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1.  Role of DNA Methylation and Epigenetic Silencing of HAND2 in Endometrial Cancer Development 
PLoS Medicine  2013;10(11):e1001551.
TB filled in by Laureen
Please see later in the article for the Editors' Summary
Endometrial cancer incidence is continuing to rise in the wake of the current ageing and obesity epidemics. Much of the risk for endometrial cancer development is influenced by the environment and lifestyle. Accumulating evidence suggests that the epigenome serves as the interface between the genome and the environment and that hypermethylation of stem cell polycomb group target genes is an epigenetic hallmark of cancer. The objective of this study was to determine the functional role of epigenetic factors in endometrial cancer development.
Methods and Findings
Epigenome-wide methylation analysis of >27,000 CpG sites in endometrial cancer tissue samples (n = 64) and control samples (n = 23) revealed that HAND2 (a gene encoding a transcription factor expressed in the endometrial stroma) is one of the most commonly hypermethylated and silenced genes in endometrial cancer. A novel integrative epigenome-transcriptome-interactome analysis further revealed that HAND2 is the hub of the most highly ranked differential methylation hotspot in endometrial cancer. These findings were validated using candidate gene methylation analysis in multiple clinical sample sets of tissue samples from a total of 272 additional women. Increased HAND2 methylation was a feature of premalignant endometrial lesions and was seen to parallel a decrease in RNA and protein levels. Furthermore, women with high endometrial HAND2 methylation in their premalignant lesions were less likely to respond to progesterone treatment. HAND2 methylation analysis of endometrial secretions collected using high vaginal swabs taken from women with postmenopausal bleeding specifically identified those patients with early stage endometrial cancer with both high sensitivity and high specificity (receiver operating characteristics area under the curve = 0.91 for stage 1A and 0.97 for higher than stage 1A). Finally, mice harbouring a Hand2 knock-out specifically in their endometrium were shown to develop precancerous endometrial lesions with increasing age, and these lesions also demonstrated a lack of PTEN expression.
HAND2 methylation is a common and crucial molecular alteration in endometrial cancer that could potentially be employed as a biomarker for early detection of endometrial cancer and as a predictor of treatment response. The true clinical utility of HAND2 DNA methylation, however, requires further validation in prospective studies.
Please see later in the article for the Editors' Summary
Editors' Summary
Cancer, which is responsible for 13% of global deaths, can develop anywhere in the body, but all cancers are characterized by uncontrolled cell growth and reduced cellular differentiation (the process by which unspecialized cells such as “stem” cells become specialized during development, tissue repair, and normal cell turnover). Genetic alterations—changes in the sequence of nucleotides (DNA's building blocks) in specific genes—are required for this cellular transformation and subsequent cancer development (carcinogenesis). However, recent evidence suggests that epigenetic modifications—reversible, heritable changes in gene function that occur in the absence of nucleotide sequence changes—may also be involved in carcinogenesis. For example, the addition of methyl groups to a set of genes called stem cell polycomb group target genes (PCGTs; polycomb genes control the expression of their target genes by modifying their DNA or associated proteins) is one of the earliest molecular changes in human cancer development, and increasing evidence suggests that hypermethylation of PCGTs is an epigenetic hallmark of cancer.
Why Was This Study Done?
The methylation of PCGTs, which is triggered by age and by environmental factors that are associated with cancer development, reduces cellular differentiation and leads to the accumulation of undifferentiated cells that are susceptible to cancer development. It is unclear, however, whether epigenetic modifications have a causal role in carcinogenesis. Here, the researchers investigate the involvement of epigenetic factors in the development of endometrial (womb) cancer. The risk of endometrial cancer (which affects nearly 50,000 women annually in the United States) is largely determined by environmental and lifestyle factors. Specifically, the risk of this cancer is increased in women in whom estrogen (a hormone that drives cell proliferation in the endometrium) is functionally dominant over progesterone (a hormone that inhibits endometrial proliferation and causes cell differentiation); obese women and women who have taken estrogen-only hormone replacement therapies fall into this category. Thus, endometrial cancer is an ideal model in which to study whether epigenetic mechanisms underlie carcinogenesis.
What Did the Researchers Do and Find?
The researchers collected data on genome-wide DNA methylation at cytosine- and guanine-rich sites in endometrial cancers and normal endometrium and integrated this information with the human interactome and transcriptome (all the physical interactions between proteins and all the genes expressed, respectively, in a cell) using an algorithm called Functional Epigenetic Modules (FEM). This analysis identified HAND2 as the hub of the most highly ranked differential methylation hotspot in endometrial cancer. HAND2 is a progesterone-regulated stem cell PCGT. It encodes a transcription factor that is expressed in the endometrial stroma (the connective tissue that lies below the epithelial cells in which most endometrial cancers develop) and that suppresses the production of the growth factors that mediate the growth-inducing effects of estrogen on the endometrial epithelium. The researchers hypothesized, therefore, that epigenetic deregulation of HAND2 could be a key step in endometrial cancer development. In support of this hypothesis, the researchers report that HAND2 methylation was increased in premalignant endometrial lesions (cancer-prone, abnormal-looking tissue) compared to normal endometrium, and was associated with suppression of HAND2 expression. Moreover, a high level of endometrial HAND2 methylation in premalignant lesions predicted a poor response to progesterone treatment (which stops the growth of some endometrial cancers), and analysis of HAND2 methylation in endometrial secretions collected from women with postmenopausal bleeding (a symptom of endometrial cancer) accurately identified individuals with early stage endometrial cancer. Finally, mice in which the Hand2 gene was specifically deleted in the endometrium developed precancerous endometrial lesions with age.
What Do These Findings Mean?
These and other findings identify HAND2 methylation as a common, key molecular alteration in endometrial cancer. These findings need to be confirmed in more women, and studies are needed to determine the immediate molecular and cellular consequences of HAND2 silencing in endometrial stromal cells. Nevertheless, these results suggest that HAND2 methylation could potentially be used as a biomarker for the early detection of endometrial cancer and for predicting treatment response. More generally, these findings support the idea that methylation of HAND2 (and, by extension, the methylation of other PCGTs) is not a passive epigenetic feature of cancer but is functionally involved in cancer development, and provide a framework for identifying other genes that are epigenetically regulated and functionally important in carcinogenesis.
