[Show abstract][Hide abstract] ABSTRACT: DNA methylation can be abnormally regulated in human disease and associated with effects on gene transcription that appear to be causally related to pathogenesis. The potential to use pharmacological agents that reverse this dysregulation is therefore an attractive possibility. To test how 5-aza-2'-deoxycytidine (5-aza-CdR) influences the genome therapeutically, we exposed non-malignant cells in culture to the agent and used genome-wide assays to assess the cellular response.
We found that cells allowed to recover from 5-aza-CdR treatment only partially recover DNA methylation levels, retaining an epigenetic 'imprint' of drug exposure. We show very limited transcriptional responses to demethylation of not only protein-coding genes but also loci-encoding non-coding RNAs, with a limited proportion of the induced genes acquiring new promoter activation within gene bodies. The data revealed an uncoupling of DNA methylation effects at promoters, with demethylation mostly unaccompanied by transcriptional changes. The limited panel of genes induced by 5-aza-CdR resembles those activated in other human cell types exposed to the drug and represents loci targeted for Polycomb-mediated silencing in stem cells, suggesting a model for the therapeutic effects of the drug.
Our results do not support the hypothesis of DNA methylation having a predominant role to regulate transcriptional noise in the genome and indicate that DNA methylation acts only as part of a larger complex system of transcriptional regulation. The targeting of 5-aza-CdR effects with its clastogenic consequences to euchromatin raises concerns that the use of 5-aza-CdR has innate tumorigenic consequences, requiring its cautious use in diseases involving epigenetic dysregulation.
[Show abstract][Hide abstract] ABSTRACT: Epigenetic dysregulation in disease is increasingly studied as a potential mediator of pathophysiology. The epigenetic events are believed to occur in somatic cells, but the limited changes of DNA methylation in studies to date indicate that only subsets of the cells tested undergo epigenetic dysregulation. The recognition of this subpopulation effect indicates the need for care in design and execution of epigenome-wide association studies (EWASs), paying particular attention to confounding sources of variability. To maximize the sensitivity of the EWASs, ideally, the cell type mediating the disease should be tested, which is not always practical or ethical in human subjects. The value of using accessible cells as surrogates for the target, disease-mediating cell type has not been rigorously tested to date. In this review, participants in a workshop convened by the National Institutes of Health update EWAS design and execution guidelines to reflect new insights in the field.
[Show abstract][Hide abstract] ABSTRACT: The number of different assays that has been published to study DNA methylation is extensive, complemented by recently described assays that test modifications of cytosine other than the most abundant 5-methylcytosine (5mC) variant. In this review, we describe the considerations involved in choosing how to study 5mC throughout the genome, with an emphasis on the common application of testing for epigenetic dysregulation in human disease. While microarray studies of 5mC continue to be commonly used, these lack the additional qualitative information from sequencing-based approaches that is increasingly recognized to be valuable. When we test the representation of functional elements in the human genome by several current assay types, we find that no survey approach interrogates anything more than a small minority of the nonpromoter cis-regulatory sites where DNA methylation variability is now appreciated to influence gene expression and to be associated with human disease. However, whole-genome bisulphite sequencing (WGBS) adds a substantial representation of loci at which DNA methylation changes are unlikely to be occurring with transcriptional consequences. Our assessment is that the most effective approach to DNA methylation studies in human diseases is to use targeted bisulphite sequencing of the cis-regulatory loci in a cell type of interest, using a capture-based or comparable system, and that no single design of a survey approach will be suitable for all cell types.
