Search tips
Search criteria

Results 1-25 (1605844)

Clipboard (0)

Related Articles

1.  Contribution of Intragenic DNA Methylation in Mouse Gametic DNA Methylomes to Establish Oocyte-Specific Heritable Marks 
PLoS Genetics  2012;8(1):e1002440.
Genome-wide dynamic changes in DNA methylation are indispensable for germline development and genomic imprinting in mammals. Here, we report single-base resolution DNA methylome and transcriptome maps of mouse germ cells, generated using whole-genome shotgun bisulfite sequencing and cDNA sequencing (mRNA-seq). Oocyte genomes showed a significant positive correlation between mRNA transcript levels and methylation of the transcribed region. Sperm genomes had nearly complete coverage of methylation, except in the CpG-rich regions, and showed a significant negative correlation between gene expression and promoter methylation. Thus, these methylome maps revealed that oocytes and sperms are widely different in the extent and distribution of DNA methylation. Furthermore, a comparison of oocyte and sperm methylomes identified more than 1,600 CpG islands differentially methylated in oocytes and sperm (germline differentially methylated regions, gDMRs), in addition to the known imprinting control regions (ICRs). About half of these differentially methylated DNA sequences appear to be at least partially resistant to the global DNA demethylation that occurs during preimplantation development. In the absence of Dnmt3L, neither methylation of most oocyte-methylated gDMRs nor intragenic methylation was observed. There was also genome-wide hypomethylation, and partial methylation at particular retrotransposons, while maintaining global gene expression, in oocytes. Along with the identification of the many Dnmt3L-dependent gDMRs at intragenic regions, the present results suggest that oocyte methylation can be divided into 2 types: Dnmt3L-dependent methylation, which is required for maternal methylation imprinting, and Dnmt3L-independent methylation, which might be essential for endogenous retroviral DNA silencing. The present data provide entirely new perspectives on the evaluation of epigenetic markers in germline cells.
Author Summary
In mammals, germ-cell–specific methylation patterns and genomic imprints are established throughout large-scale de novo DNA methylation in oogenesis and spermatogenesis. These steps are required for normal germline differentiation and embryonic development; however, current DNA methylation analyses only provide us a partial picture of germ cell methylome. To the best of our knowledge, this is the first study to generate comprehensive maps of DNA methylomes and transcriptomes at single base resolution for mouse germ cells. These methylome maps revealed genome-wide opposing DNA methylation patterns and differential correlation between methylation and gene expression levels in oocyte and sperm genomes. In addition, our results indicate the presence of 2 types of methylation patterns in the oocytes: (i) methylation across the transcribed regions, which might be required for the establishment of maternal methylation imprints and normal embryogenesis, and (ii) retroviral methylation, which might be essential for silencing of retrotransposons and normal oogenesis. We believe that an extension of this work would lead to a better understanding of the epigenetic reprogramming in germline cells and of the role for gene regulations.
PMCID: PMC3252278  PMID: 22242016
2.  GADD45A Does Not Promote DNA Demethylation 
PLoS Genetics  2008;4(3):e1000013.
Although DNA methylation patterns in somatic cells are thought to be relatively stable, they undergo dramatic changes during embryonic development, gametogenesis, and during malignant transformation. The enzymology of DNA methyltransferases is well understood, but the mechanism that removes methylated cytosines from DNA (active DNA demethylation) has remained enigmatic. Recently, a role of the growth arrest and DNA damage inducible protein GADD45A in DNA demethylation has been reported [1]. We have investigated the function of GADD45A in DNA demethylation in more detail using gene reactivation and DNA methylation assays. Contrary to the previous report, we were unable to substantiate a functional role of GADD45A in DNA demethylation. The mechanism of active DNA demethylation in mammalian cells remains unknown.
Author Summary
During mammalian development, genome-wide DNA demethylation occurs both in developing germ cells and in fertilized oocytes. This rapid DNA demethylation is an active process that occurs in the absence of DNA replication. The mechanism of active DNA demethylation represents a conundrum for researchers in this field, i.e. the breakage of a carbon-carbon bond to remove a methyl group from the DNA cytosine ring appears energetically unfavorable, and the elimination of approximately 30 million 5-methylcytosine bases from both DNA strands within a short time window raises questions about the maintenance of genome stability during this process. Recently, it has been reported that the protein GADD45A, a small acidic protein that has been implicated in the DNA damage response, plays a crucial role in promoting active DNA demethylation in several mammalian cell lines. We noticed that GADD45A does not fulfill one likely requirement for a mammalian DNA demethylase factor in that it is not expressed in oocytes or zygotes. We then investigated the role of GADD45A in DNA demethylation using methylated reporter plasmids and DNA methylation analysis of several endogenous genes in cell lines overexpressing GADD45A. Contrary to the previous report, we were not able to demonstrate a role of GADD45A in DNA demethylation. The activity that promotes DNA demethylation at a genome-wide level in mammals remains to be identified.
PMCID: PMC2265528  PMID: 18369439
3.  Epigenetic Changes during Hepatic Stellate Cell Activation 
PLoS ONE  2015;10(6):e0128745.
Background and Aims
Hepatic stellate cells (HSC), which can participate in liver regeneration and fibrogenesis, have recently been identified as liver-resident mesenchymal stem cells. During their activation HSC adopt a myofibroblast-like phenotype accompanied by profound changes in the gene expression profile. DNA methylation changes at single genes have been reported during HSC activation and may participate in the regulation of this process, but comprehensive DNA methylation analyses are still missing. The aim of the present study was to elucidate the role of DNA methylation during in vitro activation of HSC.
Methods and Results
The analysis of DNA methylation changes by antibody-based assays revealed a strong decrease in the global DNA methylation level during culture-induced activation of HSC. To identify genes which may be regulated by DNA methylation, we performed a genome-wide Methyl-MiniSeq EpiQuest sequencing comparing quiescent and early culture-activated HSC. Approximately 400 differentially methylated regions with a methylation change of at least 20% were identified, showing either hypo- or hypermethylation during activation. Further analysis of selected genes for DNA methylation and expression were performed revealing a good correlation between DNA methylation changes and gene expression. Furthermore, global DNA demethylation during HSC activation was investigated by 5-bromo-2-deoxyuridine assay and L-mimosine treatment showing that demethylation was independent of DNA synthesis and thereby excluding a passive DNA demethylation mechanism.
In summary, in vitro activation of HSC initiated strong DNA methylation changes, which were associated with gene regulation. These results indicate that epigenetic mechanisms are important for the control of early HSC activation. Furthermore, the data show that global DNA demethylation during activation is based on an active DNA demethylation mechanism.
PMCID: PMC4466775  PMID: 26065684
4.  Passive and active DNA methylation and the interplay with genetic variation in gene regulation 
eLife  2013;2:e00523.
DNA methylation is an essential epigenetic mark whose role in gene regulation and its dependency on genomic sequence and environment are not fully understood. In this study we provide novel insights into the mechanistic relationships between genetic variation, DNA methylation and transcriptome sequencing data in three different cell-types of the GenCord human population cohort. We find that the association between DNA methylation and gene expression variation among individuals are likely due to different mechanisms from those establishing methylation-expression patterns during differentiation. Furthermore, cell-type differential DNA methylation may delineate a platform in which local inter-individual changes may respond to or act in gene regulation. We show that unlike genetic regulatory variation, DNA methylation alone does not significantly drive allele specific expression. Finally, inferred mechanistic relationships using genetic variation as well as correlations with TF abundance reveal both a passive and active role of DNA methylation to regulatory interactions influencing gene expression.
eLife digest
Variations occur throughout our genome. These variations can cause genes to be expressed (switched on) in slightly different ways among individuals. Moreover, the same gene can also be expressed in different ways in different cells within an individual. A third level of variation is supplied by epigenetic markers: these are molecules that bind to the DNA at specific points and can have profound effects on the expression of nearby genes. One such epigenetic marker is the addition of a methyl group to a cytosine base, a process that is known as DNA methylation.
