and relatively modest in magnitude. They frequently overlapped with lineage-associated transcription factors and their binding sites, suggesting that DNA methylation may protect cells from aberrant transcription factor activation. DNA methylation and gene expression provided complementary information, and combining the two enabled us to infer the cellular differentiation hierarchy of the blood lineage directly from genomic data. In summary, these results demonstrate that in vivo differentiation of adult stem cells is associated with small but informative changes in the genomic distribution of DNA methylation. PMID:22841485

Emerging technologies for studying DNA methylation for the molecular diagnosis of cancer

PubMed Central

Marzese, Diego M.; Hoon, Dave S.B.

2015-01-01

DNA methylation is an epigenetic mechanism that plays a key role in regulating gene expression and other functions. Although this modification is seen in different sequence contexts, the most frequently detected DNA methylation in mammals involves cytosine-guanine dinucleotides. Pathological alterations in DNA methylation patterns are described in a variety of human diseases, including cancer. Unlike genetic changes, DNA methylation is heavily influenced by subtle modifications in the cellular microenvironment. In all cancers, aberrant DNA methylation is involved in the alteration of a large number of oncological pathways with relevant theranostic utility. Several technologies for DNA methylation mapping were recently developed and successfully applied in cancer studies. The scope of these technologies varies from assessing a single cytosine-guanine locus to genome-wide distribution of DNA methylation. Here, we review the strengths and weaknesses of these approaches in the context of clinical utility for the molecular diagnosis of human cancers. PMID:25797072

MAOA promoter methylation and susceptibility to carotid atherosclerosis: role of familial factors in a monozygotic twin sample

PubMed Central

2012-01-01

Background Atherosclerosis is a complex process involving both genetic and epigenetic factors. The monoamine oxidase A (MAOA) gene regulates the metabolism of key neurotransmitters and has been associated with cardiovascular risk factors. This study investigates whether MAOA promoter methylation is associated with atherosclerosis, and whether this association is confounded by familial factors in a monozygotic (MZ) twin sample. Methods We studied 84 monozygotic (MZ) twin pairs drawn from the Vietnam Era Twin Registry. Carotid intima-media thickness (IMT) was measured by ultrasound. DNA methylation in the MAOA promoter region was quantified by bisulfite pyrosequencing using genomic DNA isolated from peripheral blood leukocytes. The association between DNA methylation and IMT was first examined by generalized estimating equation, followed by matched pair analyses to determine whether the association was confounded by familial factors. Results When twins were analyzed as individuals, increased methylation level was associated with decreased IMT at four of the seven studied CpG sites. However, this association substantially reduced in the matched pair analyses. Further adjustment for MAOA genotype also considerably attenuated this association. Conclusions The association between MAOA promoter methylation and carotid IMT is largely explained by familial factors shared by the twins. Because twins reared together share early life experience, which may leave a long-lasting epigenetic mark, aberrant MAOA methylation may represent an early biomarker for unhealthy familial environment. Clarification of familial factors associated with DNA methylation and early atherosclerosis will provide important information to uncover clinical correlates of disease. PMID:23116433

MAOA promoter methylation and susceptibility to carotid atherosclerosis: role of familial factors in a monozygotic twin sample.

PubMed

Zhao, Jinying; Forsberg, Christopher W; Goldberg, Jack; Smith, Nicholas L; Vaccarino, Viola

2012-11-02

Atherosclerosis is a complex process involving both genetic and epigenetic factors. The monoamine oxidase A (MAOA) gene regulates the metabolism of key neurotransmitters and has been associated with cardiovascular risk factors. This study investigates whether MAOA promoter methylation is associated with atherosclerosis, and whether this association is confounded by familial factors in a monozygotic (MZ) twin sample. We studied 84 monozygotic (MZ) twin pairs drawn from the Vietnam Era Twin Registry. Carotid intima-media thickness (IMT) was measured by ultrasound. DNA methylation in the MAOA promoter region was quantified by bisulfite pyrosequencing using genomic DNA isolated from peripheral blood leukocytes. The association between DNA methylation and IMT was first examined by generalized estimating equation, followed by matched pair analyses to determine whether the association was confounded by familial factors. When twins were analyzed as individuals, increased methylation level was associated with decreased IMT at four of the seven studied CpG sites. However, this association substantially reduced in the matched pair analyses. Further adjustment for MAOA genotype also considerably attenuated this association. The association between MAOA promoter methylation and carotid IMT is largely explained by familial factors shared by the twins. Because twins reared together share early life experience, which may leave a long-lasting epigenetic mark, aberrant MAOA methylation may represent an early biomarker for unhealthy familial environment. Clarification of familial factors associated with DNA methylation and early atherosclerosis will provide important information to uncover clinical correlates of disease.

Sensitive and specific detection of early gastric cancer with DNA methylation analysis of gastric washes.

PubMed

Watanabe, Yoshiyuki; Kim, Hyun Soo; Castoro, Ryan J; Chung, Woonbok; Estecio, Marcos R H; Kondo, Kimie; Guo, Yi; Ahmed, Saira S; Toyota, Minoru; Itoh, Fumio; Suk, Ki Tae; Cho, Mee-Yon; Shen, Lanlan; Jelinek, Jaroslav; Issa, Jean-Pierre J

2009-06-01

Aberrant DNA methylation is an early and frequent process in gastric carcinogenesis and could be useful for detection of gastric neoplasia. We hypothesized that methylation analysis of DNA recovered from gastric washes could be used to detect gastric cancer. We studied 51 candidate genes in 7 gastric cancer cell lines and 24 samples (training set) and identified 6 for further studies. We examined the methylation status of these genes in a test set consisting of 131 gastric neoplasias at various stages. Finally, we validated the 6 candidate genes in a different population of 40 primary gastric cancer samples and 113 nonneoplastic gastric mucosa samples. Six genes (MINT25, RORA, GDNF, ADAM23, PRDM5, MLF1) showed frequent differential methylation between gastric cancer and normal mucosa in the training, test, and validation sets. GDNF and MINT25 were most sensitive molecular markers of early stage gastric cancer, whereas PRDM5 and MLF1 were markers of a field defect. There was a close correlation (r = 0.5-0.9, P = .03-.001) between methylation levels in tumor biopsy and gastric washes. MINT25 methylation had the best sensitivity (90%), specificity (96%), and area under the receiver operating characteristic curve (0.961) in terms of tumor detection in gastric washes. These findings suggest MINT25 is a sensitive and specific marker for screening in gastric cancer. Additionally, we have developed a new method for gastric cancer detection by DNA methylation in gastric washes.

Methionine increases BDNF DNA methylation and improves memory in epilepsy.

PubMed

Parrish, R Ryley; Buckingham, Susan C; Mascia, Katherine L; Johnson, Jarvis J; Matyjasik, Michal M; Lockhart, Roxanne M; Lubin, Farah D

2015-04-01

Temporal lobe epilepsy (TLE) patients exhibit signs of memory impairments even when seizures are pharmacologically controlled. Surprisingly, the underlying molecular mechanisms involved in TLE-associated memory impairments remain elusive. Memory consolidation requires epigenetic transcriptional regulation of genes in the hippocampus; therefore, we aimed to determine how epigenetic DNA methylation mechanisms affect learning-induced transcription of memory-permissive genes in the epileptic hippocampus. Using the kainate rodent model of TLE and focusing on the brain-derived neurotrophic factor (Bdnf) gene as a candidate of DNA methylation-mediated transcription, we analyzed DNA methylation levels in epileptic rats following learning. After detection of aberrant DNA methylation at the Bdnf gene, we investigated functional effects of altered DNA methylation on hippocampus-dependent memory formation in our TLE rodent model. We found that behaviorally driven BdnfDNA methylation was associated with hippocampus-dependent memory deficits. Bisulfite sequencing revealed that decreased BdnfDNA methylation levels strongly correlated with abnormally high levels of BdnfmRNA in the epileptic hippocampus during memory consolidation. Methyl supplementation via methionine (Met) increased BdnfDNA methylation and reduced BdnfmRNA levels in the epileptic hippocampus during memory consolidation. Met administration reduced interictal spike activity, increased theta rhythm power, and reversed memory deficits in epileptic animals. The rescue effect of Met treatment on learning-induced BdnfDNA methylation, Bdnf gene expression, and hippocampus-dependent memory, were attenuated by DNA methyltransferase blockade. Our findings suggest that manipulation of DNA methylation in the epileptic hippocampus should be considered as a viable treatment option to ameliorate memory impairments associated with TLE.

The Control Region of Mitochondrial DNA Shows an Unusual CpG and Non-CpG Methylation Pattern

PubMed Central

Bellizzi, Dina; D'Aquila, Patrizia; Scafone, Teresa; Giordano, Marco; Riso, Vincenzo; Riccio, Andrea; Passarino, Giuseppe

2013-01-01

DNA methylation is a common epigenetic modification of the mammalian genome. Conflicting data regarding the possible presence of methylated cytosines within mitochondrial DNA (mtDNA) have been reported. To clarify this point, we analysed the methylation status of mtDNA control region (D-loop) on human and murine DNA samples from blood and cultured cells by bisulphite sequencing and methylated/hydroxymethylated DNA immunoprecipitation assays. We found methylated and hydroxymethylated cytosines in the L-strand of all samples analysed. MtDNA methylation particularly occurs within non-C-phosphate-G (non-CpG) nucleotides, mainly in the promoter region of the heavy strand and in conserved sequence blocks, suggesting its involvement in regulating mtDNA replication and/or transcription. We observed DNA methyltransferases within the mitochondria, but the inactivation of Dnmt1, Dnmt3a, and Dnmt3b in mouse embryonic stem (ES) cells results in a reduction of the CpG methylation, while the non-CpG methylation shows to be not affected. This suggests that D-loop epigenetic modification is only partially established by these enzymes. Our data show that DNA methylation occurs in the mtDNA control region of mammals, not only at symmetrical CpG dinucleotides, typical of nuclear genome, but in a peculiar non-CpG pattern previously reported for plants and fungi. The molecular mechanisms responsible for this pattern remain an open question. PMID:23804556

A common mutation in the 5,10-methylenetetrahydrofolate reductase gene affects genomic DNA methylation through an interaction with folate status

PubMed Central

Friso, Simonetta; Choi, Sang-Woon; Girelli, Domenico; Mason, Joel B.; Dolnikowski, Gregory G.; Bagley, Pamela J.; Olivieri, Oliviero; Jacques, Paul F.; Rosenberg, Irwin H.; Corrocher, Roberto; Selhub, Jacob

2002-01-01

DNA methylation, an essential epigenetic feature of DNA that modulates gene expression and genomic integrity, is catalyzed by methyltransferases that use the universal methyl donor S-adenosyl-l-methionine. Methylenetetrahydrofolate reductase (MTHFR) catalyzes the synthesis of 5-methyltetrahydrofolate (5-methylTHF), the methyl donor for synthesis of methionine from homocysteine and precursor of S-adenosyl-l-methionine. In the present study we sought to determine the effect of folate status on genomic DNA methylation with an emphasis on the interaction with the common C677T mutation in the MTHFR gene. A liquid chromatography/MS method for the analysis of nucleotide bases was used to assess genomic DNA methylation in peripheral blood mononuclear cell DNA from 105 subjects homozygous for this mutation (T/T) and 187 homozygous for the wild-type (C/C) MTHFR genotype. The results show that genomic DNA methylation directly correlates with folate status and inversely with plasma homocysteine (tHcy) levels (P < 0.01). T/T genotypes had a diminished level of DNA methylation compared with those with the C/C wild-type (32.23 vs.62.24 ng 5-methylcytosine/μg DNA, P < 0.0001). When analyzed according to folate status, however, only the T/T subjects with low levels of folate accounted for the diminished DNA methylation (P < 0.0001). Moreover, in T/T subjects DNA methylation status correlated with the methylated proportion of red blood cell folate and was inversely related to the formylated proportion of red blood cell folates (P < 0.03) that is known to be solely represented in those individuals. These results indicate that the MTHFR C677T polymorphism influences DNA methylation status through an interaction with folate status. PMID:11929966

Decoding the Regulatory Landscape of Ageing in Musculoskeletal Engineered Tissues Using Genome-Wide DNA Methylation and RNASeq

PubMed Central

Peffers, Mandy Jayne; Goljanek-Whysall, Katarzyna; Collins, John; Fang, Yongxiang; Rushton, Michael; Loughlin, John; Proctor, Carole; Clegg, Peter David

2016-01-01

Mesenchymal stem cells (MSC) are capable of multipotent differentiation into connective tissues and as such are an attractive source for autologous cell-based regenerative medicine and tissue engineering. Epigenetic mechanisms, like DNA methylation, contribute to the changes in gene expression in ageing. However there was a lack of sufficient knowledge of the role that differential methylation plays during chondrogenic, osteogenic and tenogenic differentiation from ageing MSCs. This study undertook genome level determination of the effects of DNA methylation on expression in engineered tissues from chronologically aged MSCs. We compiled unique DNA methylation signatures from chondrogenic, osteogenic, and tenogenic engineered tissues derived from young; n = 4 (21.8 years ± 2.4 SD) and old; n = 4 (65.5 years±8.3SD) human MSCs donors using the Illumina HumanMethylation 450 Beadchip arrays and compared these to gene expression by RNA sequencing. Unique and common signatures of global DNA methylation were identified. There were 201, 67 and 32 chondrogenic, osteogenic and tenogenic age-related DE protein-coding genes respectively. Findings inferred the nature of the transcript networks was predominantly for ‘cell death and survival’, ‘cell morphology’, and ‘cell growth and proliferation’. Further studies are required to validate if this gene expression effect translates to cell events. Alternative splicing (AS) was dysregulated in ageing with 119, 21 and 9 differential splicing events identified in chondrogenic, osteogenic and tenogenic respectively, and enrichment in genes associated principally with metabolic processes. Gene ontology analysis of differentially methylated loci indicated age-related enrichment for all engineered tissue types in ‘skeletal system morphogenesis’, ‘regulation of cell proliferation’ and ‘regulation of transcription’ suggesting that dynamic epigenetic modifications may occur in genes associated with shared and distinct pathways dependent upon engineered tissue type. An altered phenotype in engineered tissues was observed with ageing at numerous levels. These changes represent novel insights into the ageing process, with implications for stem cell therapies in older patients. In addition we have identified a number of tissue-dependant pathways, which warrant further studies. PMID:27533049

A fluorescence method for detection of DNA and DNA methylation based on graphene oxide and restriction endonuclease HpaII.

PubMed

Wei, Wei; Gao, Chunyan; Xiong, Yanxiang; Zhang, Yuanjian; Liu, Songqin; Pu, Yuepu

2015-01-01

DNA methylation plays an important role in many biological events and is associated with various diseases. Most traditional methods for detection of DNA methylation are based on the complex and expensive bisulfite method. In this paper, we report a novel fluorescence method to detect DNA and DNA methylation based on graphene oxide (GO) and restriction endonuclease HpaII. The skillfully designed probe DNA labeled with 5-carboxyfluorescein (FAM) and optimized GO concentration keep the probe/target DNA still adsorbed on the GO. After the cleavage action of HpaII the labeled FAM is released from the GO surface and its fluorescence recovers, which could be used to detect DNA in the linear range of 50 pM-50 nM with a detection limit of 43 pM. DNA methylation induced by transmethylase (Mtase) or other chemical reagents prevents HpaII from recognizing and cleaving the specific site; as a result, fluorescence cannot recover. The fluorescence recovery efficiency is closely related to the DNA methylation level, which can be used to detect DNA methylation by comparing it with the fluorescence in the presence of intact target DNA. The method for detection of DNA and DNA methylation is simple, reliable and accurate. Copyright © 2014 Elsevier B.V. All rights reserved.

Spectroscopic studies of STZ-induced methylated-DNA in both in vivo and in vitro conditions

NASA Astrophysics Data System (ADS)

Bathaie, S. Z.; Sedghgoo, F.; Jafarnejad, A.; Farzami, B.; Khayatian, M.

2008-12-01

Alkylating agents after formation of DNA adduct not only posses their harmful role on living cells but also can transfer this information to the next generation. Different techniques have been introduced to study the alkylated DNA, most of which are specific and designed for investigation of specific target DNA. But the exact differences between spectroscopic and functional properties of alkylated DNA are not seen in the literature. In the present study DNA was methylated using streptozotocin (STZ) by both in vitro and in vivo protocols, then methylated-DNA was investigated by various techniques. Our results show that (1) the binding of ethidium bromide as an intercalating dye decreases to methylated-DNA in comparison with normal DNA, (2) CD spectra of methylated-DNA show changes including a decrease in the positive band at 275 nm and a shift from 258 nm crossover to a longer wavelength, which is caused by reduction of water around it, due to the presence of additional hydrophobic methyl groups, (3) the stability of methylated-DNA against DTAB as a denaturant is decreased and (4) the enzyme-like activity of methylated-DNA in an electron transfer reaction is reduced. In conclusion, additional methyl groups not only protrude water around DNA, but also cause the loss of hydrogen bonding, loosening of conformation, preventing desired interactions and thus normal function of DNA.

Effects of the led therapy on the global DNA methylation and the expression of Dnmt1 and Dnmt3a genes in a rat model of skin wound healing.

PubMed

Gomes, Marcus Vinícius de Matos; Manfredo, Marcelo Henrique; Toffoli, Leandro Vaz; Castro-Alves, Daniellen Christine; do Nascimento, Lucas Magnoni; da Silva, Wyllian Rafael; Kashimoto, Roberto Kiyoshi; Rodrigues, Gelson Marcos; Estrada, Viviane Batista; Andraus, Rodrigo Antonio; Pelosi, Gislaine Garcia

2016-09-01

The use of light emitting diodes (LED) as a therapeutic resource for wound healing has increased over the last years; however, little is still known about the molecular pathways associated to LED exposure. In the present study, we verified the effects of LED therapy on DNA methylation and expression of the DNA methyltransferase (Dnmt) genes, Dnmt1 and Dnmt3a, in an in vivo model of epithelial wound healing. Male Wistar rats were submitted to epithelial excision in the dorsal region and subsequently distributed within the experimental groups: group 1, animals that received irradiation of 0.8 J/cm(2) of LED (604 nm); group 2, animals that received 1.6 J/cm(2) of LED (604 nm); control (CTL), animals not submitted to therapeutic intervention. LED applications were performed during 7 days, and tissues from the periphery of the wound area were obtained for molecular analysis. The Image-J software was used for analysis of the wound area. DNA methylation was evaluated by ELISA-based method and gene expressions were quantified by real-time PCR. Decrease on global DNA methylation profile was observed in all experimental groups (CTL, 1, and 2) revealing the participation of DNA methylation in the healing process. Significant decrease in the wound area accompanied by increase in the Dnmt3a expression was associated to group 2. Based on our findings, we propose that DNA methylation is an important molecular mechanism associated to wound healing and that irradiation with 1.6 J/cm(2) of LED evokes an increase in the expression of the Dnmt3a that might associates to the efficiency of the epithelial wound healing.

Age-related DNA methylation changes for forensic age-prediction.

PubMed

Yi, Shao Hua; Jia, Yun Shu; Mei, Kun; Yang, Rong Zhi; Huang, Dai Xin

2015-03-01

There is no available method of age-prediction for biological samples. The accumulating evidences indicate that DNA methylation patterns change with age. Aging resembles a developmentally regulated process that is tightly controlled by specific epigenetic modifications and age-associated methylation changes exist in human genome. In this study, three age-related methylation fragments were isolated and identified in blood of 40 donors. Age-related methylation changes with each fragment was validated and replicated in a general population sample of 65 donors over a wide age range (11-72 years). Methylation of these fragments is linearly correlated with age over a range of six decades (r = 0.80-0.88). Using average methylation of CpG sites of three fragments, a regression model that explained 95 % of the variance in age was built and is able to predict an individual's age with great accuracy (R (2 )= 0.93). The predicted value is highly correlated with the observed age in the sample (r = 0.96) and has great accuracy of average 4 years difference between predicted age and true age. This study implicates that DNA methylation can be an available biological marker of age-prediction. Further measurement of relevant markers in the genome could be a tool in routine screening to predict age of forensic biological samples.

A parallel and sensitive software tool for methylation analysis on multicore platforms.

PubMed

Tárraga, Joaquín; Pérez, Mariano; Orduña, Juan M; Duato, José; Medina, Ignacio; Dopazo, Joaquín

2015-10-01

DNA methylation analysis suffers from very long processing time, as the advent of Next-Generation Sequencers has shifted the bottleneck of genomic studies from the sequencers that obtain the DNA samples to the software that performs the analysis of these samples. The existing software for methylation analysis does not seem to scale efficiently neither with the size of the dataset nor with the length of the reads to be analyzed. As it is expected that the sequencers will provide longer and longer reads in the near future, efficient and scalable methylation software should be developed. We present a new software tool, called HPG-Methyl, which efficiently maps bisulphite sequencing reads on DNA, analyzing DNA methylation. The strategy used by this software consists of leveraging the speed of the Burrows-Wheeler Transform to map a large number of DNA fragments (reads) rapidly, as well as the accuracy of the Smith-Waterman algorithm, which is exclusively employed to deal with the most ambiguous and shortest reads. Experimental results on platforms with Intel multicore processors show that HPG-Methyl significantly outperforms in both execution time and sensitivity state-of-the-art software such as Bismark, BS-Seeker or BSMAP, particularly for long bisulphite reads. Software in the form of C libraries and functions, together with instructions to compile and execute this software. Available by sftp to anonymous@clariano.uv.es (password 'anonymous'). juan.orduna@uv.es or jdopazo@cipf.es. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Inhalation of diesel exhaust and allergen alters human bronchial epithelium DNA methylation.

PubMed

Clifford, Rachel L; Jones, Meaghan J; MacIsaac, Julia L; McEwen, Lisa M; Goodman, Sarah J; Mostafavi, Sara; Kobor, Michael S; Carlsten, Chris

2017-01-01

Allergic disease affects 30% to 40% of the world's population, and its development is determined by the interplay between environmental and inherited factors. Air pollution, primarily consisting of diesel exhaust emissions, has increased at a similar rate to allergic disease. Exposure to diesel exhaust may play a role in the development and progression of allergic disease, in particular allergic respiratory disease. One potential mechanism underlying the connection between air pollution and increased allergic disease incidence is DNA methylation, an epigenetic process with the capacity to integrate gene-environment interactions. We sought to investigate the effect of allergen and diesel exhaust exposure on bronchial epithelial DNA methylation. We performed a randomized crossover-controlled exposure study to allergen and diesel exhaust in humans, and measured single-site (CpG) resolution global DNA methylation in bronchial epithelial cells. Exposure to allergen alone, diesel exhaust alone, or allergen and diesel exhaust together (coexposure) led to significant changes in 7 CpG sites at 48 hours. However, when the same lung was exposed to allergen and diesel exhaust but separated by approximately 4 weeks, significant changes in more than 500 sites were observed. Furthermore, sites of differential methylation differed depending on which exposure was experienced first. Functional analysis of differentially methylated CpG sites found genes involved in transcription factor activity, protein metabolism, cell adhesion, and vascular development, among others. These findings suggest that specific exposures can prime the lung for changes in DNA methylation induced by a subsequent insult. Copyright © 2016 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

In vivo targeting of de novo DNA methylation by histone modifications in yeast and mouse

PubMed Central

Morselli, Marco; Pastor, William A; Montanini, Barbara; Nee, Kevin; Ferrari, Roberto; Fu, Kai; Bonora, Giancarlo; Rubbi, Liudmilla; Clark, Amander T; Ottonello, Simone; Jacobsen, Steven E; Pellegrini, Matteo

2015-01-01

Methylation of cytosines (5meC) is a widespread heritable DNA modification. During mammalian development, two global demethylation events are followed by waves of de novo DNA methylation. In vivo mechanisms of DNA methylation establishment are largely uncharacterized. Here, we use Saccharomyces cerevisiae as a system lacking DNA methylation to define the chromatin features influencing the activity of the murine DNMT3B. Our data demonstrate that DNMT3B and H3K4 methylation are mutually exclusive and that DNMT3B is co-localized with H3K36 methylated regions. In support of this observation, DNA methylation analysis in yeast strains without Set1 and Set2 shows an increase of relative 5meC levels at the transcription start site and a decrease in the gene-body, respectively. We extend our observation to the murine male germline, where H3K4me3 is strongly anti-correlated while H3K36me3 correlates with accelerated DNA methylation. These results show the importance of H3K36 methylation for gene-body DNA methylation in vivo. DOI: http://dx.doi.org/10.7554/eLife.06205.001 PMID:25848745

Dynamic DNA methylation reconfiguration during seed development and germination.

PubMed

Kawakatsu, Taiji; Nery, Joseph R; Castanon, Rosa; Ecker, Joseph R

2017-09-15

Unlike animals, plants can pause their life cycle as dormant seeds. In both plants and animals, DNA methylation is involved in the regulation of gene expression and genome integrity. In animals, reprogramming erases and re-establishes DNA methylation during development. However, knowledge of reprogramming or reconfiguration in plants has been limited to pollen and the central cell. To better understand epigenetic reconfiguration in the embryo, which forms the plant body, we compared time-series methylomes of dry and germinating seeds to publicly available seed development methylomes. Time-series whole genome bisulfite sequencing reveals extensive gain of CHH methylation during seed development and drastic loss of CHH methylation during germination. These dynamic changes in methylation mainly occur within transposable elements. Active DNA methylation during seed development depends on both RNA-directed DNA methylation and heterochromatin formation pathways, whereas global demethylation during germination occurs in a passive manner. However, an active DNA demethylation pathway is initiated during late seed development. This study provides new insights into dynamic DNA methylation reprogramming events during seed development and germination and suggests possible mechanisms of regulation. The observed sequential methylation/demethylation cycle suggests an important role of DNA methylation in seed dormancy.

Global methylation screening in the Arabidopsis thaliana and Mus musculus genome: applications of virtual image restriction landmark genomic scanning (Vi-RLGS)

PubMed Central

Matsuyama, Tomoki; Kimura, Makoto T.; Koike, Kuniaki; Abe, Tomoko; Nakano, Takeshi; Asami, Tadao; Ebisuzaki, Toshikazu; Held, William A.; Yoshida, Shigeo; Nagase, Hiroki

2003-01-01

Understanding the role of ‘epigenetic’ changes such as DNA methylation and chromatin remodeling has now become critical in understanding many biological processes. In order to delineate the global methylation pattern in a given genomic DNA, computer software has been developed to create a virtual image of restriction landmark genomic scanning (Vi-RLGS). When using a methylation- sensitive enzyme such as NotI as the restriction landmark, the comparison between real and in silico RLGS profiles of the genome provides a methylation map of genomic NotI sites. A methylation map of the Arabidopsis genome was created that could be confirmed by a methylation-sensitive PCR assay. The method has also been applied to the mouse genome. Although a complete methylation map has not been completed, a region of methylation difference between two tissues has been tested and confirmed by bisulfite sequencing. Vi-RLGS in conjunction with real RLGS will make it possible to develop a more complete map of genomic sites that are methylated or demethylated as a consequence of normal or abnormal development. PMID:12888509

Protection of CpG islands from DNA methylation is DNA-encoded and evolutionarily conserved

PubMed Central

Long, Hannah K.; King, Hamish W.; Patient, Roger K.; Odom, Duncan T.; Klose, Robert J.

2016-01-01

DNA methylation is a repressive epigenetic modification that covers vertebrate genomes. Regions known as CpG islands (CGIs), which are refractory to DNA methylation, are often associated with gene promoters and play central roles in gene regulation. Yet how CGIs in their normal genomic context evade the DNA methylation machinery and whether these mechanisms are evolutionarily conserved remains enigmatic. To address these fundamental questions we exploited a transchromosomic animal model and genomic approaches to understand how the hypomethylated state is formed in vivo and to discover whether mechanisms governing CGI formation are evolutionarily conserved. Strikingly, insertion of a human chromosome into mouse revealed that promoter-associated CGIs are refractory to DNA methylation regardless of host species, demonstrating that DNA sequence plays a central role in specifying the hypomethylated state through evolutionarily conserved mechanisms. In contrast, elements distal to gene promoters exhibited more variable methylation between host species, uncovering a widespread dependence on nucleotide frequency and occupancy of DNA-binding transcription factors in shaping the DNA methylation landscape away from gene promoters. This was exemplified by young CpG rich lineage-restricted repeat sequences that evaded DNA methylation in the absence of co-evolved mechanisms targeting methylation to these sequences, and species specific DNA binding events that protected against DNA methylation in CpG poor regions. Finally, transplantation of mouse chromosomal fragments into the evolutionarily distant zebrafish uncovered the existence of a mechanistically conserved and DNA-encoded logic which shapes CGI formation across vertebrate species. PMID:27084945

Epigenetic Studies Point to DNA Replication/Repair Genes as a Basis for the Heritable Nature of Long Term Complications in Diabetes.

PubMed

Leontovich, Alexey A; Intine, Robert V; Sarras, Michael P

2016-01-01

Metabolic memory (MM) is defined as the persistence of diabetic (DM) complications even after glycemic control is pharmacologically achieved. Using a zebrafish diabetic model that induces a MM state, we previously reported that, in this model, tissue dysfunction was of a heritable nature based on cell proliferation studies in limb tissue and this correlated with epigenetic DNA methylation changes that paralleled alterations in gene expression. In the current study, control, DM, and MM excised fin tissues were further analyzed by MeDIP sequencing and microarray techniques. Bioinformatics analysis of the data found that genes of the DNA replication/DNA metabolism process group (with upregulation of the apex1, mcm2, mcm4, orc3, lig1, and dnmt1 genes) were altered in the DM state and these molecular changes continued into MM. Interestingly, DNA methylation changes could be found as far as 6-13 kb upstream of the transcription start site for these genes suggesting potential higher levels of epigenetic control. In conclusion, DNA methylation changes in members of the DNA replication/repair process group best explain the heritable nature of cell proliferation impairment found in the zebrafish DM/MM model. These results are consistent with human diabetic epigenetic studies and provide one explanation for the persistence of long term tissue complications as seen in diabetes.

Global changes in DNA methylation in Alzheimer's disease peripheral blood mononuclear cells.

PubMed

Di Francesco, Andrea; Arosio, Beatrice; Falconi, Anastasia; Micioni Di Bonaventura, Maria Vittoria; Karimi, Mohsen; Mari, Daniela; Casati, Martina; Maccarrone, Mauro; D'Addario, Claudio

2015-03-01

Changes in epigenetic marks may help explain the late onset of Alzheimer's disease (AD). In this study we measured genome-wide DNA methylation by luminometric methylation assay, a quantitative measurement of genome-wide DNA methylation, on DNA isolated from peripheral blood mononuclear cells of 37 subjects with late-onset AD (LOAD) and 44 healthy controls (CT). We found an increase in global DNA methylation in LOAD subjects compared to CT (p=0.0122), associated with worse cognitive performances (p=0.0002). DNA hypermethylation in LOAD group was paralleled by higher DNA methyltransferase 1 (DNMT1) gene expression and protein levels. When data were stratified on the basis of the APOE polymorphisms, higher DNA methylation levels were associated with the presence of APOE ε4 allele (p=0.0043) in the global population. Among the APOE ε3 carriers, a significant increase of DNA methylation was still observed in LOAD patients compared to healthy controls (p=0.05). Our data suggest global DNA methylation in peripheral samples as a useful marker for screening individuals at risk of developing AD. Copyright © 2014 Elsevier Inc. All rights reserved.

Can 5-methylcytosine analogues with extended alkyl side chains guide DNA methylation?

PubMed

Kotandeniya, D; Seiler, C L; Fernandez, J; Pujari, S S; Curwick, L; Murphy, K; Wickramaratne, S; Yan, S; Murphy, D; Sham, Yuk Y; Tretyakova, N Y

2018-01-25

5-Methylcytosine ( Me C) is an endogenous modification of DNA that plays a crucial role in DNA-protein interactions, chromatin structure, epigenetic regulation, and DNA repair. Me C is produced via enzymatic methylation of the C-5 position of cytosine by DNA-methyltransferases (DNMT) which use S-adenosylmethionine (SAM) as a cofactor. Hemimethylated CG dinucleotides generated as a result of DNA replication are specifically recognized and methylated by maintenance DNA methyltransferase 1 (DNMT1). The accuracy of DNMT1-mediated methylation is essential for preserving tissue-specific DNA methylation and thus gene expression patterns. In the present study, we synthesized DNA duplexes containing MeC analogues with modified C-5 side chains and examined their ability to guide cytosine methylation by the human DNMT1 protein. We found that the ability of 5-alkylcytosines to direct cytosine methylation decreased with increased alkyl chain length and rigidity (methyl > ethyl > propyl ∼ vinyl). Molecular modeling studies indicated that this loss of activity may be caused by the distorted geometry of the DNA-protein complex in the presence of unnatural alkylcytosines.

A 5-mC Dot Blot Assay Quantifying the DNA Methylation Level of Chondrocyte Dedifferentiation In Vitro.

PubMed

Jia, Zhaofeng; Liang, Yujie; Ma, Bin; Xu, Xiao; Xiong, Jianyi; Duan, Li; Wang, Daping

2017-05-17

The dedifferentiation of hyaline chondrocytes into fibroblastic chondrocytes often accompanies monolayer expansion of chondrocytes in vitro. The global DNA methylation level of chondrocytes is considered to be a suitable biomarker for the loss of the chondrocyte phenotype. However, results based on different experimental methods can be inconsistent. Therefore, it is important to establish a precise, simple, and rapid method to quantify global DNA methylation levels during chondrocyte dedifferentiation. Current genome-wide methylation analysis techniques largely rely on bisulfite genomic sequencing. Due to DNA degradation during bisulfite conversion, these methods typically require a large sample volume. Other methods used to quantify global DNA methylation levels include high-performance liquid chromatography (HPLC). However, HPLC requires complete digestion of genomic DNA. Additionally, the prohibitively high cost of HPLC instruments limits HPLC's wider application. In this study, genomic DNA (gDNA) was extracted from human chondrocytes cultured with varying number of passages. The gDNA methylation level was detected using a methylation-specific dot blot assay. In this dot blot approach, a gDNA mixture containing the methylated DNA to be detected was spotted directly onto an N + membrane as a dot inside a previously drawn circular template pattern. Compared with other gel electrophoresis-based blotting approaches and other complex blotting procedures, the dot blot method saves significant time. In addition, dot blots can detect overall DNA methylation level using a commercially available 5-mC antibody. We found that the DNA methylation level differed between the monolayer subcultures, and therefore could play a key role in chondrocyte dedifferentiation. The 5-mC dot blot is a reliable, simple, and rapid method to detect the general DNA methylation level to evaluate chondrocyte phenotype.

Methylation-sensitive enrichment of minor DNA alleles using a double-strand DNA-specific nuclease.

PubMed

Liu, Yibin; Song, Chen; Ladas, Ioannis; Fitarelli-Kiehl, Mariana; Makrigiorgos, G Mike

2017-04-07

Aberrant methylation changes, often present in a minor allelic fraction in clinical samples such as plasma-circulating DNA (cfDNA), are potentially powerful prognostic and predictive biomarkers in human disease including cancer. We report on a novel, highly-multiplexed approach to facilitate analysis of clinically useful methylation changes in minor DNA populations. Methylation Specific Nuclease-assisted Minor-allele Enrichment (MS-NaME) employs a double-strand-specific DNA nuclease (DSN) to remove excess DNA with normal methylation patterns. The technique utilizes oligonucleotide-probes that direct DSN activity to multiple targets in bisulfite-treated DNA, simultaneously. Oligonucleotide probes targeting unmethylated sequences generate local double stranded regions resulting to digestion of unmethylated targets, and leaving methylated targets intact; and vice versa. Subsequent amplification of the targeted regions results in enrichment of the targeted methylated or unmethylated minority-epigenetic-alleles. We validate MS-NaME by demonstrating enrichment of RARb2, ATM, MGMT and GSTP1 promoters in multiplexed MS-NaME reactions (177-plex) using dilutions of methylated/unmethylated DNA and in DNA from clinical lung cancer samples and matched normal tissue. MS-NaME is a highly scalable single-step approach performed at the genomic DNA level in solution that combines with most downstream detection technologies including Sanger sequencing, methylation-sensitive-high-resolution melting (MS-HRM) and methylation-specific-Taqman-based-digital-PCR (digital Methylight) to boost detection of low-level aberrant methylation-changes. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Platinum-Based Chemotherapy Induces Methylation Changes in Blood DNA Associated with Overall Survival in Patients with Ovarian Cancer.

PubMed

Flanagan, James M; Wilson, Angela; Koo, Chail; Masrour, Nahal; Gallon, John; Loomis, Erick; Flower, Kirsty; Wilhelm-Benartzi, Charlotte; Hergovich, Alexander; Cunnea, Paula; Gabra, Hani; Braicu, Elena Ioana; Sehouli, Jalid; Darb-Esfahani, Silvia; Vanderstichele, Adriaan; Vergote, Ignace; Kreuzinger, Caroline; Castillo-Tong, Dan Cacsire; Wisman, G Bea A; Berns, Els Mjj; Siddiqui, Nadeem; Paul, James; Brown, Robert

2017-05-01

Purpose: DNA damage repair can lead to epigenetic changes. DNA mismatch repair proteins bind to platinum DNA adducts and at sites of DNA damage can recruit the DNA methylating enzyme DNMT1, resulting in aberrant methylation. We hypothesised that DNA damage repair during platinum-based chemotherapy may cause aberrant DNA methylation in normal tissues of patients such as blood. Experimental Design: We used Illumina 450k methylation arrays and bisulphite pyrosequencing to investigate methylation at presentation and relapse in blood DNA from patients with ovarian cancer enrolled in the SCOTROC1 trial ( n = 247) and in a cohort of ovarian tumor DNA samples collected at first relapse ( n = 46). We used an ovarian cancer cell line model to investigate the role of the DNA mismatch repair gene MLH1 in platinum-induced methylation changes. Results: Specific CpG methylation changes in blood at relapse are observed following platinum-based chemotherapy and are associated with patient survival, independent of other clinical factors [hazard ratio, 3.7; 95% confidence interval, 1.8-7.6, P = 2.8 × 10 -4 ]. Similar changes occur in ovarian tumors at relapse, also associated with patient survival (hazard ratio, 2.6; 95% confidence interval, 1.0-6.8, P = 0.048). Using an ovarian cancer cell line model, we demonstrate that functional mismatch repair increases the frequency of platinum-induced methylation. Conclusions: DNA methylation in blood at relapse following chemotherapy, and not at presentation, is informative regarding survival of patients with ovarian cancer. Functional DNA mismatch repair increases the frequency of DNA methylation changes induced by platinum. DNA methylation in blood following chemotherapy could provide a noninvasive means of monitoring patients' epigenetic responses to treatment without requiring a tumor biopsy. Clin Cancer Res; 23(9); 2213-22. ©2016 AACR . ©2016 American Association for Cancer Research.

Methylsorb: a simple method for quantifying DNA methylation using DNA-gold affinity interactions.

PubMed

Sina, Abu Ali Ibn; Carrascosa, Laura G; Palanisamy, Ramkumar; Rauf, Sakandar; Shiddiky, Muhammad J A; Trau, Matt

2014-10-21

The analysis of DNA methylation is becoming increasingly important both in the clinic and also as a research tool to unravel key epigenetic molecular mechanisms in biology. Current methodologies for the quantification of regional DNA methylation (i.e., the average methylation over a region of DNA in the genome) are largely affected by comprehensive DNA sequencing methodologies which tend to be expensive, tedious, and time-consuming for many applications. Herein, we report an alternative DNA methylation detection method referred to as "Methylsorb", which is based on the inherent affinity of DNA bases to the gold surface (i.e., the trend of the affinity interactions is adenine > cytosine ≥ guanine > thymine).1 Since the degree of gold-DNA affinity interaction is highly sequence dependent, it provides a new capability to detect DNA methylation by simply monitoring the relative adsorption of bisulfite treated DNA sequences onto a gold chip. Because the selective physical adsorption of DNA fragments to gold enable a direct read-out of regional DNA methylation, the current requirement for DNA sequencing is obviated. To demonstrate the utility of this method, we present data on the regional methylation status of two CpG clusters located in the EN1 and MIR200B genes in MCF7 and MDA-MB-231 cells. The methylation status of these regions was obtained from the change in relative mass on gold surface with respect to relative adsorption of an unmethylated DNA source and this was detected using surface plasmon resonance (SPR) in a label-free and real-time manner. We anticipate that the simplicity of this method, combined with the high level of accuracy for identifying the methylation status of cytosines in DNA, could find broad application in biology and diagnostics.

Polycyclic aromatic hydrocarbon (PAH)-DNA adducts and breast cancer: modification by gene promoter methylation in a population-based study.

PubMed

White, Alexandra J; Chen, Jia; McCullough, Lauren E; Xu, Xinran; Cho, Yoon Hee; Teitelbaum, Susan L; Neugut, Alfred I; Terry, Mary Beth; Hibshoosh, Hanina; Santella, Regina M; Gammon, Marilie D

2015-12-01

Polycyclic aromatic hydrocarbon (PAH)-DNA adducts have been associated with breast cancer incidence. Aberrant changes in DNA methylation may be an early event in carcinogenesis. However, possible relations between PAH-DNA adducts, methylation, and breast cancer are unknown. The objectives of this study were to (1) assess associations between PAH-DNA adducts, and breast cancer, stratified by DNA methylation markers and (2) examine interactions between adducts and DNA methylation in association with breast cancer and tumor subtype. In a population-based case-control study, promoter methylation of 13 breast cancer-related genes was measured in tumor tissue (n = 765-851 cases). Blood DNA from breast cancer cases (n = 873) and controls (n = 941) was used to assess PAH-DNA adducts and global methylation. Logistic regression was used to estimate adjusted odds ratios (ORs) and 95% confidence intervals (CI); and the ratio of the OR (ROR) was used to assess heterogeneity. Women with detectable PAH-DNA adducts and methylated RARβ (ROR 2.69, 95% CI 1.02-7.12; p for interaction = 0.03) or APC (ROR 1.76, 95% CI 0.87-3.58; p for interaction = 0.09) genes were more likely to have hormone receptor-positive tumors than other subtypes. Interactions with other methylation markers were not apparent (p ≥ 0.10). The association between adducts and breast cancer did not vary by methylation status of the tumor nor did adducts associate with global methylation in the controls. Gene-specific methylation of RARβ, and perhaps APC, may interact with PAH-DNA adducts to increase risk of hormone receptor-positive breast cancer. There was little evidence that adducts were associated with or interacted with other methylation markers of interest.

Understanding the relationship between DNA methylation and histone lysine methylation☆

PubMed Central

Rose, Nathan R.; Klose, Robert J.

2014-01-01

DNA methylation acts as an epigenetic modification in vertebrate DNA. Recently it has become clear that the DNA and histone lysine methylation systems are highly interrelated and rely mechanistically on each other for normal chromatin function in vivo. Here we examine some of the functional links between these systems, with a particular focus on several recent discoveries suggesting how lysine methylation may help to target DNA methylation during development, and vice versa. In addition, the emerging role of non-methylated DNA found in CpG islands in defining histone lysine methylation profiles at gene regulatory elements will be discussed in the context of gene regulation. This article is part of a Special Issue entitled: Methylation: A Multifaceted Modification — looking at transcription and beyond. PMID:24560929

Impact of folic acid fortification on global DNA methylation and one-carbon biomarkers in the Women's Health Initiative Observational Study cohort.

PubMed

Bae, Sajin; Ulrich, Cornelia M; Bailey, Lynn B; Malysheva, Olga; Brown, Elissa C; Maneval, David R; Neuhouser, Marian L; Cheng, Ting-Yuan David; Miller, Joshua W; Zheng, Yingye; Xiao, Liren; Hou, Lifang; Song, Xiaoling; Buck, Katharina; Beresford, Shirley A A; Caudill, Marie A

2014-03-01

DNA methylation is an epigenetic mechanism that regulates gene expression and can be modified by one-carbon nutrients. The objective of this study was to investigate the impact of folic acid (FA) fortification of the US food supply on leukocyte global DNA methylation and the relationship between DNA methylation, red blood cell (RBC) folate, and other one-carbon biomarkers among postmenopausal women enrolled in the Women's Health Initiative Observational Study. We selected 408 women from the highest and lowest tertiles of RBC folate distribution matching on age and timing of the baseline blood draw, which spanned the pre- (1994-1995), peri- (1996-1997), or post-fortification (1998) periods. Global DNA methylation was assessed by liquid chromatography-tandem mass spectrometry and expressed as a percentage of total cytosine. We observed an interaction (P = 0.02) between fortification period and RBC folate in relation to DNA methylation. Women with higher (vs. lower) RBC folate had higher mean DNA methylation (5.12 vs. 4.99%; P = 0.05) in the pre-fortification period, but lower (4.95 vs. 5.16%; P = 0.03) DNA methylation in the post-fortification period. We also observed significant correlations between one-carbon biomarkers and DNA methylation in the pre-fortification period, but not in the peri- or post-fortification period. The correlation between plasma homocysteine and DNA methylation was reversed from an inverse relationship during the pre-fortification period to a positive relationship during the post-fortification period. Our data suggest that (1) during FA fortification, higher RBC folate status is associated with a reduction in leukocyte global DNA methylation among postmenopausal women and; (2) the relationship between one-carbon biomarkers and global DNA methylation is dependent on folate availability.

Densely ionizing radiation affects DNA methylation of selective LINE-1 elements

DOE Office of Scientific and Technical Information (OSTI.GOV)

Prior, Sara; Miousse, Isabelle R.