[Show abstract][Hide abstract] ABSTRACT: ABSTRACT Osteosarcoma is the most common primary malignant bone tumor in children. Validated biological markers for disease prognosis available at diagnosis are lacking. No genome-wide DNA methylation studies linked to clinical outcomes have been reported in osteosarcoma to the best of our knowledge. To address this, we tested the methylome at over 1.1 million loci in 15 osteosarcoma biopsy samples obtained prior to the initiation of therapy and correlated these molecular data with disease outcomes. At more than 17% of the tested loci, samples obtained from patients who experienced disease relapse were more methylated than those from patients who did not have recurrence while patients who did not experience disease relapse had more DNA methylation at fewer than 1%. In samples from patients who went on to have recurrent disease, increased DNA methylation was found at gene bodies, intergenic regions and empirically-annotated candidate enhancers, whereas candidate gene promoters were unusual for a more balanced distribution of increased and decreased DNA methylation with 6.6% of gene promoter loci being more methylated and 2% of promoter loci being less methylated in patients with disease relapse. A locus at the TLR4 gene demonstrates one of strongest associations between DNA methylation and five year event-free survival (p-value = 1.7 x10(-6)), with empirical annotation of this locus showing promoter characteristics. Our data indicate that DNA methylation information has potential to be predictive of outcome in pediatric osteosarcoma, and that both promoters and non-promoter loci are potentially informative in DNA methylation studies.
Epigenetics: official journal of the DNA Methylation Society 12/2014; 10(1). DOI:10.4161/15592294.2014.989084 · 5.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The mechanism and significance of epigenetic variability in the same cell type between healthy individuals are not clear. Here we purify human CD34+ haematopoietic stem and progenitor cells (HSPCs) from different individuals and find that there is increased variability of DNA methylation at loci with properties of promoters and enhancers. The variability is especially enriched at candidate enhancers near genes transitioning between silent and expressed states, and encoding proteins with leukocyte differentiation properties. Our findings of increased variability at loci with intermediate DNA methylation values, at candidate 'poised' enhancers and at genes involved in HSPC lineage commitment suggest that CD34+ cell subtype heterogeneity between individuals is a major mechanism for the variability observed. Epigenomic studies performed on cell populations, even when purified, are testing collections of epigenomes, or meta-epigenomes. Our findings show that meta-epigenomic approaches to data analysis can provide insights into cell subpopulation structure.
[Show abstract][Hide abstract] ABSTRACT: Extreme fetal growth is associated with increased susceptibility to a range of adult diseases through an unknown mechanism of cellular memory. We tested whether heritable epigenetic processes in long-lived CD34(+) haematopoietic stem/progenitor cells showed evidence for re-programming associated with the extremes of fetal growth. Here we show that both fetal growth restriction and over-growth are associated with global shifts towards DNA hypermethylation, targeting cis-regulatory elements in proximity to genes involved in glucose homeostasis and stem cell function. We find a sexually dimorphic response; intrauterine growth restriction is associated with substantially greater epigenetic dysregulation in males, whereas large for gestational age growth predominantly affects females. The findings are consistent with extreme fetal growth interacting with variable fetal susceptibility to influence cellular ageing and metabolic characteristics through epigenetic mechanisms, potentially generating biomarkers that could identify infants at higher risk for chronic disease later in life.
[Show abstract][Hide abstract] ABSTRACT: Purpose: Even though recent studies have shown that genetic changes at enhancers can influence carcinogenesis, most methylomic studies have focused on changes at promoters. We used renal cell carcinoma (RCC), an incurable malignancy associated with mutations in epigenetic regulators, as a model to study genome wide patterns of DNA methylation at a high resolution. Experimental Design and Results: Analysis of 1.3 million CpGs by the HELP assay in RCC and healthy microdissected renal tubular controls demonstrated that the RCC samples were characterized by widespread hypermethylation that preferentially affected gene bodies. Aberrant methylation was particularly enriched in kidney specific enhancer regions associated with H3K4Me1 marks. Various important underexpressed genes such as SMAD6 were associated with aberrantly methylated, intronic enhancers and these changes were validated in an independent cohort. MOTIF analysis of aberrantly hypermethylated regions revealed enrichment for binding sites of AP2alpha, AHR, HAIRY, ARNT and HIF-1 transcription factors, reflecting contributions of dysregulated hypoxia signaling pathways in RCC. The functional importance of this aberrant hypermethylation was demonstrated by selective sensitivity of RCC cells to low levels of decitabine. Most importantly, methylation of enhancers was predictive of adverse prognosis in 405 cases of RCC in multivariate analysis. Additionally, parallel copy number analysis from MspI representations demonstrated novel cnvs that were validated in independent cohort of patients. Conclusions: Our study is the first high resolution methylome analysis of RCC; demonstrates that many kidney specific enhancers are targeted by aberrant hypermethylation and reveals the prognostic importance of these epigenetic changes in an independent cohort.