DNA methylation usually happens when a cytosine base is next to a guanine base, forming a CpG site. In mammals, most CpG sites have methyl groups attached, although regions with a lot of CpG sites (called CpG islands) are mostly unmethylated. Initial studies suggested that methylation prevented particular genes from being expressed, but more recent work has indicated that methylation can be associated with both reduced and increased expression of genes. Moreover, it is not clear if this association is active (i.e., changes in methylation drive changes in gene expression) or passive (DNA methylation is the result of gene regulation).
Now, Gutierrez-Arcelus et al. have carried out a large-scale study to clarify the relationships between three different types of gene-related variations among individuals. They extracted fibroblasts, T-cells and lymphoblastoid cells from the umbilical cords of 204 babies, and analysed them for variations in DNA sequence, gene expression and DNA methylation. Their results show that the associations between the three are more complex than was previously thought.
Gutierrez-Arcelus et al. show that the mechanisms that control the association between the variations in DNA methylation and gene expression in individuals are likely to be different to those that are responsible for the establishment of methylation patterns during the process of cell differentiation. They also find that the association between DNA methylation and gene expression can be either active or passive, and can depend on the context in which they occur in our genome. Finally, where the two copies or alleles of a gene are not equally expressed in a given cell, the difference in expression is primarily regulated by DNA sequence variation, with DNA methylation having little or no role on its own. Equally complex interactions and effects are expected in further studies of genetic and epigenetic variation.
PMCID: PMC3673336  PMID: 23755361
methylation; gene regulation; epigenetics; genome variation; Human
5.  Transcription Factor Occupancy Can Mediate Active Turnover of DNA Methylation at Regulatory Regions 
PLoS Genetics  2013;9(12):e1003994.
Distal regulatory elements, including enhancers, play a critical role in regulating gene activity. Transcription factor binding to these elements correlates with Low Methylated Regions (LMRs) in a process that is poorly understood. Here we ask whether and how actual occupancy of DNA-binding factors is linked to DNA methylation at the level of individual molecules. Using CTCF as an example, we observe that frequency of binding correlates with the likelihood of a demethylated state and sites of low occupancy display heterogeneous DNA methylation within the CTCF motif. In line with a dynamic model of binding and DNA methylation turnover, we find that 5-hydroxymethylcytosine (5hmC), formed as an intermediate state of active demethylation, is enriched at LMRs in stem and somatic cells. Moreover, a significant fraction of changes in 5hmC during differentiation occurs at these regions, suggesting that transcription factor activity could be a key driver for active demethylation. Since deletion of CTCF is lethal for embryonic stem cells, we used genetic deletion of REST as another DNA-binding factor implicated in LMR formation to test this hypothesis. The absence of REST leads to a decrease of hydroxymethylation and a concomitant increase of DNA methylation at its binding sites. These data support a model where DNA-binding factors can mediate turnover of DNA methylation as an integral part of maintenance and reprogramming of regulatory regions.
Author Summary
Cell identity is determined by differential gene expression, which in turn is controlled by the combined activity of proximal and distal regulatory elements such as enhancers. DNA within active enhancer elements is marked by a hypomethylated state as a result of transcription factor (TF) binding. Here, using CTCF as an example for a DNA-binding factor, we explore the relationship between binding and DNA methylation at the level of single molecules by enriching for CTCF occupied DNA. To our surprise, methylation at molecules which are bound by CTCF does not differ from the average methylation levels at the binding sites defined by whole-genome bisulfite sequencing. We find that binding strength inversely correlates with DNA methylation within the CTCF motif with heterogenic methylation levels at low occupancy sites, suggesting that CTCF can bind to molecules with different methylation states. Moreover, we observed enrichment of 5-hydroxymethylcytosines at constitutive and cell-type specific TF binding sites indicative of an active demethylation process. To test the requirement of TF binding for the observed hydroxymethylation, and as CTCF deletion is incompatible with the survival of embryonic stem cells, we made use of cells in which REST – a factor which was previously shown to be involved in LMR formation - was genetically deleted. This deletion leads to loss of hydroxymethylation at its binding sites, suggesting that binding is necessary for turnover. Our data support a model in which TF occupancy mediates a continuous turnover of DNA methylation during maintenance and formation of active regulatory regions.
PMCID: PMC3868540  PMID: 24367273
6.  Genome-Wide DNA Methylation Analysis of Systemic Lupus Erythematosus Reveals Persistent Hypomethylation of Interferon Genes and Compositional Changes to CD4+ T-cell Populations 
PLoS Genetics  2013;9(8):e1003678.
Systemic lupus erythematosus (SLE) is an autoimmune disease with known genetic, epigenetic, and environmental risk factors. To assess the role of DNA methylation in SLE, we collected CD4+ T-cells, CD19+ B-cells, and CD14+ monocytes from 49 SLE patients and 58 controls, and performed genome-wide DNA methylation analysis with Illumina Methylation450 microarrays. We identified 166 CpGs in B-cells, 97 CpGs in monocytes, and 1,033 CpGs in T-cells with highly significant changes in DNA methylation levels (p<1×10−8) among SLE patients. Common to all three cell-types were widespread and severe hypomethylation events near genes involved in interferon signaling (type I). These interferon-related changes were apparent in patients collected during active and quiescent stages of the disease, suggesting that epigenetically-mediated hypersensitivity to interferon persists beyond acute stages of the disease and is independent of circulating interferon levels. This interferon hypersensitivity was apparent in memory, naïve and regulatory T-cells, suggesting that this epigenetic state in lupus patients is established in progenitor cell populations. We also identified a widespread, but lower amplitude shift in methylation in CD4+ T-cells (>16,000 CpGs at FDR<1%) near genes involved in cell division and MAPK signaling. These cell type-specific effects are consistent with disease-specific changes in the composition of the CD4+ population and suggest that shifts in the proportion of CD4+ subtypes can be monitored at CpGs with subtype-specific DNA methylation patterns.
Author Summary
We have analyzed DNA methylation, an epigenetic modification that influences gene expression, in lupus patients and control subjects. Our analysis was run in three different immune cell types, T-cells, B-cells, and monocytes, to discern common epigenetic effects in lupus from cell type-specific effects. We have identified a lupus-related reduction in methylation around genes that respond to interferon, a cytokine that induces inflammation in response to pathogens. This hypomethylation suggests that lupus patients are hypersensitive to interferon, as DNA methylation is typically an inhibitor of gene expression. We also find that this hypersensitivity is preserved in lupus patients beyond active stages of the disease, and this may help explain the chronic, recurrent nature of the disease. In addition, we have identified DNA methylation changes in T-cells that suggest an alteration in the proportions of these cells in lupus patients, which may help explain the disease process.
PMCID: PMC3738443  PMID: 23950730
7.  Global Mapping of DNA Methylation in Mouse Promoters Reveals Epigenetic Reprogramming of Pluripotency Genes 
PLoS Genetics  2008;4(6):e1000116.
