Gene Body Methylation Research Articles

TGFB2 and BCL2L11 methylation in male laryngeal cancer patients.

DNA methylation is a major regulatory mechanism of gene expression. The aim of the present study was to test the association of transforming growth factor β2 (TGFB2) and B cell lymphoma 2-like 11 (BCL2L11) gene methylation with the risk of laryngeal squamous cell carcinoma (LSCC). Using bisulfite pyrosequencing technology, DNA methylation levels of TGFB2 promoter and BCL2L11 gene-body CpG cytosines were measured in 90 LSCC tissues and 90 adjacent normal tissues. Analysis of variance and paired sample t-test were used to determine the association of gene methylation and the risk of LSCC. Our results revealed that there were no differences in TGFB2 and BCL2L11 methylation levels between the LSCC tissues and the paired normal tissues (P>0.05). Further breakdown analyses demonstrated that the association results of the two gene methylation levels and LSCC remained unchanged with the age, smoking history, histological differentiation or clinical stage of the LSCC patients (all adjusted P>0.05). In conclusion, there is no association of TGFB2 promoter and BCL2L11 gene-body methylation with the risk of LSCC in males.

On the origin and evolutionary consequences of gene body DNA methylation.

In plants, CG DNA methylation is prevalent in the transcribed regions of many constitutively expressed genes (gene body methylation; gbM), but the origin and function of gbM remain unknown. Here we report the discovery that Eutrema salsugineum has lost gbM from its genome, to our knowledge the first instance for an angiosperm. Of all known DNA methyltransferases, only CHROMOMETHYLASE 3 (CMT3) is missing from E. salsugineum Identification of an additional angiosperm, Conringia planisiliqua, which independently lost CMT3 and gbM, supports that CMT3 is required for the establishment of gbM. Detailed analyses of gene expression, the histone variant H2A.Z, and various histone modifications in E. salsugineum and in Arabidopsis thaliana epigenetic recombinant inbred lines found no evidence in support of any role for gbM in regulating transcription or affecting the composition and modification of chromatin over evolutionary timescales.

DNA Methylation Variation Trends during the Embryonic Development of Chicken.

The embryogenesis period is critical for epigenetic reprogramming and is thus of great significance in the research field of poultry epigenetics for elucidation of the trends in DNA methylation variations during the embryonic development of birds, particularly due to differences in embryogenesis between birds and mammals. Here, we first examined the variations in genomic DNA methylation during chicken embryogenesis through high-performance liquid chromatography using broilers as the model organism. We then identified the degree of DNA methylation of the promoters and gene bodies involved in two specific genes (IGF2 and TNF-α) using the bisulfite sequencing polymerase chain reaction method. In addition, we measured the expression levels of IGF2, TNF-α and DNA methyltransferase (DNMT) 1, 3a and 3b. Our results showed that the genomic DNA methylation levels in the liver, heart and muscle increased during embryonic development and that the methylation level of the liver was significantly higher in mid-anaphase. In both the muscle and liver, the promoter methylation levels of TNF-α first increased and then decreased, whereas the gene body methylation levels remained lower at embryonic ages E8, 11 and 14 before increasing notably at E17. The promoter methylation level of IGF2 decreased persistently, whereas the methylation levels in the gene body showed a continuous increase. No differences in the expression of TNF-α were found among E8, 11 and 14, whereas a significant increase was observed at E17. IGF2 showed increasing expression level during the examined embryonic stages. In addition, the mRNA and protein levels of DNMTs increased with increasing embryonic ages. These results suggest that chicken shows increasing genomic DNA methylation patterns during the embryonic period. Furthermore, the genomic DNA methylation levels in tissues are closely related to the genes expression levels, and gene expression may be simultaneously regulated by promoter hypomethylation and gene body hypermethylation.

Limited Contribution of DNA Methylation Variation to Expression Regulation in Arabidopsis thaliana.