Clinical Cancer Research 06/2014; 20(16). DOI:10.1158/1078-0432.CCR-14-0494 · 8.19 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The protein MeCP2 mediates epigenetic regulation by binding methyl-CpG (mCpG) sites on chromatin. MeCP2 consists of six domains of which one, MBD, binds mCpG sites in duplex DNA. We show that solution conditions of physiological or greater salt concentration or the presence of nonspecific competitor DNA are necessary for MDB to discriminate mCpG from CpG with high specificity. The specificity for mCpG over CpG is greater than a hundred fold under these solution conditions. In contrast, MBD does not discriminate hydroxymethyl-CpG from CpG. MBD is unusual among site-specific DNA binding proteins in that i) specificity is not conferred by enhanced affinity for the specific site but rather by suppression of its affinity for generic DNA, ii) its specific binding to mCpG is highly electrostatic and iii) it takes up as well as displaces monovalent cations upon DNA binding. MBD displays an unusually high affinity for single stranded DNA independent of modification or sequence. In addition, MBD forms a discrete dimer on DNA via a noncooperative-binding pathway. Since affinity of the second monomer is an order of magnitude greater than nonspecific binding, the MBD dimer is a unique molecular complex. The significance of these results in the context of neuronal function and development and MeCP2 related developmental disorders such as Rett Syndrome are discussed.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Background
DNA methylation is a major epigenetic mechanism altering gene expression in development and disease. However, its role in the regulation of gene expression during heart development is incompletely understood. The aim of this study is to reveal DNA methylation in mouse embryonic hearts and its role in regulating gene expression during heart development.
Methods and Results
We performed the genome‐wide DNA methylation profiling of mouse embryonic hearts using methyl‐sensitive, tiny fragment enrichment/massively parallel sequencing to determine methylation levels at ACGT sites. The results showed that while global methylation of 1.64 million ACGT sites in developing hearts remains stable between embryonic day (E) 11.5 and E14.5, a small fraction (2901) of them exhibit differential methylation. Gene Ontology analysis revealed that these sites are enriched at genes involved in heart development. Quantitative real‐time PCR analysis of 350 genes with differential DNA methylation showed that the expression of 181 genes is developmentally regulated, and 79 genes have correlative changes between methylation and expression, including hyaluronan synthase 2 (Has2). Required for heart valve formation, Has2 expression in the developing heart valves is downregulated at E14.5, accompanied with increased DNA methylation in its enhancer. Genetic knockout further showed that the downregulation of Has2 expression is dependent on DNA methyltransferase 3b, which is co‐expressed with Has2 in the forming heart valve region, indicating that the DNA methylation change may contribute to the Has2 enhancer's regulating function.
DNA methylation is developmentally regulated for genes essential to heart development, and abnormal DNA methylation may contribute to congenital heart disease.
Journal of the American Heart Association 04/2014; 3(3). DOI:10.1161/JAHA.114.000976 · 2.88 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Acute myeloid leukemia (AML) is characterized by disruption of HSC and progenitor cell differentiation. Frequently, AML is associated with mutations in genes encoding epigenetic modifiers. We hypothesized that analysis of alterations in DNA methylation patterns during healthy HSC commitment and differentiation would yield epigenetic signatures that could be used to identify stage-specific prognostic subgroups of AML. We performed a nano HpaII-tiny-fragment-enrichment-by-ligation-mediated-PCR (nanoHELP) assay to compare genome-wide cytosine methylation profiles between highly purified human long-term HSC, short-term HSC, common myeloid progenitors, and megakaryocyte-erythrocyte progenitors. We observed that the most striking epigenetic changes occurred during the commitment of short-term HSC to common myeloid progenitors and these alterations were predominantly characterized by loss of methylation. We developed a metric of the HSC commitment-associated methylation pattern that proved to be highly prognostic of overall survival in 3 independent large AML patient cohorts, regardless of patient treatment and epigenetic mutations. Application of the epigenetic signature metric for AML prognosis was superior to evaluation of commitment-based gene expression signatures. Together, our data define a stem cell commitment-associated methylome that is independently prognostic of poorer overall survival in AML.