DNA methylation patterns are reprogrammed in primordial germ cells and in preimplantation embryos by demethylation and subsequent de novo methylation. It has been suggested that epigenetic reprogramming may be necessary for the embryonic genome to return to a pluripotent state. We have carried out a genome-wide promoter analysis of DNA methylation in mouse embryonic stem (ES) cells, embryonic germ (EG) cells, sperm, trophoblast stem (TS) cells, and primary embryonic fibroblasts (pMEFs). Global clustering analysis shows that methylation patterns of ES cells, EG cells, and sperm are surprisingly similar, suggesting that while the sperm is a highly specialized cell type, its promoter epigenome is already largely reprogrammed and resembles a pluripotent state. Comparisons between pluripotent tissues and pMEFs reveal that a number of pluripotency related genes, including Nanog, Lefty1 and Tdgf1, as well as the nucleosome remodeller Smarcd1, are hypomethylated in stem cells and hypermethylated in differentiated cells. Differences in promoter methylation are associated with significant differences in transcription levels in more than 60% of genes analysed. Our comparative approach to promoter methylation thus identifies gene candidates for the regulation of pluripotency and epigenetic reprogramming. While the sperm genome is, overall, similarly methylated to that of ES and EG cells, there are some key exceptions, including Nanog and Lefty1, that are highly methylated in sperm. Nanog promoter methylation is erased by active and passive demethylation after fertilisation before expression commences in the morula. In ES cells the normally active Nanog promoter is silenced when targeted by de novo methylation. Our study suggests that reprogramming of promoter methylation is one of the key determinants of the epigenetic regulation of pluripotency genes. Epigenetic reprogramming in the germline prior to fertilisation and the reprogramming of key pluripotency genes in the early embryo is thus crucial for transmission of pluripotency.
Author Summary
Large scale epigenetic reprogramming occurs in mammalian germ cells and the early embryo. The biological purpose of this reprogramming is largely unknown, although it has been suggested that it may be required for the embryonic genome to return to a pluripotent state. We carried out a genome-wide screen of promoter methylation in the mouse, comparing germ cells with pluripotent cells, multipotent cells, and more differentiated cell types. We find that promoter methylation is an epigenetic signature of developmental potency. Genes linked to pluripotency are generally hypomethylated in stem cells and hypermethylated (and silenced) in more differentiated cell types, and our genome-wide screen provides new candidates for the regulation of pluripotency. Importantly, germ cells resemble pluripotent cell types in that most promoters have been reprogrammed. However, a small group of key pluripotency regulators (including Nanog), are methylated in mature germ cells, presumably in order to suppress pluripotency at critical stages of germ cell differentiation. Indeed, methylation in these genes becomes reprogrammed after fertilisation so that the embryo can regain totipotency. This work, therefore, shows for the first time that epigenetic reprogramming is crucial for maintaining the pluripotency of germ and embryonic stem cells.
PMCID: PMC2432031  PMID: 18584034
8.  A Six Months Exercise Intervention Influences the Genome-wide DNA Methylation Pattern in Human Adipose Tissue 
PLoS Genetics  2013;9(6):e1003572.
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.
Author Summary
Given the important role of epigenetics in gene regulation and disease development, we here present the genome-wide DNA methylation pattern of 476,753 CpG sites in adipose tissue obtained from healthy men. Since environmental factors potentially change metabolism through epigenetic modifications, we examined if a six months exercise intervention alters the DNA methylation pattern as well as gene expression in human adipose tissue. Our results show that global DNA methylation changes and 17,975 individual CpG sites alter the levels of DNA methylation in response to exercise. We also found differential DNA methylation of 39 candidate genes for obesity and type 2 diabetes in human adipose tissue after exercise. Additionally, we provide functional proof that genes, which exhibit both differential DNA methylation and gene expression in human adipose tissue in response to exercise, influence adipocyte metabolism. Together, this study provides the first detailed map of the genome-wide DNA methylation pattern in human adipose tissue and links exercise to altered adipose tissue DNA methylation, potentially affecting adipocyte metabolism.
PMCID: PMC3694844  PMID: 23825961
9.  DNA Methylation Analysis of Chromosome 21 Gene Promoters at Single Base Pair and Single Allele Resolution 
PLoS Genetics  2009;5(3):e1000438.
Differential DNA methylation is an essential epigenetic signal for gene regulation, development, and disease processes. We mapped DNA methylation patterns of 190 gene promoter regions on chromosome 21 using bisulfite conversion and subclone sequencing in five human cell types. A total of 28,626 subclones were sequenced at high accuracy using (long-read) Sanger sequencing resulting in the measurement of the DNA methylation state of 580427 CpG sites. Our results show that average DNA methylation levels are distributed bimodally with enrichment of highly methylated and unmethylated sequences, both for amplicons and individual subclones, which represent single alleles from individual cells. Within CpG-rich sequences, DNA methylation was found to be anti-correlated with CpG dinucleotide density and GC content, and methylated CpGs are more likely to be flanked by AT-rich sequences. We observed over-representation of CpG sites in distances of 9, 18, and 27 bps in highly methylated amplicons. However, DNA sequence alone is not sufficient to predict an amplicon's DNA methylation status, since 43% of all amplicons are differentially methylated between the cell types studied here. DNA methylation in promoter regions is strongly correlated with the absence of gene expression and low levels of activating epigenetic marks like H3K4 methylation and H3K9 and K14 acetylation. Utilizing the single base pair and single allele resolution of our data, we found that i) amplicons from different parts of a CpG island frequently differ in their DNA methylation level, ii) methylation levels of individual cells in one tissue are very similar, and iii) methylation patterns follow a relaxed site-specific distribution. Furthermore, iv) we identified three cases of allele-specific DNA methylation on chromosome 21. Our data shed new light on the nature of methylation patterns in human cells, the sequence dependence of DNA methylation, and its function as epigenetic signal in gene regulation. Further, we illustrate genotype–epigenotype interactions by showing novel examples of allele-specific methylation.
Author Summary
Epigenetics is defined as the inheritance of changes in gene function without changing the DNA sequence. Epigenetic signals comprise methylation of cytosine bases of the DNA and chemical modifications of the histone proteins. DNA methylation plays important roles in development and disease processes. To investigate the biological role of DNA methylation, we analyzed DNA methylation patterns of 190 gene promoter regions on chromosome 21 in five human cell types. Our results show that average DNA methylation levels are distributed bimodally with enrichment of highly methylated and unmethylated sequences, indicating that DNA methylation acts in a switch-like manner. Consistent with the well-established role of DNA methylation in gene silencing, we found DNA methylation in promoter regions strongly correlated with absence of gene expression and low levels of additional activating epigenetic marks. Although methylation levels of individual cells in one tissue are very similar, we observed differences in DNA methylation when comparing different cell types in 43% of all regions analyzed. This finding is in agreement with a role of DNA methylation in cellular development. We identified three cases of genes that are differentially methylated in both alleles that illustrate the tight interplay of genetic and epigenetic processes.
PMCID: PMC2653639  PMID: 19325872
10.  MicroRNA Mediates DNA De-methylation Events Triggered By Retinoic Acid During Neuroblastoma Cell Differentiation 
Cancer research  2010;70(20):7874-7881.
Neuroblastoma is an often fatal pediatric cancer arising from precursor cells of the sympathetic nervous system. 13-Cis retinoic acid is included in the treatment regime for patients with high-risk disease, and a similar derivative, all-trans retinoic acid (ATRA) causes neuroblastoma cell lines to undergo differentiation. The molecular signaling pathways involved with ATRA induced differentiation are complex, and the role that DNA methylation changes might play are unknown. The purpose of this study was to evaluate the genome-wide effects of ATRA on DNA methylation using methylated DNA immunoprecipitation applied to microarrays representing all known promoter and CpG islands. 402 gene promoters became demethylated, while 88 were hypermethylated post-ATRA. mRNA expression microarrays revealed that 82 of the demethylated genes were over-expressed by >2 fold, while 13 of the hyper methylated genes were under-expressed. Gene ontology analysis indicated that de-methylated and re-expressed genes were enriched for signal transduction pathways, including NOS1, which is required for neural cell differentiation. As a potential mechanism for the DNA methylation changes, we demonstrate the down-regulation of methyltransferases, DNMT1 and DNMT3B, along with the up-regulation of endogenous microRNAs targeting them. Ectopic over-expression of miR-152, targeting DNMT1, also negatively impacted cell invasiveness and anchorage independent growth, contributing in part to the differentiated phenotype. We conclude that functionally important, miRNA-mediated DNA de-methylation changes contribute to the process of ATRA induced differentiation resulting in the activation of NOS1, a critical determinant of neural cell differentiation. Our findings illustrate the plasticity and dynamic nature of the epigenome during cancer cell differentiation.