The extent to which epigenetic variation affects complex traits in natural populations is not known. We addressed this question using transcriptome and DNA methylation data from a sample of 135 sequenced A. thaliana accessions. Across individuals, expression was significantly associated with cis-methylation for hundreds of genes, and many of these associations remained significant after taking SNP effects into account. The pattern of correlations differed markedly between gene body methylation and transposable element methylation. The former was usually positively correlated with expression, and the latter usually negatively correlated, although exceptions were found in both cases. Finally, we developed graphical models of causality that adapt to a sample with heavy population structure, and used them to show that while methylation appears to affect gene expression more often than expression affects methylation, there is also strong support for both being independently controlled. In conclusion, although we find clear evidence for epigenetic regulation, both the number of loci affected and the magnitude of the effects appear to be small compared to the effect of SNPs.

Allele-Specific Transcriptome and Methylome Analysis Reveals Stable Inheritance and Cis-Regulation of DNA Methylation in Nasonia.

Gene expression divergence between closely related species could be attributed to both cis- and trans- DNA sequence changes during evolution, but it is unclear how the evolutionary dynamics of epigenetic marks are regulated. In eutherian mammals, biparental DNA methylation marks are erased and reset during gametogenesis, resulting in paternal or maternal imprints, which lead to genomic imprinting. Whether DNA methylation reprogramming exists in insects is not known. Wasps of the genus Nasonia are non-social parasitoids that are emerging as a model for studies of epigenetic processes in insects. In this study, we quantified allele-specific expression and methylation genome-wide in Nasonia vitripennis and Nasonia giraulti and their reciprocal F1 hybrids. No parent-of-origin effect in allelic expression was found for >8,000 covered genes, suggesting a lack of genomic imprinting in adult Nasonia. As we expected, both significant cis- and trans- effects are responsible for the expression divergence between N. vitripennis and N. giraulti. Surprisingly, all 178 differentially methylated genes are also differentially methylated between the two alleles in F1 hybrid offspring, recapitulating the parental methylation status with nearly 100% fidelity, indicating the presence of strong cis-elements driving the target of gene body methylation. In addition, we discovered that total and allele-specific expression are positively correlated with allele-specific methylation in a subset of the differentially methylated genes. The 100% cis-regulation in F1 hybrids suggests the methylation machinery is conserved and DNA methylation is targeted by cis features in Nasonia. The lack of genomic imprinting and parent-of-origin differentially methylated regions in Nasonia, together with the stable inheritance of methylation status between generations, suggests either a cis-regulatory motif for methylation at the DNA level or highly stable inheritance of an epigenetic signal in Nasonia.

ITPKA Gene Body Methylation Regulates Gene Expression and Serves as an Early Diagnostic Marker in Lung and Other Cancers

IntroductionDespite recent advances in cancer therapy, the overall 5-year survival rate of patients with lung cancer remains low. The aim of our study was to search for novel markers for early diagnosis in patients with lung cancer. MethodsComplementary DNA microarray analysis was performed in primary lung adenocarcinomas and cell lines to search for differentially expressed genes, followed by in vivo and in vitro tumorigenic assays to characterize the oncogenic potential of the candidate genes. Gene body methylation was analyzed by 450K methylation array, bisulfite sequencing, and quantitative methylation-specific polymerase chain reaction assays. In silico analysis of The Cancer Genome Atlas data set was also performed. ResultsInositol-trisphosphate 3-kinase A gene (ITPKA), a kinase with limited tissue distribution, was identified as a potential oncogene. We showed that ITPKA expression is up-regulated in many forms of cancers, including lung and breast cancers, and that overexpressed ITPKA contributes to tumorigenesis. We also demonstrated that ITPKA expression is regulated by epigenetic DNA methylation of ITPKA gene body through modulation of the binding of SP1 transcription factor to the ITPKA promoter. ITPKA gene body displayed low or absent levels of methylation in most normal tissue but was significantly methylated in malignant tumors. In lung cancer, ITPKA gene body methylation first appeared at the in situ carcinoma stage and progressively increased after invasion. ConclusionsITPKA is a potential oncogene that it is overexpressed in most tumors, and its overexpression promotes tumorigenesis. ITPKA gene body methylation regulates its expression and thus serves as a novel and potential biomarker for early cancer detection.

