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Characterising associations between the epigenome, proteome and phenome may provide insight into molecular regulation of biological pathways governing health. However, epigenetic signatures for many neurologically-associated plasma protein markers remain uncharacterised. Here, we report an epigenome and phenome-wide association study of the circulating proteome in relation to brain health. We perform epigenome-wide studies of 4,235 plasma proteins (n=778), identifying 2,895 CpG-protein associations (protein quantitative trait methylation loci; pQTMs) after stringent correction for multiple testing. These were independent of known genetic protein quantitative trait loci (pQTL) and common lifestyle effects, extending current knowledge by analysing a further 3,286 protein measurements with 2,854 novel pQTMs. We then perform a phenome-wide study of each protein in relation to neurological traits in 1,065 individuals, identifying 644 proteins related to cognitive, brain imaging phenotypes or APOE status. By integrating our pQTM dataset with our phenome association study, we uncovered 88 epigenetic associations for protein markers of neurological traits, 83 of which were previously unreported. These data are pertinent to understanding heterogeneity in brain health.

Purpose: Beckwith-Wiedemann syndrome (BWS) is a developmental disorder caused by dysregulation of the imprinted gene cluster of chromosome 11p15.5 and often associated with loss of methylation (LOM) of the imprinting center 2 (IC2) located in KCNQ1 intron 10. To unravel the etiological mechanisms underlying these epimutations, we searched for genetic variants associated with IC2 LOM.Methods: We looked for cases showing the clinical features of both BWS and long QT syndrome (LQTS), which is often associated with KCNQ1 variants. Pathogenic variants were identified by genomic analysis and targeted sequencing. Functional experiments were performed to link these pathogenic variants to the imprinting defect.Results: We found three rare cases in which complete IC2 LOM is associated with maternal transmission of KCNQ1 variants, two of which were demonstrated to affect KCNQ1 transcription upstream of IC2. As a consequence of KCNQ1 haploinsufficiency, these variants also cause LQTS on both maternal and paternal transmission.Conclusion: These results are consistent with the hypothesis that, similar to what has been demonstrated in mouse, lack of transcription across IC2 results in failure of methylation establishment in the female germline and BWS later in development, and also suggest a new link between LQTS and BWS that is important for genetic counseling.

Inhibitors of DNA methylation and orexin type-1 receptor antagonists modulate the neurobiological effects driving drugs of abuse and natural reinforcers by activating common brain structures of the mesolimbic reward system. In this study, we applied a self-administration paradigm to assess the involvement of factors regulating DNA methylation processes and satiety or appetite signals. These factors include Dnmts and Tets, miR-212/132, orexins, and orx-R1 genes. The study focused on dopamine projection areas such as the prefrontal cortex (PFCx) and caudate putamen (CPu) and in the hypothalamus (HP) that is interconnected with the reward system. Striking changes were observed in response to both reinforcers, but differed depending on contingent and non-contingent delivery. Expression also differed in the PFCx and the CPu. Cocaine and food induced opposite effects on Dnmt3a expression in both brain structures, whereas they repressed both miRs to a different extent, without affecting their primary transcript in the CPu. Unexpectedly, orexin mRNAs were found in the CPu, suggesting a transport from their transcription site in the HP. The orexin receptor1 gene was found to be induced by cocaine in the PFCx, consistent with a regulation by DNA methylation. Global levels of 5-methylcytosines in the PFCx were not significantly altered by cocaine, suggesting that it is rather their distribution that contributes to long-lasting behaviors. Together, our data demonstrate that DNA methylation regulating factors are differentially altered by cocaine and food. At the molecular level, they support the idea that neural circuits activated by both reinforcers do not completely overlap.

DNA methylation (5mC) is central to cellular identity. The global erasure of 5mC from the parental genomes during preimplantation mammalian development is critical to reset the methylome of gametes to the cells in the blastocyst. While active and passive modes of demethylation have both been suggested to play a role in this process, the relative contribution of these two mechanisms to 5mC erasure remains unclear. Here, we report a single-cell method (scMspJI-seq) that enables strand-specific quantification of 5mC, allowing us to systematically probe the dynamics of global demethylation. When applied to mouse embryonic stem cells, we identified substantial cell-to-cell strand-specific 5mC heterogeneity, with a small group of cells displaying asymmetric levels of 5mCpG between the two DNA strands of a chromosome suggesting loss of maintenance methylation. Next, in preimplantation mouse embryos, we discovered that methylation maintenance is active till the 16-cell stage followed by passive demethylation in a fraction of cells within the early blastocyst at the 32-cell stage of development. Finally, human preimplantation embryos qualitatively show temporally delayed yet similar demethylation dynamics as mouse embryos. Collectively, these results demonstrate that scMspJI-seq is a sensitive and cost-effective method to map the strand-specific genome-wide patterns of 5mC in single cells., Erasure of DNA methylation from the parental genomes is critical to reset the methylome of differentiated gametes to pluripotent cells in the blastocyst. Here, the authors present a high-throughput single-cell method that enables strand-specific quantification of DNA methylation and identify distinct modes of DNA demethylation dynamics during early mammalian development.