PMCID: PMC2955783  PMID: 20841484
DNA Hypermethylation; MYCN; ATRA; Neuroblastoma; miRNA
11.  Development of a unique epigenetic signature during in vivo Th17 differentiation 
Nucleic Acids Research  2015;43(3):1537-1548.
Activated naive CD4+ T cells are highly plastic cells that can differentiate into various T helper (Th) cell fates characterized by the expression of effector cytokines like IFN-γ (Th1), IL-4 (Th2) or IL-17A (Th17). Although previous studies have demonstrated that epigenetic mechanisms including DNA demethylation can stabilize effector cytokine expression, a comprehensive analysis of the changes in the DNA methylation pattern during differentiation of naive T cells into Th cell subsets is lacking. Hence, we here performed a genome-wide methylome analysis of ex vivo isolated naive CD4+ T cells, Th1 and Th17 cells. We could demonstrate that naive CD4+ T cells share more demethylated regions with Th17 cells when compared to Th1 cells, and that overall Th17 cells display the highest number of demethylated regions, findings which are in line with the previously reported plasticity of Th17 cells. We could identify seven regions located in Il17a, Zfp362, Ccr6, Acsbg1, Dpp4, Rora and Dclk1 showing pronounced demethylation selectively in ex vivo isolated Th17 cells when compared to other ex vivo isolated Th cell subsets and in vitro generated Th17 cells, suggesting that this unique epigenetic signature allows identifying and functionally characterizing in vivo generated Th17 cells.
PMCID: PMC4330377  PMID: 25593324
12.  The Dynamics of DNA Methylation Covariation Patterns in Carcinogenesis 
PLoS Computational Biology  2014;10(7):e1003709.
Recently it has been observed that cancer tissue is characterised by an increased variability in DNA methylation patterns. However, how the correlative patterns in genome-wide DNA methylation change during the carcinogenic progress has not yet been explored. Here we study genome-wide inter-CpG correlations in DNA methylation, in addition to single site variability, during cervical carcinogenesis. We demonstrate how the study of changes in DNA methylation covariation patterns across normal, intra-epithelial neoplasia and invasive cancer allows the identification of CpG sites that indicate the risk of neoplastic transformation in stages prior to neoplasia. Importantly, we show that the covariation in DNA methylation at these risk CpG loci is maximal immediately prior to the onset of cancer, supporting the view that high epigenetic diversity in normal cells increases the risk of cancer. Consistent with this, we observe that invasive cancers exhibit increased covariation in DNA methylation at the risk CpG sites relative to normal tissue, but lower levels relative to pre-cancerous lesions. We further show that the identified risk CpG sites undergo preferential DNA methylation changes in relation to human papilloma virus infection and age. Results are validated in independent data including prospectively collected samples prior to neoplastic transformation. Our data are consistent with a phase transition model of carcinogenesis, in which epigenetic diversity is maximal prior to the onset of cancer. The model and algorithm proposed here may allow, in future, network biomarkers predicting the risk of neoplastic transformation to be identified.
Author Summary
DNA methylation is a covalent modification of DNA which can regulate how active genes are. DNA methylation is altered at many genomic loci in cancer cells, leading to widespread functional disruption. Importantly, DNA methylation alterations across the genome are seen even in early carcinogenesis. Although the pattern of DNA methylation change during carcinogenesis has been studied at individual genomic loci, no study has yet analysed how these patterns change at a systems-level, specifically how do DNA methylation patterns at pairs of genomic sites change during disease progression. Doing so can shed light on how the epigenetic diversity of cell populations changes during the carcinogenic process. This study performs a systems-level analysis of the dynamic changes in DNA methylation correlation pattern during cervical carcinogenesis, demonstrating that epigenetic diversity is maximal just prior to the onset of cancer. Importantly, this supports the view that the risk of cancer development is closely related to an increase in epigenetic diversity in apparently healthy cells. In addition, the study provides a computational algorithm which successfully identifies the altered genomic sites confering the risk of cervical cancer.
PMCID: PMC4091688  PMID: 25010556
13.  Locus-Specific DNA Methylation Reprogramming During Early Porcine Embryogenesis1 
Biology of Reproduction  2013;88(2):48.
During early mammalian embryogenesis, there is a wave of DNA demethylation postfertilization and de novo methylation around implantation. The paternal genome undergoes active DNA demethylation, whereas the maternal genome is passively demethylated after fertilization in most mammals except for sheep and rabbits. However, the emerging genome-wide DNA methylation landscape has revealed a regulatory and locus-specific DNA methylation reprogramming pattern in mammalian preimplantation embryos. Here we optimized a bisulfite sequencing protocol to draw base-resolution DNA methylation profiles of several selected genes in gametes, early embryos, and somatic tissue. We observed locus-specific DNA methylation reprogramming in early porcine embryos. First, some pluripotency genes (POU5F1 and NANOG) followed a typical wave of DNA demethylation and remethylation, whereas CpG-rich regions of SOX2 and CDX2 loci were hypomethylated throughout development. Second, a differentially methylated region of an imprint control region in the IGF2/H19 locus exhibited differential DNA methylation which was maintained in porcine early embryos. Third, a centromeric repeat element retained a moderate DNA methylation level in gametes, early embryos, and somatic tissue. The diverse DNA methylation reprogramming during early embryogenesis is thought to be possibly associated with the multiple functions of DNA methylation in transcriptional regulation, genome stability and genomic imprinting. The latest technology such as oxidative bisulfite sequencing to identify 5-hydroxymethylcytosine will further clarify the DNA methylation reprogramming during porcine embryonic development.
DNA methylation reprogramming in early porcine embryos is locus-specific and associated with multiple functions of DNA methylation in transcriptional regulation, genomic imprinting, and genome stability.
PMCID: PMC3589235  PMID: 23303676
CDX2; DNA methylation; NANOG; porcine preimplantation embryos; POU5F1; reprogramming; SOX2
14.  Rapid turnover of DNA methylation in human cells 
Epigenetics  2012;7(2):141-145.
Recent studies demonstrated that cytosine methylation in the genome can be reversed without DNA replication by enzymatic mechanisms based on base excision-repair pathways. Both enzymatic methylation and demethylation mechanisms are active in the cell nucleus at the same time. One can hypothesize that the actual level of CpG methylation could be the result of a balance between the two antagonistic processes with a rapid turnover. In the present study, we used mass spectrometry to measure the total methyl-cytosine content of the genome in cultured human cells after short incubation with the known methyltransferase inhibitor 5-deoxy-azacytidine. A significant decrease of the DNA methylation was observed. Indeed, the inhibition of the methylation can only result in a rapid reduction of the overall methyl-cytosine level if the process of demethylation is simultaneous. These observations suggest that the enzymatic mechanisms responsible of the opposing reactions of DNA methylation and demethylation act simultaneously and may result in a continuous and rapid turnover of methylated cytosines. This conclusion is supported by the observation that 5-deoxy-azacytidine was incorporated in the genomic DNA of non-dividing cells and could be detected as soon as after two hours of incubation, hence providing a mechanistic explanation to the inhibition of methyltransferases. The observations are compatible with the idea that the enzymatic mechanisms that bring together of the opposing reactions of DNA methylation and demethylation act simultaneously and may result in a continuous and unsuspected rapid turnover of DNA methylation. This conclusion is at odds with the generally accepted view of high stability of cytosine methylation where the role of enzymatic demethylation is considered as limited to some special situations such as transcription. It places DNA methylation in the same category as other epigenetic modifications with covalent modifications dynamically added to and removed from the chromatin with high turnover rate.