Evolutionary Consequences of DNA Methylation in a Basal Metazoan.

Gene body methylation (gbM) is an ancestral and widespread feature in Eukarya, yet its adaptive value and evolutionary implications remain unresolved. The occurrence of gbM within protein-coding sequences is particularly puzzling, because methylation causes cytosine hypermutability and hence is likely to produce deleterious amino acid substitutions. We investigate this enigma using an evolutionarily basal group of Metazoa, the stony corals (order Scleractinia, class Anthozoa, phylum Cnidaria). We show that patterns of coral gbM are similar to other invertebrate species, predicting wide and active transcription and slower sequence evolution. We also find a strong correlation between gbM and codon bias, resulting from systematic replacement of CpG bearing codons. We conclude that gbM has strong effects on codon evolution and speculate that this may influence establishment of optimal codons.

Etiology matters - Genomic DNA Methylation Patterns in Three Rat Models of Acquired Epilepsy.

This study tested the hypothesis that acquired epileptogenesis is accompanied by DNA methylation changes independent of etiology. We investigated DNA methylation and gene expression in the hippocampal CA3/dentate gyrus fields at 3 months following epileptogenic injury in three experimental models of epilepsy: focal amygdala stimulation, systemic pilocarpine injection, or lateral fluid-percussion induced traumatic brain injury (TBI) in rats. In the models studies, DNA methylation and gene expression profiles distinguished controls from injured animals. We observed consistent increased methylation in gene bodies and hypomethylation at non-genic regions. We did not find a common methylation signature in all three different models and few regions common to any two models. Our data provide evidence that genome-wide alteration of DNA methylation signatures is a general pathomechanism associated with epileptogenesis and epilepsy in experimental animal models, but the broad pathophysiological differences between models (i.e. pilocarpine, amygdala stimulation, and post-TBI) are reflected in distinct etiology-dependent DNA methylation patterns.

Simultaneous profiling of transcriptome and DNA methylome from a single cell.

BackgroundSingle-cell transcriptome and single-cell methylome technologies have become powerful tools to study RNA and DNA methylation profiles of single cells at a genome-wide scale. A major challenge has been to understand the direct correlation of DNA methylation and gene expression within single-cells. Due to large cell-to-cell variability and the lack of direct measurements of transcriptome and methylome of the same cell, the association is still unclear.ResultsHere, we describe a novel method (scMT-seq) that simultaneously profiles both DNA methylome and transcriptome from the same cell. In sensory neurons, we consistently identify transcriptome and methylome heterogeneity among single cells but the majority of the expression variance is not explained by proximal promoter methylation, with the exception of genes that do not contain CpG islands. By contrast, gene body methylation is positively associated with gene expression for only those genes that contain a CpG island promoter. Furthermore, using single nucleotide polymorphism patterns from our hybrid mouse model, we also find positive correlation of allelic gene body methylation with allelic expression.ConclusionsOur method can be used to detect transcriptome, methylome, and single nucleotide polymorphism information within single cells to dissect the mechanisms of epigenetic gene regulation.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-016-0950-z) contains supplementary material, which is available to authorized users.

DNMT3B isoforms without catalytic activity stimulate gene body methylation as accessory proteins in somatic cells.