DNA methylation (DNAm) is shaped by genetic and environmental factors and modulated by aging. Here, we examine interrelations between epigenetic aging, body weight (BW), and life span in 12 isogenic strains from the BXD family of mice that exhibit over twofold variation in longevity. Genome‐wide DNAm was assayed in 70 liver specimens from predominantly female cases, 6–25 months old, that were maintained on normal chow or high‐fat diet (HFD). We defined subsets of CpG regions associated with age, BW at young adulthood, and strain‐by‐diet‐dependent life span. These age‐associated differentially methylated CpG regions (age‐DMRs) featured distinct genomic characteristics, with DNAm gains over time occurring in sites such as promoters and exons that have high CpG density and low average methylation. CpG regions associated with BW were enriched in introns, tended to have lower methylation in mice with higher BW, and were inversely correlated with gene expression (i.e., higher mRNA levels in mice with higher BW). CpG regions associated with life span were linked to genes involved in life span modulation, including the telomerase reverse transcriptase gene, Tert, which had both lower methylation and higher expression in long‐lived strains. An epigenetic clock defined from age‐DMRs revealed accelerated aging in mice belonging to strains with shorter life spans. Both higher BW and the HFD were associated with accelerated epigenetic aging. Our results highlight the age‐accelerating effect of heavier BW. Furthermore, we demonstrate that the measure of epigenetic aging derived from age‐DMRs can predict genotype and diet‐induced differences in life span among female BXD members., The epigenetic clock, also referred to as DNA methylation age, provides a measure of biological aging. This clock can predict life expectancy in genetically distinct mouse stains, and is accelerated by higher body weight and high‐fat diet.

Epigenetic processes that regulate gene expression, such as DNA methylation (DNAm), have been linked to individual differences in physical aggression. Yet, it is currently unclear whether: (a) DNAm patterns in humans associate with physical aggression independently of other co-occurring psychiatric and behavioral symptoms; (b) whether these patterns are observable across multiple tissues; and (c) whether they may function as a causal versus noncausal biomarker of physical aggression. Here, we used a multisample, cross-tissue design to address these questions. First, we examined genome-wide DNAm patterns (buccal swabs; Illumina 450k) associated with engagement in physical fights in a sample of high-risk youth (n = 119; age = 16-24 years; 53% female). We identified one differentially methylated region in DRD4, which survived genome-wide correction, associated with physical aggression above and beyond co-occurring symptomatology (e.g., ADHD, substance use), and showed strong cross-tissue concordance with both blood and brain. Second, we found that DNAm sites within this region were also differentially methylated in an independent sample of young adults, between individuals with a history of chronic-high versus low physical aggression (peripheral T cells; ages 26-28). Finally, we ran a Mendelian randomization analysis using GWAS data from the EAGLE consortium to test for a causal association of DRD4 methylation with physical aggression. Only one genetic instrument was eligible for the analysis, and results provided no evidence for a causal association. Overall, our findings lend support for peripheral DRD4 methylation as a potential biomarker of physically aggressive behavior, with no evidence yet of a causal relationship.

ObjectiveEpigenetic changes contribute to pancreatic neuroendocrine tumor (PanNET) development. Hypermethylation of promoter DNA as a cause of tumor suppressor gene silencing is a well-established oncogenic mechanism that is potentially reversible and therefore an interesting therapeutic target. Multiple endocrine neoplasia type 1 (MEN1) is the most frequent cause of inherited PanNETs. The aim of this study was to determine promoter methylation profiles in MEN1-related PanNETs.Design and methodsMethylation-specific multiplex ligation-dependent probe amplification was used to assess promoter methylation of 56 tumor suppressor genes in MEN1-related (n = 61) and sporadic (n = 34) PanNETs. Differences in cumulative methylation index (CMI), individual methylation percentages and frequency of promoter hypermethylation between subgroups were analyzed.ResultsWe found promoter methylation of a large number of potential tumor suppressor genes. CMI (median CMI: 912 vs 876,P = 0.207) was the same in MEN1-related and sporadic PanNETs. We found higher methylation percentages ofCASP8in MEN1-related PanNETs (median: 59% vs 16.5%,P = 0.002). In MEN1-related non-functioning PanNETs, the CMI was higher in larger PanNETs (>2 cm) (median: 969.5 vs 838.5;P = 0.021) and in PanNETs with liver metastases (median: 1036 vs 869;P = 0.013). Hypermethylation ofMGMT2was more frequent in non-functioning PanNETs compared to insulinomas (median: 44.7% vs 8.3%;P = 0.022). Hypermethylation of the Von Hippel–Lindau gene promoter was observed in one MEN1-related PanNET and was associated with loss of protein expression.ConclusionPromoter hypermethylation is a frequent event in MEN1-related and sporadic PanNETs. Targeting DNA methylation could be of therapeutic value in MEN1 patients with advanced PanNETs.