PMCID: PMC3335907  PMID: 22395463
DNA methylation; turnover; 5-deoxy-azacytidine
15.  Quantitative analysis of DNA methylation at all human imprinted regions reveals preservation of epigenetic stability in adult somatic tissue 
Genes subject to genomic imprinting are mono-allelically expressed in a parent-of-origin dependent manner. Each imprinted locus has at least one differentially methylated region (DMR) which has allele specific DNA methylation and contributes to imprinted gene expression. Once DMRs are established, they are potentially able to withstand normal genome reprogramming events that occur during cell differentiation and germ-line DMRs are stably maintained throughout development. These DMRs, in addition to being either maternally or paternally methylated, have differences in whether methylation was acquired in the germ-line or post fertilization and are present in a variety of genomic locations with different Cytosine-phosphate guanine (CpG) densities and CTCF binding capacities. We therefore examined the stability of maintenance of DNA methylation imprints and determined the normal baseline DNA methylation levels in several adult tissues for all imprinted genes. In order to do this, we first developed and validated 50 highly specific, quantitative DNA methylation pyrosequencing assays for the known DMRs associated with human imprinted genes.
Remarkable stability of the DNA methylation imprint was observed in all germ-line DMRs and paternally methylated somatic DMRs (which maintained average methylation levels of between 35% - 65% in all somatic tissues, independent of gene expression). Maternally methylated somatic DMRs were found to have more variation with tissue specific methylation patterns. Most DMRs, however, showed some intra-individual variability for DNA methylation levels in peripheral blood, suggesting that more than one DMR needs to be examined in order to get an overall impression of the epigenetic stability in a tissue. The plasticity of DNA methylation at imprinted genes was examined in a panel of normal and cancer cell lines. All cell lines showed changes in DNA methylation, especially at the paternal germ-line and the somatic DMRs.
Our validated pyrosequencing methylation assays can be widely used as a tool to investigate DNA methylation levels of imprinted genes in clinical samples. This first comprehensive analysis of normal methylation levels in adult somatic tissues at human imprinted regions confirm that, despite intra-individual variability and tissue specific expression, imprinted genes faithfully maintain their DNA methylation in healthy adult tissue. DNA methylation levels of a selection of imprinted genes are, therefore, a valuable indicator for epigenetic stability.
PMCID: PMC3038880  PMID: 21281512
16.  A panel of genes methylated with high frequency in colorectal cancer 
BMC Cancer  2014;14:54.
The development of colorectal cancer (CRC) is accompanied by extensive epigenetic changes, including frequent regional hypermethylation particularly of gene promoter regions. Specific genes, including SEPT9, VIM1 and TMEFF2 become methylated in a high fraction of cancers and diagnostic assays for detection of cancer-derived methylated DNA sequences in blood and/or fecal samples are being developed. There is considerable potential for the development of new DNA methylation biomarkers or panels to improve the sensitivity and specificity of current cancer detection tests.
Combined epigenomic methods – activation of gene expression in CRC cell lines following DNA demethylating treatment, and two novel methods of genome-wide methylation assessment – were used to identify candidate genes methylated in a high fraction of CRCs. Multiplexed amplicon sequencing of PCR products from bisulfite-treated DNA of matched CRC and non-neoplastic tissue as well as healthy donor peripheral blood was performed using Roche 454 sequencing. Levels of DNA methylation in colorectal tissues and blood were determined by quantitative methylation specific PCR (qMSP).
Combined analyses identified 42 candidate genes for evaluation as DNA methylation biomarkers. DNA methylation profiles of 24 of these genes were characterised by multiplexed bisulfite-sequencing in ten matched tumor/normal tissue samples; differential methylation in CRC was confirmed for 23 of these genes. qMSP assays were developed for 32 genes, including 15 of the sequenced genes, and used to quantify methylation in tumor, adenoma and non-neoplastic colorectal tissue and from healthy donor peripheral blood. 24 of the 32 genes were methylated in >50% of neoplastic samples, including 11 genes that were methylated in 80% or more CRCs and a similar fraction of adenomas.
This study has characterised a panel of 23 genes that show elevated DNA methylation in >50% of CRC tissue relative to non-neoplastic tissue. Six of these genes (SOX21, SLC6A15, NPY, GRASP, ST8SIA1 and ZSCAN18) show very low methylation in non-neoplastic colorectal tissue and are candidate biomarkers for stool-based assays, while 11 genes (BCAT1, COL4A2, DLX5, FGF5, FOXF1, FOXI2, GRASP, IKZF1, IRF4, SDC2 and SOX21) have very low methylation in peripheral blood DNA and are suitable for further evaluation as blood-based diagnostic markers.
PMCID: PMC3924905  PMID: 24485021
Colorectal cancer; DNA methylation; Biomarker
17.  Loss of chloroplast DNA methylation during dedifferentiation of Chlamydomonas reinhardi gametes. 
Molecular and Cellular Biology  1984;4(10):2103-2108.
In Chlamydomonas reinhardi the chloroplast DNA (ch;DNA) of mating type plus cells undergoes cyclical methylation and demethylation during the life cycle. Methylation occurs during gametogenesis, and fully differentiated gametes can be dedifferentiated back to vegetative cells which contain nonmethylated chlDNA by the addition of a nitrogen source for growth. We examined the dedifferentiation process and found that the mating ability of gametes was lost rapidly after the start of dedifferentiation at a time when the chlDNA was still methylated. The enzymatic activity of the 200-kilodalton DNA methyltransferase was lost at a rate consistent with the rate of dilution during cell division. Methylation of chlDNA decreased at a slower rate than was expected from cell division alone but was consistent with the continuing activity of the preexisting methyltransferase so long as it was present. These results support the hypothesis that demethylation of chlDNA occurs by dilution out of enzymatic methylating activity rather than by enzymatic demethylation.
PMCID: PMC369028  PMID: 6095040
18.  Convergence of Mutation and Epigenetic Alterations Identifies Common Genes in Cancer That Predict for Poor Prognosis  
PLoS Medicine  2008;5(5):e114.
The identification and characterization of tumor suppressor genes has enhanced our understanding of the biology of cancer and enabled the development of new diagnostic and therapeutic modalities. Whereas in past decades, a handful of tumor suppressors have been slowly identified using techniques such as linkage analysis, large-scale sequencing of the cancer genome has enabled the rapid identification of a large number of genes that are mutated in cancer. However, determining which of these many genes play key roles in cancer development has proven challenging. Specifically, recent sequencing of human breast and colon cancers has revealed a large number of somatic gene mutations, but virtually all are heterozygous, occur at low frequency, and are tumor-type specific. We hypothesize that key tumor suppressor genes in cancer may be subject to mutation or hypermethylation.
Methods and Findings
Here, we show that combined genetic and epigenetic analysis of these genes reveals many with a higher putative tumor suppressor status than would otherwise be appreciated. At least 36 of the 189 genes newly recognized to be mutated are targets of promoter CpG island hypermethylation, often in both colon and breast cancer cell lines. Analyses of primary tumors show that 18 of these genes are hypermethylated strictly in primary cancers and often with an incidence that is much higher than for the mutations and which is not restricted to a single tumor-type. In the identical breast cancer cell lines in which the mutations were identified, hypermethylation is usually, but not always, mutually exclusive from genetic changes for a given tumor, and there is a high incidence of concomitant loss of expression. Sixteen out of 18 (89%) of these genes map to loci deleted in human cancers. Lastly, and most importantly, the reduced expression of a subset of these genes strongly correlates with poor clinical outcome.