Promoter DNA methylation is a key epigenetic mechanism for stable gene silencing, but is correlated with expression when located in gene bodies. Maintenance and de novo DNA methylation by catalytically active DNA methyltransferases (DNMT1 and DNMT3A/B) require accessory proteins such as UHRF1 and DNMT3L. DNMT3B isoforms are widely expressed, although some do not have active catalytic domains and their expression can be altered during cell development and tumourigenesis, questioning their biological roles. Here, we show that DNMT3B isoforms stimulate gene body methylation and re-methylation after methylation-inhibitor treatment. This occurs independently of the isoforms' catalytic activity, demonstrating a similar functional role to the accessory protein DNMT3L, which is only expressed in undifferentiated cells and recruits DNMT3A to initiate DNA methylation. This unexpected role for DNMT3B suggests that it might substitute for the absent accessory protein DNMT3L to recruit DNMT3A in somatic cells.

Gene Body Methylation Patterns in Daphnia Are Associated with Gene Family Size.

The relation between gene body methylation and gene function remains elusive. Yet, our understanding of this relationship can contribute significant knowledge on how and why organisms target specific gene bodies for methylation. Here, we studied gene body methylation patterns in two Daphnia species. We observed both highly methylated genes and genes devoid of methylation in a background of low global methylation levels. A small but highly significant number of genes was highly methylated in both species. Remarkably, functional analyses indicate that variation in methylation within and between Daphnia species is primarily targeted to small gene families whereas large gene families tend to lack variation. The degree of sequence similarity could not explain the observed pattern. Furthermore, a significant negative correlation between gene family size and the degree of methylation suggests that gene body methylation may help regulate gene family expansion and functional diversification of gene families leading to phenotypic variation.

Tissue-independent and tissue-specific patterns of DNA methylation alteration in cancer.

Background There is growing evidence that DNA methylation alterations contribute to carcinogenesis. While cancer tissue exhibits widespread DNA methylation changes, the proportion of tissue-specific versus tissue-independent DNA methylation alterations in cancer is unclear. In addition, it is unknown which factors determine the patterns of aberrant DNA methylation in cancer.ResultsUsing HumanMethylation450 BeadChips (450k), we here analyze genome-wide DNA methylation patterns of ten types of fetal tissue, in addition to matched normal-cancer data for corresponding tissue types, encompassing over 3000 samples. We demonstrate that the level of aberrant cancer DNA methylation in gene promoters and gene bodies is highly correlated between cancer types. We estimate that up to 60 % of the DNA methylation variation in a cancer genome of a given tissue type is explained by the corresponding variation in a cancer genome of another type, implying that much of the cancer DNA methylation landscape is tissue independent. We further show that histone marks in normal cells are better predictors of aberrant cancer DNA methylation than the corresponding signals in human embryonic stem cells. We build predictors of cancer DNA methylation patterns and show that although inclusion of three histone marks (H3K4me3, H3K27me3 and H3K36me3) improves model accuracy, the bivalent marks are the most predictive. Finally, we show that chromatin accessibility of gene promoters in normal tissue dictates the promoter’s propensity to acquire aberrant DNA methylation in cancer in so far as it determines its level of DNA methylation in normal tissue.ConclusionsOur data show that a considerable fraction of the aberrant cancer DNA methylation landscape results from a mechanism that is largely tissue specific. Histone marks as specified in the normal cell of origin provide highly predictive models of aberrant cancer DNA methylation and outperform those derived from the same marks in hESCs.Electronic supplementary materialThe online version of this article (doi:10.1186/s13072-016-0058-4) contains supplementary material, which is available to authorized users.

Roles of Distal and Genic Methylation in the Development of Prostate Tumorigenesis Revealed by Genome-wide DNA Methylation Analysis.