In human embryonic stem cells (ESCs) the transcription factor Zeb2 regulates neuroectoderm versus mesendoderm formation, but it is unclear how Zeb2 affects the global transcriptional regulatory network in these cell-fate decisions. We generated Zeb2 knockout (KO) mouse ESCs, subjected them as embryoid bodies (EBs) to neural and general differentiation and carried out temporal RNA-sequencing (RNA-seq) and reduced representation bisulfite sequencing (RRBS) analysis in neural differentiation. This shows that Zeb2 acts preferentially as a transcriptional repressor associated with developmental progression and that Zeb2 KO ESCs can exit from their naïve state. However, most cells in these EBs stall in an early epiblast-like state and are impaired in both neural and mesendodermal differentiation. Genes involved in pluripotency, epithelial-to-mesenchymal transition (EMT), and DNA-(de)methylation, including Tet1, are deregulated in the absence of Zeb2. The observed elevated Tet1 levels in the mutant cells and the knowledge of previously mapped Tet1-binding sites correlate with loss-of-methylation in neural-stimulating conditions, however, after the cells initially acquired the correct DNA-methyl marks. Interestingly, cells from such Zeb2 KO EBs maintain the ability to re-adapt to 2i + LIF conditions even after prolonged differentiation, while knockdown of Tet1 partially rescues their impaired differentiation. Hence, in addition to its role in EMT, Zeb2 is critical in ESCs for exit from the epiblast state, and links the pluripotency network and DNA-methylation with irreversible commitment to differentiation.

Lymphopoiesis requires the activation of lineage-specific genes embedded in naive, inaccessible chromatin or in primed, accessible chromatin. The mechanisms responsible for de novo gain of chromatin accessibility, known as "pioneer" function, remain poorly defined. Here, we showed that the EBF1 C-terminal domain (CTD) is required for the regulation of a specific gene set involved in B cell fate decision and differentiation, independently of activation and repression functions. Using genome-wide analysis of DNaseI hypersensitivity and DNA methylation in multipotent Ebf1(-/-) progenitors and derivative EBF1wt- or EBF1ΔC-expressing cells, we found that the CTD promoted chromatin accessibility and DNA demethylation in previously naive chromatin. The CTD allowed EBF1 to bind at inaccessible genomic regions that offer limited co-occupancy by other transcription factors, whereas the CTD was dispensable for EBF1 binding at regions that are occupied by multiple transcription factors. Thus, the CTD enables EBF1 to confer permissive lineage-specific changes in progenitor chromatin landscape.

Aberrant methylation of multiple promoter CpG islands could be related to the biology of ovarian tumors and its determination could help to improve treatment strategies. DNA methylation profiling was performed using the Methylation Ligation-dependent Macroarray (MLM), an array-based analysis. Promoter regions of 41 genes were analyzed in 102 ovarian tumors and 17 normal ovarian samples. An average of 29% of hypermethylated promoter genes was observed in normal ovarian tissues. This percentage increased slightly in serous, endometrioid, and mucinous carcinomas (32%, 34%, and 45%, respectively), but decreased in germ cell tumors (20%). Ovarian tumors had methylation profiles that were more heterogeneous than other epithelial cancers. Unsupervised hierarchical clustering identified four groups that are very close to the histological subtypes of ovarian tumors. Aberrant methylation of three genes ( BRCA1 , MGMT , and MLH1 ), playing important roles in the different DNA repair mechanisms, were dependent on the tumor subtype and represent powerful biomarkers for precision therapy. Furthermore, a promising relationship between hypermethylation of MGMT , OSMR , ESR1 , and FOXL2 and overall survival was observed. Our study of DNA methylation profiling indicates that the different histotypes of ovarian cancer should be treated as separate diseases both clinically and in research for the development of targeted therapies.