Long Interspersed Nucleotide Element 1 (LINE-1) retrotransposons are heavily methylated and are the most abundant transposable elements in mammalian genomes. Here, we investigated the differential DNA methylation within the LINE-1 under normal conditions and in response to environmentally relevant doses of sparsely and densely ionizing radiation. We demonstrate that DNA methylation of LINE-1 elements in the lungs of C57BL6 mice is dependent on their evolutionary age, where the elder age of the element is associated with the lower extent of DNA methylation. Exposure to 5-aza-2′-deoxycytidine and methionine-deficient diet affected DNA methylation of selective LINE-1 elements in an age- and promotermore » type-dependent manner. Exposure to densely IR, but not sparsely IR, resulted in DNA hypermethylation of older LINE-1 elements, while the DNA methylation of evolutionary younger elements remained mostly unchanged. We also demonstrate that exposure to densely IR increased mRNA and protein levels of LINE-1 via the loss of the histone H3K9 dimethylation and an increase in the H3K4 trimethylation at the LINE-1 5′-untranslated region, independently of DNA methylation. Our findings suggest that DNA methylation is important for regulation of LINE-1 expression under normal conditions, but histone modifications may dictate the transcriptional activity of LINE-1 in response to exposure to densely IR. - Highlights: • DNA methylation of LINE-1 elements is dependent on their evolutionary age. • Densely ionizing radiation affects DNA methylation of selective LINE-1 elements. • Radiation-induced reactivation of LINE-1 is DNA methylation-independent. • Histone modifications dictate the transcriptional activity of LINE-1.« less

Curcumin modulates DNA methylation in colorectal cancer cells.

PubMed

Link, Alexander; Balaguer, Francesc; Shen, Yan; Lozano, Juan Jose; Leung, Hon-Chiu E; Boland, C Richard; Goel, Ajay

2013-01-01

Recent evidence suggests that several dietary polyphenols may exert their chemopreventive effect through epigenetic modifications. Curcumin is one of the most widely studied dietary chemopreventive agents for colon cancer prevention, however, its effects on epigenetic alterations, particularly DNA methylation, remain unclear. Using systematic genome-wide approaches, we aimed to elucidate the effect of curcumin on DNA methylation alterations in colorectal cancer cells. To evaluate the effect of curcumin on DNA methylation, three CRC cell lines, HCT116, HT29 and RKO, were treated with curcumin. 5-aza-2'-deoxycytidine (5-aza-CdR) and trichostatin A treated cells were used as positive and negative controls for DNA methylation changes, respectively. Methylation status of LINE-1 repeat elements, DNA promoter methylation microarrays and gene expression arrays were used to assess global methylation and gene expression changes. Validation was performed using independent microarrays, quantitative bisulfite pyrosequencing, and qPCR. As expected, genome-wide methylation microarrays revealed significant DNA hypomethylation in 5-aza-CdR-treated cells (mean β-values of 0.12), however, non-significant changes in mean β-values were observed in curcumin-treated cells. In comparison to mock-treated cells, curcumin-induced DNA methylation alterations occurred in a time-dependent manner. In contrast to the generalized, non-specific global hypomethylation observed with 5-aza-CdR, curcumin treatment resulted in methylation changes at selected, partially-methylated loci, instead of fully-methylated CpG sites. DNA methylation alterations were supported by corresponding changes in gene expression at both up- and down-regulated genes in various CRC cell lines. Our data provide previously unrecognized evidence for curcumin-mediated DNA methylation alterations as a potential mechanism of colon cancer chemoprevention. In contrast to non-specific global hypomethylation induced by 5-aza-CdR, curcumin-induced methylation changes occurred only in a subset of partially-methylated genes, which provides additional mechanistic insights into the potent chemopreventive effect of this dietary nutraceutical.

Curcumin Modulates DNA Methylation in Colorectal Cancer Cells

PubMed Central

Link, Alexander; Balaguer, Francesc; Shen, Yan; Lozano, Juan Jose; Leung, Hon-Chiu E.; Boland, C. Richard; Goel, Ajay

2013-01-01

Aim Recent evidence suggests that several dietary polyphenols may exert their chemopreventive effect through epigenetic modifications. Curcumin is one of the most widely studied dietary chemopreventive agents for colon cancer prevention, however, its effects on epigenetic alterations, particularly DNA methylation, remain unclear. Using systematic genome-wide approaches, we aimed to elucidate the effect of curcumin on DNA methylation alterations in colorectal cancer cells. Materials and Methods To evaluate the effect of curcumin on DNA methylation, three CRC cell lines, HCT116, HT29 and RKO, were treated with curcumin. 5-aza-2′-deoxycytidine (5-aza-CdR) and trichostatin A treated cells were used as positive and negative controls for DNA methylation changes, respectively. Methylation status of LINE-1 repeat elements, DNA promoter methylation microarrays and gene expression arrays were used to assess global methylation and gene expression changes. Validation was performed using independent microarrays, quantitative bisulfite pyrosequencing, and qPCR. Results As expected, genome-wide methylation microarrays revealed significant DNA hypomethylation in 5-aza-CdR-treated cells (mean β-values of 0.12), however, non-significant changes in mean β-values were observed in curcumin-treated cells. In comparison to mock-treated cells, curcumin-induced DNA methylation alterations occurred in a time-dependent manner. In contrast to the generalized, non-specific global hypomethylation observed with 5-aza-CdR, curcumin treatment resulted in methylation changes at selected, partially-methylated loci, instead of fully-methylated CpG sites. DNA methylation alterations were supported by corresponding changes in gene expression at both up- and down-regulated genes in various CRC cell lines. Conclusions Our data provide previously unrecognized evidence for curcumin-mediated DNA methylation alterations as a potential mechanism of colon cancer chemoprevention. In contrast to non-specific global hypomethylation induced by 5-aza-CdR, curcumin-induced methylation changes occurred only in a subset of partially-methylated genes, which provides additional mechanistic insights into the potent chemopreventive effect of this dietary nutraceutical. PMID:23460897

MethylMeter(®): bisulfite-free quantitative and sensitive DNA methylation profiling and mutation detection in FFPE samples.

PubMed

McCarthy, David; Pulverer, Walter; Weinhaeusel, Andreas; Diago, Oscar R; Hogan, Daniel J; Ostertag, Derek; Hanna, Michelle M

2016-06-01

Development of a sensitive method for DNA methylation profiling and associated mutation detection in clinical samples. Formalin-fixed and paraffin-embedded tumors received by clinical laboratories often contain insufficient DNA for analysis with bisulfite or methylation sensitive restriction enzymes-based methods. To increase sensitivity, methyl-CpG DNA capture and Coupled Abscription PCR Signaling detection were combined in a new assay, MethylMeter(®). Gliomas were analyzed for MGMT methylation, glioma CpG island methylator phenotype and IDH1 R132H. MethylMeter had 100% assay success rate measuring all five biomarkers in formalin-fixed and paraffin-embedded tissue. MGMT methylation results were supported by survival and mRNA expression data. MethylMeter is a sensitive and quantitative method for multitarget DNA methylation profiling and associated mutation detection. The MethylMeter-based GliomaSTRAT assay measures methylation of four targets and one mutation to simultaneously grade gliomas and predict their response to temozolomide. This information is clinically valuable in management of gliomas.

A Bayesian deconvolution strategy for immunoprecipitation-based DNA methylome analysis

PubMed Central

Down, Thomas A.; Rakyan, Vardhman K.; Turner, Daniel J.; Flicek, Paul; Li, Heng; Kulesha, Eugene; Gräf, Stefan; Johnson, Nathan; Herrero, Javier; Tomazou, Eleni M.; Thorne, Natalie P.; Bäckdahl, Liselotte; Herberth, Marlis; Howe, Kevin L.; Jackson, David K.; Miretti, Marcos M.; Marioni, John C.; Birney, Ewan; Hubbard, Tim J. P.; Durbin, Richard; Tavaré, Simon; Beck, Stephan

2009-01-01

DNA methylation is an indispensible epigenetic modification of mammalian genomes. Consequently there is great interest in strategies for genome-wide/whole-genome DNA methylation analysis, and immunoprecipitation-based methods have proven to be a powerful option. Such methods are rapidly shifting the bottleneck from data generation to data analysis, necessitating the development of better analytical tools. Until now, a major analytical difficulty associated with immunoprecipitation-based DNA methylation profiling has been the inability to estimate absolute methylation levels. Here we report the development of a novel cross-platform algorithm – Bayesian Tool for Methylation Analysis (Batman) – for analyzing Methylated DNA Immunoprecipitation (MeDIP) profiles generated using arrays (MeDIP-chip) or next-generation sequencing (MeDIP-seq). The latter is an approach we have developed to elucidate the first high-resolution whole-genome DNA methylation profile (DNA methylome) of any mammalian genome. MeDIP-seq/MeDIP-chip combined with Batman represent robust, quantitative, and cost-effective functional genomic strategies for elucidating the function of DNA methylation. PMID:18612301

MethHC: a database of DNA methylation and gene expression in human cancer.

PubMed

Huang, Wei-Yun; Hsu, Sheng-Da; Huang, Hsi-Yuan; Sun, Yi-Ming; Chou, Chih-Hung; Weng, Shun-Long; Huang, Hsien-Da

2015-01-01

We present MethHC (http://MethHC.mbc.nctu.edu.tw), a database comprising a systematic integration of a large collection of DNA methylation data and mRNA/microRNA expression profiles in human cancer. DNA methylation is an important epigenetic regulator of gene transcription, and genes with high levels of DNA methylation in their promoter regions are transcriptionally silent. Increasing numbers of DNA methylation and mRNA/microRNA expression profiles are being published in different public repositories. These data can help researchers to identify epigenetic patterns that are important for carcinogenesis. MethHC integrates data such as DNA methylation, mRNA expression, DNA methylation of microRNA gene and microRNA expression to identify correlations between DNA methylation and mRNA/microRNA expression from TCGA (The Cancer Genome Atlas), which includes 18 human cancers in more than 6000 samples, 6548 microarrays and 12 567 RNA sequencing data. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

Stability of DNA methylation patterns in mouse spermatogonia under conditions of MTHFR deficiency and methionine supplementation.

PubMed

Garner, Justine L; Niles, Kirsten M; McGraw, Serge; Yeh, Jonathan R; Cushnie, Duncan W; Hermo, Louis; Nagano, Makoto C; Trasler, Jacquetta M

2013-11-01

Little is known about the conditions contributing to the stability of DNA methylation patterns in male germ cells. Altered folate pathway enzyme activity and methyl donor supply are two clinically significant factors that can affect the methylation of DNA. 5,10-Methylenetetrahydrofolate reductase (MTHFR) is a key folate pathway enzyme involved in providing methyl groups from dietary folate for DNA methylation. Mice heterozygous for a targeted mutation in the Mthfr gene (Mthfr(+/-)) are a good model for humans homozygous for the MTHFR 677C>T polymorphism, which is found in 10% of the population and is associated with decreased MTHFR activity and infertility. High-dose folic acid is administered as an empirical treatment for male infertility. Here, we examined MTHFR expression in developing male germ cells and evaluated DNA methylation patterns and effects of a range of methionine concentrations in spermatogonia from Mthfr(+/-) as compared to wild-type, Mthfr(+/+) mice. MTHFR was expressed in prospermatogonia and spermatogonia at times of DNA methylation acquisition in the male germline; its expression was also found in early spermatocytes and Sertoli cells. DNA methylation patterns were similar at imprinted genes and intergenic sites across chromosome 9 in neonatal Mthfr(+/+) and Mthfr(+/-) spermatogonia. Using spermatogonia from Mthfr(+/+) and Mthfr(+/-) mice in the spermatogonial stem cell (SSC) culture system, we examined the stability of DNA methylation patterns and determined effects of low or high methionine concentrations. No differences were detected between early and late passages, suggesting that DNA methylation patterns are generally stable in culture. Twenty-fold normal concentrations of methionine resulted in an overall increase in the levels of DNA methylation across chromosome 9, suggesting that DNA methylation can be perturbed in culture. Mthfr(+/-) cells showed a significantly increased variance of DNA methylation at multiple loci across chromosome 9 compared to Mthfr(+/+) cells when cultured with 0.25- to 2-fold normal methionine concentrations. Taken together, our results indicate that DNA methylation patterns in undifferentiated spermatogonia, including SSCs, are relatively stable in culture over time under conditions of altered methionine and MTHFR levels.

Evolutionary Transition of Promoter and Gene Body DNA Methylation across Invertebrate-Vertebrate Boundary.

PubMed

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 of the Society for Molecular Biology and Evolution.

Genomic DNA Methylation Changes in Response to Folic Acid Supplementation in a Population-Based Intervention Study among Women of Reproductive Age

PubMed Central

Berry, Robert J.; Hao, Ling; Li, Zhu; Maneval, David; Yang, Thomas P.; Rasmussen, Sonja A.; Yang, Quanhe; Zhu, Jiang-Hui; Hu, Dale J.; Bailey, Lynn B.

2011-01-01

Folate is a source of one-carbons necessary for DNA methylation, a critical epigenetic modification necessary for genomic structure and function. The use of supplemental folic acid is widespread however; the potential influence on DNA methylation is unclear. We measured global DNA methylation using DNA extracted from samples from a population-based, double-blind randomized trial of folic acid supplementation (100, 400, 4000 µg per day) taken for 6 months; including a 3 month post-supplementation sample. We observed no changes in global DNA methylation in response to up to 4,000 µg/day for 6 months supplementation in DNA extracted from uncoagulated blood (approximates circulating blood). However, when DNA methylation was determined in coagulated samples from the same individuals at the same time, significant time, dose, and MTHFR genotype-dependent changes were observed. The baseline level of DNA methylation was the same for uncoagulated and coagulated samples; marked differences between sample types were observed only after intervention. In DNA from coagulated blood, DNA methylation decreased (−14%; P<0.001) after 1 month of supplementation and 3 months after supplement withdrawal, methylation decreased an additional 23% (P<0.001) with significant variation among individuals (max+17%; min-94%). Decreases in methylation of ≥25% (vs. <25%) after discontinuation of supplementation were strongly associated with genotype: MTHFR CC vs. TT (adjusted odds ratio [aOR] 12.9, 95%CI 6.4, 26.0). The unexpected difference in DNA methylation between DNA extracted from coagulated and uncoagulated samples in response to folic acid supplementation is an important finding for evaluating use of folic acid and investigating the potential effects of folic acid supplementation on coagulation. PMID:22163281

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

PubMed

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.

Future potential of the Human Epigenome Project.

PubMed

Eckhardt, Florian; Beck, Stephan; Gut, Ivo G; Berlin, Kurt

2004-09-01

Deciphering the information encoded in the human genome is key for the further understanding of human biology, physiology and evolution. With the draft sequence of the human genome completed, elucidation of the epigenetic information layer of the human genome becomes accessible. Epigenetic mechanisms are mediated by either chemical modifications of the DNA itself or by modifications of proteins that are closely associated with DNA. Defects of the epigenetic regulation involved in processes such as imprinting, X chromosome inactivation, transcriptional control of genes, as well as mutations affecting DNA methylation enzymes, contribute fundamentally to the etiology of many human diseases. Headed by the Human Epigenome Consortium, the Human Epigenome Project is a joint effort by an international collaboration that aims to identify, catalog and interpret genome-wide DNA methylation patterns of all human genes in all major tissues. Methylation variable positions are thought to reflect gene activity, tissue type and disease state, and are useful epigenetic markers revealing the dynamic state of the genome. Like single nucleotide polymorphisms, methylation variable positions will greatly advance our ability to elucidate and diagnose the molecular basis of human diseases.

DNA methylation changes detected by methylation-sensitive amplified polymorphism in two contrasting rice genotypes under salt stress.

PubMed

Wang, Wensheng; Zhao, Xiuqin; Pan, Yajiao; Zhu, Linghua; Fu, Binying; Li, Zhikang

2011-09-20

DNA methylation, one of the most important epigenetic phenomena, plays a vital role in tuning gene expression during plant development as well as in response to environmental stimuli. In the present study, a methylation-sensitive amplified polymorphism (MSAP) analysis was performed to profile DNA methylation changes in two contrasting rice genotypes under salt stress. Consistent with visibly different phenotypes in response to salt stress, epigenetic markers classified as stable inter-cultivar DNA methylation differences were determined between salt-tolerant FL478 and salt-sensitive IR29. In addition, most tissue-specific DNA methylation loci were conserved, while many of the growth stage-dependent DNA methylation loci were dynamic between the two genotypes. Strikingly, salt stress induced a decrease in DNA methylation specifically in roots at the seedling stage that was more profound in IR29 than in the FL478. This result may indicate that demethylation of genes is an active epigenetic response to salt stress in roots at the seedling stage, and helps to further elucidate the implications of DNA methylation in crop growth and development. Copyright © 2011. Published by Elsevier Ltd.

Electrochemical biosensing strategies for DNA methylation analysis.

PubMed

Hossain, Tanvir; Mahmudunnabi, Golam; Masud, Mostafa Kamal; Islam, Md Nazmul; Ooi, Lezanne; Konstantinov, Konstantin; Hossain, Md Shahriar Al; Martinac, Boris; Alici, Gursel; Nguyen, Nam-Trung; Shiddiky, Muhammad J A

2017-08-15

DNA methylation is one of the key epigenetic modifications of DNA that results from the enzymatic addition of a methyl group at the fifth carbon of the cytosine base. It plays a crucial role in cellular development, genomic stability and gene expression. Aberrant DNA methylation is responsible for the pathogenesis of many diseases including cancers. Over the past several decades, many methodologies have been developed to detect DNA methylation. These methodologies range from classical molecular biology and optical approaches, such as bisulfite sequencing, microarrays, quantitative real-time PCR, colorimetry, Raman spectroscopy to the more recent electrochemical approaches. Among these, electrochemical approaches offer sensitive, simple, specific, rapid, and cost-effective analysis of DNA methylation. Additionally, electrochemical methods are highly amenable to miniaturization and possess the potential to be multiplexed. In recent years, several reviews have provided information on the detection strategies of DNA methylation. However, to date, there is no comprehensive evaluation of electrochemical DNA methylation detection strategies. Herein, we address the recent developments of electrochemical DNA methylation detection approaches. Furthermore, we highlight the major technical and biological challenges involved in these strategies and provide suggestions for the future direction of this important field. Copyright © 2017 Elsevier B.V. All rights reserved.

Protection of CpG islands from DNA methylation is DNA-encoded and evolutionarily conserved.

PubMed

Long, Hannah K; King, Hamish W; Patient, Roger K; Odom, Duncan T; Klose, Robert J

2016-08-19

DNA methylation is a repressive epigenetic modification that covers vertebrate genomes. Regions known as CpG islands (CGIs), which are refractory to DNA methylation, are often associated with gene promoters and play central roles in gene regulation. Yet how CGIs in their normal genomic context evade the DNA methylation machinery and whether these mechanisms are evolutionarily conserved remains enigmatic. To address these fundamental questions we exploited a transchromosomic animal model and genomic approaches to understand how the hypomethylated state is formed in vivo and to discover whether mechanisms governing CGI formation are evolutionarily conserved. Strikingly, insertion of a human chromosome into mouse revealed that promoter-associated CGIs are refractory to DNA methylation regardless of host species, demonstrating that DNA sequence plays a central role in specifying the hypomethylated state through evolutionarily conserved mechanisms. In contrast, elements distal to gene promoters exhibited more variable methylation between host species, uncovering a widespread dependence on nucleotide frequency and occupancy of DNA-binding transcription factors in shaping the DNA methylation landscape away from gene promoters. This was exemplified by young CpG rich lineage-restricted repeat sequences that evaded DNA methylation in the absence of co-evolved mechanisms targeting methylation to these sequences, and species specific DNA binding events that protected against DNA methylation in CpG poor regions. Finally, transplantation of mouse chromosomal fragments into the evolutionarily distant zebrafish uncovered the existence of a mechanistically conserved and DNA-encoded logic which shapes CGI formation across vertebrate species. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

[Applications of DNA methylation markers in forensic medicine].

PubMed

Zhao, Gui-sen; Yang, Qing-en

2005-02-01

DNA methylation is a post-replication modification that is predominantly found in cytosines of the dinucleotide sequence CpG. Epigenetic information is stored in the distribution of the modified base 5-methylcytosine. DNA methylation profiles represent a more chemically and biologically stable source of molecular diagnostic information than RNA or most proteins. Recent advances attest to the great promise of DNA methylation markers as powerful future tools in the clinic. In the past decade, DNA methylation analysis has been revolutionized by two technological advances--bisulphite modification of DNA and methylation-specific polymerase chain reaction (MSP). The methylation pattern of human genome is space-time specific, sex-specific, parent-of-origin specific and disease specific, providing us an alternative way to solve forensic problems.

Twenty-four-nucleotide siRNAs produce heritable trans-chromosomal methylation in F1 Arabidopsis hybrids.

PubMed

Greaves, Ian K; Eichten, Steven R; Groszmann, Michael; Wang, Aihua; Ying, Hua; Peacock, W James; Dennis, Elizabeth S

2016-11-01

Hybrid Arabidopsis plants undergo epigenetic reprogramming producing decreased levels of 24-nt siRNAs and altered patterns of DNA methylation that can affect gene expression. Driving the changes in methylation are the processes trans-chromosomal methylation (TCM) and trans-chromosomal demethylation (TCdM). In TCM/TCdM the methylation state of one allele is altered to resemble the other allele. We show that Pol IV-dependent sRNAs are required to establish TCM events. The changes in DNA methylation and the associated changes in sRNA levels in the F1 hybrid can be maintained in subsequent generations and affect hundreds of regions in the F2 epigenome. The inheritance of these altered epigenetic states varies in F2 individuals, resulting in individuals with genetically identical loci displaying different epigenetic states and gene expression profiles. The change in methylation at these regions is associated with the presence of sRNAs. Loci without any sRNA activity can have altered methylation states, suggesting that a sRNA-independent mechanism may also contribute to the altered methylation state of the F1 and F2 generations.

Dynamic integrated analysis of DNA methylation and gene expression profiles in in vivo and in vitro fertilized mouse post-implantation extraembryonic and placental tissues.

PubMed

Tan, Kun; Zhang, Zhenni; Miao, Kai; Yu, Yong; Sui, Linlin; Tian, Jianhui; An, Lei

2016-07-01

How does in vitro fertilization (IVF) alter promoter DNA methylation patterns and its subsequent effects on gene expression profiles during placentation in mice? IVF-induced alterations in promoter DNA methylation might have functional consequences in a number of biological processes and functions during IVF placentation, including actin cytoskeleton organization, hematopoiesis, vasculogenesis, energy metabolism and nutrient transport. During post-implantation embryonic development, both embryonic and extraembryonic tissues undergo de novo DNA methylation, thereby establishing a global DNA methylation pattern, and influencing gene expression profiles. Embryonic and placental tissues of IVF conceptuses can have aberrant morphology and functions, resulting in adverse pregnancy outcomes such as pregnancy loss, low birthweight, and long-term health effects. To date, the IVF-induced global profiling of DNA methylation alterations, and their functional consequences on aberrant gene expression profiles in IVF placentas have not been systematically studied. Institute for Cancer Research mice (6 week-old females and 8-9 week-old males) were used to generate in vivo fertilization (IVO) and IVF blastocysts. After either IVO and development (IVO group as control) or in vitro fertilization and culture (IVF group), blastocysts were collected and transferred to pseudo-pregnant recipient mice. Extraembryonic (ectoplacental cone and extraembryonic ectoderm) and placental tissues from both groups were sampled at embryonic day (E) 7.5 (IVO, n = 822; IVF, n = 795) and E10.5 (IVO, n = 324; IVF, n = 278), respectively. The collected extraembryonic (E7.5) and placental tissues (E10.5) were then used for high-throughput RNA sequencing (RNA-seq) and methylated DNA immunoprecipitation sequencing (MeDIP-seq). The main dysfunctions indicated by bioinformatic analyses were further validated using molecular detection, and morphometric and phenotypic analyses. Dynamic functional profiling of high-throughput data, together with molecular detection, and morphometric and phenotypic analyses, showed that differentially expressed genes dysregulated by DNA methylation were functionally involved in: (i) actin cytoskeleton disorganization in IVF extraembryonic tissues, which may impair allantois or chorion formation, and chorioallantoic fusion; (ii) disturbed hematopoiesis and vasculogenesis, which may lead to abnormal placenta labyrinth formation and thereby impairing nutrition transport in IVF placentas; (iii) dysregulated energy and amino acid metabolism, which may cause placental dysfunctions, leading to delayed embryonic development or even lethality; (iv) disrupted genetic information processing, which can further influence gene transcriptional and translational processes. Findings in mouse placental tissues may not be fully representative of human placentas. Further studies are necessary to confirm these findings and determine their clinical significance. Our study is the first to provide the genome-wide analysis of gene expression dysregulation caused by DNA methylation during IVF placentation. Systematic understanding of the molecular mechanisms implicated in IVF placentation can be useful for the improvement of existing assisted conception systems to prevent these IVF-associated safety concerns. This work was supported by grants from the National Natural Science Foundation of China (No. 31472092), and the National High-Tech R&D Program (Nos. 2011|AA100303, 2013AA102506). There was no conflict of interest. © The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Quantitative profiling of CpG island methylation in human stool for colorectal cancer detection.

PubMed

Elliott, Giles O; Johnson, Ian T; Scarll, Jane; Dainty, Jack; Williams, Elizabeth A; Garg, D; Coupe, Amanda; Bradburn, David M; Mathers, John C; Belshaw, Nigel J

2013-01-01

The aims of this study were to investigate the use of quantitative CGI methylation data from stool DNA to classify colon cancer patients and to relate stool CGI methylation levels to those found in corresponding tissue samples. We applied a quantitative methylation-specific PCR assay to determine CGI methylation levels of six genes, previously shown to be aberrantly methylated during colorectal carcinogenesis. Assays were performed on DNA from biopsies of "normal" mucosa and stool samples from 57 patients classified as disease-free, adenoma, or cancer by endoscopy, and in tumour tissue from cancer patients. Additionally, CGI methylation was analysed in stool DNA from an asymptomatic population of individuals covering a broad age range (mean = 47 ± 24 years) CGI methylation levels in stool DNA were significantly higher than in DNA from macroscopically normal mucosa, and a significant correlation between stool and mucosa was observed for ESR1 only. Multivariate statistical analyses using the methylation levels of each CGI in stool DNA as a continuous variable revealed a highly significant (p = 0.003) classification of cancer vs. non-cancer (adenoma + disease-free) patients (sensitivity = 65 %, specificity = 81 %). CGI methylation profiling of stool DNA successfully identified patients with cancer despite the methylation status of CGIs in stool DNA not generally reflecting those in DNA from the colonic mucosa.

A lifelong exposure to a Western-style diet, but not aging, alters global DNA methylation in mouse colon

PubMed Central

Tammen, Stephanie A; Liu, Zhenhua; Friso, Simonetta

2015-01-01

BACKGROUND/OBJECTIVES Previous studies have indicated that when compared to young mice, old mice have lower global DNA methylation and higher p16 promoter methylation in colonic mucosa, which is a common finding in colon cancer. It is also known that a Western-style diet (WSD) high in fat and calories, and low in calcium, vitamin D, fiber, methionine and choline (based on the AIN 76A diet) is tumorigenic in colons of mice. Because DNA methylation is modifiable by diet, we investigate whether a WSD disrupts DNA methylation patterns, creating a tumorigenic environment. SUBJECTVIES/METHODS We investigated the effects of a WSD and aging on global and p16 promoter DNA methylation in the colon. Two month old male C57BL/6 mice were fed either a WSD or a control diet (AIN76A) for 6, 12 or 17 months. Global DNA methylation, p16 promoter methylation and p16 expression were determined by LC/MS, methyl-specific PCR and real time RT-PCR, respectively. RESULTS The WSD group demonstrated significantly decreased global DNA methylation compared with the control at 17 months (4.05 vs 4.31%, P = 0.019). While both diets did not change global DNA methylation over time, mice fed the WSD had lower global methylation relative to controls when comparing all animals (4.13 vs 4.30%, P = 0.0005). There was an increase in p16 promoter methylation from 6 to 17 months in both diet groups (P < 0.05) but no differences were observed between diet groups. Expression of p16 increased with age in both control and WSD groups. CONCLUSIONS In this model a WSD reduces global DNA methylation, whereas aging itself has no affect. Although the epigenetic effect of aging was not strong enough to alter global DNA methylation, changes in promoter-specific methylation and gene expression occurred with aging regardless of diet, demonstrating the complexity of epigenetic patterns. PMID:26244073

Links between DNA methylation and nucleosome occupancy in the human genome.

PubMed

Collings, Clayton K; Anderson, John N

2017-01-01

DNA methylation is an epigenetic modification that is enriched in heterochromatin but depleted at active promoters and enhancers. However, the debate on whether or not DNA methylation is a reliable indicator of high nucleosome occupancy has not been settled. For example, the methylation levels of DNA flanking CTCF sites are higher in linker DNA than in nucleosomal DNA, while other studies have shown that the nucleosome core is the preferred site of methylation. In this study, we make progress toward understanding these conflicting phenomena by implementing a bioinformatics approach that combines MNase-seq and NOMe-seq data and by comprehensively profiling DNA methylation and nucleosome occupancy throughout the human genome. The results demonstrated that increasing methylated CpG density is correlated with nucleosome occupancy in the total genome and within nearly all subgenomic regions. Features with elevated methylated CpG density such as exons, SINE-Alu sequences, H3K36-trimethylated peaks, and methylated CpG islands are among the highest nucleosome occupied elements in the genome, while some of the lowest occupancies are displayed by unmethylated CpG islands and unmethylated transcription factor binding sites. Additionally, outside of CpG islands, the density of CpGs within nucleosomes was shown to be important for the nucleosomal location of DNA methylation with low CpG frequencies favoring linker methylation and high CpG frequencies favoring core particle methylation. Prominent exceptions to the correlations between methylated CpG density and nucleosome occupancy include CpG islands marked by H3K27me3 and CpG-poor heterochromatin marked by H3K9me3, and these modifications, along with DNA methylation, distinguish the major silencing mechanisms of the human epigenome. Thus, the relationship between DNA methylation and nucleosome occupancy is influenced by the density of methylated CpG dinucleotides and by other epigenomic components in chromatin.

Global DNA hypomethylation in peripheral blood mononuclear cells as a biomarker of cancer risk.

PubMed

Friso, Simonetta; Udali, Silvia; Guarini, Patrizia; Pellegrini, Camilla; Pattini, Patrizia; Moruzzi, Sara; Girelli, Domenico; Pizzolo, Francesca; Martinelli, Nicola; Corrocher, Roberto; Olivieri, Oliviero; Choi, Sang-Woon

2013-03-01

Global DNA hypomethylation is an early molecular event in carcinogenesis. Whether methylation measured in peripheral blood mononuclear cells (PBMCs) DNA is a clinically reliable biomarker for early detection or cancer risk assessment is to be established. From an original sample-set of 753 male and female adults (ages 64.8 ± 7.3 years), PBMCs DNA methylation was measured in 68 subjects with history of cancer at time of enrollment and 62 who developed cancer during follow-up. Age- and sex-matched controls for prevalent and incident cancer cases (n = 68 and 58, respectively) were also selected. Global DNA methylation was assessed by liquid chromatography/mass spectrometry (LC/MS). Methylenetetrahydrofolate reductase (MTHFR) 677C>T genotype and plasma folate concentrations were also determined for the known gene-nutrient interaction affecting DNA methylation. Cancer subjects had significantly lower PBMCs-DNA methylation than controls [4.39 (95% confidence intervals (CI), 4.25-4.53) vs. 5.13 (95% CI, 5.03-5.21) %mCyt/(mCyt+Cyt); P < 0.0001]. A DNA methylation threshold of 4.74% clearly categorized patients with cancer from controls so that those with DNA methylation less than 4.74% showed an increased prevalence of cancer than those with higher levels (91.5% vs. 19%; P < 0.001). Subjects with cancer at follow-up had, already at enrollment, reduced DNA methylation as compared with controls [4.34 (95% CI, 4.24-4.51) vs. 5.08 (95% CI, 5.05-5.22) %mCyt/(mCyt+Cyt); P < 0.0001]. Moreover, MTHFR677C>T genotype and folate interact for determining DNA methylation, so that MTHFR677TT carriers with low folate had the lowest DNA methylation and concordantly showed a higher prevalence of cancer history (OR, 7.04; 95% CI, 1.52-32.63; P = 0.013). Genomic PBMCs-DNA methylation may be a useful epigenetic biomarker for early detection and cancer risk estimation. This study identifies a threshold for PBMCs-DNA methylation to detect cancer-affected from cancer-free subjects and an at-risk condition for cancer based on genomic DNA methylation and MTHFR677C>T-folate status. ©2012 AACR.

The interaction between cytosine methylation and processes of DNA replication and repair shape the mutational landscape of cancer genomes

PubMed Central

Poulos, Rebecca C.

2017-01-01

Abstract Methylated cytosines (5mCs) are frequently mutated in the genome. However, no studies have yet comprehensively analysed mutation–methylation associations across cancer types. Here we analyse 916 cancer genomes, together with tissue type-specific methylation and replication timing data. We describe a strong mutation–methylation association across colorectal cancer subtypes, most interestingly in samples with microsatellite instability (MSI) or Polymerase epsilon (POLE) exonuclease domain mutations. By analysing genomic regions with differential mismatch repair (MMR) efficiency, we suggest a possible role for MMR in the correction of 5mC deamination events, potentially accounting for the high rate of 5mC mutation accumulation in MSI tumours. Additionally, we propose that mutant POLE asserts a mutator phenotype specifically at 5mCs, and we find coding mutation hotspots in POLE-mutant cancers at highly-methylated CpGs in the tumour-suppressor genes APC and TP53. Finally, using multivariable regression models, we demonstrate that different cancers exhibit distinct mutation–methylation associations, with DNA repair influencing such associations in certain cancer genomes. Taken together, we find differential associations with methylation that are vital for accurately predicting expected mutation loads across cancer types. Our findings reveal links between methylation and common mutation and repair processes, with these mechanisms defining a key part of the mutational landscape of cancer genomes. PMID:28531315

A DNA methylation microarray-based study identifies ERG as a gene commonly methylated in prostate cancer.

PubMed

Schwartzman, Jacob; Mongoue-Tchokote, Solange; Gibbs, Angela; Gao, Lina; Corless, Christopher L; Jin, Jennifer; Zarour, Luai; Higano, Celestia; True, Lawrence D; Vessella, Robert L; Wilmot, Beth; Bottomly, Daniel; McWeeney, Shannon K; Bova, G Steven; Partin, Alan W; Mori, Motomi; Alumkal, Joshi

2011-10-01

DNA methylation of promoter regions is a common event in prostate cancer, one of the most common cancers in men worldwide. Because prior reports demonstrating that DNA methylation is important in prostate cancer studied a limited number of genes, we systematically quantified the DNA methylation status of 1505 CpG dinucleotides for 807 genes in 78 paraffin-embedded prostate cancer samples and three normal prostate samples. The ERG gene, commonly repressed in prostate cells in the absence of an oncogenic fusion to the TMPRSS2 gene, was one of the most commonly methylated genes, occurring in 74% of prostate cancer specimens. In an independent group of patient samples, we confirmed that ERG DNA methylation was common, occurring in 57% of specimens, and cancer-specific. The ERG promoter is marked by repressive chromatin marks mediated by polycomb proteins in both normal prostate cells and prostate cancer cells, which may explain ERG's predisposition to DNA methylation and the fact that tumors with ERG DNA methylation were more methylated, in general. These results demonstrate that bead arrays offer a high-throughput method to discover novel genes with promoter DNA methylation such as ERG, whose measurement may improve our ability to more accurately detect prostate cancer.

MSAP markers and global cytosine methylation in plants: a literature survey and comparative analysis for a wild-growing species.

PubMed

Alonso, Conchita; Pérez, Ricardo; Bazaga, Pilar; Medrano, Mónica; Herrera, Carlos M

2016-01-01

Methylation of DNA cytosines affects whether transposons are silenced and genes are expressed, and is a major epigenetic mechanism whereby plants respond to environmental change. Analyses of methylation-sensitive amplification polymorphism (MS-AFLP or MSAP) have been often used to assess methyl-cytosine changes in response to stress treatments and, more recently, in ecological studies of wild plant populations. MSAP technique does not require a sequenced reference genome and provides many anonymous loci randomly distributed over the genome for which the methylation status can be ascertained. Scoring of MSAP data, however, is not straightforward, and efforts are still required to standardize this step to make use of the potential to distinguish between methylation at different nucleotide contexts. Furthermore, it is not known how accurately MSAP infers genome-wide cytosine methylation levels in plants. Here, we analyse the relationship between MSAP results and the percentage of global cytosine methylation in genomic DNA obtained by HPLC analysis. A screening of literature revealed that methylation of cytosines at cleavage sites assayed by MSAP was greater than genome-wide estimates obtained by HPLC, and percentages of methylation at different nucleotide contexts varied within and across species. Concurrent HPLC and MSAP analyses of DNA from 200 individuals of the perennial herb Helleborus foetidus confirmed that methyl-cytosine was more frequent in CCGG contexts than in the genome as a whole. In this species, global methylation was unrelated to methylation at the inner CG site. We suggest that global HPLC and context-specific MSAP methylation estimates provide complementary information whose combination can improve our current understanding of methylation-based epigenetic processes in nonmodel plants. © 2015 John Wiley & Sons Ltd.

Genome-wide analysis of DNA methylation changes induced by gestational arsenic exposure in liver tumors.

PubMed

Suzuki, Takehiro; Yamashita, Satoshi; Ushijima, Toshikazu; Takumi, Shota; Sano, Tomoharu; Michikawa, Takehiro; Nohara, Keiko

2013-12-01

Inorganic arsenic is known to be a human carcinogen. Previous studies have reported that DNA methylation changes are involved in arsenic-induced carcinogenesis, therefore, DNA methylation changes that are specific to arsenic-induced tumors would be useful to distinguish tumors induced by arsenic from tumors caused by other factors and to dissect arsenic carcinogenesis. Previous studies have shown that gestational arsenic exposure of C3H mice, which tend to spontaneously develop liver tumors, increases the incidence of tumors in male offspring. In this study we used the same experimental protocol as in those previous studies and searched for DNA regions where methylation status was specifically altered in the liver tumors of arsenic-exposed offspring by using methylated DNA immunoprecipitation-CpG island microarrays. The methylation levels of the DNA regions selected were measured by quantitative methylation-specific PCR and bisulfite sequencing. The results of this study clarified a number of regions where DNA methylation status was altered in the liver tumors in the C3H mice compared to normal liver tissues. Among such regions, we showed that a gene body region of the oncogene Fosb underwent alteration in DNA methylation by gestational arsenic exposure. We also showed that Fosb expression significantly increased corresponding to the DNA methylation level of the gene body in the arsenic-exposed group. These findings suggest that the DNA methylation status can be used to identify tumors increased by gestational arsenic exposure. © 2013 Japanese Cancer Association.

A DNA methylation fingerprint of 1628 human samples

PubMed Central

Fernandez, Agustin F.; Assenov, Yassen; Martin-Subero, Jose Ignacio; Balint, Balazs; Siebert, Reiner; Taniguchi, Hiroaki; Yamamoto, Hiroyuki; Hidalgo, Manuel; Tan, Aik-Choon; Galm, Oliver; Ferrer, Isidre; Sanchez-Cespedes, Montse; Villanueva, Alberto; Carmona, Javier; Sanchez-Mut, Jose V.; Berdasco, Maria; Moreno, Victor; Capella, Gabriel; Monk, David; Ballestar, Esteban; Ropero, Santiago; Martinez, Ramon; Sanchez-Carbayo, Marta; Prosper, Felipe; Agirre, Xabier; Fraga, Mario F.; Graña, Osvaldo; Perez-Jurado, Luis; Mora, Jaume; Puig, Susana; Prat, Jaime; Badimon, Lina; Puca, Annibale A.; Meltzer, Stephen J.; Lengauer, Thomas; Bridgewater, John; Bock, Christoph; Esteller, Manel

2012-01-01

Most of the studies characterizing DNA methylation patterns have been restricted to particular genomic loci in a limited number of human samples and pathological conditions. Herein, we present a compromise between an extremely comprehensive study of a human sample population with an intermediate level of resolution of CpGs at the genomic level. We obtained a DNA methylation fingerprint of 1628 human samples in which we interrogated 1505 CpG sites. The DNA methylation patterns revealed show this epigenetic mark to be critical in tissue-type definition and stemness, particularly around transcription start sites that are not within a CpG island. For disease, the generated DNA methylation fingerprints show that, during tumorigenesis, human cancer cells underwent a progressive gain of promoter CpG-island hypermethylation and a loss of CpG methylation in non-CpG-island promoters. Although transformed cells are those in which DNA methylation disruption is more obvious, we observed that other common human diseases, such as neurological and autoimmune disorders, had their own distinct DNA methylation profiles. Most importantly, we provide proof of principle that the DNA methylation fingerprints obtained might be useful for translational purposes by showing that we are able to identify the tumor type origin of cancers of unknown primary origin (CUPs). Thus, the DNA methylation patterns identified across the largest spectrum of samples, tissues, and diseases reported to date constitute a baseline for developing higher-resolution DNA methylation maps and provide important clues concerning the contribution of CpG methylation to tissue identity and its changes in the most prevalent human diseases. PMID:21613409

Guidelines for whole genome bisulphite sequencing of intact and FFPET DNA on the Illumina HiSeq X Ten.

PubMed

Nair, Shalima S; Luu, Phuc-Loi; Qu, Wenjia; Maddugoda, Madhavi; Huschtscha, Lily; Reddel, Roger; Chenevix-Trench, Georgia; Toso, Martina; Kench, James G; Horvath, Lisa G; Hayes, Vanessa M; Stricker, Phillip D; Hughes, Timothy P; White, Deborah L; Rasko, John E J; Wong, Justin J-L; Clark, Susan J

2018-05-28

Comprehensive genome-wide DNA methylation profiling is critical to gain insights into epigenetic reprogramming during development and disease processes. Among the different genome-wide DNA methylation technologies, whole genome bisulphite sequencing (WGBS) is considered the gold standard for assaying genome-wide DNA methylation at single base resolution. However, the high sequencing cost to achieve the optimal depth of coverage limits its application in both basic and clinical research. To achieve 15× coverage of the human methylome, using WGBS, requires approximately three lanes of 100-bp-paired-end Illumina HiSeq 2500 sequencing. It is important, therefore, for advances in sequencing technologies to be developed to enable cost-effective high-coverage sequencing. In this study, we provide an optimised WGBS methodology, from library preparation to sequencing and data processing, to enable 16-20× genome-wide coverage per single lane of HiSeq X Ten, HCS 3.3.76. To process and analyse the data, we developed a WGBS pipeline (METH10X) that is fast and can call SNPs. We performed WGBS on both high-quality intact DNA and degraded DNA from formalin-fixed paraffin-embedded tissue. First, we compared different library preparation methods on the HiSeq 2500 platform to identify the best method for sequencing on the HiSeq X Ten. Second, we optimised the PhiX and genome spike-ins to achieve higher quality and coverage of WGBS data on the HiSeq X Ten. Third, we performed integrated whole genome sequencing (WGS) and WGBS of the same DNA sample in a single lane of HiSeq X Ten to improve data output. Finally, we compared methylation data from the HiSeq 2500 and HiSeq X Ten and found high concordance (Pearson r > 0.9×). Together we provide a systematic, efficient and complete approach to perform and analyse WGBS on the HiSeq X Ten. Our protocol allows for large-scale WGBS studies at reasonable processing time and cost on the HiSeq X Ten platform.

Hemi-methylated DNA regulates DNA methylation inheritance through allosteric activation of H3 ubiquitylation by UHRF1.

PubMed

Harrison, Joseph S; Cornett, Evan M; Goldfarb, Dennis; DaRosa, Paul A; Li, Zimeng M; Yan, Feng; Dickson, Bradley M; Guo, Angela H; Cantu, Daniel V; Kaustov, Lilia; Brown, Peter J; Arrowsmith, Cheryl H; Erie, Dorothy A; Major, Michael B; Klevit, Rachel E; Krajewski, Krzysztof; Kuhlman, Brian; Strahl, Brian D; Rothbart, Scott B

2016-09-06

The epigenetic inheritance of DNA methylation requires UHRF1, a histone- and DNA-binding RING E3 ubiquitin ligase that recruits DNMT1 to sites of newly replicated DNA through ubiquitylation of histone H3. UHRF1 binds DNA with selectivity towards hemi-methylated CpGs (HeDNA); however, the contribution of HeDNA sensing to UHRF1 function remains elusive. Here, we reveal that the interaction of UHRF1 with HeDNA is required for DNA methylation but is dispensable for chromatin interaction, which is governed by reciprocal positive cooperativity between the UHRF1 histone- and DNA-binding domains. HeDNA recognition activates UHRF1 ubiquitylation towards multiple lysines on the H3 tail adjacent to the UHRF1 histone-binding site. Collectively, our studies are the first demonstrations of a DNA-protein interaction and an epigenetic modification directly regulating E3 ubiquitin ligase activity. They also define an orchestrated epigenetic control mechanism involving modifications both to histones and DNA that facilitate UHRF1 chromatin targeting, H3 ubiquitylation, and DNA methylation inheritance.

Detecting differential DNA methylation from sequencing of bisulfite converted DNA of diverse species.

PubMed

Huh, Iksoo; Wu, Xin; Park, Taesung; Yi, Soojin V

2017-07-21

DNA methylation is one of the most extensively studied epigenetic modifications of genomic DNA. In recent years, sequencing of bisulfite-converted DNA, particularly via next-generation sequencing technologies, has become a widely popular method to study DNA methylation. This method can be readily applied to a variety of species, dramatically expanding the scope of DNA methylation studies beyond the traditionally studied human and mouse systems. In parallel to the increasing wealth of genomic methylation profiles, many statistical tools have been developed to detect differentially methylated loci (DMLs) or differentially methylated regions (DMRs) between biological conditions. We discuss and summarize several key properties of currently available tools to detect DMLs and DMRs from sequencing of bisulfite-converted DNA. However, the majority of the statistical tools developed for DML/DMR analyses have been validated using only mammalian data sets, and less priority has been placed on the analyses of invertebrate or plant DNA methylation data. We demonstrate that genomic methylation profiles of non-mammalian species are often highly distinct from those of mammalian species using examples of honey bees and humans. We then discuss how such differences in data properties may affect statistical analyses. Based on these differences, we provide three specific recommendations to improve the power and accuracy of DML and DMR analyses of invertebrate data when using currently available statistical tools. These considerations should facilitate systematic and robust analyses of DNA methylation from diverse species, thus advancing our understanding of DNA methylation. © The Author 2017. Published by Oxford University Press.