Using an unbiased genome-wide approach, our analysis has enabled the discovery of a number of clinically significant genes targeted by multiple modes of inactivation in breast and colon cancer. Importantly, we demonstrate that a subset of these genes predict strongly for poor clinical outcome. Our data define a set of genes that are targeted by both genetic and epigenetic events, predict for clinical prognosis, and are likely fundamentally important for cancer initiation or progression.
Stephen Baylin and colleagues show that a combined genetic and epigenetic analysis of breast and colon cancers identifies a number of clinically significant genes targeted by multiple modes of inactivation.
Editors' Summary
Cancer is one of the developed world's biggest killers—over half a million Americans die of cancer each year, for instance. As a result, there is great interest in understanding the genetic and environmental causes of cancer in order to improve cancer prevention, diagnosis, and treatment.
Cancer begins when cells begin to multiply out of control. DNA is the sequence of coded instructions—genes—for how to build and maintain the body. Certain “tumor suppressor” genes, for instance, help to prevent cancer by preventing tumors from developing, but changes that alter the DNA code sequence—mutations—can profoundly affect how a gene works. Modern techniques of genetic analysis have identified genes such as tumor suppressors that, when mutated, are linked to the development of certain cancers.
Why Was This Study Done?
However, in recent years, it has become increasingly apparent that mutations are neither necessary nor sufficient to explain every case of cancer. This has led researchers to look at so-called epigenetic factors, which also alter how a gene works without altering its DNA sequence. An example of this is “methylation,” which prevents a gene from being expressed—deactivates it—by a chemical tag. Methylation of genes is part of the normal functioning of DNA, but abnormal methylation has been linked with cancer, aging, and some rare birth abnormalities.
Previous analysis of DNA from breast and colon cancer cells had revealed 189 “candidate cancer genes”—mutated genes that were linked to the development of breast and colon cancer. However, it was not clear how those mutations gave rise to cancer, and individual mutations were present in only 5% to 15% of specific tumors. The authors of this study wanted to know whether epigenetic factors such as methylation contributed to causing the cancers.
What Did the Researchers Do and Find?
The researchers first identified 56 of the 189 candidate cancer genes as likely tumor suppressors and then determined that 36 of these genes were methylated and deactivated, often in both breast and colon (laboratory-grown) cancer cells. In nearly all cases, the methylated genes were not active but could be reactivated by being demethylated. They further showed that, in normal colon and breast tissue samples, 18 of the 36 genes were unmethylated and functioned normally, but in cells taken from breast and colon cancer tumors they were methylated.
In contrast to the genetic mutations, the 18 genes were frequently methylated across a range of tumor types, and eight genes were methylated in both the breast and colon cancers. The authors found by reviewing the genetics and epigenetics of those 18 genes in breast and colon cancer that they were either mutated, methylated, or both. A literature review showed that at least six of the 18 genes were known to have tumor suppressor properties, and the authors determined that 16 were located in parts of DNA known to be missing from cells taken from a range of cancer tumors.
Finally, the researchers analyzed data on cancer cases to show that methylation of these 18 genes was correlated with reduced function of these genes in tumors and with a greater likelihood that a cancer will be terminal or spread to other parts of the body.
What Do These Findings Mean?
The researchers considered only the 189 candidate cancer genes found in one previous study and not other genes identified elsewhere. They also did not consider the biological effects of the individual mutations found in those genes. Despite this, they have demonstrated that methylation of specific genes is likely to play a role in the development of breast and/or colon cancer cells either together with mutations or independently, most likely by turning off their tumor suppression function.
More broadly, however, the study adds to the evidence that future analysis of the role of genes in cancer should include epigenetic as well as genetic factors. In addition, the authors have also shown that a number of these genes may be useful for predicting clinical outcomes for a range of tumor types.
Additional Information.
Please access these Web sites via the online version of this summary at
A December 2006 PLoS Medicine Perspective article reviews the value of examining methylation as a factor in common cancers and its use for early detection
The Web site of the American Cancer Society has a wealth of information and resources on a variety of cancers, including breast and colon cancer is a nonprofit organization providing information about breast cancer on the Web, including research news
Cancer Research UK provides information on cancer research
The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins publishes background information on the authors' research on methylation, setting out its potential for earlier diagnosis and better treatment of cancer
PMCID: PMC2429944  PMID: 18507500
19.  The Importance Of Epigenetic Alterations In The Development Of Epstein-Barr Virus-Related Lymphomas 
Epstein-Barr virus (EBV), a human gammaherpesvirus, is associated with a series of malignant tumors. These include lymphomas (Burkitt’s lymphoma, Hodgkin’s disease, T/NK-cell lymphoma, post-transplant lymphoproliferative disease, AIDS-associated lymphoma, X-linked lymphoproliferative syndrome), carcinomas (nasopharyngeal carcinoma, gastric carcinoma, carcinomas of major salivary glands, thymic carcinoma, mammary carcinoma) and a sarcoma (leiomyosarcoma). The latent EBV genomes persist in the tumor cells as circular episomes, co-replicating with the cellular DNA once per cell cycle. The expression of latent EBV genes is cell type specific due to the strict epigenetic control of their promoters. DNA methylation, histone modifications and binding of key cellular regulatory proteins contribute to the regulation of alternative promoters for transcripts encoding the nuclear antigens EBNA1 to 6 and affect the activity of promoters for transcripts encoding transmembrane proteins (LMP1, LMP2A, LMP2B). In addition to genes transcribed by RNA polymerase II, there are also two RNA polymerase III transcribed genes in the EBV genome (EBER 1 and 2). The 5′ and internal regulatory sequences of EBER 1 and 2 transcription units are invariably unmethylated. The highly abundant EBER 1 and 2 RNAs are not translated to protein. Based on the cell type specific epigenetic marks associated with latent EBV genomes one can distinguish between viral epigenotypes that differ in transcriptional activity in spite of having an identical (or nearly identical) DNA sequence. Whereas latent EBV genomes are regularly targeted by epigenetic control mechanisms in different cell types, EBV encoded proteins may, in turn, affect the activity of a set of cellular promoters by interacting with the very same epigenetic regulatory machinery. There are EBNA1 binding sites in the human genome. Because high affinity binding of EBNA1 to its recognition sites is known to specify sites of DNA demethylation, we suggest that binding of EBNA1 to its cellular target sites may elicit local demethylation and contribute thereby to the activation of silent cellular promoters. EBNA2 interacts with histone acetyltransferases, and EBNALP (EBNA5) coactivates transcription by displacing histone deacetylase 4 from EBNA2-bound promoter sites. EBNA3C (EBNA6) seems to be associated both with histone acetylases and deacetylases, although in separate complexes. LMP1, a transmembrane protein involved in malignant transformation, can affect both alternative systems of epigenetic memory, DNA methylation and the Polycomb-trithorax group of protein complexes. In epithelial cells LMP1 can up-regulate DNA methyltransferases and, in Hodgkin lymphoma cells, induce the Polycomb group protein Bmi-1. In addition, LMP1 can also modulate cellular gene expression programs by affecting, via the NF-κB pathway, levels of cellular microRNAs miR-146a and miR-155. These interactions may result in epigenetic dysregulation and subsequent cellular dysfunctions that may manifest in or contribute to the development of pathological changes (e.g. initiation and progression of malignant neoplasms, autoimmune phenomena, immunodeficiency). Thus, Epstein-Barr virus, similarly to other viruses and certain bacteria, may induce pathological changes by epigenetic reprogramming of host cells. Elucidation of the epigenetic consequences of EBV-host interactions (within the framework of the emerging new field of patho-epigenetics) may have important implications for therapy and disease prevention, because epigenetic processes are reversible and continuous silencing of EBV genes contributing to patho-epigenetic changes may prevent disease development.