Aberrant DNA methylation at promoters is often linked to tumorigenesis. But many aspects of DNA methylation remain unexplored, including the individual roles of distal and gene body methylation, as well as their collaborative roles with promoter methylation. Here we performed a MBD-seq analysis on prostate specimens classified into low, high, and very high risk group based on Gleason score and TNM stages. We identified gene sets with differential methylation regions (DMRs) in Distal, TSS, gene body and TES. To understand the collaborative roles, TSS was compared with the other three DMRs, resulted in 12 groups of genes with collaborative differential methylation patterns (CDMPs). We found several groups of genes that show opposite methylation patterns in Distal and Genic regions compared to TSS region, and in general they are differentially expressed genes (DEGs) in tumors in TCGA RNA-seq data. IPA (Ingenuity Pathway Analysis) reveals AR/TP53 signaling network to be a major signaling pathway, and survival analysis indicates genes subsets significantly associated with prostate cancer recurrence. Our results suggest that DNA methylation in Distal and Genic regions also plays critical roles in contributing to prostate tumorigenesis, and may act either positively or negatively with TSSs to alter gene regulation in tumors.

Germline DNA methylation in reef corals: patterns and potential roles in response to environmental change.

DNA methylation is an epigenetic mark that plays an inadequately understood role in gene regulation, particularly in nonmodel species. Because it can be influenced by the environment, DNA methylation may contribute to the ability of organisms to acclimatize and adapt to environmental change. We evaluated the distribution of gene body methylation in reef-building corals, a group of organisms facing significant environmental threats. Gene body methylation in six species of corals was inferred from in silico transcriptome analysis of CpG O/E, an estimate of germline DNA methylation that is highly correlated with patterns of methylation enrichment. Consistent with what has been documented in most other invertebrates, all corals exhibited bimodal distributions of germline methylation suggestive of distinct fractions of genes with high and low levels of methylation. The hypermethylated fractions were enriched with genes with housekeeping functions, while genes with inducible functions were highly represented in the hypomethylated fractions. High transcript abundance was associated with intermediate levels of methylation. In three of the coral species, we found that genes differentially expressed in response to thermal stress and ocean acidification exhibited significantly lower levels of methylation. These results support a link between gene body hypomethylation and transcriptional plasticity that may point to a role of DNA methylation in the response of corals to environmental change.

Chilling-Mediated DNA Methylation Changes during Dormancy and Its Release Reveal the Importance of Epigenetic Regulation during Winter Dormancy in Apple (Malus x domestica Borkh.).

Winter dormancy is a well known mechanism adopted by temperate plants, to mitigate the chilling temperature of winters. However, acquisition of sufficient chilling during winter dormancy ensures the normal phenological traits in subsequent growing period. Thus, low temperature appears to play crucial roles in growth and development of temperate plants. Apple, being an important temperate fruit crop, also requires sufficient chilling to release winter dormancy and normal phenological traits, which are often associated with yield and quality of fruits. DNA cytosine methylation is one of the important epigenetic modifications which remarkably affect the gene expression during various developmental and adaptive processes. In present study, methylation sensitive amplified polymorphism was employed to assess the changes in cytosine methylation during dormancy, active growth and fruit set in apple, under differential chilling conditions. Under high chill conditions, total methylation was decreased from 27.2% in dormant bud to 21.0% in fruit set stage, while no significant reduction was found under low chill conditions. Moreover, the demethylation was found to be decreased, while methylation increased from dormant bud to fruit set stage under low chill as compared to high chill conditions. In addition, RNA-Seq analysis showed high expression of DNA methyltransferases and histone methyltransferases during dormancy and fruit set, and low expression of DNA glcosylases during active growth under low chill conditions, which was in accordance with changes in methylation patterns. The RNA-Seq data of 47 genes associated with MSAP fragments involved in cellular metabolism, stress response, antioxidant system and transcriptional regulation showed correlation between methylation and their expression. Similarly, bisulfite sequencing and qRT-PCR analysis of selected genes also showed correlation between gene body methylation and gene expression. Moreover, significant association between chilling and methylation changes was observed, which suggested that chilling acquisition during dormancy in apple is likely to affect the epigenetic regulation through DNA methylation.