Additional Information
Please access these websites via the online version of this summary at
The US National Cancer Institute provides information on all aspects of cancer and has detailed information about endometrial cancer for patients and professionals (in English and Spanish)
The not-for-profit organization American Cancer Society provides information on cancer and how it develops and specific information on endometrial cancer (in several languages)
The UK National Health Service Choices website includes an introduction to cancer, a page on endometrial cancer, and a personal story about endometrial cancer
The not-for-profit organization Cancer Research UK provides general information about cancer and specific information about endometrial cancer
Wikipedia has a page on cancer epigenetics (note: Wikipedia is a free online encyclopedia that anyone can edit; available in several languages)
The Eve Appeal charity that supported this research provides useful information on gynecological cancers
PMCID: PMC3825654  PMID: 24265601
2.  Human-specific epigenetic variation in the immunological Leukotriene B4 Receptor (LTB4R/BLT1) implicated in common inflammatory diseases 
Genome Medicine  2014;6(3):19.
Common human diseases are caused by the complex interplay of genetic susceptibility as well as environmental factors. Due to the environment’s influence on the epigenome, and therefore genome function, as well as conversely the genome’s facilitative effect on the epigenome, analysis of this level of regulation may increase our knowledge of disease pathogenesis.
In order to identify human-specific epigenetic influences, we have performed a novel genome-wide DNA methylation analysis comparing human, chimpanzee and rhesus macaque.
We have identified that the immunological Leukotriene B4 receptor (LTB4R, BLT1 receptor) is the most epigenetically divergent human gene in peripheral blood in comparison with other primates. This difference is due to the co-ordinated active state of human-specific hypomethylation in the promoter and human-specific increased gene body methylation. This gene is significant in innate immunity and the LTB4/LTB4R pathway is involved in the pathogenesis of the spectrum of human inflammatory diseases. This finding was confirmed by additional neutrophil-only DNA methylome and lymphoblastoid H3K4me3 chromatin comparative data. Additionally we show through functional analysis that this receptor has increased expression and a higher response to the LTB4 ligand in human versus rhesus macaque peripheral blood mononuclear cells. Genome-wide we also find human species-specific differentially methylated regions (human s-DMRs) are more prevalent in CpG island shores than within the islands themselves, and within the latter are associated with the CTCF motif.
This result further emphasises the exclusive nature of the human immunological system, its divergent adaptation even from very closely related primates, and the power of comparative epigenomics to identify and understand human uniqueness.
PMCID: PMC4062055  PMID: 24598577
3.  Molecular Pathological Epidemiology of Epigenetics: Emerging Integrative Science to Analyze Environment, Host, and Disease 
Epigenetics acts as an interface between environmental / exogenous factors, cellular responses and pathological processes. Aberrant epigenetic signatures are a hallmark of complex multifactorial diseases, including non-neoplastic disorders (e.g., cardiovascular diseases, hypertension, diabetes mellitus, autoimmune diseases, and some infectious diseases) and neoplasms (e.g., leukemias, lymphomas, sarcomas, and breast, lung, prostate, liver and colorectal cancers). Epigenetic signatures (DNA methylation, mRNA and microRNA expression, etc.) may serve as biomarkers for risk stratification, early detection, and disease classification, as well as targets for therapy and chemoprevention. DNA methylation assays are widely applied to formalin-fixed paraffin-embedded archival tissue specimens as clinical pathology tests. To better understand the interplay between etiologic factors, cellular molecular characteristics, and disease evolution, the field of “Molecular Pathological Epidemiology (MPE)” has emerged as an interdisciplinary integration of “molecular pathology” and “epidemiology”, with a similar conceptual framework to systems biology and network medicine. In contrast to traditional epidemiologic research including genome-wide association studies (GWAS), MPE is founded on the unique disease principle; that is, each disease process results from unique profiles of exposomes, epigenomes, transcriptomes, proteomes, metabolomes, microbiomes, and interactomes in relation to the macro-environment and tissue microenvironment. The widespread application of epigenomics (e.g., methylome) analyses will enhance our understanding of disease heterogeneity, epigenotypes (CpG island methylator phenotype, LINE-1 hypomethylation, etc.), and host-disease interactions. MPE may represent a logical evolution of GWAS, termed “GWAS-MPE approach”. Though epigenome-wide association study attracts increasing attention, currently, it has a fundamental problem in that each cell within one individual has a unique, time-varying epigenome. This article will illustrate increasing contribution of modern pathology to broader public health sciences, which attests pivotal roles of pathologists in the new integrated MPE science towards our ultimate goal of personalized medicine and prevention.
PMCID: PMC3637979  PMID: 23307060
molecular pathologic epidemiology; genetics; omics; hypermethylation; hypomethylation; personalized therapy; unique tumor principle; CIMP; long interspersed nucleotide element
4.  Natural Single-Nucleosome Epi-Polymorphisms in Yeast 
PLoS Genetics  2010;6(4):e1000913.
Epigenomes commonly refer to the sequence of presence/absence of specific epigenetic marks along eukaryotic chromatin. Complete histone-borne epigenomes have now been described at single-nucleosome resolution from various organisms, tissues, developmental stages, or diseases, yet their intra-species natural variation has never been investigated. We describe here that the epigenomic sequence of histone H3 acetylation at Lysine 14 (H3K14ac) differs greatly between two unrelated strains of the yeast Saccharomyces cerevisiae. Using single-nucleosome chromatin immunoprecipitation and mapping, we interrogated 58,694 nucleosomes and found that 5,442 of them differed in their level of H3K14 acetylation, at a false discovery rate (FDR) of 0.0001. These Single Nucleosome Epi-Polymorphisms (SNEPs) were enriched at regulatory sites and conserved non-coding DNA sequences. Surprisingly, higher acetylation in one strain did not imply higher expression of the relevant gene. However, SNEPs were enriched in genes of high transcriptional variability and one SNEP was associated with the strength of gene activation upon stimulation. Our observations suggest a high level of inter-individual epigenomic variation in natural populations, with essential questions on the origin of this diversity and its relevance to gene x environment interactions.
Author Summary
Nucleosomes are the basic units of chromatin, with part of the long DNA molecule wrapped around a multiprotein core, which makes unpacked chromatin often portrayed as a string of pearls. This string can carry three types of sequences: DNA, methyl groups on cytosines, and, on every pearl, the presence-or-absence of histone post-translational modifications such as acetylation of lysines (nucleosomal epigenome). These latter sequences can change dynamically, and the mechanisms involved are heavily studied as they participate in many physiological processes (pluripotency, disease…). However, nothing is known about the natural diversity of nucleosomal epigenomes in natural populations. As a model, we compared two unrelated yeast strains for their epigenome of one histone modification. We found a high divergence, which was enriched at regulatory sites and often carried on specific nucleosomes. Although this nucleosome modification is usually associated with high transcription, higher acetylation in one strain did not necessarily imply higher expression of the corresponding gene. However, one nucleosomal variation was associated with a stronger gene activation upon stimulation. These results suggest that nucleosomal epigenomes largely differ between individuals, raising essential questions on the origin of these differences and their contribution to personal responses to environmental changes (such as clinical treatments).