PMCID: PMC3033174  PMID: 21416002
20.  DNA Methylation Changes Separate Allergic Patients from Healthy Controls and May Reflect Altered CD4+ T-Cell Population Structure 
PLoS Genetics  2014;10(1):e1004059.
Altered DNA methylation patterns in CD4+ T-cells indicate the importance of epigenetic mechanisms in inflammatory diseases. However, the identification of these alterations is complicated by the heterogeneity of most inflammatory diseases. Seasonal allergic rhinitis (SAR) is an optimal disease model for the study of DNA methylation because of its well-defined phenotype and etiology. We generated genome-wide DNA methylation (Npatients = 8, Ncontrols = 8) and gene expression (Npatients = 9, Ncontrols = 10) profiles of CD4+ T-cells from SAR patients and healthy controls using Illumina's HumanMethylation450 and HT-12 microarrays, respectively. DNA methylation profiles clearly and robustly distinguished SAR patients from controls, during and outside the pollen season. In agreement with previously published studies, gene expression profiles of the same samples failed to separate patients and controls. Separation by methylation (Npatients = 12, Ncontrols = 12), but not by gene expression (Npatients = 21, Ncontrols = 21) was also observed in an in vitro model system in which purified PBMCs from patients and healthy controls were challenged with allergen. We observed changes in the proportions of memory T-cell populations between patients (Npatients = 35) and controls (Ncontrols = 12), which could explain the observed difference in DNA methylation. Our data highlight the potential of epigenomics in the stratification of immune disease and represents the first successful molecular classification of SAR using CD4+ T cells.
Author Summary
T-cells, a type of white blood cell, are an important part of the immune-system in humans. T-cells allow us to adapt our immune-response to the various infectious agents we encounter during life. However, T-cells can also cause disease when they target the body's own cells, e.g. Psoriasis, or when they react to a harmless particle or ‘antigen’, i.e. allergy. Much evidence supports an environmental, or ‘epigenetic’, component to allergy. Surprisingly, although allergy is viewed as a T-cell disease with an epigenetic component, no studies have identified epigenetic differences between healthy individuals and allergic individuals. Using a state-of-the-art genome-wide approach, we found that we could clearly and robustly separate allergic patients from healthy controls. It is often assumed that these changes reflect changes in DNA methylation in a given type of cell; however such differences can also result from different mixtures of T-cell subtypes in the samples. Indeed, we found that allergic patients had different proportions of T-cell sub-types compared to healthy controls. These changes in T-cell proportions may explain the difference in DNA methylation profile we observed between patients and controls. Our study is the first successful molecular classification of allergy using CD4+ T cells.
PMCID: PMC3879208  PMID: 24391521
21.  Identification of radiation-induced aberrant hypomethylation in colon cancer 
BMC Genomics  2015;16(1):56.
Exposure to ionizing radiation (IR) results in the simultaneous activation or downregulation of multiple signaling pathways that play critical roles in cell type-specific control of survival or death. IR is a well-known genotoxic agent and human carcinogen that induces cellular damage through direct and indirect mechanisms. However, its impact on epigenetic mechanisms has not been elucidated, and more specifically, little information is available regarding genome-wide DNA methylation changes in cancer cells after IR exposure. Recently, genome-wide DNA methylation profiling technology using the Illumina HumanMethylation450K platform has emerged that allows us to query >450,000 loci within the genome. This improved technology is capable of identifying genome-wide DNA methylation changes in CpG islands and other CpG island-associated regions.
In this study, we employed this technology to test the hypothesis that exposure to IR not only induces differential DNA methylation patterns at a genome-wide level, but also results in locus- and gene-specific DNA methylation changes. We screened for differential DNA methylation changes in colorectal cancer cells after IR exposure with 2 and 5 Gy. Twenty-nine genes showed radiation-induced hypomethylation in colon cancer cells, and of those, seven genes showed a corresponding increase in gene expression by reverse transcriptase polymerase chain reaction (RT-PCR). In addition, we performed chromatin immunoprecipitation (ChIP) to confirm that the DNA-methyltransferase 1 (DNMT1) level associated with the promoter regions of these genes correlated with their methylation level and gene expression changes. Finally, we used a gene ontology (GO) database to show that a handful of hypomethylated genes induced by IR are associated with a variety of biological pathways related to cancer.
We identified alterations in global DNA methylation patterns and hypomethylation at specific cancer-related genes following IR exposure, which suggests that radiation exposure plays a critical role in conferring epigenetic alterations in cancer.
Electronic supplementary material
The online version of this article (doi:10.1186/s12864-015-1229-6) contains supplementary material, which is available to authorized users.
PMCID: PMC4342812  PMID: 25887185
Ionizing radiation; 5-aza-2′-deoxycytidine; DNA hypomethylation; Methylation profiling; Gene ontology; Colon cancer
22.  The Honey Bee Epigenomes: Differential Methylation of Brain DNA in Queens and Workers 
PLoS Biology  2010;8(11):e1000506.
Using genome-wide methylation profiles in honey bee queen and worker brains to understand how contrasting organismal outputs are generated from the same genotype.
In honey bees (Apis mellifera) the behaviorally and reproductively distinct queen and worker female castes derive from the same genome as a result of differential intake of royal jelly and are implemented in concert with DNA methylation. To determine if these very different diet-controlled phenotypes correlate with unique brain methylomes, we conducted a study to determine the methyl cytosine (mC) distribution in the brains of queens and workers at single-base-pair resolution using shotgun bisulfite sequencing technology. The whole-genome sequencing was validated by deep 454 sequencing of selected amplicons representing eight methylated genes. We found that nearly all mCs are located in CpG dinucleotides in the exons of 5,854 genes showing greater sequence conservation than non-methylated genes. Over 550 genes show significant methylation differences between queens and workers, revealing the intricate dynamics of methylation patterns. The distinctiveness of the differentially methylated genes is underscored by their intermediate CpG densities relative to drastically CpG-depleted methylated genes and to CpG-richer non-methylated genes. We find a strong correlation between methylation patterns and splicing sites including those that have the potential to generate alternative exons. We validate our genome-wide analyses by a detailed examination of two transcript variants encoded by one of the differentially methylated genes. The link between methylation and splicing is further supported by the differential methylation of genes belonging to the histone gene family. We propose that modulation of alternative splicing is one mechanism by which DNA methylation could be linked to gene regulation in the honey bee. Our study describes a level of molecular diversity previously unknown in honey bees that might be important for generating phenotypic flexibility not only during development but also in the adult post-mitotic brain.
Author Summary
The queen honey bee and her worker sisters do not seem to have much in common. Workers are active and intelligent, skillfully navigating the outside world in search of food for the colony. They never reproduce; that task is left entirely to the much larger and longer-lived queen, who is permanently ensconced within the colony and uses a powerful chemical influence to exert control. Remarkably, these two female castes are generated from identical genomes. The key to each female's developmental destiny is her diet as a larva: future queens are raised on royal jelly. This specialized diet is thought to affect a particular chemical modification, methylation, of the bee's DNA, causing the same genome to be deployed differently. To document differences in this epigenomic setting and hypothesize about its effects on behavior, we performed high-resolution bisulphite sequencing of whole genomes from the brains of queen and worker honey bees. In contrast to the heavily methylated human genome, we found that only a small and specific fraction of the honey bee genome is methylated. Most methylation occurred within conserved genes that provide critical cellular functions. Over 550 genes showed significant methylation differences between the queen and the worker, which may contribute to the profound divergence in behavior. How DNA methylation works on these genes remains unclear, but it may change their accessibility to the cellular machinery that controls their expression. We found a tantalizing clue to a mechanism in the clustering of methylation within parts of genes where splicing occurs, suggesting that methylation could control which of several versions of a gene is expressed. Our study provides the first documentation of extensive molecular differences that may allow honey bees to generate different phenotypes from the same genome.
