Extensive DNA Methylation Changes Research Articles

Epigenetic responses to heat: From adaptation to maladaptation.

What is the topic of this review? This review outlines the history of research on epigenetic adaptations to heat exposure. The perspective taken is that adaptations reflect properties of hormesis, whereby low, repeated doses of heat induce adaptation (acclimation/acclimatization); whereas brief, life-threatening exposures can induce maladaptive responses. What advances does it highlight? The epigenetic mechanisms underlying acclimation/acclimatization comprise specific molecular programmes on histones that regulate heat shock proteins transcriptionally and protect the organism from subsequent heat exposures, even after long delays. The epigenetic signalling underlying maladaptive responses might rely, in part, on extensive changes in DNA methylation that are sustained over time and might contribute to later health challenges. Epigenetics plays a strong role in molecular adaptations to heat by producing a molecular memory of past environmental exposures. Moderate heat, over long periods of time, induces an 'adaptive' epigenetic memory, resulting in a condition of 'resilience' to future heat exposures or cross-tolerance to other forms of toxic stress. In contrast, intense, life-threatening heat exposures, such as severe heat stroke, can result in a 'maladaptive' epigenetic memory that can place an organism at risk of later health complications. These cellular memories are coded by post-translational modifications of histones on the nucleosomes and/or by changes in DNA methylation. They operate by inducing changes in the level of gene transcription and therefore phenotype. The adaptive response to heat acclimation functions, in part, by facilitating transcription of essential heat shock proteins and exhibits a biphasic short programme (maintaining DNA integrity, followed by a long-term consolidation). The latter accelerates acclimation responses after de-acclimation. Although less studied, the maladaptive responses to heat stroke appear to be coded in long-lasting changes in DNA methylation near the promoter region of genes involved with basic cell function. Whether these memories are also encoded in histone modifications is not yet known. There is considerable evidence that both adaptive and maladaptive epigenetic responses to heat can be inherited, although most evidence comes from lower organisms. Future challenges include understanding the signalling mechanisms responsible and discovering new ways to promote adaptive responses while suppressing maladaptive responses to heat, as all life forms adapt to life on a warming planet.

Intra- and intergenerational changes in the cortical DNA methylome in response to therapeutic intermittent hypoxia in mice.

Recent evidence from our laboratory documents functional resilience to retinal ischemic injury in untreated mice derived from parents exposed to repetitive hypoxic conditioning (RHC) before breeding. To begin to understand the epigenetic basis of this intergenerational protection, we used methylated DNA immunoprecipitation and sequencing to identify genes with differentially methylated promoters (DMGPs) in the prefrontal cortex of mice treated directly with the same RHC stimulus (F0-RHC) and in the prefrontal cortex of their untreated F1-generation offspring (F1-*RHC). Subsequent bioinformatic analyses provided key mechanistic insights into how changes in gene expression secondary to promoter hypo- and hypermethylation might afford such protection within and across generations. We found extensive changes in DNA methylation in both generations consistent with the expression of many survival-promoting genes, with twice the number of DMGPs in the cortex of F1*RHC mice relative to their F0 parents that were directly exposed to RHC. In contrast to our hypothesis that similar epigenetic modifications would be realized in the cortices of both F0-RHC and F1-*RHC mice, we instead found relatively few DMGPs common to both generations; in fact, each generation manifested expected injury resilience via distinctly unique gene expression profiles. Whereas in the cortex of F0-RHC mice, predicted protein-protein interactions reflected activation of an anti-ischemic phenotype, networks activated in F1-*RHC cortex comprised networks indicative of a much broader cytoprotective phenotype. Altogether, our results suggest that the intergenerational transfer of an acquired phenotype to offspring does not necessarily require the faithful recapitulation of the conditioning-modified DNA methylome of the parent.

PM2.5-induced extensive DNA methylation changes in the heart of zebrafish embryos and the protective effect of folic acid

We previously found that folic acid (FA) attenuated cardiac defects in zebrafish embryos exposed to extractable organic matter (EOM) from PM2.5, but the underlining mechanisms remain to be elucidated. Since DNA methylation is crucial to cardiac development, we hypothesized that EOM-induced aberrant DNA methylation changes could be diminished by FA supplementation. In this study, zebrafish embryos were exposed to EOM in the absence or presence of FA. Genomic-wide DNA methylation analysis identified both DNA hypo- and hyper-methylation changes in CCGG sites in zebrafish embryos exposed to EOM, which were attenuated by FA supplementation. We identified a total of 316 genes with extensive DNA methylation changes in EOM samples but little or no DNA methylation changes in EOM plus FA samples. The genes were involved in critical cellular processes and signaling pathways important for embryo development. In addition, the EOM-decreased SAM/SAH ratio was counteracted by FA supplementation. Furthermore, FA attenuated the EOM-induced changes in the expression of genes involved in the regulation of DNA methylation and in folate biosynthesis. In conclusion, our data suggest that FA supplementation protected zebrafish embryos from the cardiac developmental toxicity of PM2.5 by alleviating EOM-induced DNA methylation changes.