The global rise in metabolic diseases can be attributed to a complex interplay between biology, behavior and environmental factors. This article reviews the current literature concerning DNA methylation-based epigenetic inheritance (intergenerational and transgenerational) of metabolic diseases through the male germ line. Included are a presentation of the basic principles for DNA methylation in developmental programming, and a description of windows of susceptibility for the inheritance of environmentally induced aberrations in DNA methylation and their associated metabolic disease phenotypes. To this end, escapees, genomic regions with the intrinsic potential to transmit acquired paternal epigenetic information across generations by escaping the extensive programmed DNA demethylation that occurs during gametogenesis and in the zygote, are described. The ongoing descriptive and functional examinations of DNA methylation in the relevant biological samples, in conjugation with analyses of non-coding RNA and histone modifications, hold promise for improved delineation of the effect size and mechanistic background for epigenetic inheritance of metabolic diseases.

X-chromosome inactivation (XCI), i.e., the inactivation of one of the female X chromosomes, restores equal expression of X-chromosomal genes between females and males. However, ~10% of genes show variable degrees of escape from XCI between females, although little is known about the causes of variable XCI. Using a discovery data-set of 1867 females and 1398 males and a replication sample of 3351 females, we show that genetic variation at three autosomal loci is associated with female-specific changes in X-chromosome methylation. Through cis-eQTL expression analysis, we map these loci to the genes SMCHD1/METTL4, TRIM6/HBG2, and ZSCAN9. Low-expression alleles of the loci are predominantly associated with mild hypomethylation of CpG islands near genes known to variably escape XCI, implicating the autosomal genes in variable XCI. Together, these results suggest a genetic basis for variable escape from XCI and highlight the potential of a population genomics approach to identify genes involved in XCI.

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

To study the impact of epigenetic changes on biological functions, the ability to manipulate the epigenetic status of certain genomic regions artificially could be an indispensable technology. "Epigenome editing" techniques have gradually emerged that apply TALE or CRISPR/Cas9 technologies with various effector domains isolated from epigenetic code writers or erasers such as DNA methyltransferase, 5-methylcytosine oxidase, and histone modification enzymes. Here we demonstrate that a TALE recognizing a major satellite, consisting of a repeated sequence in pericentromeres, could be fused with the bacterial CpG methyltransferase, SssI. ChIP-qPCR assays demonstrated that the fusion protein TALMaj-SssI preferentially bound to major chromosomal satellites in cultured cell lines. Then, TALMaj-SssI was expressed in fertilized mouse oocytes with hypomethylated major satellites (10-20% CpG islands). Bisulfite sequencing revealed that the DNA methylation status was increased specifically in major satellites (50-60%), but not in minor satellites or other repeat elements, such as Intracisternal A-particle (IAP) or long interspersed nuclear elements-1 (Line1) when the expression level of TALMaj-SssI is optimized in the cell. At a microscopic level, distal ends of chromosomes at the first mitotic stage were dramatically highlighted by the mCherry-tagged methyl CpG binding domain of human MBD1 (mCherry-MBD-NLS). Moreover, targeted DNA methylation to major satellites did not interfere with kinetochore function during early embryonic cleavages. Co-injection of dCas9 fused with SssI and guide RNA (gRNA) recognizing major satellite sequences enabled increment of the DNA methylation in the satellites, but a few off-target effects were also observed in minor satellites and retrotransposons. Although CRISPR can be applied instead of the TALE system, technical improvements to reduce off-target effects are required. We have demonstrated a new method of introducing DNA methylation without the need of other binding partners using the CpG methyltransferase, SssI.

Deletion of the long arm of chromosome 11 (11q deletion) is one of the most frequent events that occur during the development of aggressive neuroblastoma. Clinically, 11q deletion is associated with higher disease stage and decreased survival probability. During the last 25 years, extensive efforts have been invested to identify the precise frequency of 11q aberrations in neuroblastoma, the recurrently involved genes, and to understand the molecular mechanisms of 11q deletion, but definitive answers are still unclear. In this review, it is our intent to compile and review the evidence acquired to date on 11q deletion in neuroblastoma.

Vertebrate DNA can be chemically modified by methylation of the 5 position of the cytosine base in the context of CpG dinucleotides. This modification creates a binding site for MBD (methyl-CpG-binding domain) proteins which target chromatin-modifying activities that are thought to contribute to transcriptional repression and maintain heterochromatic regions of the genome. In contrast with DNA methylation, which is found broadly across vertebrate genomes, non-methylated DNA is concentrated in regions known as CGIs (CpG islands). Recently, a family of proteins which encode a ZF-CxxC (zinc finger-CxxC) domain have been shown to specifically recognize non-methylated DNA and recruit chromatin-modifying activities to CGI elements. For example, CFP1 (CxxC finger protein 1), MLL (mixed lineage leukaemia protein), KDM (lysine demethylase) 2A and KDM2B regulate lysine methylation on histone tails, whereas TET (ten-eleven translocation) 1 and TET3 hydroxylate methylated cytosine bases. In the present review, we discuss the most recent advances in our understanding of how ZF-CxxC domain-containing proteins recognize non-methylated DNA and describe their role in chromatin modification at CGIs. © 2013 The Author(s).