PMCID: PMC2970541  PMID: 21072239
23.  DNA Methylation Restricts Lineage-specific Functions of Transcription Factor Gata4 during Embryonic Stem Cell Differentiation 
PLoS Genetics  2013;9(6):e1003574.
DNA methylation changes dynamically during development and is essential for embryogenesis in mammals. However, how DNA methylation affects developmental gene expression and cell differentiation remains elusive. During embryogenesis, many key transcription factors are used repeatedly, triggering different outcomes depending on the cell type and developmental stage. Here, we report that DNA methylation modulates transcription-factor output in the context of cell differentiation. Using a drug-inducible Gata4 system and a mouse embryonic stem (ES) cell model of mesoderm differentiation, we examined the cellular response to Gata4 in ES and mesoderm cells. The activation of Gata4 in ES cells is known to drive their differentiation to endoderm. We show that the differentiation of wild-type ES cells into mesoderm blocks their Gata4-induced endoderm differentiation, while mesoderm cells derived from ES cells that are deficient in the DNA methyltransferases Dnmt3a and Dnmt3b can retain their response to Gata4, allowing lineage conversion from mesoderm cells to endoderm. Transcriptome analysis of the cells' response to Gata4 over time revealed groups of endoderm and mesoderm developmental genes whose expression was induced by Gata4 only when DNA methylation was lost, suggesting that DNA methylation restricts the ability of these genes to respond to Gata4, rather than controlling their transcription per se. Gata4-binding-site profiles and DNA methylation analyses suggested that DNA methylation modulates the Gata4 response through diverse mechanisms. Our data indicate that epigenetic regulation by DNA methylation functions as a heritable safeguard to prevent transcription factors from activating inappropriate downstream genes, thereby contributing to the restriction of the differentiation potential of somatic cells.
Author Summary
Animal bodies are constructed from many different specialized cell types that are generated during embryogenesis from a single fertilized egg, and acquire their specific characteristics through a series of differentiation steps. After being committed to a specific cell type, it is generally difficult for differentiated cells to convert to other cell types, at least partly because the cells maintain some memory or mark of their developmental history. Such cellular memory is mediated by “epigenetic” mechanisms, which function to stabilize the cell state. DNA methylation, a chemical modification of genomic cytosine residues, is one such mechanism. Genomic DNA methylation patterns in early embryonic cells are established in a cell-type-dependent manner, and these specific patterns are propagated through cell divisions in a clonal manner. However, our understanding of how DNA methylation controls cell differentiation and developmental gene regulation is limited. In this study, using an in vitro model of differentiation, we obtained evidence that DNA methylation modulates the cell's response to DNA-binding transcription factors in a cell-type-dependent manner. These findings extend our understanding of how cellular traits are stabilized within specific lineages during development, and may contribute to advances in cellular engineering.
PMCID: PMC3694845  PMID: 23825962
24.  DNA methylation in Arabidopsis has a genetic basis and shows evidence of local adaptation 
eLife  null;4:e05255.
Epigenome modulation potentially provides a mechanism for organisms to adapt, within and between generations. However, neither the extent to which this occurs, nor the mechanisms involved are known. Here we investigate DNA methylation variation in Swedish Arabidopsis thaliana accessions grown at two different temperatures. Environmental effects were limited to transposons, where CHH methylation was found to increase with temperature. Genome-wide association studies (GWAS) revealed that the extensive CHH methylation variation was strongly associated with genetic variants in both cis and trans, including a major trans-association close to the DNA methyltransferase CMT2. Unlike CHH methylation, CpG gene body methylation (GBM) was not affected by growth temperature, but was instead correlated with the latitude of origin. Accessions from colder regions had higher levels of GBM for a significant fraction of the genome, and this was associated with increased transcription for the genes affected. GWAS revealed that this effect was largely due to trans-acting loci, many of which showed evidence of local adaptation.
eLife digest
Organisms need to adapt quickly to changes in their environment. Mutations in the DNA sequence of genes can lead to new adaptations, but this can take many generations. Instead, altering how genes are switched on by changing how the DNA is packaged in cells can allow organisms to adapt within and between generations. One way that genes are controlled in organisms is by a process known as DNA methylation, where ‘methyl’ tags are added to DNA and act as markers for other proteins involved in activating genes.
DNA is made of four different molecules called ‘nucleotides’ that are arranged in different orders to produce a vast variety of DNA sequences. One type of DNA methylation can happen at sites where a nucleotide called cytosine is followed by two other non-cytosine nucleotides. Another type of methylation can take place at sites where a cytosine is followed by a guanine nucleotide. However, it is not clear how big a role DNA methylation plays in allowing organisms to adapt to their changing environment.
Here, Dubin, Zhang, Meng, Remigereau et al. studied DNA methylation in a plant called Arabidopsis thaliana. Several different varieties of A. thaliana plants from Sweden were grown at two different temperatures. The experiments showed that the A. thaliana plants grown at higher temperatures were more likely to have methyl tags attached to sections of DNA called transposons, which are able to move around the genome. There was a lot of variety in the levels of this DNA methylation in the different plants, and some of it was shown to be associated with variation in a gene that is involved in DNA methylation.
However, not all of the DNA methylation in these plants was sensitive to the temperature the plants were grown in. Dubin, Zhang, Meng, Remigereau et al. show that the pattern of a type of DNA methylation that is found within genes depends on how far north in Sweden the plants' ancestors came from rather than the temperature the plants were grown in. Plants that originated from colder regions, farther north, had more DNA methylation within many genes and these genes were more active.
These findings suggest that genetic differences in these plants strongly influence the levels of DNA methylation, and they provide the first direct link between DNA methylation and adaption to the environment. Future studies should reveal how DNA methylation is regulated in these plants, and whether it plays a key role in adaptation, or merely reflects other changes in the genome.
PMCID: PMC4413256  PMID: 25939354
epigenetics; population genetics; local adaptation; DNA methylation; Arabidopsis
25.  5-Methylcytosine DNA glycosylase participates in the genome-wide loss of DNA methylation occurring during mouse myoblast differentiation 
Nucleic Acids Research  2001;29(21):4452-4461.
Changes in gene expression during mouse myoblast differentiation were monitored by DNA microarray hybridisation. Four days after the onset of differentiation 2.37% of the genes increased in activity from a value of zero, whereas during the same time 1.68% of total genes had decreased expression. During the first 24 h of differentiation an average of 700 000 CpG sites per haploid genome were demethylated. Maximal loss of DNA methylation is attained after 2 days of differentiation, followed by a gradual remethylation. The highest demethylation is observed in highly repeated DNA sequences, followed by single copy sequences. When DNA replication is inhibited by aphidicolin or l-mimosine this genome-wide demethylation is still observed. During the first 3 h of differentiation there is an increase in the number of hemimethylated CpG sites, which disappear rapidly during the course of genome-wide hypomethylation. Transfection of cells with an antisense morpholino oligonucleotide to 5-methylcytosine DNA glycosylase (G/T mismatch DNA glycosylase) decreases both the activity of the enzyme and genome-wide demethylation. It is concluded that the genome-wide loss of DNA methylation in differentiating mouse myoblasts occurs in part by formation of hemimethylated CpG sites, which can serve as the substrate for 5-methylcytosine-DNA glycosylase.
PMCID: PMC60186  PMID: 11691933

Results 1-25 (1605844)