DNA methylation levels analysis in four tissues of sea cucumber Apostichopus japonicus based on fluorescence-labeled methylation-sensitive amplified polymorphism (F-MSAP) during aestivation.

PubMed

Zhao, Ye; Chen, Muyan; Storey, Kenneth B; Sun, Lina; Yang, Hongsheng

2015-03-01

DNA methylation plays an important role in regulating transcriptional change in response to environmental stimuli. In the present study, DNA methylation levels of tissues of the sea cucumber Apostichopus japonicus were analyzed by the fluorescence-labeled methylation-sensitive amplified polymorphism (F-MSAP) technique over three stages of the aestivation cycle. Overall, a total of 26,963 fragments were amplified including 9112 methylated fragments among four sea cucumber tissues using 18 pairs of selective primers. Results indicated an average DNA methylation level of 33.79% for A. japonicus. The incidence of DNA methylation was different across tissue types in the non-aestivation stage: intestine (30.16%), respiratory tree (27.61%), muscle (27.94%) and body wall (56.25%). Our results show that hypermethylation accompanied deep-aestivation in A. japonicus, which suggests that DNA methylation may have an important role in regulating global transcriptional suppression during aestivation. Further analysis indicated that the main DNA modification sites were focused on intestine and respiratory tree tissues and that full-methylation but not hemi-methylation levels exhibited significant increases in the deep-aestivation stage. Copyright © 2014 Elsevier Inc. All rights reserved.

Unique DNA methylome profiles in CpG island methylator phenotype colon cancers

PubMed Central

Xu, Yaomin; Hu, Bo; Choi, Ae-Jin; Gopalan, Banu; Lee, Byron H.; Kalady, Matthew F.; Church, James M.; Ting, Angela H.

2012-01-01

A subset of colorectal cancers was postulated to have the CpG island methylator phenotype (CIMP), a higher propensity for CpG island DNA methylation. The validity of CIMP, its molecular basis, and its prognostic value remain highly controversial. Using MBD-isolated genome sequencing, we mapped and compared genome-wide DNA methylation profiles of normal, non-CIMP, and CIMP colon specimens. Multidimensional scaling analysis revealed that each specimen could be clearly classified as normal, non-CIMP, and CIMP, thus signifying that these three groups have distinctly different global methylation patterns. We discovered 3780 sites in various genomic contexts that were hypermethylated in both non-CIMP and CIMP colon cancers when compared with normal colon. An additional 2026 sites were found to be hypermethylated in CIMP tumors only; and importantly, 80% of these sites were located in CpG islands. These data demonstrate on a genome-wide level that the additional hypermethylation seen in CIMP tumors occurs almost exclusively at CpG islands and support definitively that these tumors were appropriately named. When these sites were examined more closely, we found that 25% were adjacent to sites that were also hypermethylated in non-CIMP tumors. Thus, CIMP is also characterized by more extensive methylation of sites that are already prone to be hypermethylated in colon cancer. These observations indicate that CIMP tumors have specific defects in controlling both DNA methylation seeding and spreading and serve as an important first step in delineating molecular mechanisms that control these processes. PMID:21990380

Comprehensive analysis of genome-wide DNA methylation across human polycystic ovary syndrome ovary granulosa cell.

PubMed

Xu, Jiawei; Bao, Xiao; Peng, Zhaofeng; Wang, Linlin; Du, Linqing; Niu, Wenbin; Sun, Yingpu

2016-05-10

Polycystic ovary syndrome (PCOS) affects approximately 7% of the reproductive-age women. A growing body of evidence indicated that epigenetic mechanisms contributed to the development of PCOS. The role of DNA modification in human PCOS ovary granulosa cell is still unknown in PCOS progression. Global DNA methylation and hydroxymethylation were detected between PCOS' and controls' granulosa cell. Genome-wide DNA methylation was profiled to investigate the putative function of DNA methylaiton. Selected genes expressions were analyzed between PCOS' and controls' granulosa cell. Our results showed that the granulosa cell global DNA methylation of PCOS patients was significant higher than the controls'. The global DNA hydroxymethylation showed low level and no statistical difference between PCOS and control. 6936 differentially methylated CpG sites were identified between control and PCOS-obesity. 12245 differential methylated CpG sites were detected between control and PCOS-nonobesity group. 5202 methylated CpG sites were significantly differential between PCOS-obesity and PCOS-nonobesity group. Our results showed that DNA methylation not hydroxymethylation altered genome-wide in PCOS granulosa cell. The different methylation genes were enriched in development protein, transcription factor activity, alternative splicing, sequence-specific DNA binding and embryonic morphogenesis. YWHAQ, NCF2, DHRS9 and SCNA were up-regulation in PCOS-obesity patients with no significance different between control and PCOS-nonobesity patients, which may be activated by lower DNA methylaiton. Global and genome-wide DNA methylation alteration may contribute to different genes expression and PCOS clinical pathology.

Maternal DNA hypomethylation and congenital heart defects

PubMed Central

Chowdhury, Shimul; Cleves, Mario A.; MacLeod, Stewart L.; James, S. Jill; Zhao, Weizhi; Hobbs, Charlotte A.

2011-01-01

Background Congenital heart defects (CHDs) are among the most prevalent and serious of birth defects. Multiple maternal factors are thought to contribute to CHD development including folate intake. Maternal DNA methylation, which is dependent on folate metabolism, may impact the risk of CHDs. Objective Our study was designed to determine whether maternal long interspersed nucleotide elements-1 (LINE-1) DNA hypomethylation is associated with increased occurrence of non-syndromic CHDs and whether maternal folate-dependent metabolites are correlated with DNA methylation status. Design Using a case-control study design, we measured global DNA methylation status among mothers whose pregnancies were affected by non-syndromic CHDs (n=180) and mothers of unaffected pregnancies (n=187). Methylation of LINE-1 was used as a surrogate marker of global DNA methylation status. The association between DNA methylation and CHD risk was determined while adjusting for selected lifestyle factors. Results LINE-1 DNA methylation was significantly lower in cases compared with controls (p=0.049). After covariate adjustments, a significant difference between cases and controls remained (p=0.010). Among women with LINE-1 methylation in the lowest decile of DNA methylation, the estimated risk of having a CHD-affected pregnancy was almost twice that of women in all other deciles (OR=1.91; 95% CI: 1.03, 3.58). Conclusions Our findings indicate that maternal LINE-1 DNA hypomethylation is associated with an increased risk of CHDs. Future studies investigating the association between maternal DNA methylation patterns and CHDs should be pursued. PMID:21254366

miRNA-Processing Gene Methylation and Cancer Risk.

PubMed

Joyce, Brian T; Zheng, Yinan; Zhang, Zhou; Liu, Lei; Kocherginsky, Masha; Murphy, Robert; Achenbach, Chad J; Musa, Jonah; Wehbe, Firas; Just, Allan; Shen, Jincheng; Vokonas, Pantel; Schwartz, Joel; Baccarelli, Andrea A; Hou, Lifang

2018-05-01

Background: Dysregulation of miRNA and methylation levels are epigenetic hallmarks of cancer, potentially linked via miRNA-processing genes. Studies have found genetic alterations to miRNA-processing genes in cancer cells and human population studies. Our objective was to prospectively examine changes in DNA methylation of miRNA-processing genes and their associations with cancer risk. Methods: We examined cohort data from the Department of Veterans' Affairs Normative Aging Study. Participants were assessed every 3 to 5 years starting in 1999 through 2013 including questionnaires, medical record review, and blood collection. Blood from 686 consenting participants was analyzed using the Illumina 450K BeadChip array to measure methylation at CpG sites throughout the genome. We selected 19 genes based on a literature review, with 519 corresponding CpG sites. We then used Cox proportional hazards models to examine associations with cancer incidence, and generalized estimating equations to examine associations with cancer prevalence. Associations at false discovery rate < 0.05 were considered statistically significant. Results: Methylation of three CpGs ( DROSHA : cg23230564, TNRC6B : cg06751583, and TNRC6B : cg21034183) was prospectively associated with time to cancer development (positively for cg06751583, inversely for cg23230564 and cg21034183), whereas methylation of one CpG site ( DROSHA : cg16131300) was positively associated with cancer prevalence. Conclusions: DNA methylation of DROSHA , a key miRNA-processing gene, and TNRC6B may play a role in early carcinogenesis. Impact: Changes in miRNA processing may exert multiple effects on cancer development, including protecting against it via altered global miRNAs, and may be a useful early detection biomarker of cancer. Cancer Epidemiol Biomarkers Prev; 27(5); 550-7. ©2018 AACR . ©2018 American Association for Cancer Research.

Sensitive and Specific Detection of Early Gastric Cancer Using DNA Methylation Analysis of Gastric Washes

PubMed Central

Watanabe, Yoshiyuki; Kim, Hyun Soo; Castoro, Ryan J.; Chung, Woonbok; Estecio, Marcos R. H.; Kondo, Kimie; Guo, Yi; Ahmed, Saira S.; Toyota, Minoru; Itoh, Fumio; Suk, Ki Tae; Cho, Mee-Yon; Shen, Lanlan; Jelinek, Jaroslav; Issa, Jean-Pierre J.

2009-01-01

Background & Aims Aberrant DNA methylation is an early and frequent process in gastric carcinogenesis and could be useful for detection of gastric neoplasia. We hypothesized that methylation analysis of DNA recovered from gastric washes could be used to detect gastric cancer. Methods We studied 51 candidate genes in 7 gastric cancer cell lines and 24 samples (training set) and identified 6 for further studies. We examined the methylation status of these genes in a test set consisting of 131 gastric neoplasias at various stages. Finally, we validated the 6 candidate genes in a different population of 40 primary gastric cancer samples and 113 non-neoplastic gastric mucosa samples. Results 6 genes (MINT25, RORA, GDNF, ADAM23, PRDM5, MLF1) showed frequent differential methylation between gastric cancer and normal mucosa in the training, test and validation sets. GDNF and MINT25 were most sensitive molecular markers of early stage gastric cancer while PRDM5 and MLF1 were markers of a field defect. There was a close correlation (r=0.5 to 0.9, p=0.03 to 0.001) between methylation levels in tumor biopsy and gastric washes. MINT25 methylation had the best sensitivity (90%), specificity (96%), and area under the ROC curve (0.961) in terms of tumor detection in gastric washes. Conclusions These findings suggest MINT25 is a sensitive and specific marker for screening in gastric cancer. Additionally we have developed a new methodology for gastric cancer detection by DNA methylation in gastric washes. PMID:19375421

Detection of Cytosine methylation in ancient DNA from five native american populations using bisulfite sequencing.

PubMed

Smith, Rick W A; Monroe, Cara; Bolnick, Deborah A

2015-01-01

While cytosine methylation has been widely studied in extant populations, relatively few studies have analyzed methylation in ancient DNA. Most existing studies of epigenetic marks in ancient DNA have inferred patterns of methylation in highly degraded samples using post-mortem damage to cytosines as a proxy for cytosine methylation levels. However, this approach limits the inference of methylation compared with direct bisulfite sequencing, the current gold standard for analyzing cytosine methylation at single nucleotide resolution. In this study, we used direct bisulfite sequencing to assess cytosine methylation in ancient DNA from the skeletal remains of 30 Native Americans ranging in age from approximately 230 to 4500 years before present. Unmethylated cytosines were converted to uracils by treatment with sodium bisulfite, bisulfite products of a CpG-rich retrotransposon were pyrosequenced, and C-to-T ratios were quantified for a single CpG position. We found that cytosine methylation is readily recoverable from most samples, given adequate preservation of endogenous nuclear DNA. In addition, our results indicate that the precision of cytosine methylation estimates is inversely correlated with aDNA preservation, such that samples of low DNA concentration show higher variability in measures of percent methylation than samples of high DNA concentration. In particular, samples in this study with a DNA concentration above 0.015 ng/μL generated the most consistent measures of cytosine methylation. This study presents evidence of cytosine methylation in a large collection of ancient human remains, and indicates that it is possible to analyze epigenetic patterns in ancient populations using direct bisulfite sequencing approaches.

Genome-wide association between DNA methylation and alternative splicing in an invertebrate

PubMed Central

2012-01-01

Background Gene bodies are the most evolutionarily conserved targets of DNA methylation in eukaryotes. However, the regulatory functions of gene body DNA methylation remain largely unknown. DNA methylation in insects appears to be primarily confined to exons. Two recent studies in Apis mellifera (honeybee) and Nasonia vitripennis (jewel wasp) analyzed transcription and DNA methylation data for one gene in each species to demonstrate that exon-specific DNA methylation may be associated with alternative splicing events. In this study we investigated the relationship between DNA methylation, alternative splicing, and cross-species gene conservation on a genome-wide scale using genome-wide transcription and DNA methylation data. Results We generated RNA deep sequencing data (RNA-seq) to measure genome-wide mRNA expression at the exon- and gene-level. We produced a de novo transcriptome from this RNA-seq data and computationally predicted splice variants for the honeybee genome. We found that exons that are included in transcription are higher methylated than exons that are skipped during transcription. We detected enrichment for alternative splicing among methylated genes compared to unmethylated genes using fisher’s exact test. We performed a statistical analysis to reveal that the presence of DNA methylation or alternative splicing are both factors associated with a longer gene length and a greater number of exons in genes. In concordance with this observation, a conservation analysis using BLAST revealed that each of these factors is also associated with higher cross-species gene conservation. Conclusions This study constitutes the first genome-wide analysis exhibiting a positive relationship between exon-level DNA methylation and mRNA expression in the honeybee. Our finding that methylated genes are enriched for alternative splicing suggests that, in invertebrates, exon-level DNA methylation may play a role in the construction of splice variants by positively influencing exon inclusion during transcription. The results from our cross-species homology analysis suggest that DNA methylation and alternative splicing are genetic mechanisms whose utilization could contribute to a longer gene length and a slower rate of gene evolution. PMID:22978521

Overexpression of Human-Derived DNMT3A Induced Intergenerational Inheritance of Active DNA Methylation Changes in Rat Sperm

PubMed Central

Zheng, Xiaoguo; Li, Zhenhua; Wang, Guishuan; Li, Zhengzheng; Liang, Ajuan; Wang, Hanshu; Dai, Yubing; Huang, Xingxu; Chen, Xuejin; Ma, Yuanwu; Sun, Fei

2017-01-01

DNA methylation is the major focus of studies on paternal epigenetic inheritance in mammals, but most previous studies about inheritable DNA methylation changes are passively induced by environmental factors. However, it is unclear whether the active changes mediated by variations in DNA methyltransferase activity are heritable. Here, we established human-derived DNMT3A (hDNMT3A) transgenic rats to study the effect of hDNMT3A overexpression on the DNA methylation pattern of rat sperm and to investigate whether this actively altered DNA methylation status is inheritable. Our results revealed that hDNMT3A was overexpressed in the testis of transgenic rats and induced genome-wide alterations in the DNA methylation pattern of rat sperm. Among 5438 reliable loci identified with 64 primer-pair combinations using a methylation-sensitive amplification polymorphism method, 28.01% showed altered amplified band types. Among these amplicons altered loci, 68.42% showed an altered DNA methylation status in the offspring of transgenic rats compared with wild-type rats. Further analysis based on loci which had identical DNA methylation status in all three biological replicates revealed that overexpression of hDNMT3A in paternal testis induced hypermethylation in sperm of both genotype-negative and genotype-positive offspring. Among the differentially methylated loci, 34.26% occurred in both positive and negative offspring of transgenic rats, indicating intergenerational inheritance of active DNA methylation changes in the absence of hDNM3A transmission. Furthermore, 75.07% of the inheritable loci were hyper-methylated while the remaining were hypomethylated. Distribution analysis revealed that the DNA methylation variations mainly occurred in introns and intergenic regions. Functional analysis revealed that genes related to differentially methylated loci were involved in a wide range of functions. Finally, this study demonstrated that active DNA methylation changes induced by hDNMT3A expression were intergenerationally inherited by offspring without transmission of the transgene, which provided evidence for the transmission of active endogenous-factors-induced epigenetic variations. PMID:29312436

Altered LINE-1 Methylation in Mothers of Children with Down Syndrome

PubMed Central

Babić Božović, Ivana; Stanković, Aleksandra; Živković, Maja; Vraneković, Jadranka; Kapović, Miljenko; Brajenović-Milić, Bojana

2015-01-01

Down syndrome (DS, also known as trisomy 21) most often results from chromosomal nondisjunction during oogenesis. Numerous studies sustain a causal link between global DNA hypomethylation and genetic instability. It has been suggested that DNA hypomethylation might affect the structure and dynamics of chromatin regions that are critical for chromosome stability and segregation, thus favouring chromosomal nondisjunction during meiosis. Maternal global DNA hypomethylation has not yet been analyzed as a potential risk factor for chromosome 21 nondisjunction. This study aimed to asses the risk for DS in association with maternal global DNA methylation and the impact of endogenous and exogenous factors that reportedly influence DNA methylation status. Global DNA methylation was analyzed in peripheral blood lymphocytes by quantifying LINE-1 methylation using the MethyLight method. Levels of global DNA methylation were significantly lower among mothers of children with maternally derived trisomy 21 than among control mothers (P = 0.000). The combination of MTHFR C677T genotype and diet significantly influenced global DNA methylation (R2 = 4.5%, P = 0.046). The lowest values of global DNA methylation were observed in mothers with MTHFR 677 CT+TT genotype and low dietary folate. Although our findings revealed an association between maternal global DNA hypomethylation and trisomy 21 of maternal origin, further progress and final conclusions regarding the role of global DNA methylation and the occurrence of trisomy 21 are facing major challenges. PMID:26017139

Whole DNA methylome profiling in mice exposed to secondhand smoke.

PubMed

Tommasi, Stella; Zheng, Albert; Yoon, Jae-In; Li, Arthur Xuejun; Wu, Xiwei; Besaratinia, Ahmad

2012-11-01

Aberration of DNA methylation is a prime epigenetic mechanism of carcinogenesis. Aberrant DNA methylation occurs frequently in lung cancer, with exposure to secondhand smoke (SHS) being an established risk factor. The causal role of SHS in the genesis of lung cancer, however, remains elusive. To investigate whether SHS can cause aberrant DNA methylation in vivo, we have constructed the whole DNA methylome in mice exposed to SHS for a duration of 4 mo, both after the termination of exposure and at ensuing intervals post-exposure (up to 10 mo). Our genome-wide and gene-specific profiling of DNA methylation in the lung of SHS-exposed mice revealed that all groups of SHS-exposed mice and controls share a similar pattern of DNA methylation. Furthermore, the methylation status of major repetitive DNA elements, including long-interspersed nuclear elements (LINE L1), intracisternal A particle long-terminal repeat retrotransposons (IAP-LTR), and short-interspersed nuclear elements (SINE B1), in the lung of all groups of SHS-exposed mice and controls remains comparable. The absence of locus-specific gain of DNA methylation and global loss of DNA methylation in the lung of SHS-exposed mice within a timeframe that precedes neoplastic-lesion formation underscore the challenges of lung cancer biomarker development. Identifying the initiating events that cause aberrant DNA methylation in lung carcinogenesis may help improve future strategies for prevention, early detection and treatment of this highly lethal disease.

Variation in the DNA methylation pattern of expressed and nonexpressed genes in chicken.

PubMed

Cooper, D N; Errington, L H; Clayton, R M

1983-01-01

Using methyl-sensitive and -insensitive restriction enzymes, Hpa II and Msp I, the methylation status of various chicken genes was examined in different tissues and developmental stages. Tissue-specific differences in methylation were found for the delta-crystallin, beta-tubulin, G3PDH, rDNA, and actin genes but not for the histone genes. Developmental decreases in methylation were noted for the delta-crystallin and actin genes in chicken kidney between embryo and adult. Since most of the sequences examined were housekeeping genes, transcriptional differences are apparently not a necessary accompaniment to changes in DNA methylation at the CpG sites examined. The only exception is sperm DNA where the delta-crystallin, beta-tubulin, and actin genes are highly methylated and almost certainly not transcribed. However the G3PDH genes are no more highly methylated in sperm than in other somatic tissues. Many sequences homologous to the rDNA and histone probes used are unmethylated in all tissues examined including sperm, but a methylated rDNA subfraction is more heavily methylated in sperm than in other tissues. We speculate as to the significance of these differences in sperm DNA methylation in the light of possible requirements for early gene activation and the probable deleterious mutagenic effects of heavy methylation within coding sequences.

The caste- and sex-specific DNA methylome of the termite Zootermopsis nevadensis

PubMed Central

Glastad, Karl M.; Gokhale, Kaustubh; Liebig, Jürgen; Goodisman, Michael A. D.

2016-01-01

Epigenetic inheritance plays an important role in mediating alternative phenotype in highly social species. In order to gain a greater understanding of epigenetic effects in societies, we investigated DNA methylation in the termite Zootermopsis nevadensis. Termites are the most ancient social insects, and developmentally distinct from highly-studied, hymenopteran social insects. We used replicated bisulfite-sequencing to investigate patterns of DNA methylation in both sexes and among castes of Z. nevadensis. We discovered that Z. nevadensis displayed some of the highest levels of DNA methylation found in insects. We also found strong differences in methylation between castes. Methylated genes tended to be uniformly and highly expressed demonstrating the antiquity of associations between intragenic methylation and gene expression. Differentially methylated genes were more likely to be alternatively spliced than not differentially methylated genes, and possessed considerable enrichment for development-associated functions. We further observed strong overrepresentation of multiple transcription factor binding sites and miRNA profiles associated with differential methylation, providing new insights into the possible function of DNA methylation. Overall, our results show that DNA methylation is widespread and associated with caste differences in termites. More generally, this study provides insights into the function of DNA methylation and the success of insect societies. PMID:27848993

Analysis of Chromatin Regulators Reveals Specific Features of Rice DNA Methylation Pathways.

PubMed

Tan, Feng; Zhou, Chao; Zhou, Qiangwei; Zhou, Shaoli; Yang, Wenjing; Zhao, Yu; Li, Guoliang; Zhou, Dao-Xiu

2016-07-01

Plant DNA methylation that occurs at CG, CHG, and CHH sites (H = A, C, or T) is a hallmark of the repression of repetitive sequences and transposable elements (TEs). The rice (Oryza sativa) genome contains about 40% repetitive sequence and TEs and displays specific patterns of genome-wide DNA methylation. The mechanism responsible for the specific methylation patterns is unclear. Here, we analyzed the function of OsDDM1 (Deficient in DNA Methylation 1) and OsDRM2 (Deficient in DNA Methylation 1) in genome-wide DNA methylation, TE repression, small RNA accumulation, and gene expression. We show that OsDDM1 is essential for high levels of methylation at CHG and, to a lesser extent, CG sites in heterochromatic regions and also is required for CHH methylation that mainly locates in the genic regions of the genome. In addition to a large member of TEs, loss of OsDDM1 leads to hypomethylation and up-regulation of many protein-coding genes, producing very severe growth phenotypes at the initial generation. Importantly, we show that OsDRM2 mutation results in a nearly complete loss of CHH methylation and derepression of mainly small TE-associated genes and that OsDDM1 is involved in facilitating OsDRM2-mediated CHH methylation. Thus, the function of OsDDM1 and OsDRM2 defines distinct DNA methylation pathways in the bulk of DNA methylation of the genome, which is possibly related to the dispersed heterochromatin across chromosomes in rice and suggests that DNA methylation mechanisms may vary among different plant species. © 2016 American Society of Plant Biologists. All Rights Reserved.

DNA methylation and small interference RNAs participate in the regulation of MADS-box genes involved in dormancy in sweet cherry (Prunus avium L.).

PubMed

Rothkegel, Karin; Sánchez, Evelyn; Montes, Christian; Greve, Macarena; Tapia, Sebastián; Bravo, Soraya; Prieto, Humberto; Almeida, Andréa Miyasaka

2017-12-01

Epigenetic modifications can yield information about connections between genotype, phenotype variation and environmental conditions. Bud dormancy release in temperate perennial fruit trees depends on internal and environmental signals such as cold accumulation and photoperiod. Previous investigations have noted the participation of epigenetic mechanisms in the control of this physiological process. We examined whether epigenetic modifications were modulated in MADS-box genes, potential candidates for the regulation of bud dormancy and flowering in sweet cherry (Prunus avium L.). We identified and cloned two MADS-box genes homologous to the already-characterized dormancy regulators DORMANCY-ASSOCIATED MADS-box (DAM3 and DAM5) from Prunus persica (L.) Batsch. Bisulfite sequencing of the identified genes (PavMADS1 and PavMADS2), Methylated DNA Immunoprecipitation and small RNA deep sequencing were performed to analyze the presence of DNA methylations that could be guided by non-coding RNAs in the floral buds exposed to differential chilling hours. The results obtained reveal an increase in the level of DNA methylation and abundance of matching small interference RNAs (siRNAs) in the promoter of PavMADS1 when the chilling requirement is complete. For the first intron and 5' UTR of PavMADS1, de novo DNA methylation could be associated with the increase in the abundance of 24-nt siRNA matching the promoter area. Also, in the second large intron of PavMADS1, maintenance DNA methylation in all cytosine contexts is associated with the presence of homologous siRNAs in that zone. For PavMADS2, only maintenance methylation was present in the CG context, and no matching siRNAs were detected. Silencing of PavMADS1 and PavMADS2 coincided with an increase in Flowering Locus T expression during dormancy. In conclusion, DNA methylations and siRNAs appear to be involved in the silencing of PavMADS1 during cold accumulation and dormancy release in sweet cherry. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Amnion as a surrogate tissue reporter of the effects of maternal preeclampsia on the fetus.

PubMed

Suzuki, Masako; Maekawa, Ryo; Patterson, Nicole E; Reynolds, David M; Calder, Brent R; Reznik, Sandra E; Heo, Hye J; Einstein, Francine Hughes; Greally, John M

2016-01-01

Preeclampsia, traditionally characterized by high blood pressure and proteinuria, is a common pregnancy complication, which affects 2-8 % of all pregnancies. Although children born to women with preeclampsia have a higher risk of hypertension in later life, the mechanism of this increased risk is unknown. DNA methylation is an epigenetic modification that has been studied as a mediator of cellular memory of adverse exposures in utero. Since each cell type in the body has a unique DNA profile, cell subtype composition is a major confounding factor in studies of tissues with heterogeneous cell types. The best way to avoid this confounding effect is by using purified cell types. However, using purified cell types in large cohort translational studies is difficult. The amnion, the inner layer of the fetal membranes of the placenta, is derived from the epiblast and consists of two cell types, which are easy to isolate from the delivered placenta. In this study, we demonstrate the value of using amnion samples for DNA methylation studies, revealing distinctive patterns between fetuses exposed to proteinuria or hypertension and fetuses from normal pregnancies. We performed a genome-wide DNA methylation analysis, HpaII tiny fragment Enrichment by Ligation-mediated PCR (HELP)-tagging, on 62 amnion samples from the placentas of uncomplicated, normal pregnancies and from those with complications of preeclampsia or hypertension. Using a regression model approach, we found 123, 85, and 99 loci with high-confidence hypertension-associated, proteinuria-associated, and hypertension- and proteinuria-associated DNA methylation changes, respectively. A gene ontology analysis showed DNA methylation changes to be selecting genes with different biological processes in exposure status. We also found that these differentially methylated regions overlap loci previously reported as differentially methylated regions in preeclampsia. Our findings support prior observations that preeclampsia is associated with changes of DNA methylation near genes that have previously been found to be dysregulated in preeclampsia. We propose that amniotic membranes represent a valuable surrogate fetal tissue on which to perform epigenome-wide association studies of adverse intrauterine conditions.

Mitochondrial DNA copy number is regulated in a tissue specific manner by DNA methylation of the nuclear-encoded DNA polymerase gamma A

PubMed Central

Kelly, Richard D. W.; Mahmud, Arsalan; McKenzie, Matthew; Trounce, Ian A.; St John, Justin C.

2012-01-01

DNA methylation is an essential mechanism controlling gene expression during differentiation and development. We investigated the epigenetic regulation of the nuclear-encoded, mitochondrial DNA (mtDNA) polymerase γ catalytic subunit (PolgA) by examining the methylation status of a CpG island within exon 2 of PolgA. Bisulphite sequencing identified low methylation levels (<10%) within exon 2 of mouse oocytes, blastocysts and embryonic stem cells (ESCs), while somatic tissues contained significantly higher levels (>40%). In contrast, induced pluripotent stem (iPS) cells and somatic nuclear transfer ESCs were hypermethylated (>20%), indicating abnormal epigenetic reprogramming. Real time PCR analysis of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) immunoprecipitated DNA suggests active DNA methylation and demethylation within exon 2 of PolgA. Moreover, neural differentiation of ESCs promoted de novo methylation and demethylation at the exon 2 locus. Regression analysis demonstrates that cell-specific PolgA expression levels were negatively correlated with DNA methylation within exon 2 and mtDNA copy number. Finally, using chromatin immunoprecipitation (ChIP) against RNA polymerase II (RNApII) phosphorylated on serine 2, we show increased DNA methylation levels are associated with reduced RNApII transcriptional elongation. This is the first study linking nuclear DNA epigenetic regulation with mtDNA regulation during differentiation and cell specialization. PMID:22941637

Increased methylation of repetitive elements and DNA repair genes is associated with higher DNA oxidation in children in an urbanized, industrial environment.

PubMed

Alvarado-Cruz, Isabel; Sánchez-Guerra, Marco; Hernández-Cadena, Leticia; De Vizcaya-Ruiz, Andrea; Mugica, Violeta; Pelallo-Martínez, Nadia Azenet; Solís-Heredia, María de Jesús; Byun, Hyang-Min; Baccarelli, Andrea; Quintanilla-Vega, Betzabet

2017-01-01

DNA methylation in DNA repair genes participates in the DNA damage regulation. Particulate matter (PM), which has metals and polycyclic aromatic hydrocarbons (PAHs) adsorbed, among others has been linked to adverse health outcomes and may modify DNA methylation. To evaluate PM exposure impact on repetitive elements and gene-specific DNA methylation and DNA damage, we conducted a cross-sectional study in 150 schoolchildren (7-10 years old) from an urbanized, industrial area of the metropolitan area of Mexico City (MAMC), which frequently exhibits PM concentrations above safety standards. Methylation (5mC) of long interspersed nuclear element-1 (LINE1) and DNA repair gene (OGG1, APEX, and PARP1) was assessed by pyrosequencing in peripheral mononuclear cells, DNA damage by comet assay and DNA oxidation by 8-OHdG content. PAH and metal contents in PM 10 (≤10μm aerodynamic diameter) were determined by HPLC-MS and ICP-AES, respectively. Multiple regression analysis between DNA methylation, DNA damage, and PM 10 exposure showed that PM 10 was significantly associated with oxidative DNA damage; a 1% increase in 5mC at all CpG sites in PARP1 promoter was associated with a 35% increase in 8-OHdG, while a 1% increase at 1, 2, and 3 CpG sites resulted in 38, 9, and 56% increments, respectively. An increase of 10pg/m 3 in benzo[b]fluoranthene content of PM 10 was associated with a 6% increase in LINE1 methylation. Acenaphthene, indene [1,2,3-cd] pyrene, and pyrene concentrations correlated with higher dinucleotide methylation in OGG1, APEX and PARP1 genes, respectively. Vanadium concentration correlated with increased methylation at selected APEX and PARP1 CpG sites. DNA repair gene methylation was significantly correlated with DNA damage and with specific PM 10 -associated PAHs and Vanadium. Data suggest that exposure to PM and its components are associated with differences in DNA methylation of repair genes in children, which may contribute to DNA damage. Copyright © 2016 Elsevier B.V. All rights reserved.

Mutations in ATRX, encoding a SWI/SNF-like protein, cause diverse changes in the pattern of DNA methylation.

PubMed

Gibbons, R J; McDowell, T L; Raman, S; O'Rourke, D M; Garrick, D; Ayyub, H; Higgs, D R

2000-04-01

A goal of molecular genetics is to understand the relationship between basic nuclear processes, epigenetic changes and the numerous proteins that orchestrate these effects. One such protein, ATRX, contains a highly conserved plant homeodomain (PHD)-like domain, present in many chromatin-associated proteins, and a carboxy-terminal domain which identifies it as a member of the SNF2 family of helicase/ATPases. Mutations in ATRX give rise to characteristic developmental abnormalities including severe mental retardation, facial dysmorphism, urogenital abnormalities and alpha-thalassaemia. This circumstantial evidence suggests that ATRX may act as a transcriptional regulator through an effect on chromatin. We have recently shown that ATRX is localized to pericentromeric heterochromatin during interphase and mitosis, suggesting that ATRX might exert other chromatin-mediated effects in the nucleus. Moreover, at metaphase, some ATRX is localized at or close to the ribosomal DNA (rDNA) arrays on the short arms of human acrocentric chromosomes. Here we show that mutations in ATRX give rise to changes in the pattern of methylation of several highly repeated sequences including the rDNA arrays, a Y-specific satellite and subtelomeric repeats. Our findings provide a potential link between the processes of chromatin remodelling, DNA methylation and gene expression in mammalian development.

Early In Vitro Differentiation of Mouse Definitive Endoderm Is Not Correlated with Progressive Maturation of Nuclear DNA Methylation Patterns

PubMed Central

Tajbakhsh, Jian; Gertych, Arkadiusz; Fagg, W. Samuel; Hatada, Seigo; Fair, Jeffrey H.

2011-01-01

The genome organization in pluripotent cells undergoing the first steps of differentiation is highly relevant to the reprogramming process in differentiation. Considering this fact, chromatin texture patterns that identify cells at the very early stage of lineage commitment could serve as valuable tools in the selection of optimal cell phenotypes for regenerative medicine applications. Here we report on the first-time use of high-resolution three-dimensional fluorescence imaging and comprehensive topological cell-by-cell analyses with a novel image-cytometrical approach towards the identification of in situ global nuclear DNA methylation patterns in early endodermal differentiation of mouse ES cells (up to day 6), and the correlations of these patterns with a set of putative markers for pluripotency and endodermal commitment, and the epithelial and mesenchymal character of cells. Utilizing this in vitro cell system as a model for assessing the relationship between differentiation and nuclear DNA methylation patterns, we found that differentiating cell populations display an increasing number of cells with a gain in DNA methylation load: first within their euchromatin, then extending into heterochromatic areas of the nucleus, which also results in significant changes of methylcytosine/global DNA codistribution patterns. We were also able to co-visualize and quantify the concomitant stochastic marker expression on a per-cell basis, for which we did not measure any correlation to methylcytosine loads or distribution patterns. We observe that the progression of global DNA methylation is not correlated with the standard transcription factors associated with endodermal development. Further studies are needed to determine whether the progression of global methylation could represent a useful signature of cellular differentiation. This concept of tracking epigenetic progression may prove useful in the selection of cell phenotypes for future regenerative medicine applications. PMID:21779341

Whole genome DNA methylation: beyond genes silencing.

PubMed

Tirado-Magallanes, Roberto; Rebbani, Khadija; Lim, Ricky; Pradhan, Sriharsa; Benoukraf, Touati

2017-01-17

The combination of DNA bisulfite treatment with high-throughput sequencing technologies has enabled investigation of genome-wide DNA methylation at near base pair level resolution, far beyond that of the kilobase-long canonical CpG islands that initially revealed the biological relevance of this covalent DNA modification. The latest high-resolution studies have revealed a role for very punctual DNA methylation in chromatin plasticity, gene regulation and splicing. Here, we aim to outline the major biological consequences of DNA methylation recently discovered. We also discuss the necessity of tuning DNA methylation resolution into an adequate scale to ease the integration of the methylome information with other chromatin features and transcription events such as gene expression, nucleosome positioning, transcription factors binding dynamic, gene splicing and genomic imprinting. Finally, our review sheds light on DNA methylation heterogeneity in cell population and the different approaches used for its assessment, including the contribution of single cell DNA analysis technology.

Whole genome DNA methylation: beyond genes silencing

PubMed Central

Tirado-Magallanes, Roberto; Rebbani, Khadija; Lim, Ricky; Pradhan, Sriharsa; Benoukraf, Touati

2017-01-01

The combination of DNA bisulfite treatment with high-throughput sequencing technologies has enabled investigation of genome-wide DNA methylation at near base pair level resolution, far beyond that of the kilobase-long canonical CpG islands that initially revealed the biological relevance of this covalent DNA modification. The latest high-resolution studies have revealed a role for very punctual DNA methylation in chromatin plasticity, gene regulation and splicing. Here, we aim to outline the major biological consequences of DNA methylation recently discovered. We also discuss the necessity of tuning DNA methylation resolution into an adequate scale to ease the integration of the methylome information with other chromatin features and transcription events such as gene expression, nucleosome positioning, transcription factors binding dynamic, gene splicing and genomic imprinting. Finally, our review sheds light on DNA methylation heterogeneity in cell population and the different approaches used for its assessment, including the contribution of single cell DNA analysis technology. PMID:27895318

DNA methylation profiling of asbestos-treated MeT5A cell line reveals novel pathways implicated in asbestos response.

PubMed

Casalone, E; Allione, A; Viberti, C; Pardini, B; Guarrera, S; Betti, M; Dianzani, I; Aldieri, E; Matullo, G

2018-05-01

Occupational and environmental asbestos exposure is the main determinant of malignant pleural mesothelioma (MPM), however, the mechanisms by which its fibres contribute to cell toxicity and transformation are not completely clear. Aberrant DNA methylation is a common event in cancer but epigenetic modifications involved specifically in MPM carcinogenesis need to be better clarified. To investigate asbestos-induced DNA methylation and gene expression changes, we treated Met5A mesothelial cells with different concentrations of crocidolite and chrysotile asbestos (0.5 ÷ 5.0 µg/cm 2 , 72 h incubation). Overall, we observed 243 and 302 differentially methylated CpGs (≥ 10%) between the asbestos dose at 5 µg/cm 2 and untreated control, in chrysotile and crocidolite treatment, respectively. To examine the dose-response effect, Spearman's correlation test was performed and significant CpGs located in genes involved in migration/cell adhesion processes were identified in both treatments. Moreover, we found that both crocidolite and chrysotile exposure induced a significant up-regulation of CA9 and SRGN (log2 fold change > 1.5), previously reported as associated with a more aggressive MPM phenotype. However, we found no correlation between methylation and gene expression changes, except for a moderate significant inverse correlation at the promoter region of DKK1 (Spearman rho = - 1, P value = 0.02) after chrysotile exposure. These results describe for the first time the relationship between DNA methylation modifications and asbestos exposure. Our findings provide a basis to further explore and validate asbestos-induced DNA methylation changes, that could influence MPM carcinogenesis and possibly identifying new chemopreventive target.

Protective effects of folic acid on DNA damage and DNA methylation levels induced by N-methyl- N'-nitro- N-nitrosoguanidine in Kazakh esophageal epithelial cells.

PubMed

Chen, Y; Feng, H; Chen, D; Abuduwaili, K; Li, X; Zhang, H

2018-01-01

The protective effects of folic acid on DNA damage and DNA methylation induced by N-methyl- N'-nitro- N-nitrosoguanidine (MNNG) in Kazakh esophageal epithelial cells were investigated using a 3 × 3 factorial design trial. The cells were cultured in vitro and exposed to media containing different concentrations of folic acid and MNNG, after which growth indices were detected. DNA damage levels were measured using comet assays, and genome-wide DNA methylation levels (MLs) were measured using high-performance liquid chromatography. The DNA methylation of methylenetetrahydrofolate reductase (MTHFR) and folate receptor- α (FR α) genes was detected by bisulfite sequencing polymerase chain reaction (PCR). The results showed significant increases in tail DNA concentration, tail length, and Olive tail moment ( p < 0.01); a significant reduction of genome-wide DNA MLs ( p < 0.01); and an increase in the methylation frequencies of MTHFR and FR α genes. In particular, significant differences were observed in the promoter regions of both genes ( p < 0.01). Our study indicated that a reduction in folic acid concentration promotes DNA damage and DNA methylation in Kazakh esophageal epithelial cells upon MNNG exposure. Thus, sufficient folic acid levels could play a protective role against the damage induced by this compound.

MethylMix 2.0: an R package for identifying DNA methylation genes. | Office of Cancer Genomics

Cancer.gov

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.

MethylMeter®: bisulfite-free quantitative and sensitive DNA methylation profiling and mutation detection in FFPE samples

PubMed Central

McCarthy, David; Pulverer, Walter; Weinhaeusel, Andreas; Diago, Oscar R; Hogan, Daniel J; Ostertag, Derek; Hanna, Michelle M

2016-01-01

Aim: Development of a sensitive method for DNA methylation profiling and associated mutation detection in clinical samples. Materials & methods: Formalin-fixed and paraffin-embedded tumors received by clinical laboratories often contain insufficient DNA for analysis with bisulfite or methylation sensitive restriction enzymes-based methods. To increase sensitivity, methyl-CpG DNA capture and Coupled Abscription PCR Signaling detection were combined in a new assay, MethylMeter®. Gliomas were analyzed for MGMT methylation, glioma CpG island methylator phenotype and IDH1 R132H. Results: MethylMeter had 100% assay success rate measuring all five biomarkers in formalin-fixed and paraffin-embedded tissue. MGMT methylation results were supported by survival and mRNA expression data. Conclusion: MethylMeter is a sensitive and quantitative method for multitarget DNA methylation profiling and associated mutation detection. The MethylMeter-based GliomaSTRAT assay measures methylation of four targets and one mutation to simultaneously grade gliomas and predict their response to temozolomide. This information is clinically valuable in management of gliomas. PMID:27337298

MB-COMT promoter DNA methylation is associated with working-memory processing in schizophrenia patients and healthy controls

PubMed Central

Walton, Esther; Liu, Jingyu; Hass, Johanna; White, Tonya; Scholz, Markus; Roessner, Veit; Gollub, Randy; Calhoun, Vince D; Ehrlich, Stefan

2014-01-01

Many genetic studies report mixed results both for the associations between COMT polymorphisms and schizophrenia and for the effects of COMT variants on common intermediate phenotypes of the disorder. Reasons for this may include small genetic effect sizes and the modulation of environmental influences. To improve our understanding of the role of COMT in the disease etiology, we investigated the effect of DNA methylation in the MB-COMT promoter on neural activity in the dorsolateral prefrontal cortex during working memory processing as measured by fMRI - an intermediate phenotype for schizophrenia. Imaging and epigenetic data were measured in 102 healthy controls and 82 schizophrenia patients of the Mind Clinical Imaging Consortium (MCIC) study of schizophrenia. Neural activity during the Sternberg Item Recognition Paradigm was acquired with either a 3T Siemens Trio or 1.5T Siemens Sonata and analyzed using the FMRIB Software Library (FSL). DNA methylation measurements were derived from cryo-conserved blood samples. We found a positive association between MB-COMT promoter methylation and neural activity in the left dorsolateral prefrontal cortex in a model using a region-of-interest approach and could confirm this finding in a whole-brain model. This effect was independent of disease status. Analyzing the effect of MB-COMT promoter DNA methylation on a neuroimaging phenotype can provide further evidence for the importance of COMT and epigenetic risk mechanisms in schizophrenia. The latter may represent trans-regulatory or environmental risk factors that can be measured using brain-based intermediate phenotypes. PMID:24837210

Direct comparison study of DNA methylation markers in EpCAM-positive circulating tumour cells, corresponding circulating tumour DNA, and paired primary tumours in breast cancer

PubMed Central

Chimonidou, Maria; Strati, Areti; Malamos, Nikos; Kouneli, Sophia; Georgoulias, Vassilis; Lianidou, Evi

2017-01-01

Circulating Tumour Cells (CTCs) and circulating tumour DNA (ctDNA) represent a non-invasive liquid biopsy approach for the follow-up and therapy management of cancer patients. We evaluated whether DNA methylation status in CTCs and ctDNA is comparable and whether it reflects the status of primary tumours. We compared the methylation status of three genes, SOX17, CST6 and BRMS1 in primary tumours, corresponding CTCs and ctDNA in 153 breast cancer patients and healthy individuals, by using real time methylation specific PCR. We report a clear association between the EpCAM-positive CTC-fraction and ctDNA for SOX17 promoter methylation both for patients with early (P = 0.001) and metastatic breast cancer (P = 0.046) but not for CST6 and BRMS1. In early breast cancer, SOX17 promoter methylation in the EpCAM-positive CTC-fraction was associated with CK-19 mRNA expression (P = 0.006) and worse overall survival (OS) (P = 0.044). In the metastatic setting SOX17 promoter methylation in ctDNA was highly correlated with CK-19 (P = 0.04) and worse OS (Ρ = 0.016). SOX17 methylation status in CTCs and ctDNA was comparable and was associated with CK-19 expression but was not reflecting the status of primary tumours in breast cancer. DNA methylation analysis of SOX17 in CTCs and matched ctDNA provides significant prognostic value. PMID:29069768

Direct comparison study of DNA methylation markers in EpCAM-positive circulating tumour cells, corresponding circulating tumour DNA, and paired primary tumours in breast cancer.

PubMed

Chimonidou, Maria; Strati, Areti; Malamos, Nikos; Kouneli, Sophia; Georgoulias, Vassilis; Lianidou, Evi

2017-09-22

Circulating Tumour Cells (CTCs) and circulating tumour DNA (ctDNA) represent a non-invasive liquid biopsy approach for the follow-up and therapy management of cancer patients. We evaluated whether DNA methylation status in CTCs and ctDNA is comparable and whether it reflects the status of primary tumours. We compared the methylation status of three genes, SOX17, CST6 and BRMS1 in primary tumours, corresponding CTCs and ctDNA in 153 breast cancer patients and healthy individuals, by using real time methylation specific PCR. We report a clear association between the EpCAM-positive CTC-fraction and ctDNA for SOX17 promoter methylation both for patients with early ( P = 0.001) and metastatic breast cancer ( P = 0.046) but not for CST6 and BRMS1. In early breast cancer, SOX17 promoter methylation in the EpCAM-positive CTC-fraction was associated with CK-19 mRNA expression ( P = 0.006) and worse overall survival (OS) ( P = 0.044). In the metastatic setting SOX17 promoter methylation in ctDNA was highly correlated with CK-19 ( P = 0.04) and worse OS ( Ρ = 0.016). SOX17 methylation status in CTCs and ctDNA was comparable and was associated with CK-19 expression but was not reflecting the status of primary tumours in breast cancer. DNA methylation analysis of SOX17 in CTCs and matched ctDNA provides significant prognostic value.