Abstract A45: Connecting alternative transcript usage with differential DNA methylation in aggressive prostate cancer

Abstract The information content of the human transcriptome is expanded by the presence of alternative mRNA isoforms produced by alternative transcription initiation, alternative splicing, and alternative polyadenylation. Alternative isoforms can possess distinct oncogenic and tumor suppressive functions by altering gene products and may therefore relate to aggressiveness of disease. A major challenge in prostate cancer biology is the need to distinguish indolent from aggressive disease. Elucidating the molecular determinants of aggressiveness has major implications for developing new genomic diagnostics for clinicians to use, and epigenetic regulation of gene expression that results in more aggressive cancers are compelling drug targets. Here, we hypothesize that differential DNA methylation in gene bodies regulates the production of tumor-promoting alternative transcription events in aggressive prostate cancer. DNA methylation may serve a regulatory role in concert with regulatory proteins and sequence features because transcription start site selection and alternative splicing occur co-transcriptionally. To detect interdependencies between differential DNA methylation and alternative isoforms, we utilized RNA-seq and Illumina HumanMethylation 450K array data produced by The Cancer Genome Atlas for 537 normal prostate and prostate cancer tissue specimens. Reference-guided de novo transcriptome assembly was performed, and fractional usage was computed at both the exon and isoform level for 4,734 expressed genes that contain gene-body differential methylation sites on the 450K array. We identified a set of 120 genes with correlations (|Spearman correlation| > 0.5, FDR adj. p-value < 0.05 and difference > 5% between fractional usage when grouped by the top and bottom quintile of DNA methylation) between the exon or isoform usage fraction and gene body CpG site methylation level. Of these, 53 genes contain differential DNA methylation and exon or isoform usage specific to aggressive disease. This gene set includes SEPT9, which is known to have a cell migration promoting alternative promoter regulated by with DNA methylation changes in breast cancer, as well as two genes with prior links to prostate cancer aggressiveness (MT2A and TDRD1). At the exon level, there were 6 cases where at least one differential methylation site associated with first exon usage, 7 with last exons, and 121 with internal exons. At the isoform level, 92 isoforms that associated with gene body DNA methylation contained alternative promoter usage, 80 contained alternative end usage, 71 contained alternative splice donor/acceptor sites, and 82 contained exon skipping/inclusion events. Additionally, 4 isoforms were predicted to be targets for nonsense mediated decay (NMD) by inducing message frameshifts that reveal internal stop codons. The diversity of this distribution suggests that DNA methylation may interact with a variety of different regulatory mechanisms that occur co-transcriptionally. While the 450K array only covers a small fraction of gene body CpG sites, our analysis supports the possibility of alternative isoform usage regulated by prostate cancer-specific and aggressiveness subtype-specific DNA methylation changes. Future work will interrogate the exact nature of the mechanisms by which DNA methylation changes cross talk with regulatory factors in alternative splicing, promoter usage, and polyadenylation. Such mechanisms may be targetable pharmacologically for tumor suppressive effect. Citation Format: Jeffrey M. Bhasin, Angela H. Ting. Connecting alternative transcript usage with differential DNA methylation in aggressive prostate cancer. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr A45.

DNA methylation in the pathophysiology of hyperphenylalaninemia in the PAH(enu2) mouse model of phenylketonuria.