PMCID: PMC2858693  PMID: 20421933
5.  Aging and Environmental Exposures Alter Tissue-Specific DNA Methylation Dependent upon CpG Island Context 
PLoS Genetics  2009;5(8):e1000602.
Epigenetic control of gene transcription is critical for normal human development and cellular differentiation. While alterations of epigenetic marks such as DNA methylation have been linked to cancers and many other human diseases, interindividual epigenetic variations in normal tissues due to aging, environmental factors, or innate susceptibility are poorly characterized. The plasticity, tissue-specific nature, and variability of gene expression are related to epigenomic states that vary across individuals. Thus, population-based investigations are needed to further our understanding of the fundamental dynamics of normal individual epigenomes. We analyzed 217 non-pathologic human tissues from 10 anatomic sites at 1,413 autosomal CpG loci associated with 773 genes to investigate tissue-specific differences in DNA methylation and to discern how aging and exposures contribute to normal variation in methylation. Methylation profile classes derived from unsupervised modeling were significantly associated with age (P<0.0001) and were significant predictors of tissue origin (P<0.0001). In solid tissues (n = 119) we found striking, highly significant CpG island–dependent correlations between age and methylation; loci in CpG islands gained methylation with age, loci not in CpG islands lost methylation with age (P<0.001), and this pattern was consistent across tissues and in an analysis of blood-derived DNA. Our data clearly demonstrate age- and exposure-related differences in tissue-specific methylation and significant age-associated methylation patterns which are CpG island context-dependent. This work provides novel insight into the role of aging and the environment in susceptibility to diseases such as cancer and critically informs the field of epigenomics by providing evidence of epigenetic dysregulation by age-related methylation alterations. Collectively we reveal key issues to consider both in the construction of reference and disease-related epigenomes and in the interpretation of potentially pathologically important alterations.
Author Summary
The causes and extent of tissue-specific interindividual variation in human epigenomes are underappreciated and, hence, poorly characterized. We surveyed over 200 carefully annotated human tissue samples from ten anatosites at 1,413 CpGs for methylation alterations to appraise the nature of phenotypically, and hence potentially clinically important epigenomic alterations. Within tissue types, across individuals, we found variation in methylation that was significantly related to aging and environmental exposures such as tobacco smoking. Individual variation in age- and exposure-related methylation may significantly contribute to increased susceptibility to several diseases. As the NIH–funded HapMap project is critically contributing to annotating the human reference genome defining normal genetic variability, our work raises key issues to consider in the construction of reference epigenomes. It is well recognized that understanding genetic variation is essential to understanding disease. Our work, and the known interplay of epigenetics and genetics, makes it equally clear that a more complete characterization of epigenetic variation and its sources must be accomplished to reach the goal of a complete understanding of disease. Additional research is absolutely necessary to define the mechanisms controlling epigenomic variation. We have begun to lay the foundations for essential normal tissue controls for comparison to diseased tissue, which will allow the identification of the most crucial disease-related alterations and provide more robust targets for novel treatments.
PMCID: PMC2718614  PMID: 19680444
6.  Genetic Modifiers of Chromatin Acetylation Antagonize the Reprogramming of Epi-Polymorphisms 
PLoS Genetics  2012;8(9):e1002958.
Natural populations are known to differ not only in DNA but also in their chromatin-associated epigenetic marks. When such inter-individual epigenomic differences (or “epi-polymorphisms”) are observed, their stability is usually not known: they may or may not be reprogrammed over time or upon environmental changes. In addition, their origin may be purely epigenetic, or they may result from regulatory variation encoded in the DNA. Studying epi-polymorphisms requires, therefore, an assessment of their nature and stability. Here we estimate the stability of yeast epi-polymorphisms of chromatin acetylation, and we provide a genome-by-epigenome map of their genetic control. A transient epi-drug treatment was able to reprogram acetylation variation at more than one thousand nucleosomes, whereas a similar amount of variation persisted, distinguishing “labile” from “persistent” epi-polymorphisms. Hundreds of genetic loci underlied acetylation variation at 2,418 nucleosomes either locally (in cis) or distantly (in trans), and this genetic control overlapped only partially with the genetic control of gene expression. Trans-acting regulators were not necessarily associated with genes coding for chromatin modifying enzymes. Strikingly, “labile” and “persistent” epi-polymorphisms were associated with poor and strong genetic control, respectively, showing that genetic modifiers contribute to persistence. These results estimate the amount of natural epigenomic variation that can be lost after transient environmental exposures, and they reveal the complex genetic architecture of the DNA–encoded determinism of chromatin epi-polymorphisms. Our observations provide a basis for the development of population epigenetics.
Author Summary
Chemical modifications of chromatin, such as DNA methylation, incorporation of histone variants, or post-translational modifications of histone proteins, constitute the “epigenome” and confer specific properties to genome functions. Epigenomes differ from one individual to another, opening the exciting perspective to decipher the origins of these differences and their impact on physiology. However, population epigenomics remains challenging because, unlike DNA mutations, epigenetic hallmarks are themselves regulated. They can change upon particular environmental conditions, they may be inherited epigenetically, or they may result from activities encoded in the DNA. Thus, estimating the stability of intra-species epigenomic variation and its dependence on DNA polymorphisms is essential. Using a chemical perturbation of yeast cells as an experimental model system, we found that acetylation variation was persistent at some nucleosomes and labile at other nucleosomes. By studying a segregating population, we mapped DNA polymorphisms that affected chromatin acetylation levels at numerous nucleosomes. Strikingly, nucleosomes showing persistent variation of acetylation corresponded to those for which acetylation was under genetic control. Thus, part of epigenomic variation is stabilized by a DNA–encoded determinism, and another part can be reprogrammed if environmental perturbations are experienced. These results provide a necessary basis for upcoming developments in population epigenomics.
PMCID: PMC3447955  PMID: 23028365
7.  The DNA Methylome of Human Peripheral Blood Mononuclear Cells 
PLoS Biology  2010;8(11):e1000533.
Analysis across the genome of patterns of DNA methylation reveals a rich landscape of allele-specific epigenetic modification and consequent effects on allele-specific gene expression.