Profiling the genome-wide DNA methylation pattern of porcine ovaries using reduced representation bisulfite sequencing.

PubMed

Yuan, Xiao-Long; Gao, Ning; Xing, Yan; Zhang, Hai-Bin; Zhang, Ai-Ling; Liu, Jing; He, Jin-Long; Xu, Yuan; Lin, Wen-Mian; Chen, Zan-Mou; Zhang, Hao; Zhang, Zhe; Li, Jia-Qi

2016-02-25

Substantial evidence has shown that DNA methylation regulates the initiation of ovarian and sexual maturation. Here, we investigated the genome-wide profile of DNA methylation in porcine ovaries at single-base resolution using reduced representation bisulfite sequencing. The biological variation was minimal among the three ovarian replicates. We found hypermethylation frequently occurred in regions with low gene abundance, while hypomethylation in regions with high gene abundance. The DNA methylation around transcriptional start sites was negatively correlated with their own CpG content. Additionally, the methylation level in the bodies of genes was higher than that in their 5' and 3' flanking regions. The DNA methylation pattern of the low CpG content promoter genes differed obviously from that of the high CpG content promoter genes. The DNA methylation level of the porcine ovary was higher than that of the porcine intestine. Analyses of the genome-wide DNA methylation in porcine ovaries would advance the knowledge and understanding of the porcine ovarian methylome.

On the presence and role of human gene-body DNA methylation

PubMed Central

Jjingo, Daudi; Conley, Andrew B.; Yi, Soojin V.; Lunyak, Victoria V.; Jordan, I. King

2012-01-01

DNA methylation of promoter sequences is a repressive epigenetic mark that down-regulates gene expression. However, DNA methylation is more prevalent within gene-bodies than seen for promoters, and gene-body methylation has been observed to be positively correlated with gene expression levels. This paradox remains unexplained, and accordingly the role of DNA methylation in gene-bodies is poorly understood. We addressed the presence and role of human gene-body DNA methylation using a meta-analysis of human genome-wide methylation, expression and chromatin data sets. Methylation is associated with transcribed regions as genic sequences have higher levels of methylation than intergenic or promoter sequences. We also find that the relationship between gene-body DNA methylation and expression levels is non-monotonic and bell-shaped. Mid-level expressed genes have the highest levels of gene-body methylation, whereas the most lowly and highly expressed sets of genes both have low levels of methylation. While gene-body methylation can be seen to efficiently repress the initiation of intragenic transcription, the vast majority of methylated sites within genes are not associated with intragenic promoters. In fact, highly expressed genes initiate the most intragenic transcription, which is inconsistent with the previously held notion that gene-body methylation serves to repress spurious intragenic transcription to allow for efficient transcriptional elongation. These observations lead us to propose a model to explain the presence of human gene-body methylation. This model holds that the repression of intragenic transcription by gene-body methylation is largely epiphenomenal, and suggests that gene-body methylation levels are predominantly shaped via the accessibility of the DNA to methylating enzyme complexes. PMID:22577155

DNA Methylation-a Potential Source of Mitochondria DNA Base Mismatch in the Development of Diabetic Retinopathy.

PubMed

Mishra, Manish; Kowluru, Renu A

2018-04-21

In the development of diabetic retinopathy, retinal mitochondria are dysfunctional, and mitochondrial DNA (mtDNA) is damaged with increased base mismatches and hypermethylated cytosines. DNA methylation is also a potential source of mutation, and in diabetes, the noncoding region, the displacement loop (D-loop), experiences more methylation and base mismatches than other regions of the mtDNA. Our aim was to investigate a possible crosstalk between mtDNA methylation and base mismatches in the development of diabetic retinopathy. The effect of inhibition of Dnmts (by 5-aza-2'-deoxycytidine or Dnmt1-siRNA) on glucose-induced mtDNA base mismatches was investigated in human retinal endothelial cells by surveyor endonuclease digestion and validated by Sanger sequencing. The role of deamination factors on increased base mismatches was determined in the cells genetically modulated for mitochondrial superoxide dismutase (Sod2) or cytidine-deaminase (APOBEC3A). The results were confirmed in an in vivo model using retinal microvasculature from diabetic mice overexpressing Sod2. Inhibition of DNA methylation, or regulation of cytosine deamination, significantly inhibited an increase in base mismatches at the D-loop and prevented mitochondrial dysfunction. Overexpression of Sod2 in mice also prevented diabetes-induced D-loop hypermethylation and increase in base mismatches. The crosstalk between DNA methylation and base mismatches continued even after termination of hyperglycemia, suggesting its role in the metabolic memory phenomenon associated with the progression of diabetic retinopathy. Inhibition of DNA methylation limits the availability of methylated cytosine for deamination, suggesting a crosstalk between DNA methylation and base mismatches. Thus, regulation of DNA methylation, or its deamination, should impede the development of diabetic retinopathy by preventing formation of base mismatches and mitochondrial dysfunction.

Restriction-Modification systems interplay causes avoidance of GATC site in prokaryotic genomes.

PubMed

Ershova, Anna; Rusinov, Ivan; Vasiliev, Mikhail; Spirin, Sergey; Karyagina, Anna

2016-04-01

Palindromes are frequently underrepresented in prokaryotic genomes. Palindromic 5[Formula: see text]-GATC-3[Formula: see text] site is a recognition site of different Restriction-Modification (R-M) systems, as well as solitary methyltransferase Dam. Classical GATC-specific R-M systems methylate GATC and cleave unmethylated GATC. On the contrary, methyl-directed Type II restriction endonucleases cleave methylated GATC. Methylation of GATC by Dam methyltransferase is involved in the regulation of different cellular processes. The diversity of functions of GATC-recognizing proteins makes GATC sequence a good model for studying the reasons of palindrome avoidance in prokaryotic genomes. In this work, the influence of R-M systems and solitary proteins on the GATC site avoidance is described by a mathematical model. GATC avoidance is strongly associated with the presence of alternate (methyl-directed or classical Type II R-M system) genes in different strains of the same species, as we have shown for Streptococcus pneumoniae, Neisseria meningitidis, Eubacterium rectale, and Moraxella catarrhalis. We hypothesize that GATC avoidance can result from a DNA exchange between strains with different methylation status of GATC site within the process of natural transformation. If this hypothesis is correct, the GATC avoidance is a sign of a DNA exchange between bacteria with different methylation status in a mixed population.

A survey of DNA methylation across social insect species, life stages, and castes reveals abundant and caste-associated methylation in a primitively social wasp

NASA Astrophysics Data System (ADS)

Weiner, Susan A.; Galbraith, David A.; Adams, Dean C.; Valenzuela, Nicole; Noll, Fernando B.; Grozinger, Christina M.; Toth, Amy L.

2013-08-01

DNA methylation plays an important role in the epigenetic control of developmental and behavioral plasticity, with connections to the generation of striking phenotypic differences between castes (larger, reproductive queens and smaller, non-reproductive workers) in honeybees and ants. Here, we provide the first comparative investigation of caste- and life stage-associated DNA methylation in several species of bees and vespid wasps displaying different levels of social organization. Our results reveal moderate levels of DNA methylation in most bees and wasps, with no clear relationship to the level of sociality. Strikingly, primitively social Polistes dominula paper wasps show unusually high overall DNA methylation and caste-related differences in site-specific methylation. These results suggest DNA methylation may play a role in the regulation of behavioral and physiological differences in primitively social species with more flexible caste differences.

[Research Progress on the Detection Method of DNA Methylation and Its Application in Forensic Science].

PubMed

Nie, Y C; Yu, L J; Guan, H; Zhao, Y; Rong, H B; Jiang, B W; Zhang, T

2017-06-01

As an important part of epigenetic marker, DNA methylation involves in the gene regulation and attracts a wide spread attention in biological auxology, geratology and oncology fields. In forensic science, because of the relative stable, heritable, abundant, and age-related characteristics, DNA methylation is considered to be a useful complement to the classic genetic markers for age-prediction, tissue-identification, and monozygotic twins' discrimination. Various methods for DNA methylation detection have been validated based on methylation sensitive restriction endonuclease, bisulfite modification and methylation-CpG binding protein. In recent years, it is reported that the third generation sequencing method can be used to detect DNA methylation. This paper aims to make a review on the detection method of DNA methylation and its applications in forensic science. Copyright© by the Editorial Department of Journal of Forensic Medicine.

Extensive sequence-influenced DNA methylation polymorphism in the human genome

PubMed Central

2010-01-01

Background Epigenetic polymorphisms are a potential source of human diversity, but their frequency and relationship to genetic polymorphisms are unclear. DNA methylation, an epigenetic mark that is a covalent modification of the DNA itself, plays an important role in the regulation of gene expression. Most studies of DNA methylation in mammalian cells have focused on CpG methylation present in CpG islands (areas of concentrated CpGs often found near promoters), but there are also interesting patterns of CpG methylation found outside of CpG islands. Results We compared DNA methylation patterns on both alleles between many pairs (and larger groups) of related and unrelated individuals. Direct observation and simulation experiments revealed that around 10% of common single nucleotide polymorphisms (SNPs) reside in regions with differences in the propensity for local DNA methylation between the two alleles. We further showed that for the most common form of SNP, a polymorphism at a CpG dinucleotide, the presence of the CpG at the SNP positively affected local DNA methylation in cis. Conclusions Taken together with the known effect of DNA methylation on mutation rate, our results suggest an interesting interdependence between genetics and epigenetics underlying diversity in the human genome. PMID:20497546

Association between alterations in global DNA methylation and predisposing factors in diabetes: a high pressure liquid chromatography based study.

PubMed

Maghbooli, Z; Hossein-Nezhad, A; Larijani, B; Pasalar, P; Keshtkar, A A

2015-08-01

The aim of this study was to investigate the relationship between inter-individual global DNA methylation and diabetes predisposing factors. The 5-methyl cytosine content was assessed by reverse phase high pressure liquid chromatography (RP-HPLC) of peripheral blood leukocytes obtained from 178 type 2 diabetes patients to determine individual global DNA methylation status. There was a positive significant correlation between diabetes duration and DNA methylation levels (P=0.002) with increasing levels of DNA methylation associated with age (P=0.047). There was no significant correlation between DNA methylation levels and HbA1c (P=0.15). No significant differences were observed between patients with and without diabetes predisposing factors including: hypertension (P=0.772), dyslipidemia (P=0.617), insulin resistance (homeostatic model assessment index) (P=0.156) and obesity (P=0.609). As such, the duration of diabetes (>10 years) was the most important predictor of global DNA methylation levels in diabetic patients after adjusting for age and sex (P=0.023). Our findings indicate that chronic hyperglycemic exposure plays an independent role in global DNA methylation levels in type 2 diabetes patients.

Development of an electrochemical detection system for measuring DNA methylation levels using methyl CpG-binding protein and glucose dehydrogenase-fused zinc finger protein.

PubMed

Lee, Jinhee; Yoshida, Wataru; Abe, Koichi; Nakabayashi, Kazuhiko; Wakeda, Hironobu; Hata, Kenichiro; Marquette, Christophe A; Blum, Loïc J; Sode, Koji; Ikebukuro, Kazunori

2017-07-15

DNA methylation level at a certain gene region is considered as a new type of biomarker for diagnosis and its miniaturized and rapid detection system is required for diagnosis. Here we have developed a simple electrochemical detection system for DNA methylation using methyl CpG-binding domain (MBD) and a glucose dehydrogenase (GDH)-fused zinc finger protein. This analytical system consists of three steps: (1) methylated DNA collection by MBD, (2) PCR amplification of a target genomic region among collected methylated DNA, and (3) electrochemical detection of the PCR products using a GDH-fused zinc finger protein. With this system, we have successfully measured the methylation levels at the promoter region of the androgen receptor gene in 10 6 copies of genomic DNA extracted from PC3 and TSU-PR1 cancer cell lines. Since no sequence analysis or enzymatic digestion is required for this detection system, DNA methylation levels can be measured within 3h with a simple procedure. Copyright © 2016 Elsevier B.V. All rights reserved.

Epigenetic effects of green tea polyphenols in cancer

PubMed Central

Henning, Susanne M; Wang, Piwen; Carpenter, Catherine L; Heber, David

2014-01-01

Epigenetics describes heritable alterations of gene expression and chromatin organization without changes in DNA sequence. Both hypermethylation and hypomethylation of DNA can affect gene expression and the multistep process of carcinogenesis. Epigenetic changes are reversible and may be targeted by dietary interventions. Bioactive compounds from green tea (GT) such as (–)-epigallocatechin gallate have been shown to alter DNA methyltransferase activity in studies of esophageal, oral, skin, Tregs, lung, breast and prostate cancer cells, which may contribute to the chemopreventive effect of GT. Three out of four mouse model studies have confirmed the inhibitory effect of (–)-epigallocatechin gallate on DNA methylation. A human study demonstrated that decreased methylation of CDX2 and BMP-2 in gastric carcinoma was associated with higher GT consumption. It is the goal of this review to summarize our current knowledge of the potential of GT to alter epigenetic processes, which may be useful in chemoprevention. PMID:24283885

Highly sensitive fluorescence assay of DNA methyltransferase activity by methylation-sensitive cleavage-based primer generation exponential isothermal amplification-induced G-quadruplex formation.

PubMed

Xue, Qingwang; Lv, Yanqin; Xu, Shuling; Zhang, Yuanfu; Wang, Lei; Li, Rui; Yue, Qiaoli; Li, Haibo; Gu, Xiaohong; Zhang, Shuqiu; Liu, Jifeng

2015-04-15

Site-specific identification of DNA methylation and assay of MTase activity are imperative for determining specific cancer types, provide insights into the mechanism of gene repression, and develop novel drugs to treat methylation-related diseases. Herein, we developed a highly sensitive fluorescence assay of DNA methyltransferase by methylation-sensitive cleavage-based primer generation exponential isothermal amplification (PG-EXPA) coupled with supramolecular fluorescent Zinc(II)-protoporphyrin IX (ZnPPIX)/G-quadruplex. In the presence of DNA adenine methylation (Dam) MTase, the methylation-responsive sequence of hairpin probe is methylated and cleaved by the methylation-sensitive restriction endonuclease Dpn I. The cleaved hairpin probe then functions as a signal primer to initiate the exponential isothermal amplification reaction (EXPAR) by hybridizing with a unimolecular DNA containing three functional domains as the amplification template, producing a large number of G-quadruplex nanostructures by utilizing polymerases and nicking enzymes as mechanical activators. The G-quadruplex nanostructures act as host for ZnPPIX that lead to supramolecular complexes ZnPPIX/G-quadruplex, which provides optical labels for amplified fluorescence detection of Dam MTase. While in the absence of Dam MTase, neither methylation/cleavage nor PG-EXPA reaction can be initiated and no fluorescence signal is observed. The proposed method exhibits a wide dynamic range from 0.0002 to 20U/mL and an extremely low detection limit of 8.6×10(-5)U/mL, which is superior to most conventional approaches for the MTase assay. Owing to the specific site recognition of MTase toward its substrate, the proposed sensing system was able to readily discriminate Dam MTase from other MTase such as M.SssI and even detect the target in a complex biological matrix. Furthermore, the application of the proposed sensing strategy for screening Dam MTase inhibitors was also demonstrated with satisfactory results. This novel method not only provides a promising platform for monitoring activity and inhibition of DNA MTases, but also shows great potentials in biological process researches, drugs discovery and clinical diagnostics. Copyright © 2014 Elsevier B.V. All rights reserved.

MeDIP-seq and nCpG analyses illuminate sexually dimorphic methylation of gonadal development genes with high historic methylation in turtle hatchlings with temperature-dependent sex determination.

PubMed

Radhakrishnan, Srihari; Literman, Robert; Mizoguchi, Beatriz; Valenzuela, Nicole

2017-01-01

DNA methylation alters gene expression but not DNA sequence and mediates some cases of phenotypic plasticity. Temperature-dependent sex determination (TSD) epitomizes phenotypic plasticity where environmental temperature drives embryonic sexual fate, as occurs commonly in turtles. Importantly, the temperature-specific transcription of two genes underlying gonadal differentiation is known to be induced by differential methylation in TSD fish, turtle and alligator. Yet, how extensive is the link between DNA methylation and TSD remains unclear. Here we test for broad differences in genome-wide DNA methylation between male and female hatchling gonads of the TSD painted turtle Chrysemys picta using methyl DNA immunoprecipitation sequencing, to identify differentially methylated candidates for future study. We also examine the genome-wide nCpG distribution (which affects DNA methylation) in painted turtles and test for historic methylation in genes regulating vertebrate gonadogenesis. Turtle global methylation was consistent with other vertebrates (57% of the genome, 78% of all CpG dinucleotides). Numerous genes predicted to regulate turtle gonadogenesis exhibited sex-specific methylation and were proximal to methylated repeats. nCpG distribution predicted actual turtle DNA methylation and was bimodal in gene promoters (as other vertebrates) and introns (unlike other vertebrates). Differentially methylated genes, including regulators of sexual development, had lower nCpG content indicative of higher historic methylation. Ours is the first evidence suggesting that sexually dimorphic DNA methylation is pervasive in turtle gonads (perhaps mediated by repeat methylation) and that it targets numerous regulators of gonadal development, consistent with the hypothesis that it may regulate thermosensitive transcription in TSD vertebrates. However, further research during embryogenesis will help test this hypothesis and the alternative that instead, most differential methylation observed in hatchlings is the by-product of sexual differentiation and not its cause.

Folate, colorectal cancer and the involvement of DNA methylation.

PubMed

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.

Deciphering the Epigenetic Code: An Overview of DNA Methylation Analysis Methods

PubMed Central

Umer, Muhammad

2013-01-01

Abstract Significance: Methylation of cytosine in DNA is linked with gene regulation, and this has profound implications in development, normal biology, and disease conditions in many eukaryotic organisms. A wide range of methods and approaches exist for its identification, quantification, and mapping within the genome. While the earliest approaches were nonspecific and were at best useful for quantification of total methylated cytosines in the chunk of DNA, this field has seen considerable progress and development over the past decades. Recent Advances: Methods for DNA methylation analysis differ in their coverage and sensitivity, and the method of choice depends on the intended application and desired level of information. Potential results include global methyl cytosine content, degree of methylation at specific loci, or genome-wide methylation maps. Introduction of more advanced approaches to DNA methylation analysis, such as microarray platforms and massively parallel sequencing, has brought us closer to unveiling the whole methylome. Critical Issues: Sensitive quantification of DNA methylation from degraded and minute quantities of DNA and high-throughput DNA methylation mapping of single cells still remain a challenge. Future Directions: Developments in DNA sequencing technologies as well as the methods for identification and mapping of 5-hydroxymethylcytosine are expected to augment our current understanding of epigenomics. Here we present an overview of methodologies available for DNA methylation analysis with special focus on recent developments in genome-wide and high-throughput methods. While the application focus relates to cancer research, the methods are equally relevant to broader issues of epigenetics and redox science in this special forum. Antioxid. Redox Signal. 18, 1972–1986. PMID:23121567

DNA methylation of amino acid transporter genes in the human placenta.

PubMed

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.

Metabolic, hormonal and immunological associations with global DNA methylation among postmenopausal women.

PubMed

Ulrich, Cornelia M; Toriola, Adetunji T; Koepl, Lisel M; Sandifer, Tracy; Poole, Elizabeth M; Duggan, Catherine; McTiernan, Anne; Issa, Jean-Pierre J

2012-09-01

DNA methylation is an epigenetic modification essential for the regulation of gene expression that has been implicated in many diseases, including cancer. Few studies have investigated the wide range of potential predictors of global DNA methylation, including biomarkers. Here, we investigated associations between DNA methylation and dietary factors, sex-steroid hormones, metabolic, lipid, inflammation, immune and one-carbon biomarkers. Data and baseline biomarker measurements were obtained from 173 overweight/obese postmenopausal women. Global DNA methylation in lymphocyte DNA was measured using the pyrosequencing assay for LINE-1 repeats. We used correlations and linear regression analyses to investigate associations between continuous data and DNA methylation, while t-tests were used for categorical data. Secondary analyses stratified by serum folate levels and multivitamin use were also conducted. There was little variability in LINE-1 methylation (66.3-79.5%). Mean LINE-1 methylation was significantly higher among women with elevated glucose levels. Mean LINE-1 methylation was also higher among women with high CD4+/CD8+ ratio, and lower among women with elevated vitamin B6, but neither reached statistical significance. In analyses stratified by folate status, DNA methylation was negatively associated with sex hormone concentrations (estrone, estradiol, testosterone and sex hormone binding globulin) among women with low serum folate levels (n = 53). Conversely, among women with high serum folate levels (n = 53), DNA methylation was positively associated with several immune markers (CD4/CD8 ratio, NK1656/lymphocytes and IgA). Results from this screening suggest that global DNA methylation is generally stable, with differential associations for sex hormones and immune markers depending on one-carbon status.

Targeted DNA demethylation in human cells by fusion of a plant 5-methylcytosine DNA glycosylase to a sequence-specific DNA binding domain

PubMed Central

Parrilla-Doblas, Jara Teresa; Ariza, Rafael R.; Roldán-Arjona, Teresa

2017-01-01

ABSTRACT DNA methylation is a crucial epigenetic mark associated to gene silencing, and its targeted removal is a major goal of epigenetic editing. In animal cells, DNA demethylation involves iterative 5mC oxidation by TET enzymes followed by replication-dependent dilution and/or replication-independent DNA repair of its oxidized derivatives. In contrast, plants use specific DNA glycosylases that directly excise 5mC and initiate its substitution for unmethylated C in a base excision repair process. In this work, we have fused the catalytic domain of Arabidopsis ROS1 5mC DNA glycosylase (ROS1_CD) to the DNA binding domain of yeast GAL4 (GBD). We show that the resultant GBD-ROS1_CD fusion protein binds specifically a GBD-targeted DNA sequence in vitro. We also found that transient in vivo expression of GBD-ROS1_CD in human cells specifically reactivates transcription of a methylation-silenced reporter gene, and that such reactivation requires both ROS1_CD catalytic activity and GBD binding capacity. Finally, we show that reactivation induced by GBD-ROS1_CD is accompanied by decreased methylation levels at several CpG sites of the targeted promoter. All together, these results show that plant 5mC DNA glycosylases can be used for targeted active DNA demethylation in human cells. PMID:28277978

Regulation of DNA methylation turnover at LTR retrotransposons and imprinted loci by the histone methyltransferase Setdb1.

PubMed

Leung, Danny; Du, Tingting; Wagner, Ulrich; Xie, Wei; Lee, Ah Young; Goyal, Preeti; Li, Yujing; Szulwach, Keith E; Jin, Peng; Lorincz, Matthew C; Ren, Bing

2014-05-06

During mammalian development, DNA methylation patterns need to be reset in primordial germ cells (PGCs) and preimplantation embryos. However, many LTR retrotransposons and imprinted genes are impervious to such global epigenetic reprogramming via hitherto undefined mechanisms. Here, we report that a subset of such genomic regions are resistant to widespread erasure of DNA methylation in mouse embryonic stem cells (mESCs) lacking the de novo DNA methyltransferases (Dnmts) Dnmt3a and Dnmt3b. Intriguingly, these loci are enriched for H3K9me3 in mESCs, implicating this mark in DNA methylation homeostasis. Indeed, deletion of the H3K9 methyltransferase SET domain bifurcated 1 (Setdb1) results in reduced H3K9me3 and DNA methylation levels at specific loci, concomitant with increased 5-hydroxymethylation (5hmC) and ten-eleven translocation 1 binding. Taken together, these data reveal that Setdb1 promotes the persistence of DNA methylation in mESCs, likely reflecting one mechanism by which DNA methylation is maintained at LTR retrotransposons and imprinted genes during developmental stages when DNA methylation is reprogrammed.

DNA methylation of ESR-1 and N-33 in colorectal mucosa of patients with ulcerative colitis (UC).

PubMed

Arasaradnam, Ramesh P; Khoo, Kevin; Bradburn, Mike; Mathers, John C; Kelly, Seamus B

2010-07-01

Epigenetic marking such as DNA methylation influence gene transcription and chromosomal stability and may also be affected by environmental exposures. Few studies exist on alteration in DNA methylation profiles (genomic and gene specific methylation) in patients with Ulcerative Colitis (UC) and no studies exist that assess its relationship with lifestyle exposures. The methylation level of both ESR-1 and N-33 genes were significantly higher in UC subjects compared with controls (7.9% vs. 5.9%; p = 0.015 and 66% vs. 9.3%; p < 0.001 respectively). There was no detectable difference in global DNA methylation between patients with UC and age and sex matched controls. No associations between indices of DNA methylation and anthropometric measures or smoking patterns were detected. To assess genomic methylation and promoter methylation of the ESR-1 (oestrogen receptor-1) and N-33 (tumor suppressor candidate-3) genes in the macroscopically normal mucosa of UC patients as well as to investigate effects of anthropometric and lifestyle exposures on DNA methylation. Sixty eight subjects were recruited (24 UC and 44 age and sex matched controls). Colorectal mucosal biopsies were obtained and DNA was extracted. Genomic DNA methylation was quantified using the tritium-labelled cytosine extension assay (3[H] dCTP) while gene specific methylation was quantified using the COBRA method. For the first time, we have shown increased methylation in the promoter regions of the putative tumor suppressor gene N-33 in macroscopically normal mucosa of patients with UC. In addition, we have confirmed that methylation of ESR-1 promoter is higher in UC patients compared with age and sex matched controls. These findings suggest that inactivation through methylation of the putative tumor suppressor genes N-33 and ESR-1 may not be associated with colorectal carcinogenesis in UC.

Joint-specific DNA methylation and transcriptome signatures in rheumatoid arthritis identify distinct pathogenic processes

PubMed Central

Ai, Rizi; Hammaker, Deepa; Boyle, David L.; Morgan, Rachel; Walsh, Alice M.; Fan, Shicai; Firestein, Gary S.; Wang, Wei

2016-01-01

Stratifying patients on the basis of molecular signatures could facilitate development of therapeutics that target pathways specific to a particular disease or tissue location. Previous studies suggest that pathogenesis of rheumatoid arthritis (RA) is similar in all affected joints. Here we show that distinct DNA methylation and transcriptome signatures not only discriminate RA fibroblast-like synoviocytes (FLS) from osteoarthritis FLS, but also distinguish RA FLS isolated from knees and hips. Using genome-wide methods, we show differences between RA knee and hip FLS in the methylation of genes encoding biological pathways, such as IL-6 signalling via JAK-STAT pathway. Furthermore, differentially expressed genes are identified between knee and hip FLS using RNA-sequencing. Double-evidenced genes that are both differentially methylated and expressed include multiple HOX genes. Joint-specific DNA signatures suggest that RA disease mechanisms might vary from joint to joint, thus potentially explaining some of the diversity of drug responses in RA patients. PMID:27282753

[GSTP1, APC and RASSF1 gene methylation in prostate cancer samples: comparative analysis of MS-HRM method and Infinium HumanMethylation450 BeadChip beadchiparray diagnostic value].

PubMed

Skorodumova, L O; Babalyan, K A; Sultanov, R; Vasiliev, A O; Govorov, A V; Pushkar, D Y; Prilepskaya, E A; Danilenko, S A; Generozov, E V; Larin, A K; Kostryukova, E S; Sharova, E I

2016-11-01

There is a clear need in molecular markers for prostate cancer (PC) risk stratification. Alteration of DNA methylation is one of processes that occur during ÐÑ progression. Methylation-sensitive PCR with high resolution melting curve analysis (MS-HRM) can be used for gene methylation analysis in routine laboratory practice. This method requires very small amounts of DNA for analysis. Numerous results have been accumulated on DNA methylation in PC samples analyzed by the Infinium HumanMethylation450 BeadChip (HM450). However, the consistency of MS-HRM results with chip hybridization results has not been examined yet. The aim of this study was to assess the consistency of results of GSTP1, APC and RASSF1 gene methylation analysis in ÐÑ biopsy samples obtained by MS-HRM and chip hybridization. The methylation levels of each gene determined by MS-HRM were statistically different in the group of PC tissue samples and the samples without signs of tumor growth. Chip hybridization data analysis confirmed the results obtained with the MS-HRM. Differences in methylation levels between tumor tissue and histologically intact tissue of each sample determined by MS-HRM and chip hybridization, were consistent with each other. Thus, we showed that the assessment of GSTP1, APC and RASSF1 gene methylation analysis using MS-HRM is suitable for the design of laboratory assays that will differentiate the PC tissue from the tissue without signs of tumor growth.

RNA interference knockdown of DNA methyl-transferase 3 affects gene alternative splicing in the honey bee

PubMed Central

Li-Byarlay, Hongmei; Li, Yang; Stroud, Hume; Feng, Suhua; Newman, Thomas C.; Kaneda, Megan; Hou, Kirk K.; Worley, Kim C.; Elsik, Christine G.; Wickline, Samuel A.; Jacobsen, Steven E.; Ma, Jian; Robinson, Gene E.

2013-01-01

Studies of DNA methylation from fungi, plants, and animals indicate that gene body methylation is ancient and highly conserved in eukaryotic genomes, but its role has not been clearly defined. It has been postulated that regulation of alternative splicing of transcripts was an original function of DNA methylation, but a direct experimental test of the effect of methylation on alternative slicing at the whole genome level has never been performed. To do this, we developed a unique method to administer RNA interference (RNAi) in a high-throughput and noninvasive manner and then used it to knock down the expression of DNA methyl-transferase 3 (dnmt3), which is required for de novo DNA methylation. We chose the honey bee (Apis mellifera) for this test because it has recently emerged as an important model organism for studying the effects of DNA methylation on development and social behavior, and DNA methylation in honey bees is predominantly on gene bodies. Here we show that dnmt3 RNAi decreased global genomic methylation level as expected and in addition caused widespread and diverse changes in alternative splicing in fat tissue. Four different types of splicing events were affected by dnmt3 gene knockdown, and change in two types, exon skipping and intron retention, was directly related to decreased methylation. These results demonstrate that one function of gene body DNA methylation is to regulate alternative splicing. PMID:23852726

Genome-wide comparative analysis of DNA methylation between soybean cytoplasmic male-sterile line NJCMS5A and its maintainer NJCMS5B.

PubMed

Li, Yanwei; Ding, Xianlong; Wang, Xuan; He, Tingting; Zhang, Hao; Yang, Longshu; Wang, Tanliu; Chen, Linfeng; Gai, Junyi; Yang, Shouping

2017-08-10

DNA methylation is an important epigenetic modification. It can regulate the expression of many key genes without changing the primary structure of the genomic DNA, and plays a vital role in the growth and development of the organism. The genome-wide DNA methylation profile of the cytoplasmic male sterile (CMS) line in soybean has not been reported so far. In this study, genome-wide comparative analysis of DNA methylation between soybean CMS line NJCMS5A and its maintainer NJCMS5B was conducted by whole-genome bisulfite sequencing. The results showed 3527 differentially methylated regions (DMRs) and 485 differentially methylated genes (DMGs), including 353 high-credible methylated genes, 56 methylated genes coding unknown protein and 76 novel methylated genes with no known function were identified. Among them, 25 DMRs were further validated that the genome-wide DNA methylation data were reliable through bisulfite treatment, and 9 DMRs were confirmed the relationship between DNA methylation and gene expression by qRT-PCR. Finally, 8 key DMGs possibly associated with soybean CMS were identified. Genome-wide DNA methylation profile of the soybean CMS line NJCMS5A and its maintainer NJCMS5B was obtained for the first time. Several specific DMGs which participated in pollen and flower development were further identified to be probably associated with soybean CMS. This study will contribute to further understanding of the molecular mechanism behind soybean CMS.

Whole DNA methylome profiling in mice exposed to secondhand smoke

PubMed Central

Tommasi, Stella; Zheng, Albert; Yoon, Jae-In; Li, Arthur Xuejun; Wu, Xiwei; Besaratinia, Ahmad

2012-01-01

Aberration of DNA methylation is a prime epigenetic mechanism of carcinogenesis. Aberrant DNA methylation occurs frequently in lung cancer, with exposure to secondhand smoke (SHS) being an established risk factor. The causal role of SHS in the genesis of lung cancer, however, remains elusive. To investigate whether SHS can cause aberrant DNA methylation in vivo, we have constructed the whole DNA methylome in mice exposed to SHS for a duration of 4 mo, both after the termination of exposure and at ensuing intervals post-exposure (up to 10 mo). Our genome-wide and gene-specific profiling of DNA methylation in the lung of SHS-exposed mice revealed that all groups of SHS-exposed mice and controls share a similar pattern of DNA methylation. Furthermore, the methylation status of major repetitive DNA elements, including long-interspersed nuclear elements (LINE L1), intracisternal A particle long-terminal repeat retrotransposons (IAP-LTR), and short-interspersed nuclear elements (SINE B1), in the lung of all groups of SHS-exposed mice and controls remains comparable. The absence of locus-specific gain of DNA methylation and global loss of DNA methylation in the lung of SHS-exposed mice within a timeframe that precedes neoplastic-lesion formation underscore the challenges of lung cancer biomarker development. Identifying the initiating events that cause aberrant DNA methylation in lung carcinogenesis may help improve future strategies for prevention, early detection and treatment of this highly lethal disease. PMID:23051858

LINE-1 methylation in plasma DNA as a biomarker of activity of DNA methylation inhibitors in patients with solid tumors.

PubMed

Aparicio, Ana; North, Brittany; Barske, Lindsey; Wang, Xuemei; Bollati, Valentina; Weisenberger, Daniel; Yoo, Christine; Tannir, Nizar; Horne, Erin; Groshen, Susan; Jones, Peter; Yang, Allen; Issa, Jean-Pierre

2009-04-01

Multiple clinical trials are investigating the use of the DNA methylation inhibitors azacitidine and decitabine for the treatment of solid tumors. Clinical trials in hematological malignancies have shown that optimal activity does not occur at their maximum tolerated doses but selection of an optimal biological dose and schedule for use in solid tumor patients is hampered by the difficulty of obtaining tumor tissue to measure their activity. Here we investigate the feasibility of using plasma DNA to measure the demethylating activity of the DNA methylation inhibitors in patients with solid tumors. We compared four methods to measure LINE-1 and MAGE-A1 promoter methylation in T24 and HCT116 cancer cells treated with decitabine treatment and selected Pyrosequencing for its greater reproducibility and higher signal to noise ratio. We then obtained DNA from plasma, peripheral blood mononuclear cells, buccal mucosa cells and saliva from ten patients with metastatic solid tumors at two different time points, without any intervening treatment. DNA methylation measurements were not significantly different between time point 1 and time point 2 in patient samples. We conclude that measurement of LINE-1 methylation in DNA extracted from the plasma of patients with advanced solid tumors, using Pyrosequencing, is feasible and has low within patient variability. Ongoing studies will determine whether changes in LINE-1 methylation in plasma DNA occur as a result of treatment with DNA methylation inhibitors and parallel changes in tumor tissue DNA.

Electronic transport in methylated fragments of DNA

DOE Office of Scientific and Technical Information (OSTI.GOV)

Almeida, M. L. de; Oliveira, J. I. N.; Lima Neto, J. X.

2015-11-16

We investigate the electronic transport properties of methylated deoxyribonucleic-acid (DNA) strands, a biological system in which methyl groups are added to DNA (a major epigenetic modification in gene expression), sandwiched between two metallic platinum electrodes. Our theoretical simulations apply an effective Hamiltonian based on a tight-binding model to obtain current-voltage curves related to the non-methylated/methylated DNA strands. The results suggest potential applications in the development of novel biosensors for molecular diagnostics.

Electronic transport in methylated fragments of DNA

NASA Astrophysics Data System (ADS)

de Almeida, M. L.; Oliveira, J. I. N.; Lima Neto, J. X.; Gomes, C. E. M.; Fulco, U. L.; Albuquerque, E. L.; Freire, V. N.; Caetano, E. W. S.; de Moura, F. A. B. F.; Lyra, M. L.

2015-11-01

We investigate the electronic transport properties of methylated deoxyribonucleic-acid (DNA) strands, a biological system in which methyl groups are added to DNA (a major epigenetic modification in gene expression), sandwiched between two metallic platinum electrodes. Our theoretical simulations apply an effective Hamiltonian based on a tight-binding model to obtain current-voltage curves related to the non-methylated/methylated DNA strands. The results suggest potential applications in the development of novel biosensors for molecular diagnostics.

Genome-wide DNA methylation modified by soy phytoestrogens: role for epigenetic therapeutics in prostate cancer?

PubMed

Karsli-Ceppioglu, Seher; Ngollo, Marjolaine; Adjakly, Mawussi; Dagdemir, Aslihan; Judes, Gaëlle; Lebert, André; Boiteux, Jean-Paul; Penault-LLorca, Frédérique; Bignon, Yves-Jean; Guy, Laurent; Bernard-Gallon, Dominique

2015-04-01

In prostate cancer, DNA methylation is significantly associated with tumor initiation, progression, and metastasis. Previous studies have suggested that soy phytoestrogens might regulate DNA methylation at individual candidate gene loci and that they play a crucial role as potential therapeutic agents for prostate cancer. The purpose of our study was to examine the modulation effects of phytoestrogens on a genome-wide scale in regards to DNA methylation in prostate cancer. Prostate cancer cell lines DU-145 and LNCaP were treated with 40 μM of genistein and 110 μM of daidzein. DNMT inhibitor 5-azacytidine (2 μM) and the methylating agent budesonide (2 μM) were used to compare their demethylation/methylation effects with phytoestrogens. The regulatory effects of phytoestrogens on DNA methylation were analyzed by using a methyl-DNA immunoprecipitation method coupled with Human DNA Methylation Microarrays (MeDIP-chip). We observed that the methylation profiles of 58 genes were altered by genistein and daidzein treatments in DU-145 and LNCaP prostate cancer cells. In addition, the methylation frequencies of the MAD1L1, TRAF7, KDM4B, and hTERT genes were remarkably modified by genistein treatment. Our results suggest that the modulation effects of phytoestrogens on DNA methylation essentially lead to inhibition of cell growth and induction of apoptosis. Genome-wide methylation profiling reported here suggests that epigenetic regulation mechanisms and, by extension, epigenetics-driven novel therapeutic candidates warrant further consideration in future "omics" studies of prostate cancer.

DNA methylation-based variation between human populations.

PubMed

Kader, Farzeen; Ghai, Meenu

2017-02-01

Several studies have proved that DNA methylation affects regulation of gene expression and development. Epigenome-wide studies have reported variation in methylation patterns between populations, including Caucasians, non-Caucasians (Blacks), Hispanics, Arabs, and numerous populations of the African continent. Not only has DNA methylation differences shown to impact externally visible characteristics, but is also a potential biomarker for underlying racial health disparities between human populations. Ethnicity-related methylation differences set their mark during early embryonic development. Genetic variations, such as single-nucleotide polymorphisms and environmental factors, such as age, dietary folate, socioeconomic status, and smoking, impacts DNA methylation levels, which reciprocally impacts expression of phenotypes. Studies show that it is necessary to address these external influences when attempting to differentiate between populations since the relative impacts of these factors on the human methylome remain uncertain. The present review summarises several reported attempts to establish the contribution of differential DNA methylation to natural human variation, and shows that DNA methylation could represent new opportunities for risk stratification and prevention of several diseases amongst populations world-wide. Variation of methylation patterns between human populations is an exciting prospect which inspires further valuable research to apply the concept in routine medical and forensic casework. However, trans-generational inheritance needs to be quantified to decipher the proportion of variation contributed by DNA methylation. The future holds thorough evaluation of the epigenome to understand quantification, heritability, and the effect of DNA methylation on phenotypes. In addition, methylation profiling of the same ethnic groups across geographical locations will shed light on conserved methylation differences in populations.

Forensic DNA methylation profiling from evidence material for investigative leads

PubMed Central

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

In Situ Analysis of DNA Methylation in Plants.

PubMed

Kathiria, Palak; Kovalchuk, Igor

2017-01-01

Epigenetic regulation in the plant genome is associated with the determination of expression patterns of various genes. Methylation of DNA at cytosine residues is one of the mechanisms of epigenetic regulation and has been a subject of various studies. Various techniques have been developed to analyze DNA methylation, most of which involve isolation of chromatin from cells and further in vitro studies. Limited techniques are available for in situ study of DNA methylation in plants. Here, we present such an in situ method for DNA methylation analysis which has high sensitivity and good reproducibility.

DNA methylation regulates neurophysiological spatial representation in memory formation

PubMed Central

Roth, Eric D.; Roth, Tania L.; Money, Kelli M.; SenGupta, Sonda; Eason, Dawn E.; Sweatt, J. David

2015-01-01

Epigenetic mechanisms including altered DNA methylation are critical for altered gene transcription subserving synaptic plasticity and the retention of learned behavior. Here we tested the idea that one role for activity-dependent altered DNA methylation is stabilization of cognition-associated hippocampal place cell firing in response to novel place learning. We observed that a behavioral protocol (spatial exploration of a novel environment) known to induce hippocampal place cell remapping resulted in alterations of hippocampal Bdnf DNA methylation. Further studies using neurophysiological in vivo single unit recordings revealed that pharmacological manipulations of DNA methylation decreased long-term but not short-term place field stability. Together our data highlight a role for DNA methylation in regulating neurophysiological spatial representation and memory formation. PMID:25960947

Altered DNA methylation: a secondary mechanism involved in carcinogenesis.

PubMed

Goodman, Jay I; Watson, Rebecca E

2002-01-01

This review focuses on the role that DNA methylation plays in the regulation of normal and aberrant gene expression and on how, in a hypothesis-driven fashion, altered DNA methylation may be viewed as a secondary mechanism involved in carcinogenesis. Research aimed at discerning the mechanisms by which chemicals can transform normal cells into frank carcinomas has both theoretical and practical implications. Through an increased understanding of the mechanisms by which chemicals affect the carcinogenic process, we learn more about basic biology while, at the same time, providing the type of information required to make more rational safety assessment decisions concerning their actual potential to cause cancer under particular conditions of exposure. One key question is: does the mechanism of action of the chemical in question involve a secondary mechanism and, if so, what dose may be below its threshold?

Heritable DNA methylation marks associated with susceptibility to breast cancer.

PubMed

Joo, Jihoon E; Dowty, James G; Milne, Roger L; Wong, Ee Ming; Dugué, Pierre-Antoine; English, Dallas; Hopper, John L; Goldgar, David E; Giles, Graham G; Southey, Melissa C

2018-02-28

Mendelian-like inheritance of germline DNA methylation in cancer susceptibility genes has been previously reported. We aimed to scan the genome for heritable methylation marks associated with breast cancer susceptibility by studying 25 Australian multiple-case breast cancer families. Here we report genome-wide DNA methylation measured in 210 peripheral blood DNA samples provided by family members using the Infinium HumanMethylation450. We develop and apply a new statistical method to identify heritable methylation marks based on complex segregation analysis. We estimate carrier probabilities for the 1000 most heritable methylation marks based on family structure, and we use Cox proportional hazards survival analysis to identify 24 methylation marks with corresponding carrier probabilities significantly associated with breast cancer. We replicate an association with breast cancer risk for four of the 24 marks using an independent nested case-control study. Here, we report a novel approach for identifying heritable DNA methylation marks associated with breast cancer risk.

Methylated bases in mycoplasmal DNA.

PubMed Central

Razin, A; Razin, S

1980-01-01

The DNAs of four Mycoplasma and one Acholeplasma species were found to contain methylated bases. All of the five species contained 6-methyladenine (m6Ade), the methylated base characteristic of prokaryotic DNA. The extent of methylation of adenine residues in the mycoplasmal DNA ranged from 0.2% in Mycoplasma capricolum to about 2% in Mycoplasma arginini and Mycoplasma hyorhinis with intermediate methylation values for Mycoplasma orale and Acholeplasma laidlawii DNAs. About 5.8% of the cytosine residues in M. hyorhinis DNA were methylated also. Analysis of cell culture DNA for the presence of m6Ade as a means for detection of contamination by mycoplasmas, and the phylogenetic implications of the finding of methylated bases in mycoplasmal DNAs are discussed. PMID:7433124

High fat diet and exercise lead to a disrupted and pathogenic DNA methylome in mouse liver.

PubMed

Zhou, Dan; Hlady, Ryan A; Schafer, Marissa J; White, Thomas A; Liu, Chen; Choi, Jeong-Hyeon; Miller, Jordan D; Roberts, Lewis R; LeBrasseur, Nathan K; Robertson, Keith D

2017-01-02

High-fat diet consumption and sedentary lifestyle elevates risk for obesity, non-alcoholic fatty liver disease, and cancer. Exercise training conveys health benefits in populations with or without these chronic conditions. Diet and exercise regulate gene expression by mediating epigenetic mechanisms in many tissues; however, such effects are poorly documented in the liver, a central metabolic organ. To dissect the consequences of diet and exercise on the liver epigenome, we measured DNA methylation, using reduced representation bisulfite sequencing, and transcription, using RNA-seq, in mice maintained on a fast food diet with sedentary lifestyle or exercise, compared with control diet with and without exercise. Our analyses reveal that genome-wide differential DNA methylation and expression of gene clusters are induced by diet and/or exercise. A combination of fast food and exercise triggers extensive gene alterations, with enrichment of carbohydrate/lipid metabolic pathways and muscle developmental processes. Through evaluation of putative protective effects of exercise on diet-induced DNA methylation, we show that hypermethylation is effectively prevented, especially at promoters and enhancers, whereas hypomethylation is only partially attenuated. We assessed diet-induced DNA methylation changes associated with liver cancer-related epigenetic modifications and identified significant increases at liver-specific enhancers in fast food groups, suggesting partial loss of liver cell identity. Hypermethylation at a subset of gene promoters was associated with inhibition of tissue development and promotion of carcinogenic processes. Our study demonstrates extensive reprogramming of the epigenome by diet and exercise, emphasizing the functional relevance of epigenetic mechanisms as an interface between lifestyle modifications and phenotypic alterations.

eMethylsorb: electrochemical quantification of DNA methylation at CpG resolution using DNA-gold affinity interactions.