The etiology of respiratory allergies (RA) can be partly explained by DNA methylation changes caused by adverse environmental and lifestyle factors experienced early in life. Longitudinal, prospective studies can aid in the unravelment of the epigenetic mechanisms involved in the disease development. High compliance rates can be expected in these studies when data is collected using non-invasive and convenient procedures. Saliva is an attractive biofluid to analyze changes in DNA methylation patterns. We investigated in a pilot study the differential methylation in saliva of RA (n = 5) compared to healthy controls (n = 5) using the Illumina Methylation 450K BeadChip platform. We evaluated the results against the results obtained in mononuclear blood cells from the same individuals. Differences in methylation patterns from saliva and mononuclear blood cells were clearly distinguishable (P-Adj0.1), respectively, of which 216 were in common. The methylation levels of these sites were significantly correlated between blood and saliva. The absolute levels of methylation in blood and saliva were confirmed for 3 selected DMS in the PM20D1, STK32C, and FGFR2 genes using pyrosequencing analysis. The differential methylation could only be confirmed for DMS in PM20D1 and STK32C genes in saliva. We show that saliva can be used for genome-wide methylation analysis and that it is possible to identify DMS when comparing RA cases and healthy controls. The results were replicated in blood cells of the same individuals and confirmed by pyrosequencing analysis. This study provides proof-of-concept for the applicability of saliva-based whole-genome methylation analysis in the field of respiratory allergy. SL is the beneficiary of a post-doctoral grant from the AXA Research Fund and the Cefic-LRI Innovative Science Award 2013.

Understanding how genetic variation affects distinct cellular phenotypes, such as gene expression levels, alternative splicing and DNA methylation levels, is essential for better understanding of complex diseases and traits. Furthermore, how inter-individual variation of DNA methylation is associated to gene expression is just starting to be studied. In this study, we use the GenCord cohort of 204 newborn Europeans' lymphoblastoid cell lines, T-cells and fibroblasts derived from umbilical cords. The samples were previously genotyped for 2.5 million SNPs, mRNA-sequenced, and assayed for methylation levels in 482,421 CpG sites. We observe that methylation sites associated to expression levels are enriched in enhancers, gene bodies and CpG island shores. We show that while the correlation between DNA methylation and gene expression can be positive or negative, it is very consistent across cell-types. However, this epigenetic association to gene expression appears more tissue-specific than the genetic effects on gene expression or DNA methylation (observed in both sharing estimations based on P-values and effect size correlations between cell-types). This predominance of genetic effects can also be reflected by the observation that allele specific expression differences between individuals dominate over tissue-specific effects. Additionally, we discover genetic effects on alternative splicing and interestingly, a large amount of DNA methylation correlating to alternative splicing, both in a tissue-specific manner. The locations of the SNPs and methylation sites involved in these associations highlight the participation of promoter proximal and distant regulatory regions on alternative splicing. Overall, our results provide high-resolution analyses showing how genome sequence variation has a broad effect on cellular phenotypes across cell-types, whereas epigenetic factors provide a secondary layer of variation that is more tissue-specific. Furthermore, the details of how this tissue-specificity may vary across inter-relations of molecular traits, and where these are occurring, can yield further insights into gene regulation and cellular biology as a whole.