DNA methylation plays an important role in biological processes in human health and disease. Recent technological advances allow unbiased whole-genome DNA methylation (methylome) analysis to be carried out on human cells. Using whole-genome bisulfite sequencing at 24.7-fold coverage (12.3-fold per strand), we report a comprehensive (92.62%) methylome and analysis of the unique sequences in human peripheral blood mononuclear cells (PBMC) from the same Asian individual whose genome was deciphered in the YH project. PBMC constitute an important source for clinical blood tests world-wide. We found that 68.4% of CpG sites and <0.2% of non-CpG sites were methylated, demonstrating that non-CpG cytosine methylation is minor in human PBMC. Analysis of the PBMC methylome revealed a rich epigenomic landscape for 20 distinct genomic features, including regulatory, protein-coding, non-coding, RNA-coding, and repeat sequences. Integration of our methylome data with the YH genome sequence enabled a first comprehensive assessment of allele-specific methylation (ASM) between the two haploid methylomes of any individual and allowed the identification of 599 haploid differentially methylated regions (hDMRs) covering 287 genes. Of these, 76 genes had hDMRs within 2 kb of their transcriptional start sites of which >80% displayed allele-specific expression (ASE). These data demonstrate that ASM is a recurrent phenomenon and is highly correlated with ASE in human PBMCs. Together with recently reported similar studies, our study provides a comprehensive resource for future epigenomic research and confirms new sequencing technology as a paradigm for large-scale epigenomics studies.
Author Summary
Epigenetic modifications such as addition of methyl groups to cytosine in DNA play a role in regulating gene expression. To better understand these processes, knowledge of the methylation status of all cytosine bases in the genome (the methylome) is required. DNA methylation can differ between the two gene copies (alleles) in each cell. Such allele-specific methylation (ASM) can be due to parental origin of the alleles (imprinting), X chromosome inactivation in females, and other as yet unknown mechanisms. This may significantly alter the expression profile arising from different allele combinations in different individuals. Using advanced sequencing technology, we have determined the methylome of human peripheral blood mononuclear cells (PBMC). Importantly, the PBMC were obtained from the same male Han Chinese individual whose complete genome had previously been determined. This allowed us, for the first time, to study genome-wide differences in ASM. Our analysis shows that ASM in PBMC is higher than can be accounted for by regions known to undergo parent-of-origin imprinting and frequently (>80%) correlates with allele-specific expression (ASE) of the corresponding gene. In addition, our data reveal a rich landscape of epigenomic variation for 20 genomic features, including regulatory, coding, and non-coding sequences, and provide a valuable resource for future studies. Our work further establishes whole-genome sequencing as an efficient method for methylome analysis.
PMCID: PMC2976721  PMID: 21085693
8.  Using ChIP-Seq Technology to Generate High-Resolution Profiles of Histone Modifications 
The dynamic modification of DNA and histones plays a key role in transcriptional regulation through altering the packaging of DNA and modifying the nucleosome surface. These chromatin states, also referred to as the epigenome, are distinctive for different tissues, developmental stages, and disease states and can also be altered by environmental influences. New technologies allow the genome-wide visualization of the information encoded in the epigenome. For example, the chromatin immunoprecipitation (ChIP) assay allows investigators to characterize DNA–protein interactions in vivo. ChIP followed by hybridization to microarrays (ChIP-chip) or by high-throughput sequencing (ChIP-seq) are both powerful tools to identify genome-wide profiles of transcription factors, histone modifications, DNA methylation, and nucleosome positioning. ChIP-seq technology, which can now interrogate the entire human genome at high resolution with only one lane of sequencing, has recently surpassed ChIP-chip technology for epigenomic analyses. Importantly, for the study of primary cells and tissues, epigenetic profiles can be generated using as little as 1 μg of chromatin. In this chapter, we describe in detail the steps involved in performing ChIP assays (with a focus on characterizing histone modifications in primary cells) either manually or using the IP-Star ChIP robot, followed by a detailed protocol to prepare successful libraries for Illumina sequencing. Critical quality control checkpoints are discussed. Although not a focus of this chapter, we also point the reader to several methods by which massive ChIP-seq data sets can be analyzed to extract the tremendous information contained within.
PMCID: PMC4151291  PMID: 21913086
Chromatin immunoprecipitation; ChIP-seq; Next generation sequencing; Epigenomics; Histone modifications; IP-Star; ChIP robot
9.  Mosaic Epigenetic Dysregulation of Ectodermal Cells in Autism Spectrum Disorder 
PLoS Genetics  2014;10(5):e1004402.
DNA mutational events are increasingly being identified in autism spectrum disorder (ASD), but the potential additional role of dysregulation of the epigenome in the pathogenesis of the condition remains unclear. The epigenome is of interest as a possible mediator of environmental effects during development, encoding a cellular memory reflected by altered function of progeny cells. Advanced maternal age (AMA) is associated with an increased risk of having a child with ASD for reasons that are not understood. To explore whether AMA involves covert aneuploidy or epigenetic dysregulation leading to ASD in the offspring, we tested a homogeneous ectodermal cell type from 47 individuals with ASD compared with 48 typically developing (TD) controls born to mothers of ≥35 years, using a quantitative genome-wide DNA methylation assay. We show that DNA methylation patterns are dysregulated in ectodermal cells in these individuals, having accounted for confounding effects due to subject age, sex and ancestral haplotype. We did not find mosaic aneuploidy or copy number variability to occur at differentially-methylated regions in these subjects. Of note, the loci with distinctive DNA methylation were found at genes expressed in the brain and encoding protein products significantly enriched for interactions with those produced by known ASD-causing genes, representing a perturbation by epigenomic dysregulation of the same networks compromised by DNA mutational mechanisms. The results indicate the presence of a mosaic subpopulation of epigenetically-dysregulated, ectodermally-derived cells in subjects with ASD. The epigenetic dysregulation observed in these ASD subjects born to older mothers may be associated with aging parental gametes, environmental influences during embryogenesis or could be the consequence of mutations of the chromatin regulatory genes increasingly implicated in ASD. The results indicate that epigenetic dysregulatory mechanisms may complement and interact with DNA mutations in the pathogenesis of the disorder.
Author Summary
Older mothers have a higher than expected risk of having a child with an autism spectrum disorder (ASD). The reason for this increased risk is unknown. The eggs of older mothers are more prone to abnormalities of chromosome numbers, suggesting this as one possible mechanism of the increased ASD risk. Age is also associated with a loss of control of epigenetic regulatory patterns that govern gene expression, indicating a second potential mechanism. To test both possibilities, we sampled cells from the same developmental origin as the brain, and performed genome-wide tests looking for unusual chromosome numbers and DNA methylation patterns. The studies were performed on individuals with ASD and typically developing controls, all born to mothers at least 35 years of age at the time of birth. We found the cells from individuals with ASD to have changes in DNA methylation at a number of loci, especially near genes encoding proteins known to interact with those already implicated in ASD. We conclude that epigenetic dysregulation occurring in gametes or early embryonic life may be one of the contributors to the development of ASD.