PubMed

Sina, Abu Ali Ibn; Howell, Sidney; Carrascosa, Laura G; Rauf, Sakandar; Shiddiky, Muhammad J A; Trau, Matt

2014-11-07

We report a simple electrochemical method referred to as "eMethylsorb" for the detection of DNA methylation. The method relies on the base dependent affinity interaction of DNA with gold. The methylation status of DNA is quantified by monitoring the electrochemical current as a function of the relative adsorption level of bisulphite treated DNA samples onto a bare gold electrode. This method can successfully distinguish methylated and unmethylated epigenotypes at single CpG resolution.

Studies on the Mechanism of Action of Hydrazine-Induced Methylation of DNA Guanne

DTIC Science & Technology

1984-10-03

potent methylating agent , diazomethane (-CH -N+-N). Several in vivo studies were carried out to determine the role of aldehydes in the alkylation of DNA...methylating agent available to interact with DNA. If such a mechanism occurs, it may explain why disulfiram appears to inhibit the alkylation of DNA...a much slower/poorer alkylating agent for DNA. Effect of the 1-Carbon Pool on DNA Methylation in Hydrazine Toxicity: In Vitro In vitro studies were

Highly sensitive detection of DNA methylation levels by using a quantum dot-based FRET method

NASA Astrophysics Data System (ADS)

Ma, Yunfei; Zhang, Honglian; Liu, Fangming; Wu, Zhenhua; Lu, Shaohua; Jin, Qinghui; Zhao, Jianlong; Zhong, Xinhua; Mao, Hongju

2015-10-01

DNA methylation is the most frequently studied epigenetic modification that is strongly involved in genomic stability and cellular plasticity. Aberrant changes in DNA methylation status are ubiquitous in human cancer and the detection of these changes can be informative for cancer diagnosis. Herein, we reported a facile quantum dot-based (QD-based) fluorescence resonance energy transfer (FRET) technique for the detection of DNA methylation. The method relies on methylation-sensitive restriction enzymes for the differential digestion of genomic DNA based on its methylation status. Digested DNA is then subjected to PCR amplification for the incorporation of Alexa Fluor-647 (A647) fluorophores. DNA methylation levels can be detected qualitatively through gel analysis and quantitatively by the signal amplification from QDs to A647 during FRET. Furthermore, the methylation levels of three tumor suppressor genes, PCDHGB6, HOXA9 and RASSF1A, in 20 lung adenocarcinoma and 20 corresponding adjacent nontumorous tissue (NT) samples were measured to verify the feasibility of the QD-based FRET method and a high sensitivity for cancer detection (up to 90%) was achieved. Our QD-based FRET method is a convenient, continuous and high-throughput method, and is expected to be an alternative for detecting DNA methylation as a biomarker for certain human cancers.DNA methylation is the most frequently studied epigenetic modification that is strongly involved in genomic stability and cellular plasticity. Aberrant changes in DNA methylation status are ubiquitous in human cancer and the detection of these changes can be informative for cancer diagnosis. Herein, we reported a facile quantum dot-based (QD-based) fluorescence resonance energy transfer (FRET) technique for the detection of DNA methylation. The method relies on methylation-sensitive restriction enzymes for the differential digestion of genomic DNA based on its methylation status. Digested DNA is then subjected to PCR amplification for the incorporation of Alexa Fluor-647 (A647) fluorophores. DNA methylation levels can be detected qualitatively through gel analysis and quantitatively by the signal amplification from QDs to A647 during FRET. Furthermore, the methylation levels of three tumor suppressor genes, PCDHGB6, HOXA9 and RASSF1A, in 20 lung adenocarcinoma and 20 corresponding adjacent nontumorous tissue (NT) samples were measured to verify the feasibility of the QD-based FRET method and a high sensitivity for cancer detection (up to 90%) was achieved. Our QD-based FRET method is a convenient, continuous and high-throughput method, and is expected to be an alternative for detecting DNA methylation as a biomarker for certain human cancers. Electronic supplementary information (ESI) available: Synthesis of CdSe/CdS/ZnS core/shell/shell QDs. Sequences of primers used for amplifying the promoter regions in bisulfate-modified DNA. Comparison of detected methylation levels in different gene promoters using the QD-based FRET method versus bisulfite pyrosequencing. Methylation levels of the RASSF1A gene in one pair of NT and cancer samples as indicated by pyrosequencing. Theoretical calculation of the Förster distance R0. See DOI: 10.1039/c5nr04956c

Epigenetic regulation of neuronal immediate early genes is associated with decline in their expression and memory consolidation in scopolamine-induced amnesic mice.

PubMed

Srivas, Sweta; Thakur, Mahendra K

2017-09-01

Recently, we reported a correlation of scopolamine mediated decline in memory consolidation with increase in the expression of DNA methyltransferase 1 (DNMT1) and histone deacetylase 2 (HDAC2) in the mouse hippocampus. Memory consolidation is a protein synthesis-dependent process which involves the expression of synaptic plasticity genes, particularly neuronal immediate early genes (IEGs). However, the mechanism of regulation of these genes during decline in memory is poorly understood. Therefore, we have studied the epigenetic regulation of expression of neuronal IEGs in scopolamine-induced amnesic mice. Scopolamine significantly impaired memory consolidation as tested by radial arm maze, and the expression of neuronal IEGs was downregulated in the hippocampus as revealed by qRT-PCR and Western blotting. Further, methylated DNA immunoprecipitation (MeDIP) analysis showed increase in DNA methylation, while chromatin immunoprecipitation (ChIP) revealed decrease in H3K9/14 acetylation at the promoter of neuronal IEGs. Taken together, the present study shows that increased DNA methylation and decreased histone acetylation at the promoter of neuronal IEGs are associated with decline in their expression and memory consolidation during scopolamine-induced amnesia. These findings suggest that the epigenetic regulation through altered DNA methylation and histone acetylation might be explored further to develop potential therapeutic interventions for amnesia.

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

PubMed

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.

Genome-wide Mapping Reveals Conservation of Promoter DNA Methylation Following Chicken Domestication

PubMed Central

Li, Qinghe; Wang, Yuanyuan; Hu, Xiaoxiang; Zhao, Yaofeng; Li, Ning

2015-01-01

It is well-known that environment influences DNA methylation, however, the extent of heritable DNA methylation variation following animal domestication remains largely unknown. Using meDIP-chip we mapped the promoter methylomes for 23,316 genes in muscle tissues of ancestral and domestic chickens. We systematically examined the variation of promoter DNA methylation in terms of different breeds, differentially expressed genes, SNPs and genes undergo genetic selection sweeps. While considerable changes in DNA sequence and gene expression programs were prevalent, we found that the inter-strain DNA methylation patterns were highly conserved in promoter region between the wild and domestic chicken breeds. Our data suggests a global preservation of DNA methylation between the wild and domestic chicken breeds in either a genome-wide or locus-specific scale in chick muscle tissues. PMID:25735894

Epigenetic changes in leukocytes after 8 weeks of resistance exercise training.

PubMed

Denham, Joshua; Marques, Francine Z; Bruns, Emma L; O'Brien, Brendan J; Charchar, Fadi J

2016-06-01

Regular engagement in resistance exercise training elicits many health benefits including improvement to muscular strength, hypertrophy and insulin sensitivity, though the underpinning molecular mechanisms are poorly understood. The purpose of this study was to determine the influence 8 weeks of resistance exercise training has on leukocyte genome-wide DNA methylation and gene expression in healthy young men. Eight young (21.1 ± 2.2 years) men completed one repetition maximum (1RM) testing before completing 8 weeks of supervised, thrice-weekly resistance exercise training comprising three sets of 8-12 repetitions with a load equivalent to 80 % of 1RM. Blood samples were collected at rest before and after the 8-week training intervention. Genome-wide DNA methylation and gene expression were assessed on isolated leukocyte DNA and RNA using the 450K BeadChip and HumanHT-12 v4 Expression BeadChip (Illumina), respectively. Resistance exercise training significantly improved upper and lower body strength concurrently with diverse genome-wide DNA methylation and gene expression changes (p ≤ 0. 01). DNA methylation changes occurred at multiple regions throughout the genome in context with genes and CpG islands, and in genes relating to axon guidance, diabetes and immune pathways. There were multiple genes with increased expression that were enriched for RNA processing and developmental proteins. Growth factor genes-GHRH and FGF1-showed differential methylation and mRNA expression changes after resistance training. Our findings indicate that resistance exercise training improves muscular strength and is associated with reprogramming of the leukocyte DNA methylome and transcriptome.

In silico modeling of epigenetic-induced changes in photoreceptor cis-regulatory elements.

PubMed

Hossain, Reafa A; Dunham, Nicholas R; Enke, Raymond A; Berndsen, Christopher E

2018-01-01

DNA methylation is a well-characterized epigenetic repressor of mRNA transcription in many plant and vertebrate systems. However, the mechanism of this repression is not fully understood. The process of transcription is controlled by proteins that regulate recruitment and activity of RNA polymerase by binding to specific cis-regulatory sequences. Cone-rod homeobox (CRX) is a well-characterized mammalian transcription factor that controls photoreceptor cell-specific gene expression. Although much is known about the functions and DNA binding specificity of CRX, little is known about how DNA methylation modulates CRX binding affinity to genomic cis-regulatory elements. We used bisulfite pyrosequencing of human ocular tissues to measure DNA methylation levels of the regulatory regions of RHO , PDE6B, PAX6 , and LINE1 retrotransposon repeats. To describe the molecular mechanism of repression, we used molecular modeling to illustrate the effect of DNA methylation on human RHO regulatory sequences. In this study, we demonstrate an inverse correlation between DNA methylation in regulatory regions adjacent to the human RHO and PDE6B genes and their subsequent transcription in human ocular tissues. Docking of CRX to the DNA models shows that CRX interacts with the grooves of these sequences, suggesting changes in groove structure could regulate binding. Molecular dynamics simulations of the RHO promoter and enhancer regions show changes in the flexibility and groove width upon epigenetic modification. Models also demonstrate changes in the local dynamics of CRX binding sites within RHO regulatory sequences which may account for the repression of CRX-dependent transcription. Collectively, these data demonstrate epigenetic regulation of CRX binding sites in human retinal tissue and provide insight into the mechanism of this mode of epigenetic regulation to be tested in future experiments.

Analysis of trichloroethylene-induced global DNA hypomethylation in hepatic L-02 cells by liquid chromatography-electrospray ionization tandem mass spectrometry.

PubMed

Zhang, Hang; Hong, Wen-Xu; Ye, Jinbo; Yang, Xifei; Ren, Xiaohu; Huang, Aibo; Yang, Linqing; Zhou, Li; Huang, Haiyan; Wu, Desheng; Huang, Xinfeng; Zhuang, Zhixiong; Liu, Jianjun

2014-04-04

Trichloroethylene (TCE), a major occupational and environmental pollutant, has been recently associated with aberrant epigenetic changes in experimental animals and cultured cells. TCE is known to cause severe hepatotoxicity; however, the association between epigenetic alterations and TCE-induced hepatotoxicity are not yet well explored. DNA methylation, catalyzed by enzymes known as DNA methyltransferases (DNMT), is a major epigenetic modification that plays a critical role in regulating many cellular processes. In this study, we analyzed the TCE-induced effect on global DNA methylation and DNMT enzymatic activity in human hepatic L-02 cells. A sensitive and quantitative method combined with liquid chromatography and electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) was validated and utilized for assessing the altered DNA methylation in TCE-induced L-02 cells. Quantification was accomplished in multiple reaction monitoring (MRM) mode by monitoring a transition pair of m/z 242.1 (molecular ion)/126.3 (fragment ion) for 5-mdC and m/z 268.1/152.3 for dG. The correlation coefficient of calibration curves between 5-mdC and dG was higher than 0.9990. The intra-day and inter-day relative standard derivation values (RSD) were on the range of 0.53-7.09% and 0.40-2.83%, respectively. We found that TCE exposure was able to significantly decrease the DNA methylation and inhibit DNMT activity in L-02 cells. Our results not only reveal the association between TCE exposure and epigenetic alterations, but also provide an alternative mass spectrometry-based method for rapid and accurate assessment of chemical-induced altered DNA methylation in mammal cells. Copyright © 2014 Elsevier Inc. All rights reserved.

Epigenetics insights into chronic pain: DNA hypomethylation in fibromyalgia-a controlled pilot-study.

PubMed

Ciampi de Andrade, Daniel; Maschietto, Mariana; Galhardoni, Ricardo; Gouveia, Gisele; Chile, Thais; Victorino Krepischi, Ana C; Dale, Camila S; Brunoni, André R; Parravano, Daniella C; Cueva Moscoso, Ana S; Raicher, Irina; Kaziyama, Helena H S; Teixeira, Manoel J; Brentani, Helena P

2017-08-01

To evaluate changes in DNA methylation profiles in patients with fibromyalgia (FM) compared to matched healthy controls (HCs). All individuals underwent full clinical and neurophysiological assessment by cortical excitability (CE) parameters measured by transcranial magnetic stimulation. DNA from the peripheral blood of patients with FM (n = 24) and HC (n = 24) were assessed using the Illumina-HumanMethylation450 BeadChips. We identified 1610 differentially methylated positions (DMPs) in patients with FM displaying a nonrandom distribution in regions of the genome. Sixty-nine percent of DMP in FM were hypomethylated compared to HC. Differentially methylated positions were enriched in 5 genomic regions (1p34; 6p21; 10q26; 17q25; 19q13). The functional characterization of 960 genes related to DMPs revealed an enrichment for MAPK signaling pathway (n = 18 genes), regulation of actin cytoskeleton (n = 15 genes), and focal adhesion (n = 13 genes). A gene-gene interaction network enrichment analysis revealed the participation of DNA repair pathways, mitochondria-related processes, and synaptic signaling. Even though DNA was extracted from peripheral blood, this set of genes was enriched for disorders such as schizophrenia, mood disorders, bulimia, hyperphagia, and obesity. Remarkably, the hierarchical clusterization based on the methylation levels of the 1610 DMPs showed an association with neurophysiological measurements of CE in FM and HC. Fibromyalgia has a hypomethylation DNA pattern, which is enriched in genes implicated in stress response and DNA repair/free radical clearance. These changes occurred parallel to changes in CE parameters. New epigenetic insights into the pathophysiology of FM may provide the basis for the development of biomarkers of this disorder.

A Six Months Exercise Intervention Influences the Genome-wide DNA Methylation Pattern in Human Adipose Tissue

PubMed Central

Rönn, Tina; Volkov, Petr; Davegårdh, Cajsa; Dayeh, Tasnim; Hall, Elin; Olsson, Anders H.; Nilsson, Emma; Tornberg, Åsa; Dekker Nitert, Marloes; Eriksson, Karl-Fredrik; Jones, Helena A.; Groop, Leif; Ling, Charlotte

2013-01-01

Epigenetic mechanisms are implicated in gene regulation and the development of different diseases. The epigenome differs between cell types and has until now only been characterized for a few human tissues. Environmental factors potentially alter the epigenome. Here we describe the genome-wide pattern of DNA methylation in human adipose tissue from 23 healthy men, with a previous low level of physical activity, before and after a six months exercise intervention. We also investigate the differences in adipose tissue DNA methylation between 31 individuals with or without a family history of type 2 diabetes. DNA methylation was analyzed using Infinium HumanMethylation450 BeadChip, an array containing 485,577 probes covering 99% RefSeq genes. Global DNA methylation changed and 17,975 individual CpG sites in 7,663 unique genes showed altered levels of DNA methylation after the exercise intervention (q<0.05). Differential mRNA expression was present in 1/3 of gene regions with altered DNA methylation, including RALBP1, HDAC4 and NCOR2 (q<0.05). Using a luciferase assay, we could show that increased DNA methylation in vitro of the RALBP1 promoter suppressed the transcriptional activity (p = 0.03). Moreover, 18 obesity and 21 type 2 diabetes candidate genes had CpG sites with differences in adipose tissue DNA methylation in response to exercise (q<0.05), including TCF7L2 (6 CpG sites) and KCNQ1 (10 CpG sites). A simultaneous change in mRNA expression was seen for 6 of those genes. To understand if genes that exhibit differential DNA methylation and mRNA expression in human adipose tissue in vivo affect adipocyte metabolism, we silenced Hdac4 and Ncor2 respectively in 3T3-L1 adipocytes, which resulted in increased lipogenesis both in the basal and insulin stimulated state. In conclusion, exercise induces genome-wide changes in DNA methylation in human adipose tissue, potentially affecting adipocyte metabolism. PMID:23825961

[Dynamics of LINE-1 Retrotransposon Methylation Levels in Circulating DNA from Lung Cancer Patients Undergoing Antitumor Therapy].

PubMed

Ponomaryova, A A; Cherdyntseva, N V; Bondar, A A; Dobrodeev, A Y; Zavyalov, A A; Tuzikov, S A; Vlassov, V V; Choinzonov, E L; Laktionov, P P; Rykova, E Y

2017-01-01

Malignant cell transformation is accompanied with abnormal DNA methylation, such as the hypermethylation of certain gene promoters and hypomethylation of retrotransposons. In particular, the hypomethylation of the human-specific family of LINE-1 retrotransposons was observed in lung cancer tissues. It is also known that the circulating DNA (cirDNA) of blood plasma and cell-surface-bound circulating DNA (csb-cirDNA) of cancer patients accumulate tumor-specific aberrantly methylated DNA fragments, which are currently considered to be valuable cancer markers. This work compares LINE-1 retrotransposon methylation patterns in cirDNA of 16 lung cancer patients before and after treatment. CirDNA was isolated from blood plasma, and csb-cirDNA fractions were obtained by successive elution with EDTA-containing phosphate buffered saline and trypsin. Concentrations of methylated LINE-1 region 1 copies (LINE-1-met) were assayed by real-time methylation-specific PCR. LINE-1 methylation levels were normalized to the concentration of LINE-1 region 2, which was independent of the methylation status (LINE-1-Ind). The concentrations of LINE-1-met and LINE-1-Ind in csb-cirDNA of lung cancer patients exhibited correlations before treatment (r = 0.54), after chemotherapy (r = 0.72), and after surgery (r = 0.83) (P < 0.05, Spearman rank test). In the total group of patients, the level of LINE-1 methylation (determined as the LINE-1-met/LINE-1-Ind ratio) was shown to increase significantly during the follow-up after chemotherapy (P < 0.05, paired t test) and after surgery compared to the level of methylation before treatment (P < 0.05, paired t test). The revealed association between the level of LINE-1 methylation and the effect of antitumor therapy was more pronounced in squamous cell lung cancer than in adenocarcinoma (P < 0.05 and P > 0.05, respectively). These results suggest a need for the further investigation of dynamic changes in levels of LINE-1 methylation depending on the antitumor therapy.

Aberrant DNA methylation patterns in diabetic nephropathy

PubMed Central

2014-01-01

Background The aim of this study was to evaluate whether global levels of DNA methylation status were associated with albuminuria and progression of diabetic nephropathy in a case-control study of 123 patients with type 2 diabetes- 53 patients with albuminuria and 70 patients without albuminuria. Methods The 5-methyl cytosine content was assessed by reverse phase high pressure liquid chromatography (RP-HPLC) of peripheral blood mononuclear cells to determine individual global DNA methylation status in two groups. Results Global DNA methylation levels were significantly higher in patients with albuminuria compared with those in normal range of albuminuria (p = 0.01). There were significant differences in global levels of DNA methylation in relation to albuminuria (p = 0.028) and an interesting pattern of increasing global levels of DNA methylation in terms of albuminuria severity. In patients with micro- and macro albuminuria, we found no significant correlations between global DNA methylation levels and duration of diabetes (p > 0.05). In both sub groups, there were not significant differences between global DNA methylation levels with good and poor glycaemic control (p > 0.05). In addition, in patients with albuminuria, no differences in DNA methylation levels were observed between patients with and without other risk factors including age, gender, hypertension, dyslipidaemia and obesity. Conclusions These data may be helpful in further studies to develop novel biomarkers and new strategies for clinical care of patients at risk of diabetic nephropathy. PMID:25028646

DNA methylation dynamics during early plant life.

PubMed

Bouyer, Daniel; Kramdi, Amira; Kassam, Mohamed; Heese, Maren; Schnittger, Arp; Roudier, François; Colot, Vincent

2017-09-25

Cytosine methylation is crucial for gene regulation and silencing of transposable elements in mammals and plants. While this epigenetic mark is extensively reprogrammed in the germline and early embryos of mammals, the extent to which DNA methylation is reset between generations in plants remains largely unknown. Using Arabidopsis as a model, we uncovered distinct DNA methylation dynamics over transposable element sequences during the early stages of plant development. Specifically, transposable elements and their relics show invariably high methylation at CG sites but increasing methylation at CHG and CHH sites. This non-CG methylation culminates in mature embryos, where it reaches saturation for a large fraction of methylated CHH sites, compared to the typical 10-20% methylation level observed in seedlings or adult plants. Moreover, the increase in CHH methylation during embryogenesis matches the hypomethylated state in the early endosperm. Finally, we show that interfering with the embryo-to-seedling transition results in the persistence of high CHH methylation levels after germination, specifically over sequences that are targeted by the RNA-directed DNA methylation (RdDM) machinery. Our findings indicate the absence of extensive resetting of DNA methylation patterns during early plant life and point instead to an important role of RdDM in reinforcing DNA methylation of transposable element sequences in every cell of the mature embryo. Furthermore, we provide evidence that this elevated RdDM activity is a specific property of embryogenesis.

Maternal Methyl-Group Donor Intake and Global DNA (Hydroxy)Methylation before and during Pregnancy

PubMed Central

Pauwels, Sara; Duca, Radu Corneliu; Devlieger, Roland; Freson, Kathleen; Straetmans, Dany; Van Herck, Erik; Huybrechts, Inge; Koppen, Gurdun; Godderis, Lode

2016-01-01

It is still unclear to which extent methyl-group intake during pregnancy can affect maternal global DNA (hydroxyl)methylation. Pregnancy methylation profiling and its link with methyl-group intake in a healthy population could enhance our understanding of the development of pregnancy related disorders. One hundred forty-eight women were enrolled in the MANOE (MAternal Nutrition and Offspring’s Epigenome) study. Thiry-four women were enrolled before pregnancy and 116 during the first trimester of pregnancy. Global DNA (hydroxy)methylation in blood using LC-MS/MS and dietary methyl-group intake (methionine, folate, betaine, and choline) using a food-frequency questionnaire were estimated pre-pregnancy, during each trimester, and at delivery. Global DNA (hydroxy)methylation levels were highest pre-pregnancy and at weeks 18–22 of pregnancy. We observed a positive relation between folic acid and global DNA methylation (p = 0.04) and hydroxymethylation (p = 0.04). A high intake of methionine pre-pregnancy and in the first trimester showed lower (hydroxy)methylation percentage in weeks 11–13 and weeks 18–22, respectively. Choline and betaine intake in the first weeks was negatively associated with hydroxymethylation. Women with a high intake of these three methyl groups in the second and third trimester showed higher hyrdoxymethylation/methylation levels in the third trimester. To conclude, a time trend in DNA (hydroxy)methylation was found and women with higher methyl-group intake showed higher methylation in the third trimester, and not in earlier phases of pregnancy. PMID:27509522

Maternal Methyl-Group Donor Intake and Global DNA (Hydroxy)Methylation before and during Pregnancy.

PubMed

Pauwels, Sara; Duca, Radu Corneliu; Devlieger, Roland; Freson, Kathleen; Straetmans, Dany; Van Herck, Erik; Huybrechts, Inge; Koppen, Gurdun; Godderis, Lode

2016-08-06

It is still unclear to which extent methyl-group intake during pregnancy can affect maternal global DNA (hydroxyl)methylation. Pregnancy methylation profiling and its link with methyl-group intake in a healthy population could enhance our understanding of the development of pregnancy related disorders. One hundred forty-eight women were enrolled in the MANOE (MAternal Nutrition and Offspring's Epigenome) study. Thiry-four women were enrolled before pregnancy and 116 during the first trimester of pregnancy. Global DNA (hydroxy)methylation in blood using LC-MS/MS and dietary methyl-group intake (methionine, folate, betaine, and choline) using a food-frequency questionnaire were estimated pre-pregnancy, during each trimester, and at delivery. Global DNA (hydroxy)methylation levels were highest pre-pregnancy and at weeks 18-22 of pregnancy. We observed a positive relation between folic acid and global DNA methylation (p = 0.04) and hydroxymethylation (p = 0.04). A high intake of methionine pre-pregnancy and in the first trimester showed lower (hydroxy)methylation percentage in weeks 11-13 and weeks 18-22, respectively. Choline and betaine intake in the first weeks was negatively associated with hydroxymethylation. Women with a high intake of these three methyl groups in the second and third trimester showed higher hyrdoxymethylation/methylation levels in the third trimester. To conclude, a time trend in DNA (hydroxy)methylation was found and women with higher methyl-group intake showed higher methylation in the third trimester, and not in earlier phases of pregnancy.

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

PubMed

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.

Tissue-specific DNA methylation is conserved across human, mouse, and rat, and driven by primary sequence conservation.

PubMed

Zhou, Jia; Sears, Renee L; Xing, Xiaoyun; Zhang, Bo; Li, Daofeng; Rockweiler, Nicole B; Jang, Hyo Sik; Choudhary, Mayank N K; Lee, Hyung Joo; Lowdon, Rebecca F; Arand, Jason; Tabers, Brianne; Gu, C Charles; Cicero, Theodore J; Wang, Ting

2017-09-12

Uncovering mechanisms of epigenome evolution is an essential step towards understanding the evolution of different cellular phenotypes. While studies have confirmed DNA methylation as a conserved epigenetic mechanism in mammalian development, little is known about the conservation of tissue-specific genome-wide DNA methylation patterns. Using a comparative epigenomics approach, we identified and compared the tissue-specific DNA methylation patterns of rat against those of mouse and human across three shared tissue types. We confirmed that tissue-specific differentially methylated regions are strongly associated with tissue-specific regulatory elements. Comparisons between species revealed that at a minimum 11-37% of tissue-specific DNA methylation patterns are conserved, a phenomenon that we define as epigenetic conservation. Conserved DNA methylation is accompanied by conservation of other epigenetic marks including histone modifications. Although a significant amount of locus-specific methylation is epigenetically conserved, the majority of tissue-specific DNA methylation is not conserved across the species and tissue types that we investigated. Examination of the genetic underpinning of epigenetic conservation suggests that primary sequence conservation is a driving force behind epigenetic conservation. In contrast, evolutionary dynamics of tissue-specific DNA methylation are best explained by the maintenance or turnover of binding sites for important transcription factors. Our study extends the limited literature of comparative epigenomics and suggests a new paradigm for epigenetic conservation without genetic conservation through analysis of transcription factor binding sites.

Epigenetic Variation in Monozygotic Twins: A Genome-Wide Analysis of DNA Methylation in Buccal Cells

PubMed Central

van Dongen, Jenny; Ehli, Erik A.; Slieker, Roderick C.; Bartels, Meike; Weber, Zachary M.; Davies, Gareth E.; Slagboom, P. Eline; Heijmans, Bastiaan T.; Boomsma, Dorret I.

2014-01-01

DNA methylation is one of the most extensively studied epigenetic marks in humans. Yet, it is largely unknown what causes variation in DNA methylation between individuals. The comparison of DNA methylation profiles of monozygotic (MZ) twins offers a unique experimental design to examine the extent to which such variation is related to individual-specific environmental influences and stochastic events or to familial factors (DNA sequence and shared environment). We measured genome-wide DNA methylation in buccal samples from ten MZ pairs (age 8–19) using the Illumina 450k array and examined twin correlations for methylation level at 420,921 CpGs after QC. After selecting CpGs showing the most variation in the methylation level between subjects, the mean genome-wide correlation (rho) was 0.54. The correlation was higher, on average, for CpGs within CpG islands (CGIs), compared to CGI shores, shelves and non-CGI regions, particularly at hypomethylated CpGs. This finding suggests that individual-specific environmental and stochastic influences account for more variation in DNA methylation in CpG-poor regions. Our findings also indicate that it is worthwhile to examine heritable and shared environmental influences on buccal DNA methylation in larger studies that also include dizygotic twins. PMID:24802513

De novo DNA methylation during monkey pre-implantation embryogenesis.

PubMed

Gao, Fei; Niu, Yuyu; Sun, Yi Eve; Lu, Hanlin; Chen, Yongchang; Li, Siguang; Kang, Yu; Luo, Yuping; Si, Chenyang; Yu, Juehua; Li, Chang; Sun, Nianqin; Si, Wei; Wang, Hong; Ji, Weizhi; Tan, Tao

2017-04-01

Critical epigenetic regulation of primate embryogenesis entails DNA methylome changes. Here we report genome-wide composition, patterning, and stage-specific dynamics of DNA methylation in pre-implantation rhesus monkey embryos as well as male and female gametes studied using an optimized tagmentation-based whole-genome bisulfite sequencing method. We show that upon fertilization, both paternal and maternal genomes undergo active DNA demethylation, and genome-wide de novo DNA methylation is also initiated in the same period. By the 8-cell stage, remethylation becomes more pronounced than demethylation, resulting in an increase in global DNA methylation. Promoters of genes associated with oxidative phosphorylation are preferentially remethylated at the 8-cell stage, suggesting that this mode of energy metabolism may not be favored. Unlike in rodents, X chromosome inactivation is not observed during monkey pre-implantation development. Our study provides the first comprehensive illustration of the 'wax and wane' phases of DNA methylation dynamics. Most importantly, our DNA methyltransferase loss-of-function analysis indicates that DNA methylation influences early monkey embryogenesis.

De novo DNA methylation during monkey pre-implantation embryogenesis

PubMed Central

Gao, Fei; Niu, Yuyu; Sun, Yi Eve; Lu, Hanlin; Chen, Yongchang; Li, Siguang; Kang, Yu; Luo, Yuping; Si, Chenyang; Yu, Juehua; Li, Chang; Sun, Nianqin; Si, Wei; Wang, Hong; Ji, Weizhi; Tan, Tao

2017-01-01

Critical epigenetic regulation of primate embryogenesis entails DNA methylome changes. Here we report genome-wide composition, patterning, and stage-specific dynamics of DNA methylation in pre-implantation rhesus monkey embryos as well as male and female gametes studied using an optimized tagmentation-based whole-genome bisulfite sequencing method. We show that upon fertilization, both paternal and maternal genomes undergo active DNA demethylation, and genome-wide de novo DNA methylation is also initiated in the same period. By the 8-cell stage, remethylation becomes more pronounced than demethylation, resulting in an increase in global DNA methylation. Promoters of genes associated with oxidative phosphorylation are preferentially remethylated at the 8-cell stage, suggesting that this mode of energy metabolism may not be favored. Unlike in rodents, X chromosome inactivation is not observed during monkey pre-implantation development. Our study provides the first comprehensive illustration of the 'wax and wane' phases of DNA methylation dynamics. Most importantly, our DNA methyltransferase loss-of-function analysis indicates that DNA methylation influences early monkey embryogenesis. PMID:28233770

Triplex-mediated analysis of cytosine methylation at CpA sites in DNA.

PubMed

Johannsen, Marie W; Gerrard, Simon R; Melvin, Tracy; Brown, Tom

2014-01-18

Modified triplex-forming oligonucleotides distinguish 5-methyl cytosine from unmethylated cytosine in DNA duplexes by differences in triplex melting temperatures. The discrimination is sequence-specific; dramatic differences in stabilisation are seen for CpA methylation, whereas CpG methylation is not detected. This direct detection of DNA methylation constitutes a new approach for epigenetic analysis.

Global DNA methylation loss associated with mercury contamination and aging in the American alligator (Alligator mississippiensis).

PubMed

Nilsen, Frances M; Parrott, Benjamin B; Bowden, John A; Kassim, Brittany L; Somerville, Stephen E; Bryan, Teresa A; Bryan, Colleen E; Lange, Ted R; Delaney, J Patrick; Brunell, Arnold M; Long, Stephen E; Guillette, Louis J

2016-03-01

Mercury is a widespread environmental contaminant with exposures eliciting a well-documented catalog of adverse effects. Yet, knowledge regarding the underlying mechanisms by which mercury exposures are translated into biological effects remains incomplete. DNA methylation is an epigenetic modification that is sensitive to environmental cues, and alterations in DNA methylation at the global level are associated with a variety of diseases. Using a liquid chromatography tandem mass spectrometry-based (LC-MS/MS) approach, global DNA methylation levels were measured in red blood cells of 144 wild American alligators (Alligator mississippiensis) from 6 sites with variable levels of mercury contamination across Florida's north-south axis. Variation in mercury concentrations measured in whole blood was highly associated with location, allowing the comparison of global DNA methylation levels across different "treatments" of mercury. Global DNA methylation in alligators across all locations was weakly associated with increased mercury exposure. However, a much more robust relationship was observed in those animals sampled from locations more highly contaminated with mercury. Also, similar to other vertebrates, global DNA methylation appears to decline with age in alligators. The relationship between age-associated loss of global DNA methylation and varying mercury exposures was examined to reveal a potential interaction. These findings demonstrate that global DNA methylation levels are associated with mercury exposure, and give insights into interactions between contaminants, aging, and epigenetics. Published by Elsevier B.V.

Densely ionizing radiation affects DNA methylation of selective LINE-1 elements1

PubMed Central

Prior, Sara; Miousse, Isabelle R.; Nzabarushimana, Etienne; Pathak, Rupak; Skinner, Charles; Kutanzi, Kristy R.; Allen, Antiño R.; Raber, Jacob; Tackett, Alan J.; Hauer-Jensen, Martin; Nelson, Gregory A.; Koturbash, Igor

2016-01-01

Long Interspersed Nucleotide Element 1 (LINE-1) retrotransposons are heavily methylated and are the most abundant transposable elements in mammalian genomes. Here, we investigated the differential DNA methylation within the LINE-1 under normal conditions and in response to environmentally relevant doses of sparsely and densely ionizing radiation. We demonstrate that DNA methylation of LINE-1 elements in the lungs of C57BL6 mice is dependent on their evolutionary age, where the elder age of the element is associated with the lower extent of DNA methylation. Exposure to 5-aza-2′-deoxycytidine and methionine-deficient diet affected DNA methylation of selective LINE-1 elements in an age- and promoter type-dependent manner. Exposure to densely IR, but not sparsely IR, resulted in DNA hypermethylation of older LINE-1 elements, while the DNA methylation of evolutionary younger elements remained mostly unchanged. We also demonstrate that exposure to densely IR increased mRNA and protein levels of LINE-1 via the loss of the histone H3K9 dimethylation and an increase in the H3K4 trimethylation at the LINE-1 5′-untranslated region, independently of DNA methylation. Our findings suggest that DNA methylation is important for regulation of LINE-1 expression under normal conditions, but histone modifications may dictate the transcriptional activity of LINE-1 in response to exposure to densely IR. PMID:27419368

The interaction between cytosine methylation and processes of DNA replication and repair shape the mutational landscape of cancer genomes.

PubMed

Poulos, Rebecca C; Olivier, Jake; Wong, Jason W H

2017-07-27

Methylated cytosines (5mCs) are frequently mutated in the genome. However, no studies have yet comprehensively analysed mutation-methylation associations across cancer types. Here we analyse 916 cancer genomes, together with tissue type-specific methylation and replication timing data. We describe a strong mutation-methylation association across colorectal cancer subtypes, most interestingly in samples with microsatellite instability (MSI) or Polymerase epsilon (POLE) exonuclease domain mutations. By analysing genomic regions with differential mismatch repair (MMR) efficiency, we suggest a possible role for MMR in the correction of 5mC deamination events, potentially accounting for the high rate of 5mC mutation accumulation in MSI tumours. Additionally, we propose that mutant POLE asserts a mutator phenotype specifically at 5mCs, and we find coding mutation hotspots in POLE-mutant cancers at highly-methylated CpGs in the tumour-suppressor genes APC and TP53. Finally, using multivariable regression models, we demonstrate that different cancers exhibit distinct mutation-methylation associations, with DNA repair influencing such associations in certain cancer genomes. Taken together, we find differential associations with methylation that are vital for accurately predicting expected mutation loads across cancer types. Our findings reveal links between methylation and common mutation and repair processes, with these mechanisms defining a key part of the mutational landscape of cancer genomes. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Regulation of DNA methylation patterns by CK2-mediated phosphorylation of Dnmt3a.

PubMed

Deplus, Rachel; Blanchon, Loïc; Rajavelu, Arumugam; Boukaba, Abdelhalim; Defrance, Matthieu; Luciani, Judith; Rothé, Françoise; Dedeurwaerder, Sarah; Denis, Hélène; Brinkman, Arie B; Simmer, Femke; Müller, Fabian; Bertin, Benjamin; Berdasco, Maria; Putmans, Pascale; Calonne, Emilie; Litchfield, David W; de Launoit, Yvan; Jurkowski, Tomasz P; Stunnenberg, Hendrik G; Bock, Christoph; Sotiriou, Christos; Fraga, Mario F; Esteller, Manel; Jeltsch, Albert; Fuks, François

2014-08-07

DNA methylation is a central epigenetic modification that is established by de novo DNA methyltransferases. The mechanisms underlying the generation of genomic methylation patterns are still poorly understood. Using mass spectrometry and a phosphospecific Dnmt3a antibody, we demonstrate that CK2 phosphorylates endogenous Dnmt3a at two key residues located near its PWWP domain, thereby downregulating the ability of Dnmt3a to methylate DNA. Genome-wide DNA methylation analysis shows that CK2 primarily modulates CpG methylation of several repeats, most notably of Alu SINEs. This modulation can be directly attributed to CK2-mediated phosphorylation of Dnmt3a. We also find that CK2-mediated phosphorylation is required for localization of Dnmt3a to heterochromatin. By revealing phosphorylation as a mode of regulation of de novo DNA methyltransferase function and by uncovering a mechanism for the regulation of methylation at repetitive elements, our results shed light on the origin of DNA methylation patterns. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

Methylation-Sensitive Expression of a DNA Demethylase Gene Serves As an Epigenetic Rheostat

PubMed Central

Williams, Ben P.; Pignatta, Daniela; Henikoff, Steven; Gehring, Mary

2015-01-01

Genomes must balance active suppression of transposable elements (TEs) with the need to maintain gene expression. In Arabidopsis, euchromatic TEs are targeted by RNA-directed DNA methylation (RdDM). Conversely, active DNA demethylation prevents accumulation of methylation at genes proximal to these TEs. It is unknown how a cellular balance between methylation and demethylation activities is achieved. Here we show that both RdDM and DNA demethylation are highly active at a TE proximal to the major DNA demethylase gene ROS1. Unexpectedly, and in contrast to most other genomic targets, expression of ROS1 is promoted by DNA methylation and antagonized by DNA demethylation. We demonstrate that inducing methylation in the ROS1 proximal region is sufficient to restore ROS1 expression in an RdDM mutant. Additionally, methylation-sensitive expression of ROS1 is conserved in other species, suggesting it is adaptive. We propose that the ROS1 locus functions as an epigenetic rheostat, tuning the level of demethylase activity in response to methylation alterations, thus ensuring epigenomic stability. PMID:25826366

Quantitative DNA Methylation Profiling in Cancer.

PubMed

Ammerpohl, Ole; Haake, Andrea; Kolarova, Julia; Siebert, Reiner

2016-01-01

Epigenetic mechanisms including DNA methylation are fundamental for the regulation of gene expression. Epigenetic alterations can lead to the development and the evolution of malignant tumors as well as the emergence of phenotypically different cancer cells or metastasis from one single tumor cell. Here we describe bisulfite pyrosequencing, a technology to perform quantitative DNA methylation analyses, to detect aberrant DNA methylation in malignant tumors.

Using logic programming for modeling the one-carbon metabolism network to study the impact of folate deficiency on methylation processes.

PubMed

Gnimpieba, Etienne Z; Eveillard, Damien; Guéant, Jean-Louis; Chango, Abalo

2011-08-01

Dynamical modeling is an accurate tool for describing the dynamic regulation of one-carbon metabolism (1CM) with emphasis on the alteration of DNA methylation and/or dUMP methylation into dTMP. Using logic programming we present a comprehensive and adaptative mathematical model to study the impact of folate deficiency, including folate transport and enzymes activities. 5-Methyltetrahydrofolate (5mTHF) uptake and DNA and dUMP methylation were studied by simulating nutritional 5mTHF deficiency and methylenetetrahydrofolate reductase (MTHFR) gene defects. Both conditions had distinct effects on 1CM metabolite synthesis. Simulating severe 5mTHF deficiency (25% of normal levels) modulated 11 metabolites. However, simulating a severe decrease in MTHFR activity (25% of normal activity) modulated another set of metabolites. Two oscillations of varying amplitude were observed at the steady state for DNA methylation with severe 5mTHF deficiency, and the dUMP/dTMP ratio reached a steady state after 2 h, compared to 2.5 h for 100% 5mTHF. MTHFR activity with 25% of V(max) resulted in an increased methylated DNA pool after half an hour. We observed a deviation earlier in the profile compared to 50% and 100% V(max). For dUMP methylation, the highest level was observed with 25%, suggesting a low rate of dUMP methylation into dTMP with 25% of MTHFR activity. In conclusion, using logic programming we were able to construct the 1CM for analyzing the dynamic system behavior. This model may be used to refine biological interpretations of data or as a tool that can provide new hypotheses for pathogenesis.

DNA Replication Origin Function Is Promoted by H3K4 Di-methylation in Saccharomyces cerevisiae

PubMed Central

Rizzardi, Lindsay F.; Dorn, Elizabeth S.; Strahl, Brian D.; Cook, Jeanette Gowen

2012-01-01

DNA replication is a highly regulated process that is initiated from replication origins, but the elements of chromatin structure that contribute to origin activity have not been fully elucidated. To identify histone post-translational modifications important for DNA replication, we initiated a genetic screen to identify interactions between genes encoding chromatin-modifying enzymes and those encoding proteins required for origin function in the budding yeast Saccharomyces cerevisiae. We found that enzymes required for histone H3K4 methylation, both the histone methyltransferase Set1 and the E3 ubiquitin ligase Bre1, are required for robust growth of several hypomorphic replication mutants, including cdc6-1. Consistent with a role for these enzymes in DNA replication, we found that both Set1 and Bre1 are required for efficient minichromosome maintenance. These phenotypes are recapitulated in yeast strains bearing mutations in the histone substrates (H3K4 and H2BK123). Set1 functions as part of the COMPASS complex to mono-, di-, and tri-methylate H3K4. By analyzing strains lacking specific COMPASS complex members or containing H2B mutations that differentially affect H3K4 methylation states, we determined that these replication defects were due to loss of H3K4 di-methylation. Furthermore, histone H3K4 di-methylation is enriched at chromosomal origins. These data suggest that H3K4 di-methylation is necessary and sufficient for normal origin function. We propose that histone H3K4 di-methylation functions in concert with other histone post-translational modifications to support robust genome duplication. PMID:22851644

DNA replication origin function is promoted by H3K4 di-methylation in Saccharomyces cerevisiae.

PubMed

Rizzardi, Lindsay F; Dorn, Elizabeth S; Strahl, Brian D; Cook, Jeanette Gowen

2012-10-01

DNA replication is a highly regulated process that is initiated from replication origins, but the elements of chromatin structure that contribute to origin activity have not been fully elucidated. To identify histone post-translational modifications important for DNA replication, we initiated a genetic screen to identify interactions between genes encoding chromatin-modifying enzymes and those encoding proteins required for origin function in the budding yeast Saccharomyces cerevisiae. We found that enzymes required for histone H3K4 methylation, both the histone methyltransferase Set1 and the E3 ubiquitin ligase Bre1, are required for robust growth of several hypomorphic replication mutants, including cdc6-1. Consistent with a role for these enzymes in DNA replication, we found that both Set1 and Bre1 are required for efficient minichromosome maintenance. These phenotypes are recapitulated in yeast strains bearing mutations in the histone substrates (H3K4 and H2BK123). Set1 functions as part of the COMPASS complex to mono-, di-, and tri-methylate H3K4. By analyzing strains lacking specific COMPASS complex members or containing H2B mutations that differentially affect H3K4 methylation states, we determined that these replication defects were due to loss of H3K4 di-methylation. Furthermore, histone H3K4 di-methylation is enriched at chromosomal origins. These data suggest that H3K4 di-methylation is necessary and sufficient for normal origin function. We propose that histone H3K4 di-methylation functions in concert with other histone post-translational modifications to support robust genome duplication.

Cannabis use by women during pregnancy does not influence infant DNA methylation of the dopamine receptor DRD4.

PubMed

Fransquet, Peter D; Hutchinson, Delyse; Olsson, Craig A; Allsop, Steve; Elliott, Elizabeth J; Burns, Lucinda; Mattick, Richard; Saffery, Richard; Ryan, Joanne

2017-11-01

Maternal cannabis use in pregnancy is linked with long-term adverse behavioral outcomes in offspring. Epigenetic processes established in utero that affect dopaminergic (reward) signaling may mediate risks. Associations between cannabis use and offspring DNA methylation have not been investigated; however, maternal tobacco smoking in pregnancy is associated with distinct patterns of DNA methylation at birth and beyond. To determine whether maternal cannabis use is associated with methylation of the dopamine receptor gene DRD4 promoter in infants. Mothers in the Triple B study provided detailed information on drug use in each trimester of pregnancy. Buccal swabs were collected from neonates at 8 weeks (n = 804, 51.7% male, and 48.3% female). DRD4 promoter DNA methylation was measured using SEQUENOM MassARRAY. Fifty-seven of the women in the study reported drug use during pregnancy, of whom 44 used cannabis. Of 19 cytosine-phosphate-guanine dinucleotides (CpG) units tested in DRD4, gestational cannabis use was associated with offspring methylation at 1 CpG unit in multivariate models (β + 1.48, CI: 0.02 to 2.93, and p = 0.047). At another site there was weak evidence that both cannabis and other drug use were independently associated with increased methylation, while the association with tobacco was in the reverse direction (cannabis use β + 0.67, CI: -0.12 to 1.46, and p = 0.09; other drug use β + 1.11, CI: 0.17 to 2.05, and p = 0.02; tobacco use β -0.41, CI: -0.85 to 0.03, and p = 0.07). None of the associations would remain significant after correction for multiple testing. There is no strong evidence that maternal cannabis use in pregnancy is associated with offspring DRD4 methylation.