Phenylalanine hydroxylase deficient phenylketonuria (PKU) is the paradigm for a treatable inborn error of metabolism where maintaining plasma phenylalanine (Phe) in the therapeutic range relates to improved clinical outcomes. While Phe is the presumed intoxicating analyte causal in neurologic damage, the mechanism(s) of Phe toxicity has remained elusive. Altered DNA methylation is a recognized response associated with exposure to numerous small molecule toxic agents. Paralleling this effect, we hypothesized that chronic Phe over-exposure in the brain would lead to aberrant DNA methylation with secondary influence upon gene regulation that would ultimately contribute to PKU neuropathology. The PAH(enu2) mouse models human PKU with intrinsic hyperphenylalaninemia, abnormal response to Phe challenge, and neurologic deficit. To examine this hypothesis, we assessed DNA methylation patterns in brain tissues using methylated DNA immunoprecipitation and paired end sequencing in adult PAH(enu2) animals maintained under either continuous dietary Phe restriction or chronic hyperphenylalaninemia. Heterozygous PAH(enu2/WT) litter mates served as controls for normal Phe exposure. Extensive repatterning of DNA methylation was observed in brain tissue of hyperphenylalaninemic animals while Phe restricted animals displayed an attenuated pattern of aberrant DNA methylation. Affected gene coding regions displayed aberrant hypermethylation and hypomethylation. Gene body methylation of noncoding RNA genes was observed and among these microRNA genes were prominent. Of particular note, observed only in hyperphenylalaninemic animals, was hypomethylation of miRNA genes within the imprinted Dlk1-Dio3 locus on chromosome 12. Aberrant methylation of microRNA genes influenced their expression which has secondary effects upon the expression of targeted protein coding genes. Differential hypermethylation of gene promoters was exclusive to hyperphenylalaninemic PAH(enu2) animals. Genes with synaptic involvement were targets of promoter hypermethylation that resulted in down-regulation of their expression. Gene dysregulation secondary to abnormal DNA methylation may be contributing to PKU neuropathology. These results suggest drugs that prevent or correct aberrant DNA methylation may offer a novel therapeutic option to management of neurological symptoms in PKU patients.

DNA Methylation and Gene Regulation in Honeybees: From Genome-Wide Analyses to Obligatory Epialleles.

In contrast to heavily methylated mammalian genomes, invertebrate genomes are only sparsely methylated in a 'mosaic' fashion with the majority of methylated CpG dinucleotides found across gene bodies. Importantly, this gene body methylation is frequently associated with active transcription, and studies in the honeybee have shown that there are strong links between gene body methylation and alternative splicing. Additional work also highlights that obligatory methylated epialleles influence transcriptional changes in a context-specific manner. Here we discuss the current knowledge in this emerging field and highlight both similarities and differences between DNA methylation systems in mammals and invertebrates. Finally, we argue that the relationship between genetic variation, differential DNA methylation, other epigenetic modifications and the transcriptome must be further explored to fully understand the role of DNA methylation in converting genomic sequences into phenotypes.

Combination Treatment of AML Cells with Dnmt and HDAC Inhibitors: Synergistic Target Gene Downregulation Associated with Gene Body Demethylation