PMCID: PMC4038484  PMID: 24875834
10.  A Sustained Dietary Change Increases Epigenetic Variation in Isogenic Mice 
PLoS Genetics  2011;7(4):e1001380.
Epigenetic changes can be induced by adverse environmental exposures, such as nutritional imbalance, but little is known about the nature or extent of these changes. Here we have explored the epigenomic effects of a sustained nutritional change, excess dietary methyl donors, by assessing genomic CpG methylation patterns in isogenic mice exposed for one or six generations. We find stochastic variation in methylation levels at many loci; exposure to methyl donors increases the magnitude of this variation and the number of variable loci. Several gene ontology categories are significantly overrepresented in genes proximal to these methylation-variable loci, suggesting that certain pathways are susceptible to environmental influence on their epigenetic states. Long-term exposure to the diet (six generations) results in a larger number of loci exhibiting epigenetic variability, suggesting that some of the induced changes are heritable. This finding presents the possibility that epigenetic variation within populations can be induced by environmental change, providing a vehicle for disease predisposition and possibly a substrate for natural selection.
Author Summary
Epigenetic changes to gene expression that do not involve changes to DNA sequence can be influenced by the environment and provide one candidate mechanism by which early nutrition can influence adult disease risk. Here, we examined epigenetic changes across the genome in response to short- and long-term exposure to a dietary supplement in genetically identical mice. We find that the supplement induces small but widespread epigenetic changes in exposed mice. These changes increase the epigenetic variability among exposed mice, and this effect is magnified in mice exposed long-term. The epigenetic changes are overrepresented in gene functions involved in cell and organ development and in gene expression. Our data is consistent with the external environment having pervasive effects on the epigenome and suggests that some genetic pathways may be more susceptible to environmental influence than others.
PMCID: PMC3080854  PMID: 21541011
Mitochondrion  2009;10(1):12-31.
The epigenome has been hypothesized to provide the interface between the environment and the nuclear DNA (nDNA) genes. Key factors in the environment are the availability of calories and demands on the organism’s energetic capacity. Energy is funneled through glycolysis and mitochondrial oxidative phosphorylation (OXPHOS), the cellular bioenergetic systems. Since there are thousands of bioenergetic genes dispersed across the chromosomes and mitochondrial DNA (mtDNA), both cis and trans regulation of the nDNA genes is required. The bioenergetic systems convert environmental calories into ATP, acetyl-Coenzyme A (acetyl-CoA), S-adenosyl-methionine (SAM), and reduced NAD+. When calories are abundant, ATP and acetyl-CoA phosphorylate and acetylate chromatin, opening the nDNA for transcription and replication. When calories are limiting, chromatin phosphorylation and acetylation are lost and gene expression is suppressed. DNA methylaton via SAM can also be modulated by mitochondrial function. Phosphorylation and acetylation are also pivotal to regulating cellular signal transduction pathways. Therefore, bioenergetics provides the interface between the environment and the epigenome. Consistent with this conclusion, the clinical phenotypes of bioenergetic diseases are strikingly similar to those observed in epigenetic diseases (Angelman, Rett, Fragile X Syndromes, the laminopathies, cancer, etc.), and an increasing number of epigenetic diseases are being associated with mitochondrial dysfunction. This bioenergetic-epigenomic hypothesis has broad implications for the etiology, pathophysiology, and treatment of a wide range of common diseases.
PMCID: PMC3245717  PMID: 19796712
12.  Environmental chemical exposures and human epigenetics 
Every year more than 13 million deaths worldwide are due to environmental pollutants, and approximately 24% of diseases are caused by environmental exposures that might be averted through preventive measures. Rapidly growing evidence has linked environmental pollutants with epigenetic variations, including changes in DNA methylation, histone modifications and microRNAs.
Environ mental chemicals and epigenetic changes All of these mechanisms are likely to play important roles in disease aetiology, and their modifications due to environmental pollutants might provide further understanding of disease aetiology, as well as biomarkers reflecting exposures to environmental pollutants and/or predicting the risk of future disease. We summarize the findings on epigenetic alterations related to environmental chemical exposures, and propose mechanisms of action by means of which the exposures may cause such epigenetic changes. We discuss opportunities, challenges and future directions for future epidemiology research in environmental epigenomics. Future investigations are needed to solve methodological and practical challenges, including uncertainties about stability over time of epigenomic changes induced by the environment, tissue specificity of epigenetic alterations, validation of laboratory methods, and adaptation of bioinformatic and biostatistical methods to high-throughput epigenomics. In addition, there are numerous reports of epigenetic modifications arising following exposure to environmental toxicants, but most have not been directly linked to disease endpoints. To complete our discussion, we also briefly summarize the diseases that have been linked to environmental chemicals-related epigenetic changes.
PMCID: PMC3304523  PMID: 22253299
Environmental chemicals; epigenetics; disease susceptibility
13.  Environmental Epigenetics 
Heredity  2010;105(1):105-112.
Purpose of the review
Epigenetics investigates heritable changes in gene expression occurring without changes in DNA sequence. Several epigenetic mechanisms, including DNA methylation and histone modifications, can change genome function under exogenous influence. We review current evidence indicating that epigenetic alterations mediate effects from exposure to environmental toxicants.
Recent findings
Results from animal models indicate that in-utero or early-life environmental exposures produce effects that can be inherited transgenerationally and are accompanied by epigenetic alterations. The search for human equivalents of the epigenetic mechanisms identified in animal models is under way. Recent investigations have identified a number of environmental toxicants that cause altered methylation of human repetitive elements or genes. Some exposures can alter epigenetic states and the same and/or similar epigenetic alterations can be found in patients with the disease of concern. Based on current evidence, we propose possible models for the interplay between environmental exposures and the human epigenome.
Several investigations have examined the relation between exposure to environmental chemicals and epigenetics, and identified toxicants that modify epigenetic states. Whether environmental exposures have transgenerational epigenetic effects in humans remains to be elucidated. In spite of the current limitations, available evidence supports the concept that epigenetics holds substantial potential for furthering our understanding of the molecular mechanisms of environmental toxicants, as well as for predicting health-related risks due to conditions of environmental exposure and individual susceptibility.