Differential DNA methylation profile of key genes in malignant prostate epithelial cells transformed by inorganic arsenic or cadmium.

PubMed

Pelch, Katherine E; Tokar, Erik J; Merrick, B Alex; Waalkes, Michael P

2015-08-01

Previous work shows altered methylation patterns in inorganic arsenic (iAs)- or cadmium (Cd)-transformed epithelial cells. Here, the methylation status near the transcriptional start site was assessed in the normal human prostate epithelial cell line (RWPE-1) that was malignantly transformed by 10μM Cd for 11weeks (CTPE) or 5μM iAs for 29weeks (CAsE-PE), at which time cells showed multiple markers of acquired cancer phenotype. Next generation sequencing of the transcriptome of CAsE-PE cells identified multiple dysregulated genes. Of the most highly dysregulated genes, five genes that can be relevant to the carcinogenic process (S100P, HYAL1, NTM, NES, ALDH1A1) were chosen for an in-depth analysis of the DNA methylation profile. DNA was isolated, bisulfite converted, and combined bisulfite restriction analysis was used to identify differentially methylated CpG sites, which was confirmed with bisulfite sequencing. Four of the five genes showed differential methylation in transformants relative to control cells that was inversely related to altered gene expression. Increased expression of HYAL1 (>25-fold) and S100P (>40-fold) in transformants was correlated with hypomethylation near the transcriptional start site. Decreased expression of NES (>15-fold) and NTM (>1000-fold) in transformants was correlated with hypermethylation near the transcriptional start site. ALDH1A1 expression was differentially expressed in transformed cells but was not differentially methylated relative to control. In conclusion, altered gene expression observed in Cd and iAs transformed cells may result from altered DNA methylation status. Published by Elsevier Inc.

The Epigenomic Landscape of Prokaryotes

DOE PAGES

Blow, Matthew J.; Clark, Tyson A.; Daum, Chris G.; ...

2016-02-12

DNA methylation acts in concert with restriction enzymes to protect the integrity of prokaryotic genomes. Studies in a limited number of organisms suggest that methylation also contributes to prokaryotic genome regulation, but the prevalence and properties of such non-restriction-associated methylation systems remain poorly understood. Here, we used single molecule, real-time sequencing to map DNA modifications including m6A, m4C, and m5C across the genomes of 230 diverse bacterial and archaeal species. We observed DNA methylation in nearly all (93%) organisms examined, and identified a total of 834 distinct reproducibly methylated motifs. This data enabled annotation of the DNA binding specificities ofmore » 620 DNA Methyltransferases (MTases), doubling known specificities for previously hard to study Type I, IIG and III MTases, and revealing their extraordinary diversity. Strikingly, 48% of organisms harbor active Type II MTases with no apparent cognate restriction enzyme. These active ‘orphan’ MTases are present in diverse bacterial and archaeal phyla and show motif specificities and methylation patterns consistent with functions in gene regulation and DNA replication. Our results reveal the pervasive presence of DNA methylation throughout the prokaryotic kingdoms, as well as the diversity of sequence specificities and potential functions of DNA methylation systems.« less

The Epigenomic Landscape of Prokaryotes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Blow, Matthew J.; Clark, Tyson A.; Daum, Chris G.

DNA methylation acts in concert with restriction enzymes to protect the integrity of prokaryotic genomes. Studies in a limited number of organisms suggest that methylation also contributes to prokaryotic genome regulation, but the prevalence and properties of such non-restriction-associated methylation systems remain poorly understood. Here, we used single molecule, real-time sequencing to map DNA modifications including m6A, m4C, and m5C across the genomes of 230 diverse bacterial and archaeal species. We observed DNA methylation in nearly all (93%) organisms examined, and identified a total of 834 distinct reproducibly methylated motifs. This data enabled annotation of the DNA binding specificities ofmore » 620 DNA Methyltransferases (MTases), doubling known specificities for previously hard to study Type I, IIG and III MTases, and revealing their extraordinary diversity. Strikingly, 48% of organisms harbor active Type II MTases with no apparent cognate restriction enzyme. These active ‘orphan’ MTases are present in diverse bacterial and archaeal phyla and show motif specificities and methylation patterns consistent with functions in gene regulation and DNA replication. Our results reveal the pervasive presence of DNA methylation throughout the prokaryotic kingdoms, as well as the diversity of sequence specificities and potential functions of DNA methylation systems.« less

Effects of DNA Methylation and Chromatin State on Rates of Molecular Evolution in Insects.

PubMed

Glastad, Karl M; Goodisman, Michael A D; Yi, Soojin V; Hunt, Brendan G

2015-12-04

Epigenetic information is widely appreciated for its role in gene regulation in eukaryotic organisms. However, epigenetic information can also influence genome evolution. Here, we investigate the effects of epigenetic information on gene sequence evolution in two disparate insects: the fly Drosophila melanogaster, which lacks substantial DNA methylation, and the ant Camponotus floridanus, which possesses a functional DNA methylation system. We found that DNA methylation was positively correlated with the synonymous substitution rate in C. floridanus, suggesting a key effect of DNA methylation on patterns of gene evolution. However, our data suggest the link between DNA methylation and elevated rates of synonymous substitution was explained, in large part, by the targeting of DNA methylation to genes with signatures of transcriptionally active chromatin, rather than the mutational effect of DNA methylation itself. This phenomenon may be explained by an elevated mutation rate for genes residing in transcriptionally active chromatin, or by increased structural constraints on genes in inactive chromatin. This result highlights the importance of chromatin structure as the primary epigenetic driver of genome evolution in insects. Overall, our study demonstrates how different epigenetic systems contribute to variation in the rates of coding sequence evolution. Copyright © 2016 Glastad et al.

Changes in DNA methylation over the growing season differ between North Carolina farmworkers and non-farmworkers.

PubMed

Howard, Timothy D; Hsu, Fang-Chi; Chen, Haiying; Quandt, Sara A; Talton, Jennifer W; Summers, Phillip; Arcury, Thomas A

2016-10-01

The occupational risk to farmworkers, particularly chronic exposure to pesticides, is an acknowledged environmental and work-related health problem. Epigenetics has recently been shown to contribute to a number of complex diseases and traits, including measures of cognitive function and preclinical neurodegenerative disease. We sought to determine whether changes in DNA methylation existed between farmworker and non-farmworker populations and to identify the genes most likely involved in those changes. Eighty-three farmworkers and 60 non-farmworkers were selected from PACE4, a community-based, participatory research project comparing occupational exposures between immigrant Latino farmworker and non-farmworker manual workers. Measurements of DNA methylation were performed with the Infinium HumanMethylation450 BeadChip, at the beginning and end of the 2012 growing season. Bonferroni adjustment was used to identify significant findings (p = 1.03 × 10(-7), based on 485,000 tested methylation sites), although less stringent criteria (i.e., p ≤ 1 × 10(-6)) were used to identify sites of interest. Expression quantitative trait locus (eQTL) databases were used to help identify the most likely functional genes for each associated methylation site. Methylation at 36 CpG sites, located in or near 72 genes, differed between the two groups (p ≤ 1 × 10(-6)). The difference between the two groups was generally due to an increase in methylation in the farmworkers and a slight decrease in methylation in the non-farmworkers. Enrichment was observed in several biological pathways, including those involved in the immune response, as well as growth hormone signaling, role of BRCA1 in DNA damage response, p70S6K signaling, and PI3K signaling in B lymphocytes. We identified considerable changes in DNA methylation at 36 CpG sites over the growing season that differed between farmworkers and non-farmworkers. Dominant pathways included immune-related (HLA) processes, as well as a number of diverse biological systems. Further studies are necessary to determine which exposures or behaviors are responsible for the observed changes, and whether these changes eventually lead to disease-related phenotypes in this population.

Intraindividual dynamics of transcriptome and genome-wide stability of DNA methylation

PubMed Central

Furukawa, Ryohei; Hachiya, Tsuyoshi; Ohmomo, Hideki; Shiwa, Yuh; Ono, Kanako; Suzuki, Sadafumi; Satoh, Mamoru; Hitomi, Jiro; Sobue, Kenji; Shimizu, Atsushi

2016-01-01

Cytosine methylation at CpG dinucleotides is an epigenetic mechanism that affects the gene expression profiles responsible for the functional differences in various cells and tissues. Although gene expression patterns are dynamically altered in response to various stimuli, the intraindividual dynamics of DNA methylation in human cells are yet to be fully understood. Here, we investigated the extent to which DNA methylation contributes to the dynamics of gene expression by collecting 24 blood samples from two individuals over a period of 3 months. Transcriptome and methylome association analyses revealed that only ~2% of dynamic changes in gene expression could be explained by the intraindividual variation of DNA methylation levels in peripheral blood mononuclear cells and purified monocytes. These results showed that DNA methylation levels remain stable for at least several months, suggesting that disease-associated DNA methylation markers are useful for estimating the risk of disease manifestation. PMID:27192970

Simultaneous Profiling of DNA Mutation and Methylation by Melting Analysis Using Magnetoresistive Biosensor Array.

PubMed

Rizzi, Giovanni; Lee, Jung-Rok; Dahl, Christina; Guldberg, Per; Dufva, Martin; Wang, Shan X; Hansen, Mikkel F

2017-09-26

Epigenetic modifications, in particular DNA methylation, are gaining increasing interest as complementary information to DNA mutations for cancer diagnostics and prognostics. We introduce a method to simultaneously profile DNA mutation and methylation events for an array of sites with single site specificity. Genomic (mutation) or bisulphite-treated (methylation) DNA is amplified using nondiscriminatory primers, and the amplicons are then hybridized to a giant magnetoresistive (GMR) biosensor array followed by melting curve measurements. The GMR biosensor platform offers scalable multiplexed detection of DNA hybridization, which is insensitive to temperature variation. The melting curve approach further enhances the assay specificity and tolerance to variations in probe length. We demonstrate the utility of this method by simultaneously profiling five mutation and four methylation sites in human melanoma cell lines. The method correctly identified all mutation and methylation events and further provided quantitative assessment of methylation density validated by bisulphite pyrosequencing.

Epigenetic Pattern on the Human Y Chromosome Is Evolutionarily Conserved

PubMed Central

Meng, Hao; Agbagwa, Ikechukwu O.; Wang, Ling-Xiang; Wang, Yingzhi; Yan, Shi; Ren, Shancheng; Sun, Yinghao; Pei, Gang; Liu, Xin; Liu, Jiang; Jin, Li; Li, Hui; Sun, Yingli

2016-01-01

DNA methylation plays an important role for mammalian development. However, it is unclear whether the DNA methylation pattern is evolutionarily conserved. The Y chromosome serves as a powerful tool for the study of human evolution because it is transferred between males. In this study, based on deep-rooted pedigrees and the latest Y chromosome phylogenetic tree, we performed epigenetic pattern analysis of the Y chromosome from 72 donors. By comparing their respective DNA methylation level, we found that the DNA methylation pattern on the Y chromosome was stable among family members and haplogroups. Interestingly, two haplogroup-specific methylation sites were found, which were both genotype-dependent. Moreover, the African and Asian samples also had similar DNA methylation pattern with a remote divergence time. Our findings indicated that the DNA methylation pattern on the Y chromosome was conservative during human male history. PMID:26760298

DNA methylation profiles distinguish different subtypes of gastroenteropancreatic neuroendocrine tumors.

PubMed

How-Kit, Alexandre; Dejeux, Emelyne; Dousset, Bertrand; Renault, Victor; Baudry, Marion; Terris, Benoit; Tost, Jörg

2015-01-01

Most studies have considered gastroenteropancreatic neuroendocrine tumors (GEP-NETs) as a homogenous group of samples or distinguish only gastrointestinal from pancreatic endocrine tumors. This article investigates if DNA methylation patterns could distinguish subtypes of GEP-NETs. The DNA methylation level of 807 cancer-related genes was investigated in insulinomas, gastrinomas, non-functioning pancreatic endocrine tumors and small intestine endocrine tumors. DNA methylation patterns were found to be tumor type specific for each of the pancreatic tumor subtypes and identified two distinct methylation-based groups in small intestine endocrine tumors. Differences of DNA methylation levels were validated by pyrosequencing for 20 candidate genes and correlated with differences at the transcriptional level for four candidate genes. The heterogeneity of DNA methylation patterns in the different subtypes of gastroenteropancreatic neuroendocrine tumors suggests different underlying pathways and, therefore, these tumors should be considered as distinct entities in molecular and clinical studies.

Dnmts and Tet target memory-associated genes after appetitive olfactory training in honey bees

PubMed Central

Biergans, Stephanie D.; Giovanni Galizia, C.; Reinhard, Judith; Claudianos, Charles

2015-01-01

DNA methylation and demethylation are epigenetic mechanisms involved in memory formation. In honey bees DNA methyltransferase (Dnmt) function is necessary for long-term memory to be stimulus specific (i.e. to reduce generalization). So far, however, it remains elusive which genes are targeted and what the time-course of DNA methylation is during memory formation. Here, we analyse how DNA methylation affects memory retention, gene expression, and differential methylation in stimulus-specific olfactory long-term memory formation. Out of 30 memory-associated genes investigated here, 9 were upregulated following Dnmt inhibition in trained bees. These included Dnmt3 suggesting a negative feedback loop for DNA methylation. Within these genes also the DNA methylation pattern changed during the first 24 hours after training. Interestingly, this was accompanied by sequential activation of the DNA methylation machinery (i.e. Dnmts and Tet). In sum, memory formation involves a temporally complex epigenetic regulation of memory-associated genes that facilitates stimulus specific long-term memory in the honey bee. PMID:26531238

Hydration properties of natural and synthetic DNA sequences with methylated adenine or cytosine bases in the R.DpnI target and BDNF promoter studied by molecular dynamics simulations

NASA Astrophysics Data System (ADS)

Shanak, Siba; Helms, Volkhard

2014-12-01

Adenine and cytosine methylation are two important epigenetic modifications of DNA sequences at the levels of the genome and transcriptome. To characterize the differential roles of methylating adenine or cytosine with respect to their hydration properties, we performed conventional MD simulations and free energy perturbation calculations for two particular DNA sequences, namely the brain-derived neurotrophic factor (BDNF) promoter and the R.DpnI-bound DNA that are known to undergo methylation of C5-methyl cytosine and N6-methyl adenine, respectively. We found that a single methylated cytosine has a clearly favorable hydration free energy over cytosine since the attached methyl group has a slightly polar character. In contrast, capping the strongly polar N6 of adenine with a methyl group gives a slightly unfavorable contribution to its free energy of solvation. Performing the same demethylation in the context of a DNA double-strand gave quite similar results for the more solvent-accessible cytosine but much more unfavorable results for the rather buried adenine. Interestingly, the same demethylation reactions are far more unfavorable when performed in the context of the opposite (BDNF or R.DpnI target) sequence. This suggests a natural preference for methylation in a specific sequence context. In addition, free energy calculations for demethylating adenine or cytosine in the context of B-DNA vs. Z-DNA suggest that the conformational B-Z transition of DNA transition is rather a property of cytosine methylated sequences but is not preferable for the adenine-methylated sequences investigated here.

Hydration properties of natural and synthetic DNA sequences with methylated adenine or cytosine bases in the R.DpnI target and BDNF promoter studied by molecular dynamics simulations.

PubMed

Shanak, Siba; Helms, Volkhard

2014-12-14

Adenine and cytosine methylation are two important epigenetic modifications of DNA sequences at the levels of the genome and transcriptome. To characterize the differential roles of methylating adenine or cytosine with respect to their hydration properties, we performed conventional MD simulations and free energy perturbation calculations for two particular DNA sequences, namely the brain-derived neurotrophic factor (BDNF) promoter and the R.DpnI-bound DNA that are known to undergo methylation of C5-methyl cytosine and N6-methyl adenine, respectively. We found that a single methylated cytosine has a clearly favorable hydration free energy over cytosine since the attached methyl group has a slightly polar character. In contrast, capping the strongly polar N6 of adenine with a methyl group gives a slightly unfavorable contribution to its free energy of solvation. Performing the same demethylation in the context of a DNA double-strand gave quite similar results for the more solvent-accessible cytosine but much more unfavorable results for the rather buried adenine. Interestingly, the same demethylation reactions are far more unfavorable when performed in the context of the opposite (BDNF or R.DpnI target) sequence. This suggests a natural preference for methylation in a specific sequence context. In addition, free energy calculations for demethylating adenine or cytosine in the context of B-DNA vs. Z-DNA suggest that the conformational B-Z transition of DNA transition is rather a property of cytosine methylated sequences but is not preferable for the adenine-methylated sequences investigated here.

Altered DNA Methylation Patterns Associated With Clinically Relevant Increases in PTSD Symptoms and PTSD Symptom Profiles in Military Personnel.

PubMed

Martin, Christiana; Cho, Young-Eun; Kim, Hyungsuk; Yun, Sijung; Kanefsky, Rebekah; Lee, Hyunhwa; Mysliwiec, Vincent; Cashion, Ann; Gill, Jessica

2018-05-01

Military personnel experience posttraumatic stress disorder (PTSD), which is associated with differential DNA methylation across the whole genome. However, the relationship between these DNA methylation patterns and clinically relevant increases in PTSD severity is not yet clearly understood. The purpose of this study was to identify differences in DNA methylation associated with PTSD symptoms and investigate DNA methylation changes related to increases in the severity of PTSD in military personnel. In this pilot study, a cross-sectional comparison was made between military personnel with PTSD (n = 8) and combat-matched controls without PTSD (n = 6). Symptom measures were obtained, and genome-wide DNA methylation was measured using methylated DNA immunoprecipitation (MeDIP-seq) from whole blood samples at baseline and 3 months later. A longitudinal comparison measured DNA methylation changes in military personnel with clinically relevant increases in PTSD symptoms between time points (PTSD onset) and compared methylation patterns to controls with no clinical changes in PTSD. In military personnel with elevated PTSD symptoms 3 months following baseline, 119 genes exhibited reduced methylation and 8 genes exhibited increased methylation. Genes with reduced methylation in the PTSD-onset group relate to the canonical pathways of netrin signaling, Wnt/Ca + pathway, and axonal guidance signaling. These gene pathways relate to neurological disorders, and the current findings suggest that these epigenetic changes potentially relate to PTSD symptomology. This study provides some novel insights into the role of epigenetic changes in PTSD symptoms and the progression of PTSD symptoms in military personnel.

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

PubMed

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.

Toxicological effects of benzo[a]pyrene on DNA methylation of whole genome in ICR mice.

PubMed

Zhao, L; Zhang, S; An, X; Tan, W; Pang, D; Ouyang, H

2015-10-30

It has been well known that alterations in DNA methylation - an important regulator of gene transcription - lead to cancer. Therefore a change in the level of DNA methylation of whole genome has been considered as a biomarker of carcinogenesis. Previously, a large number of experimental results in genetic toxicology have showed that benzo[a]pyrene could cause DNA mutation and fragmentation. However, there was little to no studies on alterations in DNA methylation of genome directly result from exposure to benzo[a]pyrene. In this paper, possible mechanisms of alterations in whole genomic DNA methylation by benzo[a]pyrene were investigated using ICR mice after benzo[a]pyrene exposure. The blood, liver, pancreas, skin, lung and bladder of ICR mice were removed and checked after a fixed time interval (6 hours) of benzo[a]pyrene exposure, and whole genomic DNA methylation level was determined by high performance liquid chromatography (HPLC). The results exhibited tissue specificity, that is, the level of whole genomic DNA methylation decreases significantly in blood and liver, rather than pancreas, lung, skin and bladder of ICR mice. This study investigated the direct relationship between aberrant DNA methylation level and benzo[a]pyrene exposure, which might be helpful to clarify the toxicological mechanism of benzo[a]pyrene in epigenetic perspectives.

Twenty-four–nucleotide siRNAs produce heritable trans-chromosomal methylation in F1 Arabidopsis hybrids

PubMed Central

Greaves, Ian K.; Eichten, Steven R.; Groszmann, Michael; Wang, Aihua; Ying, Hua; Peacock, W. James; Dennis, Elizabeth S.

2016-01-01

Hybrid Arabidopsis plants undergo epigenetic reprogramming producing decreased levels of 24-nt siRNAs and altered patterns of DNA methylation that can affect gene expression. Driving the changes in methylation are the processes trans-chromosomal methylation (TCM) and trans-chromosomal demethylation (TCdM). In TCM/TCdM the methylation state of one allele is altered to resemble the other allele. We show that Pol IV-dependent sRNAs are required to establish TCM events. The changes in DNA methylation and the associated changes in sRNA levels in the F1 hybrid can be maintained in subsequent generations and affect hundreds of regions in the F2 epigenome. The inheritance of these altered epigenetic states varies in F2 individuals, resulting in individuals with genetically identical loci displaying different epigenetic states and gene expression profiles. The change in methylation at these regions is associated with the presence of sRNAs. Loci without any sRNA activity can have altered methylation states, suggesting that a sRNA-independent mechanism may also contribute to the altered methylation state of the F1 and F2 generations. PMID:27791153

Methylation pattern of fish lymphocystis disease virus DNA.

PubMed

Wagner, H; Simon, D; Werner, E; Gelderblom, H; Darai, C; Flügel, R M

1985-03-01

The content and distribution of 5-methylcytosine in DNA from fish lymphocystis disease virus was analyzed by high-pressure liquid chromatography, nearest-neighbor analysis, and with restriction endonucleases. We found that 22% of all C residues were methylated, including methylation of the following dinucleotide sequences: CpG to 75%, CpC to ca. 1%, and CpA to 2 to 5%. Comparison of relative digestion of viral DNA with MspI and HpaII indicated that CCGG sequences were almost completely methylated at the inner C. The degree of methylation of GCGC was much lower. The methylation pattern of fish lymphocystis disease virus DNA differed from that of the host cell DNA.

Methylation pattern of fish lymphocystis disease virus DNA.

PubMed Central

Wagner, H; Simon, D; Werner, E; Gelderblom, H; Darai, C; Flügel, R M

1985-01-01

The content and distribution of 5-methylcytosine in DNA from fish lymphocystis disease virus was analyzed by high-pressure liquid chromatography, nearest-neighbor analysis, and with restriction endonucleases. We found that 22% of all C residues were methylated, including methylation of the following dinucleotide sequences: CpG to 75%, CpC to ca. 1%, and CpA to 2 to 5%. Comparison of relative digestion of viral DNA with MspI and HpaII indicated that CCGG sequences were almost completely methylated at the inner C. The degree of methylation of GCGC was much lower. The methylation pattern of fish lymphocystis disease virus DNA differed from that of the host cell DNA. Images PMID:3973962

Direct bisulfite sequencing for examination of DNA methylation with gene and nucleotide resolution from brain tissues.

PubMed

Parrish, R Ryley; Day, Jeremy J; Lubin, Farah D

2012-07-01

DNA methylation is an epigenetic modification that is essential for the development and mature function of the central nervous system. Due to the relevance of this modification to the transcriptional control of gene expression, it is often necessary to examine changes in DNA methylation patterns with both gene and single-nucleotide resolution. Here, we describe an in-depth basic protocol for direct bisulfite sequencing of DNA isolated from brain tissue, which will permit direct assessment of methylation status at individual genes as well as individual cytosine molecules/nucleotides within a genomic region. This method yields analysis of DNA methylation patterns that is robust, accurate, and reproducible, thereby allowing insights into the role of alterations in DNA methylation in brain tissue.

Dynamic and static maintenance of epigenetic memory in pluripotent and somatic cells.

PubMed

Shipony, Zohar; Mukamel, Zohar; Cohen, Netta Mendelson; Landan, Gilad; Chomsky, Elad; Zeliger, Shlomit Reich; Fried, Yael Chagit; Ainbinder, Elena; Friedman, Nir; Tanay, Amos

2014-09-04

Stable maintenance of gene regulatory programs is essential for normal function in multicellular organisms. Epigenetic mechanisms, and DNA methylation in particular, are hypothesized to facilitate such maintenance by creating cellular memory that can be written during embryonic development and then guide cell-type-specific gene expression. Here we develop new methods for quantitative inference of DNA methylation turnover rates, and show that human embryonic stem cells preserve their epigenetic state by balancing antagonistic processes that add and remove methylation marks rather than by copying epigenetic information from mother to daughter cells. In contrast, somatic cells transmit considerable epigenetic information to progenies. Paradoxically, the persistence of the somatic epigenome makes it more vulnerable to noise, since random epimutations can accumulate to massively perturb the epigenomic ground state. The rate of epigenetic perturbation depends on the genomic context, and, in particular, DNA methylation loss is coupled to late DNA replication dynamics. Epigenetic perturbation is not observed in the pluripotent state, because the rapid turnover-based equilibrium continuously reinforces the canonical state. This dynamic epigenetic equilibrium also explains how the epigenome can be reprogrammed quickly and to near perfection after induced pluripotency.

Insights into the Pathogenesis of Anaplastic Large-Cell Lymphoma through Genome-wide DNA Methylation Profiling.

PubMed

Hassler, Melanie R; Pulverer, Walter; Lakshminarasimhan, Ranjani; Redl, Elisa; Hacker, Julia; Garland, Gavin D; Merkel, Olaf; Schiefer, Ana-Iris; Simonitsch-Klupp, Ingrid; Kenner, Lukas; Weisenberger, Daniel J; Weinhaeusel, Andreas; Turner, Suzanne D; Egger, Gerda

2016-10-04

Aberrant DNA methylation patterns in malignant cells allow insight into tumor evolution and development and can be used for disease classification. Here, we describe the genome-wide DNA methylation signatures of NPM-ALK-positive (ALK+) and NPM-ALK-negative (ALK-) anaplastic large-cell lymphoma (ALCL). We find that ALK+ and ALK- ALCL share common DNA methylation changes for genes involved in T cell differentiation and immune response, including TCR and CTLA-4, without an ALK-specific impact on tumor DNA methylation in gene promoters. Furthermore, we uncover a close relationship between global ALCL DNA methylation patterns and those in distinct thymic developmental stages and observe tumor-specific DNA hypomethylation in regulatory regions that are enriched for conserved transcription factor binding motifs such as AP1. Our results indicate similarity between ALCL tumor cells and thymic T cell subsets and a direct relationship between ALCL oncogenic signaling and DNA methylation through transcription factor induction and occupancy. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

Basic procedures for epigenetic analysis in plant cell and tissue culture.

PubMed

Rodríguez, José L; Pascual, Jesús; Viejo, Marcos; Valledor, Luis; Meijón, Mónica; Hasbún, Rodrigo; Yrei, Norma Yague; Santamaría, María E; Pérez, Marta; Fernández Fraga, Mario; Berdasco, María; Rodríguez Fernández, Roberto; Cañal, María J

2012-01-01

In vitro culture is one of the most studied techniques, and it is used to study many developmental processes, especially in forestry species, because of growth timing and easy manipulation. Epigenetics has been shown as an important influence on many research analyses such as cancer in mammals and developmental processes in plants such as flowering, but regarding in vitro culture, techniques to study DNA methylation or chromatin modifications were mainly limited to identify somaclonal variation of the micropropagated material. Because in vitro culture is not only a way to generate plant material but also a bunch of differentially induced developmental processes, an approach of techniques and some research carried out to study the different changes regarding DNA methylation and chromatin and translational modifications that take place during these processes is reviewed.

Dynamic changes in methylome and transcriptome patterns in response to methyltransferase inhibitor 5-azacytidine treatment in citrus

PubMed Central

Xu, Jidi; Wang, Xia; Cao, Hongbo; Xu, Haidan; Xu, Qiang

2017-01-01

Abstract DNA methylation is known to play an important role in various developmental processes in plants. However, there is a general lack of understanding about the possible functions of DNA methylation in fruit trees. Using callus as a model, methylome, transcriptome and metabolite changes were assessed after treatment with the DNA methyltransferase inhibitor 5-azacytidine (5azaC). Genome-wide methylome analysis revealed the demethylation of a diverse of genes, including many genes encoding transcription factors (TFs), genes involved in biological processes, and the up-regulation of a wide range of transposable elements (TEs). Combined with the RNA-seq data, we observed no obvious genome-wide correlation between the changes in methylation status and expression levels. Furthermore, 5azaC treatment induced carotenoid degradation along with strong activation of carotenoid cleavage dioxygenases 1 (CpCCD1). Functional complementation analysis in bacterial system showed that CpCCD1 exhibited strong catalytic activities toward zeaxanthin, β-carotene and lycopene. In summary, 5azaC treatments induced carotenoid degradation by CpCCD1 activation and led to a genome-wide demethylation effect. PMID:28575160

DNA methylation polymorphism in a set of elite rice cultivars and its possible contribution to inter-cultivar differential gene expression.

PubMed

Wang, Yongming; Lin, Xiuyun; Dong, Bo; Wang, Yingdian; Liu, Bao

2004-01-01

RAPD (randomly amplified polymorphic DNA) and ISSR (inter-simple sequence repeat) fingerprinting on HpaII/MspI-digested genomic DNA of nine elite japonica rice cultivars implies inter-cultivar DNA methylation polymorphism. Using both DNA fragments isolated from RAPD or ISSR gels and selected low-copy sequences as probes, methylation-sensitive Southern blot analysis confirms the existence of extensive DNA methylation polymorphism in both genes and DNA repeats among the rice cultivars. The cultivar-specific methylation patterns are stably maintained, and can be used as reliable molecular markers. Transcriptional analysis of four selected sequences (RdRP, AC9, HSP90 and MMR) on leaves and roots from normal and 5-azacytidine-treated seedlings of three representative cultivars shows an association between the transcriptional activity of one of the genes, the mismatch repair (MMR) gene, and its CG methylation patterns.

The DNA methylation profile of liver tumors in C3H mice and identification of differentially methylated regions involved in the regulation of tumorigenic genes.

PubMed

Matsushita, Junya; Okamura, Kazuyuki; Nakabayashi, Kazuhiko; Suzuki, Takehiro; Horibe, Yu; Kawai, Tomoko; Sakurai, Toshihiro; Yamashita, Satoshi; Higami, Yoshikazu; Ichihara, Gaku; Hata, Kenichiro; Nohara, Keiko

2018-03-22

C3H mice have been frequently used in cancer studies as animal models of spontaneous liver tumors and chemically induced hepatocellular carcinoma (HCC). Epigenetic modifications, including DNA methylation, are among pivotal control mechanisms of gene expression leading to carcinogenesis. Although information on somatic mutations in liver tumors of C3H mice is available, epigenetic aspects are yet to be clarified. We performed next generation sequencing-based analysis of DNA methylation and microarray analysis of gene expression to explore genes regulated by DNA methylation in spontaneous liver tumors of C3H mice. Overlaying these data, we selected cancer-related genes whose expressions are inversely correlated with DNA methylation levels in the associated differentially methylated regions (DMRs) located around transcription start sites (TSSs) (promoter DMRs). We further assessed mutuality of the selected genes for expression and DNA methylation in human HCC using the Cancer Genome Atlas (TCGA) database. We obtained data on genome-wide DNA methylation profiles in the normal and tumor livers of C3H mice. We identified promoter DMRs of genes which are reported to be related to cancer and whose expressions are inversely correlated with the DNA methylation, including Mst1r, Slpi and Extl1. The association between DNA methylation and gene expression was confirmed using a DNA methylation inhibitor 5-aza-2'-deoxycytidine (5-aza-dC) in Hepa1c1c7 cells and Hepa1-6 cells. Overexpression of Mst1r in Hepa1c1c7 cells illuminated a novel downstream pathway via IL-33 upregulation. Database search indicated that gene expressions of Mst1r and Slpi are upregulated and the TSS upstream regions are hypomethylated also in human HCC. These results suggest that DMRs, including those of Mst1r and Slpi, are involved in liver tumorigenesis in C3H mice, and also possibly in human HCC. Our study clarified genome wide DNA methylation landscape of C3H mice. The data provide useful information for further epigenetic studies of mice models of HCC. The present study particularly proposed novel DNA methylation-regulated pathways for Mst1r and Slpi, which may be applied not only to mouse HCC but also to human HCC.

DNA methylation profiling of genomic DNA isolated from urine in diabetic chronic kidney disease: A pilot study

PubMed Central

Sexton-Oates, Alexandra; Carmody, Jake; Ekinci, Elif I.; Dwyer, Karen M.; Saffery, Richard

2018-01-01

Aim To characterise the genomic DNA (gDNA) yield from urine and quality of derived methylation data generated from the widely used Illuminia Infinium MethylationEPIC (HM850K) platform and compare this with buffy coat samples. Background DNA methylation is the most widely studied epigenetic mark and variations in DNA methylation profile have been implicated in diabetes which affects approximately 415 million people worldwide. Methods QIAamp Viral RNA Mini Kit and QIAamp DNA micro kit were used to extract DNA from frozen and fresh urine samples as well as increasing volumes of fresh urine. Matched buffy coats to the frozen urine were also obtained and DNA was extracted from the buffy coats using the QIAamp DNA Mini Kit. Genomic DNA of greater concentration than 20μg/ml were used for methylation analysis using the HM850K array. Results Irrespective of extraction technique or the use of fresh versus frozen urine samples, limited genomic DNA was obtained using a starting sample volume of 5ml (0–0.86μg/mL). In order to optimize the yield, we increased starting volumes to 50ml fresh urine, which yielded only 0–9.66μg/mL A different kit, QIAamp DNA Micro Kit, was trialled in six fresh urine samples and ten frozen urine samples with inadequate DNA yields from 0–17.7μg/mL and 0–1.6μg/mL respectively. Sufficient genomic DNA was obtained from only 4 of the initial 41 frozen urine samples (10%) for DNA methylation profiling. In comparison, all four buffy coat samples (100%) provided sufficient genomic DNA. Conclusion High quality data can be obtained provided a sufficient yield of genomic DNA is isolated. Despite optimizing various extraction methodologies, the modest amount of genomic DNA derived from urine, may limit the generalisability of this approach for the identification of DNA methylation biomarkers of chronic diabetic kidney disease. PMID:29462136

DNA methylation profiling of genomic DNA isolated from urine in diabetic chronic kidney disease: A pilot study.

PubMed

Lecamwasam, Ashani; Sexton-Oates, Alexandra; Carmody, Jake; Ekinci, Elif I; Dwyer, Karen M; Saffery, Richard

2018-01-01

To characterise the genomic DNA (gDNA) yield from urine and quality of derived methylation data generated from the widely used Illuminia Infinium MethylationEPIC (HM850K) platform and compare this with buffy coat samples. DNA methylation is the most widely studied epigenetic mark and variations in DNA methylation profile have been implicated in diabetes which affects approximately 415 million people worldwide. QIAamp Viral RNA Mini Kit and QIAamp DNA micro kit were used to extract DNA from frozen and fresh urine samples as well as increasing volumes of fresh urine. Matched buffy coats to the frozen urine were also obtained and DNA was extracted from the buffy coats using the QIAamp DNA Mini Kit. Genomic DNA of greater concentration than 20μg/ml were used for methylation analysis using the HM850K array. Irrespective of extraction technique or the use of fresh versus frozen urine samples, limited genomic DNA was obtained using a starting sample volume of 5ml (0-0.86μg/mL). In order to optimize the yield, we increased starting volumes to 50ml fresh urine, which yielded only 0-9.66μg/mL A different kit, QIAamp DNA Micro Kit, was trialled in six fresh urine samples and ten frozen urine samples with inadequate DNA yields from 0-17.7μg/mL and 0-1.6μg/mL respectively. Sufficient genomic DNA was obtained from only 4 of the initial 41 frozen urine samples (10%) for DNA methylation profiling. In comparison, all four buffy coat samples (100%) provided sufficient genomic DNA. High quality data can be obtained provided a sufficient yield of genomic DNA is isolated. Despite optimizing various extraction methodologies, the modest amount of genomic DNA derived from urine, may limit the generalisability of this approach for the identification of DNA methylation biomarkers of chronic diabetic kidney disease.

Plasticity of DNA methylation and gene expression under zinc deficiency in Arabidopsis roots.

PubMed

Chen, Xiaochao; Schönberger, Brigitte; Menz, Jochen; Ludewig, Uwe

2018-05-25

DNA methylation is a heritable chromatin modification that maintains chromosome stability, regulates transposon silencing and appears to be involved in gene expression in response to environmental conditions. Environmental stress alters DNA methylation patterns that are correlated with gene expression differences. Here, genome-wide differential DNA-methylation was identified upon prolonged Zn deficiency, leading to hypo- and hyper-methylated chromosomal regions. Preferential CpG methylation changes occurred in gene promoters and gene bodies, but did not overlap with transcriptional start sites. Methylation changes were also prominent in transposable elements. By contrast, non-CG methylation differences were exclusively found in promoters of protein coding genes and in transposable elements. Strongly Zn deficiency-induced genes and their promoters were mostly non-methylated, irrespective of Zn supply. Differential DNA methylation in the CpG and CHG, but not in the CHH context, was found close to a few up-regulated Zn-deficiency genes. However, the transcriptional Zn-deficiency response in roots appeared little correlated with associated DNA methylation changes in promoters or gene bodies. Furthermore, under Zn deficiency, developmental defects were identified in an Arabidopsis mutant lacking non-CpG methylation. The root methylome thus responds specifically to a micro-nutrient deficiency and is important for efficient Zn utilization at low availability, but the relationship of differential methylation and differentially expressed genes is surprisingly poor.

Genome-Wide DNA Methylation Analysis and Epigenetic Variations Associated with Congenital Aortic Valve Stenosis (AVS).

PubMed

Radhakrishna, Uppala; Albayrak, Samet; Alpay-Savasan, Zeynep; Zeb, Amna; Turkoglu, Onur; Sobolewski, Paul; Bahado-Singh, Ray O

2016-01-01

Congenital heart defect (CHD) is the most common cause of death from congenital anomaly. Among several candidate epigenetic mechanisms, DNA methylation may play an important role in the etiology of CHDs. We conducted a genome-wide DNA methylation analysis using an Illumina Infinium 450k human methylation assay in a cohort of 24 newborns who had aortic valve stenosis (AVS), with gestational-age matched controls. The study identified significantly-altered CpG methylation at 59 sites in 52 genes in AVS subjects as compared to controls (either hypermethylated or demethylated). Gene Ontology analysis identified biological processes and functions for these genes including positive regulation of receptor-mediated endocytosis. Consistent with prior clinical data, the molecular function categories as determined using DAVID identified low-density lipoprotein receptor binding, lipoprotein receptor binding and identical protein binding to be over-represented in the AVS group. A significant epigenetic change in the APOA5 and PCSK9 genes known to be involved in AVS was also observed. A large number CpG methylation sites individually demonstrated good to excellent diagnostic accuracy for the prediction of AVS status, thus raising possibility of molecular screening markers for this disorder. Using epigenetic analysis we were able to identify genes significantly involved in the pathogenesis of AVS.

Genome-Wide DNA Methylation Analysis and Epigenetic Variations Associated with Congenital Aortic Valve Stenosis (AVS)

PubMed Central

Radhakrishna, Uppala; Albayrak, Samet; Alpay-Savasan, Zeynep; Zeb, Amna; Turkoglu, Onur; Sobolewski, Paul; Bahado-Singh, Ray O.

2016-01-01

Congenital heart defect (CHD) is the most common cause of death from congenital anomaly. Among several candidate epigenetic mechanisms, DNA methylation may play an important role in the etiology of CHDs. We conducted a genome-wide DNA methylation analysis using an Illumina Infinium 450k human methylation assay in a cohort of 24 newborns who had aortic valve stenosis (AVS), with gestational-age matched controls. The study identified significantly-altered CpG methylation at 59 sites in 52 genes in AVS subjects as compared to controls (either hypermethylated or demethylated). Gene Ontology analysis identified biological processes and functions for these genes including positive regulation of receptor-mediated endocytosis. Consistent with prior clinical data, the molecular function categories as determined using DAVID identified low-density lipoprotein receptor binding, lipoprotein receptor binding and identical protein binding to be over-represented in the AVS group. A significant epigenetic change in the APOA5 and PCSK9 genes known to be involved in AVS was also observed. A large number CpG methylation sites individually demonstrated good to excellent diagnostic accuracy for the prediction of AVS status, thus raising possibility of molecular screening markers for this disorder. Using epigenetic analysis we were able to identify genes significantly involved in the pathogenesis of AVS. PMID:27152866

Quantitative detection of RASSF1A DNA promoter methylation in tumors and serum of patients with serous epithelial ovarian cancer.

PubMed

Bondurant, Amy E; Huang, Zhiqing; Whitaker, Regina S; Simel, Lauren R; Berchuck, Andrew; Murphy, Susan K

2011-12-01

Detection of cell free tumor-specific DNA methylation has been proposed as a potentially useful noninvasive mechanism to detect malignancies, including ovarian cancer, and to monitor response to treatment. However, there are few easily implemented quantitative approaches available for DNA methylation analysis. Our objectives were to develop an absolute quantitative method for detection of DNA methylation using RASSF1A, a known target of promoter methylation in ovarian cancer, and test the ability to detect RASSF1A methylation in tumors and serum specimens of women with ovarian cancer. Bisulfite modified DNAs were subjected to real time PCR using nondiscriminatory PCR primers and a probe with sequence containing a single CpG site, theoretically able to capture the methylation status of that CpG for every allele within a given specimen. Input DNA was normalized to ACTB levels detected simultaneously by assay multiplexing. Methylation levels were established by comparison to results obtained from universally methylated DNA. The assay was able to detect one methylated RASSF1A allele in 100,000 unmethylated alleles. RASSF1A was methylated in 54 of 106 (51%) invasive serous ovarian cancers analyzed and methylation status was concordant in 20/20 matched preoperative serum-tumor pairs. Serial serum specimens taken over the course of treatment for 8 of 9 patients showed fluctuations in RASSF1A methylation concomitant with disease status. This novel assay provides a real-time PCR-based method for absolute quantitation of DNA methylation. Our results support feasibility of monitoring RASSF1A methylation from serum samples taken over the course of treatment from women with ovarian cancer. Copyright © 2011 Elsevier Inc. All rights reserved.

Live-cell analysis of DNA methylation during sexual reproduction in Arabidopsis reveals context and sex-specific dynamics controlled by noncanonical RdDM.

PubMed

Ingouff, Mathieu; Selles, Benjamin; Michaud, Caroline; Vu, Thiet M; Berger, Frédéric; Schorn, Andrea J; Autran, Daphné; Van Durme, Matthias; Nowack, Moritz K; Martienssen, Robert A; Grimanelli, Daniel

2017-01-01

Cytosine methylation is a key epigenetic mark in many organisms, important for both transcriptional control and genome integrity. While relatively stable during somatic growth, DNA methylation is reprogrammed genome-wide during mammalian reproduction. Reprogramming is essential for zygotic totipotency and to prevent transgenerational inheritance of epimutations. However, the extent of DNA methylation reprogramming in plants remains unclear. Here, we developed sensors reporting with single-cell resolution CG and non-CG methylation in Arabidopsis. Live imaging during reproduction revealed distinct and sex-specific dynamics for both contexts. We found that CHH methylation in the egg cell depends on DOMAINS REARRANGED METHYLASE 2 (DRM2) and RNA polymerase V (Pol V), two main actors of RNA-directed DNA methylation, but does not depend on Pol IV. Our sensors provide insight into global DNA methylation dynamics at the single-cell level with high temporal resolution and offer a powerful tool to track CG and non-CG methylation both during development and in response to environmental cues in all organisms with methylated DNA, as we illustrate in mouse embryonic stem cells. © 2017 Ingouff et al.; Published by Cold Spring Harbor Laboratory Press.

Live-cell analysis of DNA methylation during sexual reproduction in Arabidopsis reveals context and sex-specific dynamics controlled by noncanonical RdDM

PubMed Central

Ingouff, Mathieu; Selles, Benjamin; Michaud, Caroline; Vu, Thiet M.; Berger, Frédéric; Schorn, Andrea J.; Autran, Daphné; Van Durme, Matthias; Nowack, Moritz K.; Martienssen, Robert A.; Grimanelli, Daniel

2017-01-01

Cytosine methylation is a key epigenetic mark in many organisms, important for both transcriptional control and genome integrity. While relatively stable during somatic growth, DNA methylation is reprogrammed genome-wide during mammalian reproduction. Reprogramming is essential for zygotic totipotency and to prevent transgenerational inheritance of epimutations. However, the extent of DNA methylation reprogramming in plants remains unclear. Here, we developed sensors reporting with single-cell resolution CG and non-CG methylation in Arabidopsis. Live imaging during reproduction revealed distinct and sex-specific dynamics for both contexts. We found that CHH methylation in the egg cell depends on DOMAINS REARRANGED METHYLASE 2 (DRM2) and RNA polymerase V (Pol V), two main actors of RNA-directed DNA methylation, but does not depend on Pol IV. Our sensors provide insight into global DNA methylation dynamics at the single-cell level with high temporal resolution and offer a powerful tool to track CG and non-CG methylation both during development and in response to environmental cues in all organisms with methylated DNA, as we illustrate in mouse embryonic stem cells. PMID:28115468

MCT4 surpasses the prognostic relevance of the ancillary protein CD147 in clear cell renal cell carcinoma.