Signal Integration and Transcriptional Regulation of the Inflammatory Response Mediated by the GM-/M-CSF Signaling Axis in Human Monocytes

In recent years, the macrophage colony-stimulating factor (M-CSF) and granulocyte-macrophage CSF (GM-CSF) cytokines have been identified as opposing regulators of the inflammatory program. However, the two cytokines are simultaneously present in the inflammatory milieu, and it is not clear how cells integrate these signals. In order to understand the regulatory networks associated with the GM/M-CSF signaling axis, we analyzed DNA methylation in human monocytes. Our results indicate that GM-CSF induces activation of the inflammatory program and extensive DNA methylation changes, while M-CSF-polarized cells are in a less differentiated state. This inflammatory program is mediated via JAK2 associated with the GM-CSF receptor and the downstream extracellular signal-regulated (ERK) signaling. However, PI3K signaling is associated with a negative regulatory loop of the inflammatory program and M-CSF autocrine signaling in GM-CSF-polarized monocytes. Our findings describe the regulatory networks associated with the GM/M-CSF signaling axis and how they contribute to the establishment of the inflammatory program associated with monocyte activation.

\u201cDNA Methylation signatures in panic disorder\u201d

Panic disorder (PD) affects about four million Europeans, with women affected twice as likely as men, causing substantial suffering and high economic costs. The etiopathogenesis of PD remains largely unknown, but both genetic and environmental factors contribute to risk. An epigenome-wide association study (EWAS) was conducted to compare medication-free PD patients (n = 89) with healthy controls (n = 76) stratified by gender. Replication was sought in an independent sample (131 cases, 169 controls) and functional analyses were conducted in a third sample (N = 71). DNA methylation was assessed in whole blood using the Infinium HumanMethylation450 BeadChip. One genome-wide association surviving FDR of 5% (cg07308824, P = 1.094 × 10-7, P-adj = 0.046) was identified in female PD patients (N = 49) compared to controls (N = 48). The same locus, located in an enhancer region of the HECA gene, was also hypermethylated in female PD patients in the replication sample (P = 0.035) and the significance of the association improved in the meta-analysis (P-adj = 0.004). Methylation at this CpG site was associated with HECA mRNA expression in another independent female sample (N = 71) both at baseline (P = 0.046) and after induction by dexamethasone (P = 0.029). Of 15 candidates, 5 previously reported as associated with PD or anxiety traits also showed differences in DNA methylation after gene-wise correction and included SGK1, FHIT, ADCYAP1, HTR1A, HTR2A. Our study examines epigenome-wide differences in peripheral blood for PD patients. Our results point to possible sex-specific methylation changes in the HECA gene for PD but overall highlight that this disorder is not associated with extensive changes in DNA methylation in peripheral blood.

Charting the dynamic epigenome during B-cell development.

The epigenetic landscape undergoes a widespread modulation during embryonic development and cell differentiation. Within the hematopoietic system, B cells are perhaps the cell lineage with a more dynamic DNA methylome during their maturation process, which involves approximately one third of all the CpG sites of the genome. Although each B-cell maturation step displays its own DNA methylation fingerprint, the DNA methylome is more extensively modified in particular maturation transitions. These changes are gradually accumulated in specific chromatin environments as cell differentiation progresses and reflect different features and functional states of B cells. Promoters and enhancers of B-cell transcription factors acquire activation-related epigenetic marks and are sequentially expressed in particular maturation windows. These transcription factors further reconfigure the epigenetic marks and activity state of their target sites to regulate the expression of genes related to B-cell functions. Together with this observation, extensive DNA methylation changes in areas outside gene regulatory elements such as hypomethylation of heterochromatic regions and hypermethylation of CpG-rich regions, also take place in mature B cells, which intriguingly have been described as hallmarks of cancer. This process starts in germinal center B cells, a highly proliferative cell type, and becomes particularly apparent in long-lived cells such as memory and plasma cells. Overall, the characterization of the DNA methylome during B-cell differentiation not only provides insights into the complex epigenetic network of regulatory elements that mediate the maturation process but also suggests that late B cells also passively accumulate epigenetic changes related to cell proliferation and longevity.

A DNA methylation map of human cancer at single base-pair resolution

Although single base-pair resolution DNA methylation landscapes for embryonic and different somatic cell types provided important insights into epigenetic dynamics and cell-type specificity, such comprehensive profiling is incomplete across human cancer types. This prompted us to perform genome-wide DNA methylation profiling of 22 samples derived from normal tissues and associated neoplasms, including primary tumors and cancer cell lines. Unlike their invariant normal counterparts, cancer samples exhibited highly variable CpG methylation levels in a large proportion of the genome, involving progressive changes during tumor evolution. The whole-genome sequencing results from selected samples were replicated in a large cohort of 1112 primary tumors of various cancer types using genome-scale DNA methylation analysis. Specifically, we determined DNA hypermethylation of promoters and enhancers regulating tumor-suppressor genes, with potential cancer-driving effects. DNA hypermethylation events showed evidence of positive selection, mutual exclusivity and tissue specificity, suggesting their active participation in neoplastic transformation. Our data highlight the extensive changes in DNA methylation that occur in cancer onset, progression and dissemination.