PMCID: PMC3133724  PMID: 20179736
Epigenetics; DNA methylation; Histone modifications; Environmental exposures; Transgenerational effects
14.  Dynamic Epigenetic Regulation of Gene Expression during the Life Cycle of Malaria Parasite Plasmodium falciparum 
PLoS Pathogens  2013;9(2):e1003170.
Epigenetic mechanisms are emerging as one of the major factors of the dynamics of gene expression in the human malaria parasite, Plasmodium falciparum. To elucidate the role of chromatin remodeling in transcriptional regulation associated with the progression of the P. falciparum intraerythrocytic development cycle (IDC), we mapped the temporal pattern of chromosomal association with histone H3 and H4 modifications using ChIP-on-chip. Here, we have generated a broad integrative epigenomic map of twelve histone modifications during the P. falciparum IDC including H4K5ac, H4K8ac, H4K12ac, H4K16ac, H3K9ac, H3K14ac, H3K56ac, H4K20me1, H4K20me3, H3K4me3, H3K79me3 and H4R3me2. While some modifications were found to be associated with the vast majority of the genome and their occupancy was constant, others showed more specific and highly dynamic distribution. Importantly, eight modifications displaying tight correlations with transcript levels showed differential affinity to distinct genomic regions with H4K8ac occupying predominantly promoter regions while others occurred at the 5′ ends of coding sequences. The promoter occupancy of H4K8ac remained unchanged when ectopically inserted at a different locus, indicating the presence of specific DNA elements that recruit histone modifying enzymes regardless of their broad chromatin environment. In addition, we showed the presence of multivalent domains on the genome carrying more than one histone mark, highlighting the importance of combinatorial effects on transcription. Overall, our work portrays a substantial association between chromosomal locations of various epigenetic markers, transcriptional activity and global stage-specific transitions in the epigenome.
Author Summary
Malaria is a devastating parasitic disease caused by the protozoan protist Plasmodium falciparum. The complex life cycle of P. falciparum comprises various morphological and functionally distinct forms and is completed in two different hosts. Various regulatory mechanisms are employed by these parasites to complete their life cycle and survive in human hosts. Epigenetic mechanisms, though not fully explored, have been implicated as one of the key players in gene regulation, morphological differentiation and antigenic variation. Here, we present a comprehensive epigenetic map of 12 histone post-translational modifications during the intraerythrocytic life cycle of P. falciparum. We have been able to identify at least eight histone modifications whose dynamic patterns correlate with the transcriptional regulation across the life cycle. In particular, we have shown that a set of euchromatic histone marks work in synergy, creating a dynamic unique histone code that is linked with gene expression during the progression of the Plasmodium intraerythrocytic developmental cycle. These findings enhance our knowledge of complex gene regulation and will help to identify novel targets for fighting malaria.
PMCID: PMC3585154  PMID: 23468622
15.  Genome-Wide Quantitative Analysis of Histone H3 Lysine 4 Trimethylation in Wild House Mouse Liver: Environmental Change Causes Epigenetic Plasticity 
PLoS ONE  2014;9(5):e97568.
In mammals, exposure to toxic or disease-causing environments can change epigenetic marks that are inherited independently of the intrauterine environment. Such inheritance of molecular phenotypes may be adaptive. However, studies demonstrating molecular evidence for epigenetic inheritance have so far relied on extreme treatments, and are confined to inbred animals. We therefore investigated whether epigenomic changes could be detected after a non-drastic change in the environment of an outbred organism. We kept two populations of wild-caught house mice (Mus musculus domesticus) for several generations in semi-natural enclosures on either standard diet and light cycle, or on an energy-enriched diet with longer daylight to simulate summer. As epigenetic marker for active chromatin we quantified genome-wide histone-3 lysine-4 trimethylation (H3K4me3) from liver samples by chromatin immunoprecipitation and high-throughput sequencing as well as by quantitative polymerase chain reaction. The treatment caused a significant increase of H3K4me3 at metabolic genes such as lipid and cholesterol regulators, monooxygenases, and a bile acid transporter. In addition, genes involved in immune processes, cell cycle, and transcription and translation processes were also differently marked. When we transferred young mice of both populations to cages and bred them under standard conditions, most of the H3K4me3 differences were lost. The few loci with stable H3K4me3 changes did not cluster in metabolic functional categories. This is, to our knowledge, the first quantitative study of an epigenetic marker in an outbred mammalian organism. We demonstrate genome-wide epigenetic plasticity in response to a realistic environmental stimulus. In contrast to disease models, the bulk of the epigenomic changes we observed were not heritable.
PMCID: PMC4029994  PMID: 24849289
16.  NCBI Epigenomics: a new public resource for exploring epigenomic data sets 
Nucleic Acids Research  2010;39(Database issue):D908-D912.
The Epigenomics database at the National Center for Biotechnology Information (NCBI) is a new resource that has been created to serve as a comprehensive public resource for whole-genome epigenetic data sets ( Epigenetics is the study of stable and heritable changes in gene expression that occur independently of the primary DNA sequence. Epigenetic mechanisms include post-translational modifications of histones, DNA methylation, chromatin conformation and non-coding RNAs. It has been observed that misregulation of epigenetic processes has been associated with human disease. We have constructed the new resource by selecting the subset of epigenetics-specific data from general-purpose archives, such as the Gene Expression Omnibus, and Sequence Read Archives, and then subjecting them to further review, annotation and reorganization. Raw data is processed and mapped to genomic coordinates to generate ‘tracks’ that are a visual representation of the data. These data tracks can be viewed using popular genome browsers or downloaded for local analysis. The Epigenomics resource also provides the user with a unique interface that allows for intuitive browsing and searching of data sets based on biological attributes. Currently, there are 69 studies, 337 samples and over 1100 data tracks from five well-studied species that are viewable and downloadable in Epigenomics.
PMCID: PMC3013719  PMID: 21075792
17.  DNA–Methylome Analysis of Mouse Intestinal Adenoma Identifies a Tumour-Specific Signature That Is Partly Conserved in Human Colon Cancer 
PLoS Genetics  2013;9(2):e1003250.