PubMed

Fisel, Pascale; Stühler, Viktoria; Bedke, Jens; Winter, Stefan; Rausch, Steffen; Hennenlotter, Jörg; Nies, Anne T; Stenzl, Arnulf; Scharpf, Marcus; Fend, Falko; Kruck, Stephan; Schwab, Matthias; Schaeffeler, Elke

2015-10-13

Cluster of differentiation 147 (CD147/BSG) is a transmembrane glycoprotein mediating oncogenic processes partly through its role as binding partner for monocarboxylate transporter MCT4/SLC16A3. As demonstrated for MCT4, CD147 is proposed to be associated with progression in clear cell renal cell carcinoma (ccRCC). In this study, we evaluated the prognostic relevance of CD147 in comparison to MCT4/SLC16A3 expression and DNA methylation. CD147 protein expression was assessed in two independent ccRCC-cohorts (n = 186, n = 59) by immunohistochemical staining of tissue microarrays and subsequent manual as well as automated software-supported scoring (Tissue Studio, Definien sAG). Epigenetic regulation of CD147 was investigated using RNAseq and DNA methylation data of The Cancer Genome Atlas. These results were validated in our cohort. Relevance of prognostic models for cancer-specific survival, comprising CD147 and MCT4 expression or SLC16A3 DNA methylation, was compared using chi-square statistics. CD147 protein expression generated with Tissue Studio correlated significantly with those from manual scoring (P < 0.0001, rS = 0.85), indicating feasibility of software-based evaluation exemplarily for the membrane protein CD147 in ccRCC. Association of CD147 expression with patient outcome differed between cohorts. DNA methylation in the CD147/BSG promoter was not associated with expression. Comparison of prognostic relevance of CD147/BSG and MCT4/SLC16A3, showed higher significance for MCT4 expression and superior prognostic power for DNA methylation at specific CpG-sites in the SLC16A3 promoter (e.g. CD147 protein: P = 0.7780,Harrell's c-index = 53.7% vs. DNA methylation: P = 0.0076, Harrell's c-index = 80.0%). Prognostic significance of CD147 protein expression could not surpass that of MCT4, especially of SLC16A3 DNA methylation, corroborating the role of MCT4 as prognostic biomarker for ccRCC.

MCT4 surpasses the prognostic relevance of the ancillary protein CD147 in clear cell renal cell carcinoma

PubMed Central

Winter, Stefan; Rausch, Steffen; Hennenlotter, Jörg; Nies, Anne T.; Stenzl, Arnulf; Scharpf, Marcus; Fend, Falko; Kruck, Stephan; Schwab, Matthias; Schaeffeler, Elke

2015-01-01

Cluster of differentiation 147 (CD147/BSG) is a transmembrane glycoprotein mediating oncogenic processes partly through its role as binding partner for monocarboxylate transporter MCT4/SLC16A3. As demonstrated for MCT4, CD147 is proposed to be associated with progression in clear cell renal cell carcinoma (ccRCC). In this study, we evaluated the prognostic relevance of CD147 in comparison to MCT4/SLC16A3 expression and DNA methylation. Methods CD147 protein expression was assessed in two independent ccRCC-cohorts (n = 186, n = 59) by immunohistochemical staining of tissue microarrays and subsequent manual as well as automated software-supported scoring (Tissue Studio, Definien sAG). Epigenetic regulation of CD147 was investigated using RNAseq and DNA methylation data of The Cancer Genome Atlas. These results were validated in our cohort. Relevance of prognostic models for cancer-specific survival, comprising CD147 and MCT4 expression or SLC16A3 DNA methylation, was compared using chi-square statistics. Results CD147 protein expression generated with Tissue Studio correlated significantly with those from manual scoring (P < 0.0001, rS = 0.85), indicating feasibility of software-based evaluation exemplarily for the membrane protein CD147 in ccRCC. Association of CD147 expression with patient outcome differed between cohorts. DNA methylation in the CD147/BSG promoter was not associated with expression. Comparison of prognostic relevance of CD147/BSG and MCT4/SLC16A3, showed higher significance for MCT4 expression and superior prognostic power for DNA methylation at specific CpG-sites in the SLC16A3 promoter (e.g. CD147 protein: P = 0.7780, Harrell's c-index = 53.7% vs. DNA methylation: P = 0.0076, Harrell's c-index = 80.0%). Conclusions Prognostic significance of CD147 protein expression could not surpass that of MCT4, especially of SLC16A3 DNA methylation, corroborating the role of MCT4 as prognostic biomarker for ccRCC. PMID:26384346

Environmental Deflection: The Impact of Toxicant Exposures on the Aging Epigenome.

PubMed

Kochmanski, Joseph; Montrose, Luke; Goodrich, Jaclyn M; Dolinoy, Dana C

2017-04-01

Epigenetic drift and age-related methylation have both been used in the literature to describe changes in DNA methylation that occurs with aging. However, ambiguity remains regarding the exact definition of both of these terms, and neither of these fields of study explicitly considers the impact of environmental factors on the aging epigenome. Recent twin studies have demonstrated longitudinal, pair-specific discordance in DNA methylation patterns, suggesting an effect of the environment on age-related methylation and/or epigenetic drift. Supporting this idea, other new reports have shown clear environment- and toxicant-mediated shifts away from the baseline rates of age-related methylation and epigenetic drift within an organism, a process we now term "environmental deflection." By defining and delineating environmental deflection, this contemporary review aims to highlight the effects of specific toxicological factors on the rate of DNA methylation changes that occur over the life course. In an effort to inform future epigenetics-based toxicology studies, a field of research now classified as toxicoepigenetics, we provide clear definitions and examples of "epigenetic drift" and "age-related methylation," summarize the recent evidence for environmental deflection of the aging epigenome, and discuss the potential functional effects of environmental deflection. © The Author 2017. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

The Enzymatic Release of O6-methylguanine and 3-methyladenine from DNA Reacted with the Carcinogen N-methyl-N-nitrosourea

PubMed Central

Kirtikar, D. M.; Goldthwait, D. A.

1974-01-01

Endonuclease II (deoxyribonucleate oligonucleotidohydrolase, EC 3.1.4.30) of Escherichia coli has been shown to break phosphodiester bonds in alkylated DNA and depurinated DNA. The hypothesis that depurination is a step in the mechanism of the reaction with alkylated DNA is supported by in vitro experiments with DNA reacted with N-methyl-N-nitrosourea. Endonuclease II releases O6-methylguanine and 3-methyladenine, but not 7-methylguanine, from DNA that has been methylated by the carcinogen N-methyl-N-nitrosourea. PMID:4600266

A simple method for semi-random DNA amplicon fragmentation using the methylation-dependent restriction enzyme MspJI.

PubMed

Shinozuka, Hiroshi; Cogan, Noel O I; Shinozuka, Maiko; Marshall, Alexis; Kay, Pippa; Lin, Yi-Han; Spangenberg, German C; Forster, John W

2015-04-11

Fragmentation at random nucleotide locations is an essential process for preparation of DNA libraries to be used on massively parallel short-read DNA sequencing platforms. Although instruments for physical shearing, such as the Covaris S2 focused-ultrasonicator system, and products for enzymatic shearing, such as the Nextera technology and NEBNext dsDNA Fragmentase kit, are commercially available, a simple and inexpensive method is desirable for high-throughput sequencing library preparation. MspJI is a recently characterised restriction enzyme which recognises the sequence motif CNNR (where R = G or A) when the first base is modified to 5-methylcytosine or 5-hydroxymethylcytosine. A semi-random enzymatic DNA amplicon fragmentation method was developed based on the unique cleavage properties of MspJI. In this method, random incorporation of 5-methyl-2'-deoxycytidine-5'-triphosphate is achieved through DNA amplification with DNA polymerase, followed by DNA digestion with MspJI. Due to the recognition sequence of the enzyme, DNA amplicons are fragmented in a relatively sequence-independent manner. The size range of the resulting fragments was capable of control through optimisation of 5-methyl-2'-deoxycytidine-5'-triphosphate concentration in the reaction mixture. A library suitable for sequencing using the Illumina MiSeq platform was prepared and processed using the proposed method. Alignment of generated short reads to a reference sequence demonstrated a relatively high level of random fragmentation. The proposed method may be performed with standard laboratory equipment. Although the uniformity of coverage was slightly inferior to the Covaris physical shearing procedure, due to efficiencies of cost and labour, the method may be more suitable than existing approaches for implementation in large-scale sequencing activities, such as bacterial artificial chromosome (BAC)-based genome sequence assembly, pan-genomic studies and locus-targeted genotyping-by-sequencing.

Multi-tissue DNA methylation age: Molecular relationships and perspectives for advancing biomarker utility.

PubMed

Nwanaji-Enwerem, Jamaji C; Weisskopf, Marc G; Baccarelli, Andrea A

2018-04-23

The multi-tissue DNA methylation estimator of chronological age (DNAm-age) has been associated with a wide range of exposures and health outcomes. Still, it is unclear how DNAm-age can have such broad relationships and how it can be best utilized as a biomarker. Understanding DNAm-age's molecular relationships is a promising approach to address this critical knowledge gap. In this review, we discuss the existing literature regarding DNAm-age's molecular relationships in six major categories: animal model systems, cancer processes, cellular aging processes, immune system processes, metabolic processes, and nucleic acid processes. We also present perspectives regarding the future of DNAm-age research, including the need to translate a greater number of ongoing research efforts to experimental and animal model systems. Copyright © 2018 Elsevier B.V. All rights reserved.

Effects of TET2 mutations on DNA methylation in chronic myelomonocytic leukemia

USDA-ARS?s Scientific Manuscript database

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...

Targeting DNA methylation to the genome.

PubMed

Lo, Patrick C H

2014-01-01

Proving direct relationships between DNA alterations and phenotypes is challenging. For epigenetics researchers, linking DNA methylation with human disease is no exception. But Patrick Lo looks at how two researchers are developing new methods to try to trace the road from DNA methylation to human biology.

Kaempferol Modulates DNA Methylation and Downregulates DNMT3B in Bladder Cancer.

PubMed

Qiu, Wei; Lin, Jun; Zhu, Yichen; Zhang, Jian; Zeng, Liping; Su, Ming; Tian, Ye

2017-01-01

Genomic DNA methylation plays an important role in both the occurrence and development of bladder cancer. Kaempferol (Kae), a natural flavonoid that is present in many fruits and vegetables, exhibits potent anti-cancer effects in bladder cancer. Similar to other flavonoids, Kae possesses a flavan nucleus in its structure. This structure was reported to inhibit DNA methylation by suppressing DNA methyltransferases (DNMTs). However, whether Kae can inhibit DNA methylation remains unclear. Nude mice bearing bladder cancer were treated with Kae for 31 days. The genomic DNA was extracted from xenografts and the methylation changes was determined using an Illumina Infinium HumanMethylation 450 BeadChip Array. The ubiquitination was detected using immuno-precipitation assay. Our data indicated that Kae modulated DNA methylation in bladder cancer, inducing 103 differential DNA methylation positions (dDMPs) associated with genes (50 hyper-methylated and 53 hypo-methylated). DNA methylation is mostly relied on the levels of DNMTs. We observed that Kae specifically inhibited the protein levels of DNMT3B without altering the expression of DNMT1 or DNMT3A. However, Kae did not downregulate the transcription of DNMT3B. Interestingly, we observed that Kae induced a premature degradation of DNMT3B by inhibiting protein synthesis with cycloheximide (CHX). By blocking proteasome with MG132, we observed that Kae induced an increased ubiquitination of DNMT3B. These results suggested that Kae could induce the degradation of DNMT3B through ubiquitin-proteasome pathway. Our data indicated that Kae is a novel DNMT3B inhibitor, which may promote the degradation of DNMT3B in bladder cancer. © 2017 The Author(s)Published by S. Karger AG, Basel.

Environment, diet and CpG island methylation: epigenetic signals in gastrointestinal neoplasia.

PubMed

Johnson, Ian T; Belshaw, Nigel J

2008-04-01

The epithelial surfaces of the mammalian alimentary tract are characterised by very high rates of cell proliferation and DNA synthesis, and in humans they are highly susceptible to cancer. The role of somatic mutations as drivers of carcinogenesis in the alimentary tract is well established, but the importance of gene silencing by epigenetic mechanisms is increasingly recognised. Methylation of CpG islands is an important component of the epigenetic code that regulates gene expression during development and normal cellular differentiation, and a number of genes are well known to become abnormally methylated during the development of tumours of the oesophagus, stomach and colorectum. Aberrant patterns of DNA methylation develop as a result of pathological processes such as chronic inflammation, and in response to various dietary factors, including imbalances in the supply of methyl donors, particularly folates, and exposure to DNA methyltransferase inhibitors, which include polyphenols and possibly isothiocyanates from plant foods. However the importance of these environmental interactions in human health and disease remains to be established. Recent moves to modify the exposure of human populations to folate, by mandatory supplementation of cereal foods, emphasise the importance of understanding the susceptibility of the human epigenome to dietary and other environmental effects.

The RNAs of RNA-directed DNA methylation

PubMed Central

Wendte, Jered M.; Pikaard, Craig S.

2016-01-01

Summary RNA-directed chromatin modification that includes cytosine methylation silences transposable elements in both plants and mammals, contributing to genome defense and stability. In Arabidopsis thaliana, most RNA-directed DNA methylation (RdDM) is guided by small RNAs derived from double-stranded precursors synthesized at cytosine-methylated loci by nuclear multisubunit RNA Polymerase IV (Pol IV), in close partnership with the RNA-dependent RNA polymerase, RDR2. These small RNAs help keep transposons transcriptionally inactive. However, if transposons escape silencing, and are transcribed by multisubunit RNA polymerase II (Pol II), their mRNAs can be recognized and degraded, generating small RNAs that can also guide initial DNA methylation, thereby enabling subsequent Pol IV-RDR2 recruitment. In both pathways, the small RNAs find their target sites by interacting with longer noncoding RNAs synthesized by multisubunit RNA Polymerase V (Pol V). Despite a decade of progress, numerous questions remain concerning the initiation, synthesis, processing, size and features of the RNAs that drive RdDM. Here, we review recent insights, questions and controversies concerning RNAs produced by Pols IV and V, and their functions in RdDM. We also provide new data concerning Pol V transcript 5’ and 3’ ends. PMID:27521981

Search for methylation-sensitive amplification polymorphisms in mutant figs.

PubMed

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.

Zebrafish as a model to study the role of DNA methylation in environmental toxicology.

PubMed

Kamstra, Jorke H; Aleström, Peter; Kooter, Jan M; Legler, Juliette

2015-11-01

Environmental epigenetics is a rapidly growing field which studies the effects of environmental factors such as nutrition, stress, and exposure to compounds on epigenetic gene regulation. Recent studies have shown that exposure to toxicants in vertebrates is associated with changes in DNA methylation, a major epigenetic mechanism affecting gene transcription. Zebra fish, a well-known model in toxicology and developmental biology, are emerging as a model species in environmental epigenetics despite their evolutionary distance to rodents and humans. In this review, recent insights in DNA methylation during zebra fish development are discussed and compared to mammalian models in order to evaluate zebra fish as a model to study the role of DNA methylation in environmental toxicology. Differences exist in DNA methylation reprogramming during early development, whereas in later developmental stages, tissue distribution of both 5-methylcytosine and 5-hydroxymethylcytosine seems more conserved between species, as well as basic DNA (de)methylation mechanisms. All DNA methyl transferases identified so far in mammals are present in zebra fish, as well as a number of major demethylation pathways. However, zebra fish appear to lack some methylation pathways present in mammals, such as parental imprinting. Several studies report effects on DNA methylation in zebra fish following exposure to environmental contaminants, such as arsenic, benzo[a]pyrene, and tris(1,3-dichloro-2-propyl)phosphate. Though more research is needed to examine heritable effects of contaminant exposure on DNA methylation, recent data suggests the usefulness of the zebra fish as a model in environmental epigenetics.

Concordance of DNA methylation profiles between breast core biopsy and surgical excision specimens containing ductal carcinoma in situ (DCIS).

PubMed

Chen, Youdinghuan; Marotti, Jonathan D; Jenson, Erik G; Onega, Tracy L; Johnson, Kevin C; Christensen, Brock C

2017-08-01

The utility and reliability of assessing molecular biomarkers for translational applications on pre-operative core biopsy specimens assume consistency of molecular profiles with larger surgical specimens. Whether DNA methylation in ductal carcinoma in situ (DCIS), measured in core biopsy and surgical specimens are similar, remains unclear. Here, we compared genome-scale DNA methylation measured in matched core biopsy and surgical specimens from DCIS, including specific DNA methylation biomarkers of subsequent invasive cancer. DNA was extracted from guided 2mm cores of formalin fixed paraffin embedded (FFPE) specimens, bisulfite-modified, and measured on the Illumina HumanMethylation450 BeadChip. DNA methylation profiles of core biopsies exhibited high concordance with matched surgical specimens. Within-subject variability in DNA methylation was significantly lower than between-subject variability (all P<2.20E-16). In 641 CpGs whose methylation was related with increased hazard of invasive breast cancer, lower within-subject than between-subject variability was observed in 92.3% of the study participants (P<0.05). Between patient-matched core biopsy and surgical specimens, <0.6% of CpGs measured had changes in median DNA methylation >15%, and a pathway analysis of these CpGs indicated enrichment for genes related with wound healing. Our results indicate that DNA methylation measured in core biopsies are representative of the matched surgical specimens and suggest that DCIS biomarkers measured in core biopsies can inform clinical decision-making. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Gene-Specific DNA Methylation Changes Predict Remission in Patients with ANCA-Associated Vasculitis

PubMed Central

Jones, Britta E.; Yang, Jiajin; Muthigi, Akhil; Hogan, Susan L.; Hu, Yichun; Starmer, Joshua; Henderson, Candace D.; Poulton, Caroline J.; Brant, Elizabeth J.; Pendergraft, William F.; Jennette, J. Charles; Falk, Ronald J.

2017-01-01

ANCA-associated vasculitis is an autoimmune condition characterized by vascular inflammation and organ damage. Pharmacologically induced remission of this condition is complicated by relapses. Potential triggers of relapse are immunologic challenges and environmental insults, both of which associate with changes in epigenetic silencing modifications. Altered histone modifications implicated in gene silencing associate with aberrant autoantigen expression. To establish a link between DNA methylation, a model epigenetic gene silencing modification, and autoantigen gene expression and disease status in ANCA-associated vasculitis, we measured gene-specific DNA methylation of the autoantigen genes myeloperoxidase (MPO) and proteinase 3 (PRTN3) in leukocytes of patients with ANCA-associated vasculitis observed longitudinally (n=82) and of healthy controls (n=32). Patients with active disease demonstrated hypomethylation of MPO and PRTN3 and increased expression of the autoantigens; in remission, DNA methylation generally increased. Longitudinal analysis revealed that patients with ANCA-associated vasculitis could be divided into two groups, on the basis of whether DNA methylation increased or decreased from active disease to remission. In patients with increased DNA methylation, MPO and PRTN3 expression correlated with DNA methylation. Kaplan–Meier estimate of relapse revealed patients with increased DNA methylation at the PRTN3 promoter had a significantly greater probability of a relapse-free period (P<0.001), independent of ANCA serotype. Patients with decreased DNA methylation at the PRTN3 promoter had a greater risk of relapse (hazard ratio, 4.55; 95% confidence interval, 2.09 to 9.91). Thus, changes in the DNA methylation status of the PRTN3 promoter may predict the likelihood of stable remission and explain autoantigen gene regulation. PMID:27821628

Differential epigenome-wide DNA methylation patterns in childhood obesity-associated asthma

PubMed Central

Rastogi, Deepa; Suzuki, Masako; Greally, John M.

2013-01-01

While DNA methylation plays a role in T-helper (Th) cell maturation, its potential dysregulation in the non-atopic Th1-polarized systemic inflammation observed in obesity-associated asthma is unknown. We studied DNA methylation epigenome-wide in peripheral blood mononuclear cells (PBMCs) from 8 obese asthmatic pre-adolescent children and compared it to methylation in PBMCs from 8 children with asthma alone, obesity alone and healthy controls. Differentially methylated loci implicated certain biologically relevant molecules and pathways. PBMCs from obese asthmatic children had distinctive DNA methylation patterns, with decreased promoter methylation of CCL5, IL2RA and TBX21, genes encoding proteins linked with Th1 polarization, and increased promoter methylation of FCER2, a low-affinity receptor for IgE, and of TGFB1, inhibitor of Th cell activation. T-cell signaling and macrophage activation were the two primary pathways that were selectively hypomethylated in obese asthmatics. These findings suggest that dysregulated DNA methylation is associated with non-atopic inflammation observed in pediatric obesity-associated asthma. PMID:23857381

Intra-Gene DNA Methylation Variability Is a Clinically Independent Prognostic Marker in Women’s Cancers

PubMed Central

Bartlett, Thomas E.; Jones, Allison; Goode, Ellen L.; Fridley, Brooke L.; Cunningham, Julie M.; Berns, Els M. J. J.; Wik, Elisabeth; Salvesen, Helga B.; Davidson, Ben; Trope, Claes G.; Lambrechts, Sandrina; Vergote, Ignace; Widschwendter, Martin

2015-01-01

We introduce a novel per-gene measure of intra-gene DNA methylation variability (IGV) based on the Illumina Infinium HumanMethylation450 platform, which is prognostic independently of well-known predictors of clinical outcome. Using IGV, we derive a robust gene-panel prognostic signature for ovarian cancer (OC, n = 221), which validates in two independent data sets from Mayo Clinic (n = 198) and TCGA (n = 358), with significance of p = 0.004 in both sets. The OC prognostic signature gene-panel is comprised of four gene groups, which represent distinct biological processes. We show the IGV measurements of these gene groups are most likely a reflection of a mixture of intra-tumour heterogeneity and transcription factor (TF) binding/activity. IGV can be used to predict clinical outcome in patients individually, providing a surrogate read-out of hard-to-measure disease processes. PMID:26629914

Intra-Gene DNA Methylation Variability Is a Clinically Independent Prognostic Marker in Women's Cancers.

PubMed

Bartlett, Thomas E; Jones, Allison; Goode, Ellen L; Fridley, Brooke L; Cunningham, Julie M; Berns, Els M J J; Wik, Elisabeth; Salvesen, Helga B; Davidson, Ben; Trope, Claes G; Lambrechts, Sandrina; Vergote, Ignace; Widschwendter, Martin

2015-01-01

We introduce a novel per-gene measure of intra-gene DNA methylation variability (IGV) based on the Illumina Infinium HumanMethylation450 platform, which is prognostic independently of well-known predictors of clinical outcome. Using IGV, we derive a robust gene-panel prognostic signature for ovarian cancer (OC, n = 221), which validates in two independent data sets from Mayo Clinic (n = 198) and TCGA (n = 358), with significance of p = 0.004 in both sets. The OC prognostic signature gene-panel is comprised of four gene groups, which represent distinct biological processes. We show the IGV measurements of these gene groups are most likely a reflection of a mixture of intra-tumour heterogeneity and transcription factor (TF) binding/activity. IGV can be used to predict clinical outcome in patients individually, providing a surrogate read-out of hard-to-measure disease processes.

Epigenetic regulation and chromatin remodeling in learning and memory.

PubMed

Kim, Somi; Kaang, Bong-Kiun

2017-01-13

Understanding the underlying mechanisms of memory formation and maintenance has been a major goal in the field of neuroscience. Memory formation and maintenance are tightly controlled complex processes. Among the various processes occurring at different levels, gene expression regulation is especially crucial for proper memory processing, as some genes need to be activated while some genes must be suppressed. Epigenetic regulation of the genome involves processes such as DNA methylation and histone post-translational modifications. These processes edit genomic properties or the interactions between the genome and histone cores. They then induce structural changes in the chromatin and lead to transcriptional changes of different genes. Recent studies have focused on the concept of chromatin remodeling, which consists of 3D structural changes in chromatin in relation to gene regulation, and is an important process in learning and memory. In this review, we will introduce three major epigenetic processes involved in memory regulation: DNA methylation, histone methylation and histone acetylation. We will also discuss general mechanisms of long-term memory storage and relate the epigenetic control of learning and memory to chromatin remodeling. Finally, we will discuss how epigenetic mechanisms can contribute to the pathologies of neurological disorders and cause memory-related symptoms.

Novel epigenetic changes unveiled by monozygotic twins discordant for smoking habits.

PubMed

Allione, Alessandra; Marcon, Francesca; Fiorito, Giovanni; Guarrera, Simonetta; Siniscalchi, Ester; Zijno, Andrea; Crebelli, Riccardo; Matullo, Giuseppe

2015-01-01

Exposure to cigarette smoking affects the epigenome and could increase the risk of developing diseases such as cancer and cardiovascular disorders. Changes in DNA methylation associated with smoking may help to identify molecular pathways that contribute to disease etiology. Previous studies are not completely concordant in the identification of differentially methylated regions in the DNA of smokers. We performed an epigenome-wide DNA methylation study in a group of monozygotic (MZ) twins discordant for smoking habits to determine the effect of smoking on DNA methylation. As MZ twins are considered genetically identical, this model allowed us to identify smoking-related DNA methylation changes independent from genetic components. We investigated the whole blood genome-wide DNA methylation profiles in 20 MZ twin pairs discordant for smoking habits by using the Illumina HumanMethylation450 BeadChip. We identified 22 CpG sites that were differentially methylated between smoker and non-smoker MZ twins by intra-pair analysis. We confirmed eight loci already described by other groups, located in AHRR, F2RL3, MYOG1 genes, at 2q37.1 and 6p21.33 regions, and also identified several new loci. Moreover, pathway analysis showed an enrichment of genes involved in GTPase regulatory activity. Our study confirmed the evidence of smoking-related DNA methylation changes, emphasizing that well-designed MZ twin models can aid the discovery of novel DNA methylation signals, even in a limited sample population.

TEA: the epigenome platform for Arabidopsis methylome study.

PubMed

Su, Sheng-Yao; Chen, Shu-Hwa; Lu, I-Hsuan; Chiang, Yih-Shien; Wang, Yu-Bin; Chen, Pao-Yang; Lin, Chung-Yen

2016-12-22

Bisulfite sequencing (BS-seq) has become a standard technology to profile genome-wide DNA methylation at single-base resolution. It allows researchers to conduct genome-wise cytosine methylation analyses on issues about genomic imprinting, transcriptional regulation, cellular development and differentiation. One single data from a BS-Seq experiment is resolved into many features according to the sequence contexts, making methylome data analysis and data visualization a complex task. We developed a streamlined platform, TEA, for analyzing and visualizing data from whole-genome BS-Seq (WGBS) experiments conducted in the model plant Arabidopsis thaliana. To capture the essence of the genome methylation level and to meet the efficiency for running online, we introduce a straightforward method for measuring genome methylation in each sequence context by gene. The method is scripted in Java to process BS-Seq mapping results. Through a simple data uploading process, the TEA server deploys a web-based platform for deep analysis by linking data to an updated Arabidopsis annotation database and toolkits. TEA is an intuitive and efficient online platform for analyzing the Arabidopsis genomic DNA methylation landscape. It provides several ways to help users exploit WGBS data. TEA is freely accessible for academic users at: http://tea.iis.sinica.edu.tw .

Comparisons of non-Gaussian statistical models in DNA methylation analysis.

PubMed

Ma, Zhanyu; Teschendorff, Andrew E; Yu, Hong; Taghia, Jalil; Guo, Jun

2014-06-16

As a key regulatory mechanism of gene expression, DNA methylation patterns are widely altered in many complex genetic diseases, including cancer. DNA methylation is naturally quantified by bounded support data; therefore, it is non-Gaussian distributed. In order to capture such properties, we introduce some non-Gaussian statistical models to perform dimension reduction on DNA methylation data. Afterwards, non-Gaussian statistical model-based unsupervised clustering strategies are applied to cluster the data. Comparisons and analysis of different dimension reduction strategies and unsupervised clustering methods are presented. Experimental results show that the non-Gaussian statistical model-based methods are superior to the conventional Gaussian distribution-based method. They are meaningful tools for DNA methylation analysis. Moreover, among several non-Gaussian methods, the one that captures the bounded nature of DNA methylation data reveals the best clustering performance.

Comparisons of Non-Gaussian Statistical Models in DNA Methylation Analysis

PubMed Central

Ma, Zhanyu; Teschendorff, Andrew E.; Yu, Hong; Taghia, Jalil; Guo, Jun

2014-01-01

As a key regulatory mechanism of gene expression, DNA methylation patterns are widely altered in many complex genetic diseases, including cancer. DNA methylation is naturally quantified by bounded support data; therefore, it is non-Gaussian distributed. In order to capture such properties, we introduce some non-Gaussian statistical models to perform dimension reduction on DNA methylation data. Afterwards, non-Gaussian statistical model-based unsupervised clustering strategies are applied to cluster the data. Comparisons and analysis of different dimension reduction strategies and unsupervised clustering methods are presented. Experimental results show that the non-Gaussian statistical model-based methods are superior to the conventional Gaussian distribution-based method. They are meaningful tools for DNA methylation analysis. Moreover, among several non-Gaussian methods, the one that captures the bounded nature of DNA methylation data reveals the best clustering performance. PMID:24937687

Heritable Epigenomic Changes to the Maize Methylome Resulting from Tissue Culture.

PubMed

Han, Zhaoxue; Crisp, Peter A; Stelpflug, Scott; Kaeppler, Shawn M; Li, Qing; Springer, Nathan M

2018-05-30

DNA methylation can contribute to the maintenance of genome integrity and regulation of gene expression. In most situations, DNA methylation patterns are inherited quite stably. However, changes in DNA methylation can occur at some loci as a result of tissue culture resulting in somaclonal variation. To investigate heritable epigenetic changes as a consequence of tissue culture, a sequence-capture bisulfite sequencing approach was implemented to monitor context-specific DNA methylation patterns in ∼15Mb of the maize genome for a population of plants that had been regenerated from tissue culture. Plants that have been regenerated from tissue culture exhibit gains and losses of DNA methylation at a subset of genomic regions. There was evidence for a high rate of homozygous changes to DNA methylation levels that occur consistently in multiple independent tissue culture lines suggesting that some loci are either targeted or hotspots for epigenetic variation. The consistent changes inherited following tissue culture include both gains and losses of DNA methylation and can affect CG, CHG or both contexts within a region. Only a subset of the tissue culture changes observed in callus plants are observed in the primary regnerants but the majority of DNA methylation changes present in primary regenerants are passed onto offspring. This study provides insights into the susceptibility of some loci and potential mechanisms that could contribute to altered DNA methylation and epigenetic state that occur during tissue culture in plant species. Copyright © 2018, Genetics.

A pooling-based approach to mapping genetic variants associated with DNA methylation

PubMed Central

Kaplow, Irene M.; MacIsaac, Julia L.; Mah, Sarah M.; McEwen, Lisa M.; Kobor, Michael S.; Fraser, Hunter B.

2015-01-01

DNA methylation is an epigenetic modification that plays a key role in gene regulation. Previous studies have investigated its genetic basis by mapping genetic variants that are associated with DNA methylation at specific sites, but these have been limited to microarrays that cover <2% of the genome and cannot account for allele-specific methylation (ASM). Other studies have performed whole-genome bisulfite sequencing on a few individuals, but these lack statistical power to identify variants associated with DNA methylation. We present a novel approach in which bisulfite-treated DNA from many individuals is sequenced together in a single pool, resulting in a truly genome-wide map of DNA methylation. Compared to methods that do not account for ASM, our approach increases statistical power to detect associations while sharply reducing cost, effort, and experimental variability. As a proof of concept, we generated deep sequencing data from a pool of 60 human cell lines; we evaluated almost twice as many CpGs as the largest microarray studies and identified more than 2000 genetic variants associated with DNA methylation. We found that these variants are highly enriched for associations with chromatin accessibility and CTCF binding but are less likely to be associated with traits indirectly linked to DNA, such as gene expression and disease phenotypes. In summary, our approach allows genome-wide mapping of genetic variants associated with DNA methylation in any tissue of any species, without the need for individual-level genotype or methylation data. PMID:25910490

A pooling-based approach to mapping genetic variants associated with DNA methylation

DOE PAGES

Kaplow, Irene M.; MacIsaac, Julia L.; Mah, Sarah M.; ...

2015-04-24

DNA methylation is an epigenetic modification that plays a key role in gene regulation. Previous studies have investigated its genetic basis by mapping genetic variants that are associated with DNA methylation at specific sites, but these have been limited to microarrays that cover <2% of the genome and cannot account for allele-specific methylation (ASM). Other studies have performed whole-genome bisulfite sequencing on a few individuals, but these lack statistical power to identify variants associated with DNA methylation. We present a novel approach in which bisulfite-treated DNA from many individuals is sequenced together in a single pool, resulting in a trulymore » genome-wide map of DNA methylation. Compared to methods that do not account for ASM, our approach increases statistical power to detect associations while sharply reducing cost, effort, and experimental variability. As a proof of concept, we generated deep sequencing data from a pool of 60 human cell lines; we evaluated almost twice as many CpGs as the largest microarray studies and identified more than 2000 genetic variants associated with DNA methylation. Here we found that these variants are highly enriched for associations with chromatin accessibility and CTCF binding but are less likely to be associated with traits indirectly linked to DNA, such as gene expression and disease phenotypes. In summary, our approach allows genome-wide mapping of genetic variants associated with DNA methylation in any tissue of any species, without the need for individual-level genotype or methylation data.« less

Complexity and Entropy Analysis of DNMT1 Gene

USDA-ARS?s Scientific Manuscript database

Background: The application of complexity information on DNA sequence and protein in biological processes are well established in this study. Available sequences for DNMT1 gene, which is a maintenance methyltransferase is responsible for copying DNA methylation patterns to the daughter strands durin...

SOX17 promoter methylation in plasma circulating tumor DNA of patients with non-small cell lung cancer.

PubMed

Balgkouranidou, Ioanna; Chimonidou, Maria; Milaki, Georgia; Tsaroucha, Emily; Kakolyris, Stylianos; Georgoulias, Vasilis; Lianidou, Evi

2016-08-01

SOX17 belongs to the high-mobility group-box transcription factor superfamily and down-regulates the Wnt pathway. The aim of our study was to evaluate the prognostic significance of SOX17 promoter methylation in circulating tumor DNA (ctDNA) in plasma of non-small cell lung cancer (NSCLC) patients. We examined the methylation status of SOX17 promoter in 57 operable NSCLC primary tumors and paired adjacent non-cancerous tissues and in ctDNA isolated from 48 corresponding plasma samples as well as in plasma from 74 patients with advanced NSCLC and 49 healthy individuals. SOX17 promoter methylation was examined by Methylation Specific PCR (MSP). In operable NSCLC, SOX17 promoter was fully methylated in primary tumors (57/57, 100%), and in corresponding ctDNA (27/48, 56.2%) while it was detected in only 1/49 (2.0%) healthy individuals. In advanced NSCLC, SOX17 promoter was methylated in ctDNA in 27/74 (36.4%) patients and OS was significantly different in favor of patients with non-methylated SOX17 promoter (p=0.012). Multivariate analysis revealed that SOX17 promoter methylation in ctDNA was an independent prognostic factor associated with OS in patients with advanced but not operable NSCLC. Our results show that SOX17 promoter is highly methylated in primary tumors and in corresponding plasma samples both in operable and advanced NSCLC. In the advanced setting, SOX17 promoter methylation in plasma ctDNA has a statistical significant influence on NSCLC patient's survival time. Detection of SOX17 promoter methylation in plasma provides prognostic information and merits to be further evaluated as a circulating tumor biomarker in patients with operable and advanced NSCLC.

Epigenetics and migraine; complex mitochondrial interactions contributing to disease susceptibility.

PubMed

Roos-Araujo, Deidré; Stuart, Shani; Lea, Rod A; Haupt, Larisa M; Griffiths, Lyn R

2014-06-10

Migraine is a common neurological disorder classified by the World Health Organisation (WHO) as one of the top twenty most debilitating diseases in the developed world. Current therapies are only effective for a proportion of sufferers and new therapeutic targets are desperately needed to alleviate this burden. Recently the role of epigenetics in the development of many complex diseases including migraine has become an emerging topic. By understanding the importance of acetylation, methylation and other epigenetic modifications, it then follows that this modification process is a potential target to manipulate epigenetic status with the goal of treating disease. Bisulphite sequencing and methylated DNA immunoprecipitation have been used to demonstrate the presence of methylated cytosines in the human D-loop of mitochondrial DNA (mtDNA), proving that the mitochondrial genome is methylated. For the first time, it has been shown that there is a difference in mtDNA epigenetic status between healthy controls and those with disease, especially for neurodegenerative and age related conditions. Given co-morbidities with migraine and the suggestive link between mitochondrial dysfunction and the lowered threshold for triggering a migraine attack, mitochondrial methylation may be a new avenue to pursue. Creative thinking and new approaches are needed to solve complex problems and a systems biology approach, where multiple layers of information are integrated is becoming more important in complex disease modelling. Copyright © 2014 Elsevier B.V. All rights reserved.

TET2 functions as a resistance factor against DNA methylation acquisition during Epstein-Barr virus infection.

PubMed

Namba-Fukuyo, Hiroe; Funata, Sayaka; Matsusaka, Keisuke; Fukuyo, Masaki; Rahmutulla, Bahityar; Mano, Yasunobu; Fukayama, Masashi; Aburatani, Hiroyuki; Kaneda, Atsushi

2016-12-06

Extensive DNA methylation is observed in gastric cancer with Epstein-Barr virus (EBV) infection, and EBV infection is the cause to induce this extensive hypermethylaton phenotype in gastric epithelial cells. However, some 5' regions of genes do not undergo de novo methylation, despite the induction of methylation in surrounding regions, suggesting the existence of a resistance factor against DNA methylation acquisition. We conducted an RNA-seq analysis of gastric epithelial cells with and without EBV infection and found that TET family genes, especially TET2, were repressed by EBV infection at both mRNA and protein levels. TET2 was found to be downregulated by EBV transcripts, e.g. BARF0 and LMP2A, and also by seven human miRNAs targeting TET2, e.g., miR-93 and miR-29a, which were upregulated by EBV infection, and transfection of which into gastric cells repressed TET2. Hydroxymethylation target genes by TET2 were detected by hydroxymethylated DNA immunoprecipitation sequencing (hMeDIP-seq) with and without TET2 overexpression, and overlapped significantly with methylation target genes in EBV-infected cells. When TET2 was knocked down by shRNA, EBV infection induced de novo methylation more severely, including even higher methylation in methylation-acquired promoters or de novo methylation acquisition in methylation-protected promoters, leading to gene repression. TET2 knockdown alone without EBV infection did not induce de novo DNA methylation. These data suggested that TET2 functions as a resistance factor against DNA methylation in gastric epithelial cells and repression of TET2 contributes to DNA methylation acquisition during EBV infection.

Maternal exposure to anti-androgenic compounds, vinclozolin, flutamide and procymidone, has no effects on spermatogenesis and DNA methylation in male rats of subsequent generations

DOE Office of Scientific and Technical Information (OSTI.GOV)

Inawaka, Kunifumi; Kawabe, Mayumi; DIMS Institute of Medical Science, Inc., Ichinomiya

To verify whether anti-androgens cause transgenerational effects on spermatogenesis and DNA methylation in rats, gravid Crl:CD(SD) female rats (4 or 5/group, gestational day (GD) 0 = day sperm detected) were intraperitoneally treated with anti-androgenic compounds, such as vinclozolin (100 mg/kg/day), procymidone (100 mg/kg/day), or flutamide (10 mg/kg/day), from GD 8 to GD 15. Testes were collected from F1 male pups at postnatal day (PND) 6 for DNA methylation analysis of the region (210 bp including 7 CpG sites) within the lysophospholipase gene by bisulfite DNA sequencing method. F0 and F1 males underwent the sperm analysis (count, motility and morphology), followedmore » by DNA methylation analysis of the sperm. Remaining F1 males were cohabited with untreated-females to obtain F2 male pups for subsequent DNA methylation analysis of the testes at PND 6. These analyses showed no effects on spermatogenesis and fertility in F1 males of any treatment group. DNA methylation status in testes (F1 and F2 pups at PND 6) or sperms (F1 males at 13 weeks old) of the treatment groups were comparable to the control at all observation points, although DNA methylation rates in testes were slightly lower than those in sperm. In F0 males, no abnormalities in the spermatogenesis, fertility and DNA methylation status of sperm were observed. No transgenerational abnormalities of spermatogenesis and DNA methylation status caused by anti-androgenic compounds were observed.« less

Maternal exposure to anti-androgenic compounds, vinclozolin, flutamide and procymidone, has no effects on spermatogenesis and DNA methylation in male rats of subsequent generations.

PubMed

Inawaka, Kunifumi; Kawabe, Mayumi; Takahashi, Satoru; Doi, Yuko; Tomigahara, Yoshitaka; Tarui, Hirokazu; Abe, Jun; Kawamura, Satoshi; Shirai, Tomoyuki

2009-06-01

To verify whether anti-androgens cause transgenerational effects on spermatogenesis and DNA methylation in rats, gravid Crl:CD(SD) female rats (4 or 5/group, gestational day (GD) 0=day sperm detected) were intraperitoneally treated with anti-androgenic compounds, such as vinclozolin (100 mg/kg/day), procymidone (100 mg/kg/day), or flutamide (10 mg/kg/day), from GD 8 to GD 15. Testes were collected from F1 male pups at postnatal day (PND) 6 for DNA methylation analysis of the region (210 bp including 7 CpG sites) within the lysophospholipase gene by bisulfite DNA sequencing method. F0 and F1 males underwent the sperm analysis (count, motility and morphology), followed by DNA methylation analysis of the sperm. Remaining F1 males were cohabited with untreated-females to obtain F2 male pups for subsequent DNA methylation analysis of the testes at PND 6. These analyses showed no effects on spermatogenesis and fertility in F1 males of any treatment group. DNA methylation status in testes (F1 and F2 pups at PND 6) or sperms (F1 males at 13 weeks old) of the treatment groups were comparable to the control at all observation points, although DNA methylation rates in testes were slightly lower than those in sperm. In F0 males, no abnormalities in the spermatogenesis, fertility and DNA methylation status of sperm were observed. No transgenerational abnormalities of spermatogenesis and DNA methylation status caused by anti-androgenic compounds were observed.

Expression and methylation of BDNF in the human brain in schizophrenia.

PubMed

Cheah, Sern-Yih; McLeay, Robert; Wockner, Leesa F; Lawford, Bruce R; Young, Ross McD; Morris, Charles P; Voisey, Joanne

2017-08-01

To examine the combined effect of the BDNF Val66Met (rs6265) polymorphism and BDNF DNA methylation on transcriptional regulation of the BDNF gene. DNA methylation profiles were generated for CpG sites proximal to Val66Met, within BDNF promoter I and exon V for prefrontal cortex samples from 25 schizophrenia and 25 control subjects. Val66Met genotypes and BDNF mRNA expression data were generated by transcriptome sequencing. Expression, methylation and genotype data were correlated and examined for association with schizophrenia. There was 43% more of the BDNF V-VIII-IX transcript in schizophrenia samples. BDNF mRNA expression and DNA methylation of seven CpG sites were not associated with schizophrenia after accounting for age and PMI effects. BDNF mRNA expression and DNA methylation were not altered by Val66Met after accounting for age and PMI effects. DNA methylation of one CpG site had a marginally significant positive correlation with mRNA expression in schizophrenia subjects. Schizophrenia risk was not associated with differential BDNF mRNA expression and DNA methylation. A larger age-matched cohort with comprehensive clinical history is required to accurately identify the effects of genotype, mRNA expression and DNA methylation on schizophrenia risk.

Pancreatic cancer patient survival correlates with DNA methylation of pancreas development genes.

PubMed

Thompson, Michael J; Rubbi, Liudmilla; Dawson, David W; Donahue, Timothy R; Pellegrini, Matteo

2015-01-01

DNA methylation is an epigenetic mark associated with regulation of transcription and genome structure. These markers have been investigated in a variety of cancer settings for their utility in differentiating normal tissue from tumor tissue. Here, we examine the direct correlation between DNA methylation and patient survival. We find that changes in the DNA methylation of key pancreatic developmental genes are strongly associated with patient survival.

Persistent organic pollutants alter DNA methylation during human adipocyte differentiation.

PubMed

van den Dungen, Myrthe W; Murk, Albertinka J; Kok, Dieuwertje E; Steegenga, Wilma T

2017-04-01

Ubiquitous persistent organic pollutants (POPs) can accumulate in humans where they might influence differentiation of adipocytes. The aim of this study was to investigate whether DNA methylation is one of the underlying mechanisms by which POPs affect adipocyte differentiation, and to what extent DNA methylation can be related to gene transcription. Adipocyte differentiation was induced in two human cell models with continuous exposure to different POPs throughout differentiation. From the seven tested POPs, perfluorooctanesulfonic acid (PFOS) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) decreased lipid accumulation, while tributyltin (TBT) increased lipid accumulation. In human mesenchymal stem cells (hMSCs), TCDD and TBT induced opposite gene expression profiles, whereas after PFOS exposure gene expression remained relatively stable. Genome-wide DNA methylation analysis showed that all three POPs affected DNA methylation patterns in adipogenic and other genes, possibly related to the phenotypic outcome, but without concomitant gene expression changes. Differential methylation was predominantly detected in intergenic regions, where the biological relevance of alterations in DNA methylation is unclear. This study demonstrates that POPs, at environmentally relevant levels, are able to induce differential DNA methylation in human differentiating adipocytes. Copyright © 2017 Wageningen University. Published by Elsevier Ltd.. All rights reserved.

Ras regulation of DNA-methylation and cancer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Patra, Samir Kumar

2008-04-01

Genome wide hypomethylation and regional hypermethylation of cancer cells and tissues remain a paradox, though it has received a convincing confirmation that epigenetic switching systems, including DNA-methylation represent a fundamental regulatory mechanism that has an impact on genome maintenance and gene transcription. Methylated cytosine residues of vertebrate DNA are transmitted by clonal inheritance through the strong preference of DNA methyltransferase, DNMT1, for hemimethylated-DNA. Maintenance of methylation patterns is necessary for normal development of mice, and aberrant methylation patterns are associated with many human tumours. DNMT1 interacts with many proteins during cell cycle progression, including PCNA, p53, EZH2 and HP1. Rasmore » family of GTPases promotes cell proliferation by its oncogenic nature, which transmits signals by multiple pathways in both lipid raft dependent and independent fashion. DNA-methylation-mediated repression of DNA-repair protein O6-methylguanine DNA methyltransferase (MGMT) gene and increased rate of K-Ras mutation at codon for amino acids 12 and 13 have been correlated with a secondary role for Ras-effector homologues (RASSFs) in tumourigenesis. Lines of evidence suggest that DNA-methylation associated repression of tumour suppressors and apoptotic genes and ceaseless proliferation of tumour cells are regulated in part by Ras-signaling. Control of Ras GTPase signaling might reduce the aberrant methylation and accordingly may reduce the risk of cancer development.« less

DNA damage and methylation induced by glyphosate in human peripheral blood mononuclear cells (in vitro study).