The Journal of clinical investigation 02/2014; 124(3). DOI:10.1172/JCI71264 · 13.77 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: DNA methylation contributes to genomic integrity by suppressing repeat-associated transposition. In addition to the canonical DNA methyltransferases, several auxillary chromatin factors are required to maintain DNA methylation at intergenic and satellite repeats. The interaction between Lsh, a chromatin helicase, and the de novo methyltransferase Dnmt3b facilitates deposition of DNA methylation at stem cell genes, which are hypomethylated in Lsh-/- embryos. We wished to determine if a similar targeting mechanism operates to maintain DNA methylation at repetitive sequences.
We mapped genome-wide DNA methylation patterns in Lsh-/- and Dnmt3b-/- somatic cells. DNA methylation is predominantly lost from specific genomic repeats in Lsh-/- cells: LTR-retrotransposons, LINE-1 repeats and mouse satellites. RNA-seq experiments demonstrate that specific IAP LTRs and satellites, but not LINE-1 elements, are aberrantly transcribed in Lsh-/- cells. LTR hypomethylation in Dnmt3b-/- cells is moderate, whereas IAP, LINE-1 and satellite elements are hypomethylated but silent. Repressed LINE-1 elements in Lsh-/- cells gain H3K4me3, but H3K9me3 levels are unaltered, indicating that DNA hypomethylation alone is not permissive for their transcriptional activation. Mis-expressed IAPs and satellites lose H3K9me3 and gain H3K4me3 in Lsh-/- cells.
Our study emphasizes that regulation of repetitive elements by Lsh and DNA methylation is selective and context dependent. Silencing of repeats in somatic cells appears not to be critically dependent on Dnmt3b function. We propose a model where Lsh is specifically required at a precise developmental window to target de novo methylation to repeat sequences, which is subsequently maintained by Dnmt1 to enforce selective repeat silencing.
[Show abstract][Hide abstract] ABSTRACT: The identification of the H3K4 trimethylase, PRDM9, as the gene responsible for recombination hotspot localization has provided considerable insight into the mechanisms by which recombination is initiated in mammals. However, uniquely amongst mammals, canids appear to lack a functional version of PRDM9 and may therefore provide a model for understanding recombination that occurs in the absence of PRDM9, and thus how PRDM9 functions to shape the recombination landscape. We have constructed a fine-scale genetic map from patterns of linkage disequilibrium assessed using high-throughput sequence data from 51 free-ranging dogs, Canis lupus familiaris. While broad-scale properties of recombination appear similar to other mammalian species, our fine-scale estimates indicate that canine highly elevated recombination rates are observed in the vicinity of CpG rich regions including gene promoter regions, but show little association with H3K4 trimethylation marks identified in spermatocytes. By comparison to genomic data from the Andean fox, Lycalopex culpaeus, we show that biased gene conversion is a plausible mechanism by which the high CpG content of the dog genome could have occurred.
[Show abstract][Hide abstract] ABSTRACT: One in eleven people is affected by chronic kidney disease, a condition characterized by kidney fibrosis and progressive loss of kidney function. Epidemiological studies indicate that adverse intrauterine and postnatal environments have a long-lasting role in chronic kidney disease development. Epigenetic information represents a plausible carrier for mediating this programming effect. Here we demonstrate that genome-wide cytosine methylation patterns of healthy and chronic kidney disease tubule samples obtained from patients show significant differences.
We identify differentially methylated regions and validate these in a large replication dataset. The differentially methylated regions are rarely observed on promoters, but mostly overlap with putative enhancer regions, and they are enriched in consensus binding sequences for important renal transcription factors. This indicates their importance in gene expression regulation. A core set of genes that are known to be related to kidney fibrosis, including genes encoding collagens, show cytosine methylation changes correlating with downstream transcript levels.
Our report raises the possibility that epigenetic dysregulation plays a role in chronic kidney disease development via influencing core pro-fibrotic pathways and can aid the development of novel biomarkers and future therapeutics.