Several regulators are involved in the control of cell cycle progression in the bacterial model system Caulobacter crescentus, which divides asymmetrically into a vegetative G1-phase (swarmer) cell and a replicative S-phase (stalked) cell. Here we report a novel functional interaction between the enigmatic cell cycle regulator GcrA and the N6-adenosine methyltransferase CcrM, both highly conserved proteins among Alphaproteobacteria, that are activated early and at the end of S-phase, respectively. As no direct biochemical and regulatory relationship between GcrA and CcrM were known, we used a combination of ChIP (chromatin-immunoprecipitation), biochemical and biophysical experimentation, and genetics to show that GcrA is a dimeric DNA–binding protein that preferentially targets promoters harbouring CcrM methylation sites. After tracing CcrM-dependent N6-methyl-adenosine promoter marks at a genome-wide scale, we show that these marks recruit GcrA in vitro and in vivo. Moreover, we found that, in the presence of a methylated target, GcrA recruits the RNA polymerase to the promoter, consistent with its role in transcriptional activation. Since methylation-dependent DNA binding is also observed with GcrA orthologs from other Alphaproteobacteria, we conclude that GcrA is the founding member of a new and conserved class of transcriptional regulators that function as molecular effectors of a methylation-dependent (non-heritable) epigenetic switch that regulates gene expression during the cell cycle., Author Summary Methylation of genomic DNA at a specific regulatory site can impact a myriad of processes in eukaryotic cells. In bacteria, methylation at the N6 position of adenosine (m6A) is known to mediate a non-adaptive immunity response to protect cells from foreign DNA. While m6A marks are not known to govern expression of cell cycle genes in Gammaproteobacteria, cell cycle transcription in the model alphaproteobacterium Caulobacter crescentus requires the m6A methyltransferase CcrM that introduces m6A marks at GAnTC sequences and the enigmatic factor GcrA. Investigating if a functional and biochemical relationship exists between CcrM and GcrA, we found that CcrM-dependent m6A marks recruit GcrA to the promoters of cell cycle genes in vitro and in vivo and is required for efficient transcription. GcrA interacts with RNA polymerase, explaining how cell cycle transcription is affected. Importantly, m6A-dependent binding is also seen in GcrA orthologs, indicating that this transcriptional regulatory mechanism by CcrM and GcrA is conserved in Alphaproteobacteria.

C13ORF18 is frequently hypermethylated in cervical cancer but not in normal cervix and might serve as a biomarker for the early detection of cervical cancer in scrapings. As hypermethylation is often observed for silenced tumor suppressor genes (TSGs), hypermethylated biomarker genes might exhibit tumor suppressive activities upon re-expression. Epigenetic drugs are successfully exploited to reverse TSG silencing, but act genome-wide. Artificial Transcription Factors (ATFs) provide a gene-specific approach for re-expression of silenced genes. Here, we investigated the potential tumor suppressive role of C13ORF18 in cervical cancer by ATF-induced re-expression.Five zinc finger proteins were engineered to bind the C13ORF18 promoter and fused to a strong transcriptional activator. C13ORF18 expression could be induced in cervical cell lines: ranging from >40-fold in positive (C13ORF18-unmethylated) cells to >110-fold in negative (C13ORF18-methylated) cells. Re-activation of C13ORF18 resulted in significant cell growth inhibition and/or induction of apoptosis. Co-treatment of cell lines with ATFs and epigenetic drugs further enhanced the ATF-induced effects. Interestingly, reactivation of C13ORF18 led to partial demethylation of the C13ORF18 promoter and decreased repressive histone methylation. These data demonstrate the potency of ATFs to re-express and potentially demethylate hypermethylated silenced genes. Concluding, we show that C13ORF18 has a TSG function in cervical cancer and may serve as a therapeutic anti-cancer target. As the amount of epimutations in cancer exceeds the number of gene mutations, ATFs provide promising tools to validate hypermethylated marker genes as therapeutic targets. (C) 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Human cancers nearly ubiquitously harbor epigenetic alterations. While such alterations in epigenetic marks, including DNA methylation, are potentially heritable, they can also be dynamically altered. Given this potential for plasticity, the degree to which epigenetic changes can be subject to selection and act as drivers of neoplasia has been questioned. Here, we carried out genome-scale analyses of DNA methylation alterations in lethal metastatic prostate cancer and created DNA methylation “cityscape” plots to visualize these complex data. We show that somatic DNA methylation alterations, despite showing marked inter-individual heterogeneity among men with lethal metastatic prostate cancer, were maintained across all metastases within the same individual. The overall extent of maintenance in DNA methylation changes was comparable to that of genetic copy number alterations. Regions that were frequently hypermethylated across individuals were markedly enriched for cancer and development/differentiation related genes. Additionally, regions exhibiting high consistency of hypermethylation across metastases within individuals, even if variably hypermethylated across individuals, showed enrichment of cancer-related genes. Interestingly, whereas some regions showed intra-individual metastatic tumor heterogeneity in promoter methylation, such methylation alterations were generally not correlated with gene expression. This was despite a general tendency for promoter methylation patterns to be strongly correlated with gene expression, particularly at regions that were variably methylated across individuals. These findings suggest that DNA methylation alterations have the potential for producing selectable driver events in carcinogenesis and disease progression and highlight the possibility of targeting such epigenome alterations for development of longitudinal markers and therapeutic strategies.

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.