PubMed

Kwiatkowska, Marta; Reszka, Edyta; Woźniak, Katarzyna; Jabłońska, Ewa; Michałowicz, Jaromir; Bukowska, Bożena

2017-07-01

Glyphosate is a very important herbicide that is widely used in the agriculture, and thus the exposure of humans to this substance and its metabolites has been noted. The purpose of this study was to assess DNA damage (determination of single and double strand-breaks by the comet assay) as well as to evaluate DNA methylation (global DNA methylation and methylation of p16 (CDKN2A) and p53 (TP53) promoter regions) in human peripheral blood mononuclear cells (PBMCs) exposed to glyphosate. PBMCs were incubated with the compound studied at concentrations ranging from 0.1 to 10 mM for 24 h. The study has shown that glyphosate induced DNA lesions, which were effectively repaired. However, PBMCs were unable to repair completely DNA damage induced by glyphosate. We also observed a decrease in global DNA methylation level at 0.25 mM of glyphosate. Glyphosate at 0.25 mM and 0.5 mM increased p53 promoter methylation, while it did not induce statistically significant changes in methylation of p16 promoter. To sum up, we have shown for the first time that glyphosate (at high concentrations from 0.5 to 10 mM) may induce DNA damage in leucocytes such as PBMCs and cause DNA methylation in human cells. Copyright © 2017 Elsevier Ltd. All rights reserved.

MIRA: An R package for DNA methylation-based inference of regulatory activity.

PubMed

Lawson, John T; Tomazou, Eleni M; Bock, Christoph; Sheffield, Nathan C

2018-03-01

DNA methylation contains information about the regulatory state of the cell. MIRA aggregates genome-scale DNA methylation data into a DNA methylation profile for independent region sets with shared biological annotation. Using this profile, MIRA infers and scores the collective regulatory activity for each region set. MIRA facilitates regulatory analysis in situations where classical regulatory assays would be difficult and allows public sources of open chromatin and protein binding regions to be leveraged for novel insight into the regulatory state of DNA methylation datasets. R package available on Bioconductor: http://bioconductor.org/packages/release/bioc/html/MIRA.html. nsheffield@virginia.edu.

Environmental pollution and DNA methylation: carcinogenesis, clinical significance, and practical applications.

PubMed

Cao, Yi

2015-09-01

Environmental pollution is one of the main causes of human cancer. Exposures to environmental carcinogens result in genetic and epigenetic alterations which induce cell transformation. Epigenetic changes caused by environmental pollution play important roles in the development and progression of environmental pollution-related cancers. Studies on DNA methylation are among the earliest and most conducted epigenetic research linked to cancer. In this review, the roles of DNA methylation in carcinogenesis and their significance in clinical medicine were summarized, and the effects of environmental pollutants, particularly air pollutants, on DNA methylation were introduced. Furthermore, prospective applications of DNA methylation to environmental pollution detection and cancer prevention were discussed.

Genome-wide DNA methylation measurements in prostate tissues uncovers novel prostate cancer diagnostic biomarkers and transcription factor binding patterns.

PubMed

Kirby, Marie K; Ramaker, Ryne C; Roberts, Brian S; Lasseigne, Brittany N; Gunther, David S; Burwell, Todd C; Davis, Nicholas S; Gulzar, Zulfiqar G; Absher, Devin M; Cooper, Sara J; Brooks, James D; Myers, Richard M

2017-04-17

Current diagnostic tools for prostate cancer lack specificity and sensitivity for detecting very early lesions. DNA methylation is a stable genomic modification that is detectable in peripheral patient fluids such as urine and blood plasma that could serve as a non-invasive diagnostic biomarker for prostate cancer. We measured genome-wide DNA methylation patterns in 73 clinically annotated fresh-frozen prostate cancers and 63 benign-adjacent prostate tissues using the Illumina Infinium HumanMethylation450 BeadChip array. We overlaid the most significantly differentially methylated sites in the genome with transcription factor binding sites measured by the Encyclopedia of DNA Elements consortium. We used logistic regression and receiver operating characteristic curves to assess the performance of candidate diagnostic models. We identified methylation patterns that have a high predictive power for distinguishing malignant prostate tissue from benign-adjacent prostate tissue, and these methylation signatures were validated using data from The Cancer Genome Atlas Project. Furthermore, by overlaying ENCODE transcription factor binding data, we observed an enrichment of enhancer of zeste homolog 2 binding in gene regulatory regions with higher DNA methylation in malignant prostate tissues. DNA methylation patterns are greatly altered in prostate cancer tissue in comparison to benign-adjacent tissue. We have discovered patterns of DNA methylation marks that can distinguish prostate cancers with high specificity and sensitivity in multiple patient tissue cohorts, and we have identified transcription factors binding in these differentially methylated regions that may play important roles in prostate cancer development.

H3K4me1 marks DNA regions hypomethylated during aging in human stem and differentiated cells

PubMed Central

Fernández, Agustín F.; Bayón, Gustavo F.; Urdinguio, Rocío G.; Toraño, Estela G.; García, María G.; Carella, Antonella; Petrus-Reurer, Sandra; Ferrero, Cecilia; Martinez-Camblor, Pablo; Cubillo, Isabel; García-Castro, Javier; Delgado-Calle, Jesús; Pérez-Campo, Flor M.; Riancho, José A.; Bueno, Clara; Menéndez, Pablo; Mentink, Anouk; Mareschi, Katia; Claire, Fabian; Fagnani, Corrado; Medda, Emanuela; Toccaceli, Virgilia; Brescianini, Sonia; Moran, Sebastián; Esteller, Manel; Stolzing, Alexandra; de Boer, Jan; Nisticò, Lorenza; Stazi, Maria A.

2015-01-01

In differentiated cells, aging is associated with hypermethylation of DNA regions enriched in repressive histone post-translational modifications. However, the chromatin marks associated with changes in DNA methylation in adult stem cells during lifetime are still largely unknown. Here, DNA methylation profiling of mesenchymal stem cells (MSCs) obtained from individuals aged 2 to 92 yr identified 18,735 hypermethylated and 45,407 hypomethylated CpG sites associated with aging. As in differentiated cells, hypermethylated sequences were enriched in chromatin repressive marks. Most importantly, hypomethylated CpG sites were strongly enriched in the active chromatin mark H3K4me1 in stem and differentiated cells, suggesting this is a cell type–independent chromatin signature of DNA hypomethylation during aging. Analysis of scedasticity showed that interindividual variability of DNA methylation increased during aging in MSCs and differentiated cells, providing a new avenue for the identification of DNA methylation changes over time. DNA methylation profiling of genetically identical individuals showed that both the tendency of DNA methylation changes and scedasticity depended on nongenetic as well as genetic factors. Our results indicate that the dynamics of DNA methylation during aging depend on a complex mixture of factors that include the DNA sequence, cell type, and chromatin context involved and that, depending on the locus, the changes can be modulated by genetic and/or external factors. PMID:25271306

Bisulfite Conversion of DNA: Performance Comparison of Different Kits and Methylation Quantitation of Epigenetic Biomarkers that Have the Potential to Be Used in Non-Invasive Prenatal Testing

PubMed Central

Leontiou, Chrysanthia A.; Hadjidaniel, Michael D.; Mina, Petros; Antoniou, Pavlos; Ioannides, Marios; Patsalis, Philippos C.

2015-01-01

Introduction Epigenetic alterations, including DNA methylation, play an important role in the regulation of gene expression. Several methods exist for evaluating DNA methylation, but bisulfite sequencing remains the gold standard by which base-pair resolution of CpG methylation is achieved. The challenge of the method is that the desired outcome (conversion of unmethylated cytosines) positively correlates with the undesired side effects (DNA degradation and inappropriate conversion), thus several commercial kits try to adjust a balance between the two. The aim of this study was to compare the performance of four bisulfite conversion kits [Premium Bisulfite kit (Diagenode), EpiTect Bisulfite kit (Qiagen), MethylEdge Bisulfite Conversion System (Promega) and BisulFlash DNA Modification kit (Epigentek)] regarding conversion efficiency, DNA degradation and conversion specificity. Methods Performance was tested by combining fully methylated and fully unmethylated λ-DNA controls in a series of spikes by means of Sanger sequencing (0%, 25%, 50% and 100% methylated spikes) and Next-Generation Sequencing (0%, 3%, 5%, 7%, 10%, 25%, 50% and 100% methylated spikes). We also studied the methylation status of two of our previously published differentially methylated regions (DMRs) at base resolution by using spikes of chorionic villus sample in whole blood. Results The kits studied showed different but comparable results regarding DNA degradation, conversion efficiency and conversion specificity. However, the best performance was observed with the MethylEdge Bisulfite Conversion System (Promega) followed by the Premium Bisulfite kit (Diagenode). The DMRs, EP6 and EP10, were confirmed to be hypermethylated in the CVS and hypomethylated in whole blood. Conclusion Our findings indicate that the MethylEdge Bisulfite Conversion System (Promega) was shown to have the best performance among the kits. In addition, the methylation level of two of our DMRs, EP6 and EP10, was confirmed. Finally, we showed that bisulfite amplicon sequencing is a suitable approach for methylation analysis of targeted regions. PMID:26247357

Bisulfite Conversion of DNA: Performance Comparison of Different Kits and Methylation Quantitation of Epigenetic Biomarkers that Have the Potential to Be Used in Non-Invasive Prenatal Testing.

PubMed

Leontiou, Chrysanthia A; Hadjidaniel, Michael D; Mina, Petros; Antoniou, Pavlos; Ioannides, Marios; Patsalis, Philippos C

2015-01-01

Epigenetic alterations, including DNA methylation, play an important role in the regulation of gene expression. Several methods exist for evaluating DNA methylation, but bisulfite sequencing remains the gold standard by which base-pair resolution of CpG methylation is achieved. The challenge of the method is that the desired outcome (conversion of unmethylated cytosines) positively correlates with the undesired side effects (DNA degradation and inappropriate conversion), thus several commercial kits try to adjust a balance between the two. The aim of this study was to compare the performance of four bisulfite conversion kits [Premium Bisulfite kit (Diagenode), EpiTect Bisulfite kit (Qiagen), MethylEdge Bisulfite Conversion System (Promega) and BisulFlash DNA Modification kit (Epigentek)] regarding conversion efficiency, DNA degradation and conversion specificity. Performance was tested by combining fully methylated and fully unmethylated λ-DNA controls in a series of spikes by means of Sanger sequencing (0%, 25%, 50% and 100% methylated spikes) and Next-Generation Sequencing (0%, 3%, 5%, 7%, 10%, 25%, 50% and 100% methylated spikes). We also studied the methylation status of two of our previously published differentially methylated regions (DMRs) at base resolution by using spikes of chorionic villus sample in whole blood. The kits studied showed different but comparable results regarding DNA degradation, conversion efficiency and conversion specificity. However, the best performance was observed with the MethylEdge Bisulfite Conversion System (Promega) followed by the Premium Bisulfite kit (Diagenode). The DMRs, EP6 and EP10, were confirmed to be hypermethylated in the CVS and hypomethylated in whole blood. Our findings indicate that the MethylEdge Bisulfite Conversion System (Promega) was shown to have the best performance among the kits. In addition, the methylation level of two of our DMRs, EP6 and EP10, was confirmed. Finally, we showed that bisulfite amplicon sequencing is a suitable approach for methylation analysis of targeted regions.

Impacts of Chromatin States and Long-Range Genomic Segments on Aging and DNA Methylation

PubMed Central

Sun, Dan; Yi, Soojin V.

2015-01-01

Understanding the fundamental dynamics of epigenome variation during normal aging is critical for elucidating key epigenetic alterations that affect development, cell differentiation and diseases. Advances in the field of aging and DNA methylation strongly support the aging epigenetic drift model. Although this model aligns with previous studies, the role of other epigenetic marks, such as histone modification, as well as the impact of sampling specific CpGs, must be evaluated. Ultimately, it is crucial to investigate how all CpGs in the human genome change their methylation with aging in their specific genomic and epigenomic contexts. Here, we analyze whole genome bisulfite sequencing DNA methylation maps of brain frontal cortex from individuals of diverse ages. Comparisons with blood data reveal tissue-specific patterns of epigenetic drift. By integrating chromatin state information, divergent degrees and directions of aging-associated methylation in different genomic regions are revealed. Whole genome bisulfite sequencing data also open a new door to investigate whether adjacent CpG sites exhibit coordinated DNA methylation changes with aging. We identified significant ‘aging-segments’, which are clusters of nearby CpGs that respond to aging by similar DNA methylation changes. These segments not only capture previously identified aging-CpGs but also include specific functional categories of genes with implications on epigenetic regulation of aging. For example, genes associated with development are highly enriched in positive aging segments, which are gradually hyper-methylated with aging. On the other hand, regions that are gradually hypo-methylated with aging (‘negative aging segments’) in the brain harbor genes involved in metabolism and protein ubiquitination. Given the importance of protein ubiquitination in proteome homeostasis of aging brains and neurodegenerative disorders, our finding suggests the significance of epigenetic regulation of this posttranslational modification pathway in the aging brain. Utilizing aging segments rather than individual CpGs will provide more comprehensive genomic and epigenomic contexts to understand the intricate associations between genomic neighborhoods and developmental and aging processes. These results complement the aging epigenetic drift model and provide new insights. PMID:26091484

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

PubMed

Seguin-Orlando, Andaine; Gamba, Cristina; Der Sarkissian, Clio; 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

2015-07-02

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.

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

PubMed Central

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

Global and gene specific DNA methylation changes during zebrafish development

USDA-ARS?s Scientific Manuscript database

DNA methylation is dynamic through the life of an organism. In this study, we measured the global and gene specific DNA methylation changes in zebrafish at different developmental stages. We found that the methylation percentage of cytosines was 11.75 ± 0.96% in 3.3 hour post fertilization (hpf) zeb...

Genome-wide DNA methylation drives human embryonic stem cell erythropoiesis by remodeling gene expression dynamics.

PubMed

Liu, Zhijing; Feng, Qiang; Sun, Pengpeng; Lu, Yan; Yang, Minlan; Zhang, Xiaowei; Jin, Xiangshu; Li, Yulin; Lu, Shi-Jiang; Quan, Chengshi

2017-12-01

To investigate the role of DNA methylation during erythrocyte production by human embryonic stem cells (hESCs). We employed an erythroid differentiation model from hESCs, and then tracked the genome-wide DNA methylation maps and gene expression patterns through an Infinium HumanMethylation450K BeadChip and an Ilumina Human HT-12 v4 Expression Beadchip, respectively. A negative correlation between DNA methylation and gene expression was substantially enriched during the later differentiation stage and was present in both the promoter and the gene body. Moreover, erythropoietic genes with differentially methylated CpG sites that were primarily enriched in nonisland regions were upregulated, and demethylation of their gene bodies was associated with the presence of enhancers and DNase I hypersensitive sites. Finally, the components of JAK-STAT-NF-κB signaling were DNA hypomethylated and upregulated, which targets the key genes for erythropoiesis. Erythroid lineage commitment by hESCs requires genome-wide DNA methylation modifications to remodel gene expression dynamics.

Enrichment of methylated molecules using enhanced-ice-co-amplification at lower denaturation temperature-PCR (E-ice-COLD-PCR) for the sensitive detection of disease-related hypermethylation.

PubMed

Mauger, Florence; Kernaleguen, Magali; Lallemand, Céline; Kristensen, Vessela N; Deleuze, Jean-François; Tost, Jörg

2018-05-01

The detection of specific DNA methylation patterns bears great promise as biomarker for personalized management of cancer patients. Co-amplification at lower denaturation temperature-PCR (COLD-PCR) assays are sensitive methods, but have previously only been able to analyze loss of DNA methylation. Enhanced (E)-ice-COLD-PCR reactions starting from 2 ng of bisulfite-converted DNA were developed to analyze methylation patterns in two promoters with locked nucleic acid (LNA) probes blocking amplification of unmethylated CpGs. The enrichment of methylated molecules was compared to quantitative (q)PCR and quantified using serial dilutions. E-ice-COLD-PCR allowed the multiplexed enrichment and quantification of methylated DNA. Assays were validated in primary breast cancer specimens and circulating cell-free DNA from cancer patients. E-ice-COLD-PCR could prove a useful tool in the context of DNA methylation analysis for personalized medicine.

S-adenosyl-L-methionine analogs as enhanced methyl donors: Towards novel epigenetic regulators

NASA Astrophysics Data System (ADS)

Jerbi, Jihène; Springborg, Michael; den-Haan, Helena; Cerón-Carrasco, José P.

2017-12-01

Many efforts have been devoted to discover molecules able to halt methylation processes in DNA. However, less is known about the application of methyl promoters in the framework of hypomethylation diseases. Herein, we used molecular dynamics and ab initio calculations to assess the methylation ability of the parent S-adenosyl-L-methionine cofactor (SAM) and a series of analogues. Two molecules deposited in the PubChem database are shown to be promising candidates for increasing the methyl transfer rate of the original SAM. The reported data might be consequently used to guide further steps into the search of more efficient methyl donor-based drugs.

Heterogeneity of DNA methylation in multifocal prostate cancer.

PubMed

Serenaite, Inga; Daniunaite, Kristina; Jankevicius, Feliksas; Laurinavicius, Arvydas; Petroska, Donatas; Lazutka, Juozas R; Jarmalaite, Sonata

2015-01-01

Most prostate cancer (PCa) cases are multifocal, and separate foci display histological and molecular heterogeneity. DNA hypermethylation is a frequent alteration in PCa, but interfocal heterogeneity of these changes has not been extensively investigated. Ten pairs of foci from multifocal PCa and 15 benign prostatic hyperplasia (BPH) samples were obtained from prostatectomy specimens, resulting altogether in 35 samples. Methylation-specific PCR (MSP) was used to evaluate methylation status of nine tumor suppressor genes (TSGs), and a set of selected TSGs was quantitatively analyzed for methylation intensity by pyrosequencing. Promoter sequences of the RASSF1 and ESR1 genes were methylated in all paired PCa foci, and frequent (≥75 %) DNA methylation was detected in RARB, GSTP1, and ABCB1 genes. MSP revealed different methylation status of at least one gene in separate foci in 8 out of 10 multifocal tumors. The mean methylation level of ESR1, GSTP1, RASSF1, and RARB differed between the paired foci of all PCa cases. The intensity of DNA methylation in these TSGs was significantly higher in PCa cases than in BPH (p < 0.001). Hierarchical cluster analysis revealed a divergent methylation profile of paired PCa foci, while the foci from separate cases with biochemical recurrence showed similar methylation profile and the highest mean levels of DNA methylation. Our findings suggest that PCa tissue is heterogeneous, as between paired foci differences in DNA methylation status were found. Common epigenetic profile of recurrent tumors can be inferred from our data.

Epigenomic profiling of men exposed to early-life stress reveals DNA methylation differences in association with current mental state.

PubMed

Khulan, B; Manning, J R; Dunbar, D R; Seckl, J R; Raikkonen, K; Eriksson, J G; Drake, A J

2014-09-23

Early-life stress (ELS) is known to be associated with an increased risk of neuropsychiatric and cardiometabolic disease in later life. One of the potential mechanisms underpinning this is through effects on the epigenome, particularly changes in DNA methylation. Using a well-phenotyped cohort of 83 men from the Helsinki Birth Cohort Study, who experienced ELS in the form of separation from their parents during childhood, and a group of 83 matched controls, we performed a genome-wide analysis of DNA methylation in peripheral blood. We found no differences in DNA methylation between men who were separated from their families and non-separated men; however, we did identify differences in DNA methylation in association with the development of at least mild depressive symptoms over the subsequent 5-10 years. Notably, hypomethylation was identified at a number of genes with roles in brain development and/or function in association with depressive symptoms. Pathway analysis revealed an enrichment of DNA methylation changes in pathways associated with development and morphogenesis, DNA and transcription factor binding and programmed cell death. Our results support the concept that DNA methylation differences may be important in the pathogenesis of psychiatric disease.

Terahertz molecular resonance of cancer DNA.

PubMed

Cheon, Hwayeong; Yang, Hee-Jin; Lee, Sang-Hun; Kim, Young A; Son, Joo-Hiuk

2016-11-15

Carcinogenesis involves the chemical and structural alteration of biomolecules in cells. Aberrant methylation of DNA is a well-known carcinogenic mechanism and a common chemical modification of DNA. Terahertz waves can directly observe changes in DNA because the characteristic energies lie in the same frequency region. In addition, terahertz energy levels are not high enough to damage DNA by ionization. Here, we present terahertz molecular resonance fingerprints of DNA methylation in cancer DNA. Methylated cytidine, a nucleoside, has terahertz characteristic energies that give rise to the molecular resonance of methylation in DNA. Molecular resonance is monitored in aqueous solutions of genomic DNA from cancer cell lines using a terahertz time-domain spectroscopic technique. Resonance signals can be quantified to identify the types of cancer cells with a certain degree of DNA methylation. These measurements reveal the existence of molecular resonance fingerprints of cancer DNAs in the terahertz region, which can be utilized for the early diagnosis of cancer cells at the molecular level.

Terahertz molecular resonance of cancer DNA

NASA Astrophysics Data System (ADS)

Cheon, Hwayeong; Yang, Hee-Jin; Lee, Sang-Hun; Kim, Young A.; Son, Joo-Hiuk

2016-11-01

Carcinogenesis involves the chemical and structural alteration of biomolecules in cells. Aberrant methylation of DNA is a well-known carcinogenic mechanism and a common chemical modification of DNA. Terahertz waves can directly observe changes in DNA because the characteristic energies lie in the same frequency region. In addition, terahertz energy levels are not high enough to damage DNA by ionization. Here, we present terahertz molecular resonance fingerprints of DNA methylation in cancer DNA. Methylated cytidine, a nucleoside, has terahertz characteristic energies that give rise to the molecular resonance of methylation in DNA. Molecular resonance is monitored in aqueous solutions of genomic DNA from cancer cell lines using a terahertz time-domain spectroscopic technique. Resonance signals can be quantified to identify the types of cancer cells with a certain degree of DNA methylation. These measurements reveal the existence of molecular resonance fingerprints of cancer DNAs in the terahertz region, which can be utilized for the early diagnosis of cancer cells at the molecular level.

MethSMRT: an integrative database for DNA N6-methyladenine and N4-methylcytosine generated by single-molecular real-time sequencing.

PubMed

Ye, Pohao; Luan, Yizhao; Chen, Kaining; Liu, Yizhi; Xiao, Chuanle; Xie, Zhi

2017-01-04

DNA methylation is an important type of epigenetic modifications, where 5- methylcytosine (5mC), 6-methyadenine (6mA) and 4-methylcytosine (4mC) are the most common types. Previous efforts have been largely focused on 5mC, providing invaluable insights into epigenetic regulation through DNA methylation. Recently developed single-molecule real-time (SMRT) sequencing technology provides a unique opportunity to detect the less studied DNA 6mA and 4mC modifications at single-nucleotide resolution. With a rapidly increased amount of SMRT sequencing data generated, there is an emerging demand to systematically explore DNA 6mA and 4mC modifications from these data sets. MethSMRT is the first resource hosting DNA 6mA and 4mC methylomes. All the data sets were processed using the same analysis pipeline with the same quality control. The current version of the database provides a platform to store, browse, search and download epigenome-wide methylation profiles of 156 species, including seven eukaryotes such as Arabidopsis, C. elegans, Drosophila, mouse and yeast, as well as 149 prokaryotes. It also offers a genome browser to visualize the methylation sites and related information such as single nucleotide polymorphisms (SNP) and genomic annotation. Furthermore, the database provides a quick summary of statistics of methylome of 6mA and 4mC and predicted methylation motifs for each species. MethSMRT is publicly available at http://sysbio.sysu.edu.cn/methsmrt/ without use restriction. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Genome-Wide Assessment of Differential DNA Methylation Associated with Autoantibody Production in Systemic Lupus Erythematosus.

PubMed

Chung, Sharon A; Nititham, Joanne; Elboudwarej, Emon; Quach, Hong L; Taylor, Kimberly E; Barcellos, Lisa F; Criswell, Lindsey A

2015-01-01

Systemic lupus erythematosus (SLE) is characterized by the development of autoantibodies associated with specific clinical manifestations. Previous studies have shown an association between differential DNA methylation and SLE susceptibility, but have not investigated SLE-related autoantibodies. Our goal was to determine whether DNA methylation is associated with production of clinically relevant SLE-related autoantibodies, with an emphasis on the anti-dsDNA autoantibody. In this study, we characterized the methylation status of 467,314 CpG sites in 326 women with SLE. Using a discovery and replication study design, we identified and replicated significant associations between anti-dsDNA autoantibody production and the methylation status of 16 CpG sites (pdiscovery<1.07E-07 and preplication<0.0029) in 11 genes. Associations were further investigated using multivariable regression to adjust for estimated leukocyte cell proportions and population substructure. The adjusted mean DNA methylation difference between anti-dsDNA positive and negative cases ranged from 1.2% to 19%, and the adjusted odds ratio for anti-dsDNA autoantibody production comparing the lowest and highest methylation tertiles ranged from 6.8 to 18.2. Differential methylation for these CpG sites was also associated with anti-SSA, anti-Sm, and anti-RNP autoantibody production. Overall, associated CpG sites were hypomethylated in autoantibody positive compared to autoantibody negative cases. Differential methylation of CpG sites within the major histocompatibility region was not strongly associated with autoantibody production. Genes with differentially methylated CpG sites represent multiple biologic pathways, and have not been associated with autoantibody production in genetic association studies. In conclusion, hypomethylation of CpG sites within genes from different pathways is associated with anti-dsDNA, anti-SSA, anti-Sm, and anti-RNP production in SLE, and these associations are not explained by genetic variation. Thus, studies of epigenetic mechanisms such as DNA methylation represent a complementary method to genetic association studies to identify biologic pathways that may contribute to the clinical heterogeneity of autoimmune diseases.

DNA methylation screening of primary prostate tumors identifies SRD5A2 and CYP11A1 as candidate markers for assessing risk of biochemical recurrence.

PubMed

Horning, Aaron M; Awe, Julius A; Wang, Chiou-Miin; Liu, Joseph; Lai, Zhao; Wang, Vickie Yao; Jadhav, Rohit R; Louie, Anna D; Lin, Chun-Lin; Kroczak, Tad; Chen, Yidong; Jin, Victor X; Abboud-Werner, Sherry L; Leach, Robin J; Hernandez, Javior; Thompson, Ian M; Saranchuk, Jeff; Drachenberg, Darrel; Chen, Chun-Liang; Mai, Sabine; Huang, Tim Hui-Ming

2015-11-01

Altered DNA methylation in CpG islands of gene promoters has been implicated in prostate cancer (PCa) progression and can be used to predict disease outcome. In this study, we determine whether methylation changes of androgen biosynthesis pathway (ABP)-related genes in patients' plasma cell-free DNA (cfDNA) can serve as prognostic markers for biochemical recurrence (BCR). Methyl-binding domain capture sequencing (MBDCap-seq) was used to identify differentially methylated regions (DMRs) in primary tumors of patients who subsequently developed BCR or not, respectively. Methylation pyrosequencing of candidate loci was validated in cfDNA samples of 86 PCa patients taken at and/or post-radical prostatectomy (RP) using univariate and multivariate prediction analyses. Putative DMRs in 13 of 30 ABP-related genes were found between tumors of BCR (n = 12) versus no evidence of disease (NED) (n = 15). In silico analysis of The Cancer Genome Atlas data confirmed increased DNA methylation of two loci-SRD5A2 and CYP11A1, which also correlated with their decreased expression, in tumors with subsequent BCR development. Their aberrant cfDNA methylation was also associated with detectable levels of PSA taken after patients' post-RP. Multivariate analysis of the change in cfDNA methylation at all of CpG sites measured along with patient's treatment history predicted if a patient will develop BCR with 77.5% overall accuracy. Overall, increased DNA methylation of SRD5A2 and CYP11A1 related to androgen biosynthesis functions may play a role in BCR after patients' RP. The correlation between aberrant cfDNA methylation and detectable PSA in post-RP further suggests their utility as predictive markers for PCa recurrence. . © 2015 Wiley Periodicals, Inc.

Intragenic sequences in the trophectoderm harbour the greatest proportion of methylation errors in day 17 bovine conceptuses generated using assisted reproductive technologies.

PubMed

O'Doherty, Alan M; McGettigan, Paul; Irwin, Rachelle E; Magee, David A; Gagne, Dominic; Fournier, Eric; Al-Naib, Abdullah; Sirard, Marc-André; Walsh, Colum P; Robert, Claude; Fair, Trudee

2018-06-05

Assisted reproductive technologies (ART) are widely used to treat fertility issues in humans and for the production of embryos in mammalian livestock. The use of these techniques, however, is not without consequence as they are often associated with inauspicious pre- and postnatal outcomes including premature birth, intrauterine growth restriction and increased incidence of epigenetic disorders in human and large offspring syndrome in cattle. Here, global DNA methylation profiles in the trophectoderm and embryonic discs of in vitro produced (IVP), superovulation-derived (SOV) and unstimulated, synchronised control day 17 bovine conceptuses (herein referred to as AI) were interrogated using the EmbryoGENE DNA Methylation Array (EDMA). Pyrosequencing was used to validate four loci identified as differentially methylated on the array and to assess the differentially methylated regions (DMRs) of six imprinted genes in these conceptuses. The impact of embryo-production induced DNA methylation aberrations was determined using Ingenuity Pathway Analysis, shedding light on the potential functional consequences of these differences. Of the total number of differentially methylated loci identified (3140) 77.3 and 22.7% were attributable to SOV and IVP, respectively. Differential methylation was most prominent at intragenic sequences within the trophectoderm of IVP and SOV-derived conceptuses, almost a third (30.8%) of the differentially methylated loci mapped to intragenic regions. Very few differentially methylated loci were detected in embryonic discs (ED); 0.16 and 4.9% of the differentially methylated loci were located in the ED of SOV-derived and IVP conceptuses, respectively. The overall effects of SOV and IVP on the direction of methylation changes were associated with increased methylation; 70.6% of the differentially methylated loci in SOV-derived conceptuses and 57.9% of the loci in IVP-derived conceptuses were more methylated compared to AI-conceptuses. Ontology analysis of probes associated with intragenic sequences suggests enrichment for terms associated with cancer, cell morphology and growth. By examining (1) the effects of superovulation and (2) the effects of an in vitro system (oocyte maturation, fertilisation and embryo culture) we have identified that the assisted reproduction process of superovulation alone has the largest impact on the DNA methylome of subsequent embryos.

Characteristics of DNA methylation changes induced by traffic-related air pollution.

PubMed

Ding, Rui; Jin, Yongtang; Liu, Xinneng; Zhu, Ziyi; Zhang, Yuan; Wang, Ting; Xu, Yinchun

2016-01-15

Traffic-related air pollution (TRAP) is a potential risk factor for numerous respiratory disorders, including lung cancer, while alteration of DNA methylation may be one of the underlying mechanisms. However, the effects of TRAP mixtures on DNA methylation have not been investigated. We have studied the effects of brief or prolonged TRAP exposures on DNA methylation in the rat. The exposures were performed in spring and autumn, with identical study procedures. In each season, healthy Wistar rats were exposed to TRAP at for 4 h, 7 d, 14 d, or 28 d. Global DNA methylation (LINE-1 and Alu) and specific gene methylation (p16(CDKN2A), APC, and iNOS) in the DNA from blood and lung tissues were quantified by pyrosequencing. Multiple linear regression was applied to assess the influence of air pollutants on DNA methylation levels. The levels of PM2.5, PM10, and NO2 in the high and moderate groups were significantly higher than in the control group. The DNA methylation levels were not significantly different between spring and autumn. When spring and autumn data were analyzed together, PM2.5, PM10, and NO2 exposures were associated with changes in%5mC (95% CI) in LINE-1, iNOS, p16(CDKN2A), and APC ranging from -0.088 (-0.150, -0.026) to 0.102 (0.049, 0.154) per 1 μg/m(3) increase in the pollutant concentration. Prolonged exposure to a high level of TRAP was negatively associated with LINE-1 and iNOS methylation, and positively associated with APC methylations in the DNA from lung tissues but not blood. These findings show that TRAP exposure is associated with decreased methylation of LINE-1 and iNOS, and increased methylation of p16(CDKN2A) and APC. Copyright © 2015 Elsevier B.V. All rights reserved.

DNA methylation analysis from saliva samples for epidemiological studies.

PubMed

Nishitani, Shota; Parets, Sasha E; Haas, Brian W; Smith, Alicia K

2018-06-18

Saliva is a non-invasive, easily accessible tissue, which is regularly collected in large epidemiological studies to examine genetic questions. Recently, it is becoming more common to use saliva to assess DNA methylation. However, DNA extracted from saliva is a mixture of both bacterial and human DNA derived from epithelial and immune cells in the mouth. Thus, there are unique challenges to using salivary DNA in methylation studies that can influence data quality. This study assesses: (1) quantification of human DNA after extraction; (2) delineation of human and bacterial DNA; (3) bisulfite conversion (BSC); (4) quantification of BSC DNA; (5) PCR amplification of BSC DNA from saliva and; (6) quantitation of DNA methylation with a targeted assay. The framework proposed will allow saliva samples to be more widely used in targeted epigenetic studies.

Methylation-sensitive amplified polymorphism analysis of Verticillium wilt-stressed cotton (Gossypium).

PubMed

Wang, W; Zhang, M; Chen, H D; Cai, X X; Xu, M L; Lei, K Y; Niu, J H; Deng, L; Liu, J; Ge, Z J; Yu, S X; Wang, B H

2016-10-06

In this study, a methylation-sensitive amplification polymorphism analysis system was used to analyze DNA methylation level in three cotton accessions. Two disease-sensitive near-isogenic lines, PD94042 and IL41, and one disease-resistant Gossypium mustelinum accession were exposed to Verticillium wilt, to investigate molecular disease resistance mechanisms in cotton. We observed multiple different DNA methylation types across the three accessions following Verticillium wilt exposure. These included hypomethylation, hypermethylation, and other patterns. In general, the global DNA methylation level was significantly increased in the disease-resistant accession G. mustelinum following disease exposure. In contrast, there was no significant difference in the disease-sensitive accession PD94042, and a significant decrease was observed in IL41. Our results suggest that disease-resistant cotton might employ a mechanism to increase methylation level in response to disease stress. The differing methylation patterns, together with the increase in global DNA methylation level, might play important roles in tolerance to Verticillium wilt in cotton. Through cloning and analysis of differently methylated DNA sequences, we were also able to identify several genes that may contribute to disease resistance in cotton. Our results revealed the effect of DNA methylation on cotton disease resistance, and also identified genes that played important roles, which may shed light on the future cotton disease-resistant molecular breeding.

Efficient targeted DNA methylation with chimeric dCas9–Dnmt3a–Dnmt3L methyltransferase

PubMed Central

Stepper, Peter; Kungulovski, Goran; Jurkowska, Renata Z.; Chandra, Tamir; Krueger, Felix; Reinhardt, Richard

2017-01-01

Abstract DNA methylation plays a critical role in the regulation and maintenance of cell-type specific transcriptional programs. Targeted epigenome editing is an emerging technology to specifically regulate cellular gene expression in order to modulate cell phenotypes or dissect the epigenetic mechanisms involved in their control. In this work, we employed a DNA methyltransferase Dnmt3a–Dnmt3L construct fused to the nuclease-inactivated dCas9 programmable targeting domain to introduce DNA methylation into the human genome specifically at the EpCAM, CXCR4 and TFRC gene promoters. We show that targeting of these loci with single gRNAs leads to efficient and widespread methylation of the promoters. Multiplexing of several guide RNAs does not increase the efficiency of methylation. Peaks of targeted methylation were observed around 25 bp upstream and 40 bp downstream of the PAM site, while 20–30 bp of the binding site itself are protected against methylation. Potent methylation is dependent on the multimerization of Dnmt3a/Dnmt3L complexes on the DNA. Furthermore, the introduced methylation causes transcriptional repression of the targeted genes. These new programmable epigenetic editors allow unprecedented control of the DNA methylation status in cells and will lead to further advances in the understanding of epigenetic signaling. PMID:27899645

DNA methylation biomarkers for head and neck squamous cell carcinoma.

PubMed

Zhou, Chongchang; Ye, Meng; Ni, Shumin; Li, Qun; Ye, Dong; Li, Jinyun; Shen, Zhishen; Deng, Hongxia

2018-06-21

DNA methylation plays an important role in the etiology and pathogenesis of head and neck squamous cell carcinoma (HNSCC). The current study aimed to identify aberrantly methylated-differentially expressed genes (DEGs) by a comprehensive bioinformatics analysis. In addition, we screened for DEGs affected by DNA methylation modification and further investigated their prognostic values for HNSCC. We included microarray data of DNA methylation (GSE25093 and GSE33202) and gene expression (GSE23036 and GSE58911) from Gene Expression Omnibus. Aberrantly methylated-DEGs were analyzed with R software. The Cancer Genome Atlas (TCGA) RNA sequencing and DNA methylation (Illumina HumanMethylation450) databases were utilized for validation. In total, 27 aberrantly methylated genes accompanied by altered expression were identified. After confirmation by The Cancer Genome Atlas (TCGA) database, 2 hypermethylated-low-expression genes (FAM135B and ZNF610) and 2 hypomethylated-high-expression genes (HOXA9 and DCC) were identified. A receiver operating characteristic (ROC) curve confirmed the diagnostic value of these four methylated genes for HNSCC. Multivariate Cox proportional hazards analysis showed that FAM135B methylation was a favorable independent prognostic biomarker for overall survival of HNSCC patients.

Global DNA hypomethylation in peripheral blood leukocytes as a biomarker for cancer risk: a meta-analysis.

PubMed

Woo, Hae Dong; Kim, Jeongseon

2012-01-01

Good biomarkers for early detection of cancer lead to better prognosis. However, harvesting tumor tissue is invasive and cannot be routinely performed. Global DNA methylation of peripheral blood leukocyte DNA was evaluated as a biomarker for cancer risk. We performed a meta-analysis to estimate overall cancer risk according to global DNA hypomethylation levels among studies with various cancer types and analytical methods used to measure DNA methylation. Studies were systemically searched via PubMed with no language limitation up to July 2011. Summary estimates were calculated using a fixed effects model. The subgroup analyses by experimental methods to determine DNA methylation level were performed due to heterogeneity within the selected studies (p<0.001, I(2): 80%). Heterogeneity was not found in the subgroup of %5-mC (p = 0.393, I(2): 0%) and LINE-1 used same target sequence (p = 0.097, I(2): 49%), whereas considerable variance remained in LINE-1 (p<0.001, I(2): 80%) and bladder cancer studies (p = 0.016, I(2): 76%). These results suggest that experimental methods used to quantify global DNA methylation levels are important factors in the association study between hypomethylation levels and cancer risk. Overall, cancer risks of the group with the lowest DNA methylation levels were significantly higher compared to the group with the highest methylation levels [OR (95% CI): 1.48 (1.28-1.70)]. Global DNA hypomethylation in peripheral blood leukocytes may be a suitable biomarker for cancer risk. However, the association between global DNA methylation and cancer risk may be different based on experimental methods, and region of DNA targeted for measuring global hypomethylation levels as well as the cancer type. Therefore, it is important to select a precise and accurate surrogate marker for global DNA methylation levels in the association studies between global DNA methylation levels in peripheral leukocyte and cancer risk.

The effect of inflammation-related lifestyle exposures and interactions with gene variants on long interspersed nuclear element-1 DNA methylation.

PubMed

Gogna, Priyanka; O'Sullivan, Dylan E; King, Will D

2018-06-11

To examine the relationship between inflammation-related lifestyle factors and long interspersed nuclear element-1 (LINE-1) DNA methylation, and test for interaction by gene variants involved in one-carbon metabolism. The study population consisted of 280 individuals undergoing colonoscopy screening. Multivariable linear regression was employed to examine associations of physical activity, BMI and NSAID use with LINE-1 DNA methylation and interactions with MTR and MTHFR gene variants. The highest quartile of physical activity compared with the lowest was associated with higher LINE-1 DNA methylation (p = 0.005). Long-term NSAID use and a normal BMI were associated with increased LINE-1 DNA methylation among individuals with the variant MTR allele (p = 0.02; p = 0.03). This study provides evidence that inflammation-related exposures may influence LINE-1 DNA methylation.

Epigenetic Biomarkers of Breast Cancer Risk: Across the Breast Cancer Prevention Continuum.

PubMed

Terry, Mary Beth; McDonald, Jasmine A; Wu, Hui Chen; Eng, Sybil; Santella, Regina M

2016-01-01

Epigenetic biomarkers, such as DNA methylation, can increase cancer risk through altering gene expression. The Cancer Genome Atlas (TCGA) Network has demonstrated breast cancer-specific DNA methylation signatures. DNA methylation signatures measured at the time of diagnosis may prove important for treatment options and in predicting disease-free and overall survival (tertiary prevention). DNA methylation measurement in cell free DNA may also be useful in improving early detection by measuring tumor DNA released into the blood (secondary prevention). Most evidence evaluating the use of DNA methylation markers in tertiary and secondary prevention efforts for breast cancer comes from studies that are cross-sectional or retrospective with limited corresponding epidemiologic data, raising concerns about temporality. Few prospective studies exist that are large enough to address whether DNA methylation markers add to the prediction of tertiary and secondary outcomes over and beyond standard clinical measures. Determining the role of epigenetic biomarkers in primary prevention can help in identifying modifiable pathways for targeting interventions and reducing disease incidence. The potential is great for DNA methylation markers to improve cancer outcomes across the prevention continuum. Large, prospective epidemiological studies will provide essential evidence of the overall utility of adding these markers to primary prevention efforts, screening, and clinical care.

Atypical epigenetic mark in an atypical location: cytosine methylation at asymmetric (CNN) sites within the body of a non-repetitive tomato gene.

PubMed

González, Rodrigo M; Ricardi, Martiniano M; Iusem, Norberto D

2011-05-20

Eukaryotic DNA methylation is one of the most studied epigenetic processes, as it results in a direct and heritable covalent modification triggered by external stimuli. In contrast to mammals, plant DNA methylation, which is stimulated by external cues exemplified by various abiotic types of stress, is often found not only at CG sites but also at CNG (N denoting A, C or T) and CNN (asymmetric) sites. A genome-wide analysis of DNA methylation in Arabidopsis has shown that CNN methylation is preferentially concentrated in transposon genes and non-coding repetitive elements. We are particularly interested in investigating the epigenetics of plant species with larger and more complex genomes than Arabidopsis, particularly with regards to the associated alterations elicited by abiotic stress. We describe the existence of CNN-methylated epialleles that span Asr1, a non-transposon, protein-coding gene from tomato plants that lacks an orthologous counterpart in Arabidopsis. In addition, to test the hypothesis of a link between epigenetics modifications and the adaptation of crop plants to abiotic stress, we exhaustively explored the cytosine methylation status in leaf Asr1 DNA, a model gene in our system, resulting from water-deficit stress conditions imposed on tomato plants. We found that drought conditions brought about removal of methyl marks at approximately 75 of the 110 asymmetric (CNN) sites analysed, concomitantly with a decrease of the repressive H3K27me3 epigenetic mark and a large induction of expression at the RNA level. When pinpointing those sites, we observed that demethylation occurred mostly in the intronic region. These results demonstrate a novel genomic distribution of CNN methylation, namely in the transcribed region of a protein-coding, non-repetitive gene, and the changes in those epigenetic marks that are caused by water stress. These findings may represent a general mechanism for the acquisition of new epialleles in somatic cells, which are pivotal for regulating gene expression in plants.

Circadian gene methylation profiles are associated with obesity, metabolic disturbances and carbohydrate intake.

PubMed

Ramos-Lopez, Omar; Samblas, Mirian; Milagro, Fermin I; Riezu-Boj, Jose I; Crujeiras, A B; Martinez, J Alfredo; Project, Mena

2018-03-26

The circadian clock regulates the daily rhythms of several physiological and behavioral processes. Disruptions in clock genes have been associated with obesity and related comorbidities. This study aimed to analyze the association of DNA methylation signatures at circadian rhythm pathway genes with body mass index (BMI), metabolic profiles and dietary intakes. DNA methylation profiling was determined by microarray in white blood cells from 474 adults from the Methyl Epigenome Network Association (MENA) project. Kyoto Encyclopedia of Genes and Genomes database was used to identify the genes integrating the circadian rhythm pathway. Network enrichment analyses were performed with the PathDIP platform. Associations between circadian methylation patterns with anthropometric measurements, the metabolic profile, clinical data and dietary intakes were analyzed. DNA methylation patterns of nine CpG sites at six circadian rhythm pathway genes were strongly correlated with BMI (false discovery rates <0.0001). These CpGs encompassed cg09578018 (RORA), cg20406576 (PRKAG2), cg10059324 (PER3), cg01180628 (BHLHE40), cg23871860 (FBXL3), cg16964728 (RORA), cg14129040 (CREB1), cg07012178 (PRKAG2) and cg24061580 (PRKAG2). Interestingly, network enrichment analyses revealed that the six BMI-associated genes statistically contributed to the regulation of the circadian rhythm pathway (p = 1.9E-10). In addition, methylation signatures at cg09578018 (RORA), cg24061580 (PRKAG2), cg01180628 (BHLHE40) and cg10059324 (PER3) also correlated with insulin resistance (p < 0.0001) and mean arterial blood pressure (p < 0.0001). Furthermore, relevant correlations (p < 0.05) between methylation at cg09578018 (RORA) and cg01180628 (BHLHE40) with total energy and carbohydrate intakes were found. This investigation revealed potential associations of DNA methylation profiles at circadian genes with obesity, metabolic disturbances and carbohydrate intake, with potential impact on weight homeostasis.

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

PubMed

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.