Background: Hypermethylation of CpG-rich tumor suppressor gene promoters constitutes a prototypical cancer-specific epigenetic abnormality. Recently, methylome-wide studies have provided evidence of extensive aberrant DNA methylation also in gene bodies and intergenic regions; their functional role is still under debate. DNA hypomethylating agents (HMAs) such as 5-aza-CdR (DAC) and 5-aza-CR are thought to act particularly by reactivating hypermethylated promoters of epigenetically silenced genes, and this activity is enhanced by combining HMAs with histone deacetylase inhibitors (HDACi). However, transcriptome-wide studies have shown that HMAs can also downregulate mRNAs. Recently, Yang et al. (Cancer Cell 2014) showed that gene body methylation can be strikingly reversed by DAC in human colon cancer cells, with remarkable downregulation of genes involved in metabolic processes regulated by c-MYC, implicating gene body methylation as a drug target. Therefore, we asked whether HMA-induced reversal not only of promoter but also of gene body methylation occurs in AML cells, and whether gene body demethylation is preferentially associated with gene repression rather than activation. We further asked whether the synergism between HMAs and HDACi in gene reactivation might also be observed for gene repression.Materials and Methods: U937 cells were treated with 3x24h pulses of DAC (50nM), alone or in combination with the HDACi panobinostat (Pano, 8nM) or Valproic acid (VPA, 1mM) for 24h with low-cytotoxic (>80% viability), <25% growth-inhibitory concentrations. Transcriptome and methylome profiles (biological triplicates) were generated using Affymetrix Human Gene 2.0 ST and Infinium HumanMethylation450 BeadChip arrays. Data were analyzed using R package RnBeads applying standard methods.Results: Transcriptome analyses revealed that DAC, Pano or VPA alone induced 207, 267 and 179 transcripts, with downregulation of 28, 45 and 22 transcripts, respectively (FDR<0.01; ≥2-fold change). When DAC was combined with either Pano (D+P) or VPA (D+V), a striking synergistic effect was observed for upregulated transcripts (1069 and 680). Remarkably, a synergistic effect on downregulated transcripts was even stronger (667 and 233 transcripts). As expected, DAC treatment resulted in extensive DNA demethylation: 295.6K CpGs (89% of 331.8K CpGs hypermethylated >30% in untreated U937) became >10% demethylated (FDR<0.01), with demethylation of additional 8.7K CpGs (2.6%) and 5.7K CpGs (1.7%) with D+P and D+V, respectively.Most of the differentially expressed transcripts mapped to demethylated CpGs located in both promoters and gene bodies. We focussed on up- and downregulated transcripts exclusively associated with either promoter- or gene body-demethylated CpGs. The ratio of transcripts mapping to promoter- vs. gene body-demethylated CpGs doubled from up- to downregulated transcripts (all 3 treatments). Thus, for upregulated transcripts these ratios were 1: 1.9 (DAC), 1: 3.5 (D+P) and 1: 3.4 (D+V); for downregulated transcripts 1: 4.0 (DAC), 1: 7.0 (D+P) and 1: 6.9 (D+V). Also, the combination treatments resulted in a clear shift to more transcripts mapping to gene body-demethylated CpGs than DAC treatment alone. Demethylated CpGs located in shore regions of gene bodies were particularly enriched in downregulated transcripts upon combination treatment, suggesting a possible mechanism of their regulation.Of the 667 transcripts downregulated by D+P, the 227 gene-body demethylation-associated transcripts included various putative or bona fide oncogenes (such as c-MYC) and epigenetic modifiers. Six potential candidates were chosen for validation by RT-qPCR. Gene ontology analysis revealed an enrichment for genes involved in RNA processing and transcription.Conclusions: The combination of a HMA with two different HDACi resulted not only in synergistic gene reactivation but also in massive, synergistic downregulation of genes including oncogenes, which was associated predominantly with gene body demethylation. Taken together, these findings have implications not only regarding the mechanisms of action of combined epigenetic treatment, but also for a better understanding of clinical responses in trials where this approach is tested, such as the randomized DECIDER AML trial (NCT00867672), by analyzing in vivo epigenetic drug effects on primary blasts. DisclosuresLübbert:Janssen: Honoraria, Other: travel support; Celgene: Honoraria, Other: travel support; TEVA: Other: travel support.

H3K4/H3K9me3 Bivalent Chromatin Domains Targeted by Lineage-Specific DNA Methylation Pauses Adipocyte Differentiation

Bivalent H3K4me3 and H3K27me3 chromatin domains in embryonic stem cells keep active developmental regulatory genes expressed at very low levels and poised for activation. Here, we show an alternative and previously unknown bivalent modified histone signature in lineage-committed mesenchymal stem cells and preadipocytes that pairs H3K4me3 with H3K9me3 to maintain adipogenic master regulatory genes (Cebpa and Pparg) expressed at low levels yet poised for activation when differentiation is required. We show lineage-specific gene-body DNA methylation recruits H3K9 methyltransferase SETDB1, which methylates H3K9 immediately downstream of transcription start sites marked with H3K4me3 to establish the bivalent domain. At the Cebpa locus, this prevents transcription factor C/EBPβ binding, histone acetylation, and further H3K4me3 deposition and is associated with pausing of RNA polymerase II, which limits Cebpa gene expression and adipogenesis.