Age-related changes in DNA methylation have been implicated in cellular senescence and longevity, yet the causes and functional consequences of these variants remain unclear. To elucidate the role of age-related epigenetic changes in healthy ageing and potential longevity, we tested for association between whole-blood DNA methylation patterns in 172 female twins aged 32 to 80 with age and age-related phenotypes. Twin-based DNA methylation levels at 26,690 CpG-sites showed evidence for mean genome-wide heritability of 18%, which was supported by the identification of 1,537 CpG-sites with methylation QTLs in cis at FDR 5%. We performed genome-wide analyses to discover differentially methylated regions (DMRs) for sixteen age-related phenotypes (ap-DMRs) and chronological age (a-DMRs). Epigenome-wide association scans (EWAS) identified age-related phenotype DMRs (ap-DMRs) associated with LDL (STAT5A), lung function (WT1), and maternal longevity (ARL4A, TBX20). In contrast, EWAS for chronological age identified hundreds of predominantly hyper-methylated age DMRs (490 a-DMRs at FDR 5%), of which only one (TBX20) was also associated with an age-related phenotype. Therefore, the majority of age-related changes in DNA methylation are not associated with phenotypic measures of healthy ageing in later life. We replicated a large proportion of a-DMRs in a sample of 44 younger adult MZ twins aged 20 to 61, suggesting that a-DMRs may initiate at an earlier age. We next explored potential genetic and environmental mechanisms underlying a-DMRs and ap-DMRs. Genome-wide overlap across cis-meQTLs, genotype-phenotype associations, and EWAS ap-DMRs identified CpG-sites that had cis-meQTLs with evidence for genotype–phenotype association, where the CpG-site was also an ap-DMR for the same phenotype. Monozygotic twin methylation difference analyses identified one potential environmentally-mediated ap-DMR associated with total cholesterol and LDL (CSMD1). Our results suggest that in a small set of genes DNA methylation may be a candidate mechanism of mediating not only environmental, but also genetic effects on age-related phenotypes., Author Summary Epigenetic patterns vary during healthy ageing and development. Age-related DNA methylation changes have been implicated in cellular senescence and longevity, yet the causes and functional consequences of these variants remain unclear. To understand the biological mechanisms involved in potential longevity and rate of healthy ageing, we performed genome-wide association of epigenetic and genetic variation with both chronological age and age-related phenotypes. We identified hundreds of DNA methylation variants significantly associated with age and replicated these in an independent sample of young adult twins. Only a small proportion of these variants were also associated with age-related phenotypes. Therefore, the majority of age-related epigenetic changes do not contribute to rate of healthy ageing at later stages in life. Our results suggest that age-related changes in methylation occur throughout an individual's lifespan and that a proportion of these may be initiated from an early age. Intriguingly, a fraction of the age differentially methylated regions also associated with genetic variants in our sample, suggesting that DNA methylation may be a candidate mechanism of mediating not only environmental but also genetic effects on age-related phenotypes.

During sexual reproduction, one-half of the genetic material is deposited in gametes, and a complete set of chromosomes is restored upon fertilization. Reduction of the genetic information before gametogenesis occurs in meiosis, when cross-overs (COs) between homologous chromosomes secure an exchange of their genetic information. COs are not evenly distributed along chromosomes and are suppressed in chromosomal regions encompassing compact, hypermethylated centromeric and pericentromeric DNA. Therefore, it was postulated that DNA hypermethylation is inhibitory to COs. Here, when analyzing meiotic recombination in mutant plants with hypomethylated DNA, we observed unexpected and counterintuitive effects of DNA methylation losses on CO distribution. Recombination was further promoted in the hypomethylated chromosome arms while it was inhibited in heterochromatic regions encompassing pericentromeric DNA. Importantly, the total number of COs was not affected, implying that loss of DNA methylation led to a global redistribution of COs along chromosomes. To determine by which mechanisms altered levels of DNA methylation influence recombination—whether directly in cis or indirectly in trans by changing expression of genes encoding recombination components—we analyzed CO distribution in wild-type lines with randomly scattered and well-mapped hypomethylated chromosomal segments. The results of these experiments, supported by expression profiling data, suggest that DNA methylation affects meiotic recombination in cis . Because DNA methylation exhibits significant variation even within a single species, our results imply that it may influence the evolution of plant genomes through the control of meiotic recombination.