Aberrant CpG methylation is a universal epigenetic trait of cancer cell genomes. However, human cancer samples or cell lines preclude the investigation of epigenetic changes occurring early during tumour development. Here, we have used MeDIP-seq to analyse the DNA methylome of APCMin adenoma as a model for intestinal cancer initiation, and we present a list of more than 13,000 recurring differentially methylated regions (DMRs) characterizing intestinal adenoma of the mouse. We show that Polycomb Repressive Complex (PRC) targets are strongly enriched among hypermethylated DMRs, and several PRC2 components and DNA methyltransferases were up-regulated in adenoma. We further demonstrate by bisulfite pyrosequencing of purified cell populations that the DMR signature arises de novo in adenoma cells rather than by expansion of a pre-existing pattern in intestinal stem cells or undifferentiated crypt cells. We found that epigenetic silencing of tumour suppressors, which occurs frequently in colon cancer, was rare in adenoma. Quite strikingly, we identified a core set of DMRs, which is conserved between mouse adenoma and human colon cancer, thus possibly revealing a global panel of epigenetically modified genes for intestinal tumours. Our data allow a distinction between early conserved epigenetic alterations occurring in intestinal adenoma and late stochastic events promoting colon cancer progression, and may facilitate the selection of more specific clinical epigenetic biomarkers.
Author Summary
The formation and progression of tumours to metastatic disease is driven by two major mechanisms, i.e. genetic alterations that activate oncogenes or inactivate tumour suppressor genes, and changes in the epigenome that cause variations in the expression of the genetic information. A deeper understanding of the interaction between the genetic and epigenetic mechanisms is critical for the selection of tumour biomarkers and for the future development of therapies. Human tumour specimens and cell lines contain a plethora of genetic and epigenetic changes, which complicate data analysis. In contrast, mouse tumour models such as the APCMin mouse used in this study arise by a single initiating genetic mutation, yet share key traits with human cancer. Here we show that mouse adenomas acquire a multitude of epigenetic alterations, which are recurring in mouse adenoma and in human colon cancer, representing early and advanced tumours, respectively. The use of a mouse model thus allowed us to uncover a sequence of epigenetic changes occurring in tumours, which may facilitate the identification of novel clinical colon cancer biomarkers.
PMCID: PMC3567140  PMID: 23408899
18.  PcG Complexes Set the Stage for Epigenetic Inheritance of Gene Silencing in Early S Phase before Replication 
PLoS Genetics  2011;7(11):e1002370.
Polycomb group (PcG) proteins are part of a conserved cell memory system that conveys epigenetic inheritance of silenced transcriptional states through cell division. Despite the considerable amount of information about PcG mechanisms controlling gene silencing, how PcG proteins maintain repressive chromatin during epigenome duplication is still unclear. Here we identified a specific time window, the early S phase, in which PcG proteins are recruited at BX-C PRE target sites in concomitance with H3K27me3 repressive mark deposition. Notably, these events precede and are uncoupled from PRE replication timing, which occurs in late S phase when most epigenetic signatures are reduced. These findings shed light on one of the key mechanisms for PcG–mediated epigenetic inheritance during S phase, suggesting a conserved model in which the PcG–dependent H3K27me3 mark is inherited by dilution and not by de novo methylation occurring at the time of replication.
Author Summary
During embryonic development, pluripotent cells divide and use their potential to differentiate into a variety of cells with identical genomes but different phenotypes. The emerging concept suggests that the DNA sequence information is not the sole determinant of cell identity. Indeed, epigenetic mechanisms, acting via chromatin organization, control transcriptome complexity and contribute to maintain cell fate. Polycomb-group proteins (PcG) are epigenetic transcriptional regulators that maintaining gene silencing programs through cell division. During S phase, in addition to DNA, the entire epigenome needs to be duplicated. A key question that remains to be addressed is how epigenetic marks are transmitted to subsequent generations. In this study we propose a model for PcG epigenetic inheritance during replication. We found that, during S phase, PcG engagement and characteristic H3K27me3 histone mark deposition on target sites are restricted to a brief interval occurring before DNA replication of the same regions. By increasing the dose of PcG binding the system would prevent potential weakening of silencing control, which is challenged at the time of replication, allowing proper transmission of epigenetic marks to the next generation and preservation of cell identity.
PMCID: PMC3207895  PMID: 22072989
19.  Structural and Regulatory Characterization of the Placental Epigenome at Its Maternal Interface 
PLoS ONE  2011;6(2):e14723.
Epigenetics can be loosely defined as the study of cellular “traits” that influence biological phenotype in a fashion that is not dependent on the underlying primary DNA sequence. One setting in which epigenetics is likely to have a profound influence on biological phenotype is during intrauterine development. In this context there is a defined and critical window during which balanced homeostasis is essential for normal fetal growth and development. We have carried out a detailed structural and functional analysis of the placental epigenome at its maternal interface. Specifically, we performed genome wide analysis of DNA methylation in samples of chorionic villus (CVS) and maternal blood cells (MBC) using both commercially available and custom designed microarrays. We then compared these data with genome wide transcription data for the same tissues. In addition to the discovery that CVS genomes are significantly more hypomethylated than their MBC counterparts, we identified numerous tissue-specific differentially methylated regions (T-DMRs). We further discovered that these T-DMRs are clustered spatially along the genome and are enriched for genes with tissue-specific biological functions. We identified unique patterns of DNA methylation associated with distinct genomic structures such as gene bodies, promoters and CpG islands and identified both direct and inverse relationships between DNA methylation levels and gene expression levels in gene bodies and promoters respectively. Furthermore, we found that these relationships were significantly associated with CpG content. We conclude that the early gestational placental DNA methylome is highly organized and is significantly and globally associated with transcription. These data provide a unique insight into the structural and regulatory characteristics of the placental epigenome at its maternal interface and will drive future analyses of the role of placental dysfunction in gestational disease.
PMCID: PMC3044138  PMID: 21373191
20.  Epigenome remodelling in breast cancer: insights from an early in vitro model of carcinogenesis 
Epigenetic gene regulation has influence over a diverse range of cellular functions, including the maintenance of pluripotency, differentiation, and cellular identity, and is deregulated in many diseases, including cancer. Whereas the involvement of epigenetic dysregulation in cancer is well documented, much of the mechanistic detail involved in triggering these changes remains unclear. In the current age of genomics, the development of new sequencing technologies has seen an influx of genomic and epigenomic data and drastic improvements in both resolution and coverage. Studies in cancer cell lines and clinical samples using next-generation sequencing are rapidly delivering spectacular insights into the nature of the cancer genome and epigenome. Despite these improvements in technology, the timing and relationship between genetic and epigenetic changes that occur during the process of carcinogenesis are still unclear. In particular, what changes to the epigenome are playing a driving role during carcinogenesis and what influence the temporal nature of these changes has on cancer progression are not known. Understanding the early epigenetic changes driving breast cancer has the exciting potential to provide a novel set of therapeutic targets or early-disease biomarkers or both. Therefore, it is important to find novel systems that permit the study of initial epigenetic events that potentially occur during the first stages of breast cancer. Non-malignant human mammary epithelial cells (HMECs) provide an exciting in vitro model of very early breast carcinogenesis. When grown in culture, HMECs are able to temporarily escape senescence and acquire a pre-malignant breast cancer-like phenotype (variant HMECs, or vHMECs). Cultured HMECs are composed mainly of cells from the basal breast epithelial layer. Therefore, vHMECs are considered to represent the basal-like subtype of breast cancer. The transition from HMECs to vHMECs in culture recapitulates the epigenomic phenomena that occur during the progression from normal breast to pre-malignancy. Therefore, the HMEC model system provides the unique opportunity to study the very earliest epigenomic aberrations occurring during breast carcinogenesis and can give insight into the sequence of epigenomic events that lead to breast malignancy. This review provides an overview of epigenomic research in breast cancer and discusses in detail the utility of the HMEC model system to discover early epigenomic changes involved in breast carcinogenesis.
PMCID: PMC4053120  PMID: 23168266
21.  Fine Tuning Gene Expression: The Epigenome 
Seminars in nephrology  2010;30(5):468-476.
An epigenetic trait is a stably inherited phenotype resulting from changes in a chromosome without alterations in the DNA sequence. Epigenetic modifications, such as; DNA methylation, together with covalent modification of histones, are thought to alter chromatin density and accessibility of the DNA to cellular machinery, thereby modulating the transcriptional potential of the underlying DNA sequence. As epigenetic marks under environmental influence, epigenetics provides an added layer of variation that might mediate the relationship between genotype and internal and external environmental factors. Integration of our knowledge in genetics, epigenomics and genomics with the use of systems biology tools may present investigators with new powerful tools to study many complex human diseases such as kidney disease.
PMCID: PMC3164355  PMID: 21044758
22.  Epigenetic signatures of alcohol abuse and hepatitis infection during human hepatocarcinogenesis 
Oncotarget  2014;5(19):9425-9443.
Hepatocellular carcinoma (HCC) is the second most common cause of cancer deaths worldwide. Deregulated DNA methylation landscapes are ubiquitous in human cancers. Interpretation of epigenetic aberrations in HCC is confounded by multiple etiologic drivers and underlying cirrhosis. We globally profiled the DNA methylome of 34 normal and 122 liver disease tissues arising in settings of hepatitis B (HBV) or C (HCV) viral infection, alcoholism (EtOH), and other causes to examine how these environmental agents impact DNA methylation in a manner that contributes to liver disease. Our results demonstrate that each ‘exposure’ leaves unique and overlapping signatures on the methylome. CpGs aberrantly methylated in cirrhosis-HCV and conserved in HCC were enriched for cancer driver genes, suggesting a pathogenic role for HCV-induced methylation changes. Additionally, large genomic regions displaying stepwise hypermethylation or hypomethylation during disease progression were identified. HCC-HCV/EtOH methylomes overlap highly with cryptogenic HCC, suggesting shared epigenetically deregulated pathways for hepatocarcinogenesis. Finally, overlapping methylation abnormalities between primary and cultured tumors unveil conserved epigenetic signatures in HCC. Taken together, this study reveals profound epigenome deregulation in HCC beginning during cirrhosis and influenced by common environmental agents. These results lay the foundation for defining epigenetic drivers and clinically useful methylation markers for HCC.
PMCID: PMC4253444  PMID: 25294808
hepatocellular carcinoma; cirrhosis; etiology; epigenetics; DNA methylation
23.  Epigenome Mapping in Normal and Disease States 
Circulation research  2010;107(3):327-339.
Epigenomes are comprised, in part, of all genome-wide chromatin modifications including DNA methylation and histone modifications. Unlike the genome, epigenomes are dynamic during development and differentiation in order to establish and maintain cell type-specific gene expression states that underlie cellular identity and function. Chromatin modifications are particularly labile, providing a mechanism for organisms to respond and adapt to environmental cues. Results from studies in animal models clearly demonstrate that epigenomic variability leads to phenotypic variability including susceptibility to disease that is not recognized at the DNA sequence level. Thus, capturing epigenomic information is invaluable for comprehensively understanding development, differentiation, and disease. Herein, we provide a brief overview of epigenetic processes, how they are relevant to human health, and review studies utilizing technologies that enable epigenome mapping. We conclude by describing feasible applications of epigenome mapping, focusing on epigenome-wide association studies (eGWAS), which have the potential to revolutionize current studies of human diseases and will likely promote the discovery of novel diagnostic, preventative, and treatment strategies.
PMCID: PMC2917837  PMID: 20689072
Epigenetics; genetics; gene expression; gene regulation
24.  Epigenomic strategies at the interface of genetic and environmental risk factors for autism 
Journal of human genetics  2013;58(7):396-401.
Autism spectrum disorders have been increasing in prevalence over the past two decades, primarily because of increased awareness and diagnosis. However, autism is clearly a complex human genetic disorder that involves interactions between genes and environment. Epigenetic mechanisms such as DNA methylation act at the interface of genetic and environmental risk and protective factors. Advancements in genome-wide sequencing has broadened the view of the human methylome and revealed the organization of the human genome into large-scale methylation domains that footprint over neurologically important genes involved in embryonic development. Future integrative epigenomic analyses of genetic risk factors with environmental exposures and methylome analyses are expected to be important for understanding the complex etiology of autism spectrum disorders.
PMCID: PMC3955092  PMID: 23677056
25.  Next Generation Sequencing: Advances in Characterizing the Methylome  
Genes  2010;1(2):143-165.
Epigenetic modifications play an important role in lymphoid malignancies. This has been evidenced by the large body of work published using microarray technologies to generate methylation profiles for numerous types and subtypes of lymphoma and leukemia. These studies have shown the importance of defining the epigenome so that we can better understand the biology of lymphoma. Recent advances in DNA sequencing technology have transformed the landscape of epigenomic analysis as we now have the ability to characterize the genome-wide distribution of chromatin modifications and DNA methylation using next-generation sequencing. To take full advantage of the throughput of next-generation sequencing, there are many methodologies that have been developed and many more that are currently being developed. Choosing the appropriate methodology is fundamental to the outcome of next-generation sequencing studies. In this review, published technologies and methodologies applicable to studying the methylome are presented. In addition, progress towards defining the methylome in lymphoma is discussed and prospective directions that have been made possible as a result of next-generation sequencing technology. Finally, methodologies are introduced that have not yet been published but that are being explored in the pursuit of defining the lymphoma methylome.
PMCID: PMC3954092  PMID: 24710039
lymphoma; leukemia; next-generation sequencing; methylation; epigenome

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