BackgroundDespite their well-established functional roles, histone modifications have received less attention than DNA methylation in the cancer field. In order to evaluate their importance in colorectal cancer (CRC), we generated the first genome-wide histone modification profiles in paired normal colon mucosa and tumor samples.MethodsChromatin immunoprecipitation and microarray hybridization (ChIP-chip) was used to identify promoters enriched for histone H3 trimethylated on lysine 4 (H3K4me3) and lysine 27 (H3K27me3) in paired normal colon mucosa and tumor samples from two CRC patients and for the CRC cell line HT29.ResultsBy comparing histone modification patterns in normal mucosa and tumors, we found that alterations predicted to have major functional consequences were quite rare. Furthermore, when normal or tumor tissue samples were compared to HT29, high similarities were observed for H3K4me3. However, the differences found for H3K27me3, which is important in determining cellular identity, indicates that cell lines do not represent optimal tissue models. Finally, using public expression data, we uncovered previously unknown changes in CRC expression patterns. Genes positive for H3K4me3 in normal and/or tumor samples, which are typically already active in normal mucosa, became hyperactivated in tumors, while genes with H3K27me3 in normal and/or tumor samples and which are expressed at low levels in normal mucosa, became hypersilenced in tumors.ConclusionsGenome wide histone modification profiles can be used to find epigenetic aberrations in genes associated with cancer. This strategy gives further insights into the epigenetic contribution to the oncogenic process and may identify new biomarkers.

Endogenous retroviruses (ERVs), retrovirus-like elements with long terminal repeats, are widely dispersed in the euchromatic compartment in mammalian cells, comprising approximately 10% of the mouse genome. These parasitic elements are responsible for >10% of spontaneous mutations. Whereas DNA methylation has an important role in proviral silencing in somatic and germ-lineage cells, an additional DNA-methylation-independent pathway also functions in embryonal carcinoma and embryonic stem (ES) cells to inhibit transcription of the exogenous gammaretrovirus murine leukaemia virus (MLV). Notably, a recent genome-wide study revealed that ERVs are also marked by histone H3 lysine 9 trimethylation (H3K9me3) and H4K20me3 in ES cells but not in mouse embryonic fibroblasts. However, the role that these marks have in proviral silencing remains unexplored. Here we show that the H3K9 methyltransferase ESET (also called SETDB1 or KMT1E) and the Krüppel-associated box (KRAB)-associated protein 1 (KAP1, also called TRIM28) are required for H3K9me3 and silencing of endogenous and introduced retroviruses specifically in mouse ES cells. Furthermore, whereas ESET enzymatic activity is crucial for HP1 binding and efficient proviral silencing, the H4K20 methyltransferases Suv420h1 and Suv420h2 are dispensable for silencing. Notably, in DNA methyltransferase triple knockout (Dnmt1(-/-)Dnmt3a(-/-)Dnmt3b(-/-)) mouse ES cells, ESET and KAP1 binding and ESET-mediated H3K9me3 are maintained and ERVs are minimally derepressed. We propose that a DNA-methylation-independent pathway involving KAP1 and ESET/ESET-mediated H3K9me3 is required for proviral silencing during the period early in embryogenesis when DNA methylation is dynamically reprogrammed.

Transgenerational epigenetic inheritance has been defined by the study of relatively few loci. We examined a population of recombinant inbred lines with epigenetically mosaic chromosomes consisting of wild-type and CG methylation-depleted segments (epiRILs). Surprisingly, transposons that were immobile in the parental lines displayed stochastic movement in 28% of the epiRILs. Although analysis after eight generations of inbreeding, supported by genome-wide DNA methylation profiling, identified recombined parental chromosomal segments, these were interspersed with unexpectedly high frequencies of nonparental methylation polymorphism. Hence, epigenetic inheritance in hybrids derived from parents with divergent epigenomes permits long-lasting epi-allelic interactions that violate Mendelian expectations. Such persistently “unstable” epigenetic states complicate linkage-based epigenomic mapping. Thus, future epigenomic analyses should consider possible genetic instabilities and alternative mapping strategies.

The methylation status of the O6-methylguanine-DNA methyltransferase (MGMT) gene is an important predictive biomarker for benefit from alkylating agent therapy in glioblastoma. Recent studies in anaplastic glioma suggest a prognostic value for MGMT methylation. Investigation of pathogenetic and epigenetic features of this intriguingly distinct behavior requires accurate MGMT classification to assess high throughput molecular databases. Promoter methylation-mediated gene silencing is strongly dependent on the location of the methylated CpGs, complicating classification. Using the HumanMethylation450 (HM-450K) BeadChip interrogating 176 CpGs annotated for the MGMT gene, with 14 located in the promoter, two distinct regions in the CpG island of the promoter were identified with high importance for gene silencing and outcome prediction. A logistic regression model (MGMT-STP27) comprising probes cg1243587 and cg12981137 provided good classification properties and prognostic value (kappa = 0.85; log-rank p