Aberrant epigenome in iPSC-derived dopaminergic neurons from Parkinson's disease patients.

The epigenomic landscape of Parkinson's disease (PD) remains unknown. We performed a genomewide DNA methylation and a transcriptome studies in induced pluripotent stem cell (iPSC)-derived dopaminergic neurons (DAn) generated by cell reprogramming of somatic skin cells from patients with monogenic LRRK2-associated PD (L2PD) or sporadic PD (sPD), and healthy subjects. We observed extensive DNA methylation changes in PD DAn, and of RNA expression, which were common in L2PD and sPD. No significant methylation differences were present in parental skin cells, undifferentiated iPSCs nor iPSC-derived neural cultures not-enriched-in-DAn. These findings suggest the presence of molecular defects in PD somatic cells which manifest only upon differentiation into the DAn cells targeted in PD. The methylation profile from PD DAn, but not from controls, resembled that of neural cultures not-enriched-in-DAn indicating a failure to fully acquire the epigenetic identity own to healthy DAn in PD. The PD-associated hypermethylation was prominent in gene regulatory regions such as enhancers and was related to the RNA and/or protein downregulation of a network of transcription factors relevant to PD (FOXA1, NR3C1, HNF4A, and FOSL2). Using a patient-specific iPSC-based DAn model, our study provides the first evidence that epigenetic deregulation is associated with monogenic and sporadic PD.

The DNA methylome and transcriptome of different brain regions in schizophrenia and bipolar disorder.

Extensive changes in DNA methylation have been observed in schizophrenia (SC) and bipolar disorder (BP), and may contribute to the pathogenesis of these disorders. Here, we performed genome-scale DNA methylation profiling using methylated DNA immunoprecipitation followed by sequencing (MeDIP-seq) on two brain regions (including frontal cortex and anterior cingulate) in 5 SC, 7 BP and 6 normal subjects. Comparing with normal controls, we identified substantial differentially methylated regions (DMRs) in these two brain regions of SC and BP. To our surprise, different brain regions show completely distinct distributions of DMRs across the genomes. In frontal cortex of both SC and BP subjects, we observed widespread hypomethylation as compared to normal controls, preferentially targeting the terminal ends of the chromosomes. In contrast, in anterior cingulate, both SC and BP subjects displayed extensive gain of methylation. Notably, in these two brain regions of SC and BP, only a few DMRs overlapped with promoters, whereas a greater proportion occurs in introns and intergenic regions. Functional enrichment analysis indicated that important psychiatric disorder-related biological processes such as neuron development, differentiation and projection may be altered by epigenetic changes located in the intronic regions. Transcriptome analysis revealed consistent dysfunctional processes with those determined by DMRs. Furthermore, DMRs in the same brain regions from SC and BP could successfully distinguish BP and/or SC from normal controls while differentially expressed genes could not. Overall, our results support a major role for brain-region-dependent aberrant DNA methylation in the pathogenesis of these two disorders.

Extensive changes in DNA methylation are associated with expression of mutant huntingtin

The earliest stages of Huntington disease are marked by changes in gene expression that are caused in an indirect and poorly understood manner by polyglutamine expansions in the huntingtin (HTT) protein. To explore the hypothesis that DNA methylation may be altered in cells expressing mutated HTT, we use reduced representation bisulfite sequencing (RRBS) to map sites of DNA methylation in cells carrying either wild-type or mutant HTT. We find that a large fraction of the genes that change in expression in the presence of mutant huntingtin demonstrate significant changes in DNA methylation. Regions with low CpG content, which have previously been shown to undergo methylation changes in response to neuronal activity, are disproportionately affected. On the basis of the sequence of regions that change in methylation, we identify AP-1 and SOX2 as transcriptional regulators associated with DNA methylation changes, and we confirm these hypotheses using genome-wide chromatin immunoprecipitation sequencing (ChIP-Seq). Our findings suggest new mechanisms for the effects of polyglutamine-expanded HTT. These results also raise important questions about the potential effects of changes in DNA methylation on neurogenesis and cognitive decline in patients with Huntington disease.

Regions of focal DNA hypermethylation and long-range hypomethylation in colorectal cancer coincide with nuclear lamina–associated domains

Extensive changes in DNA methylation are common in cancer and may contribute to oncogenesis through transcriptional silencing of tumor-suppressor genes. Genome-scale studies have yielded important insights into these changes but have focused on CpG islands or gene promoters. We used whole-genome bisulfite sequencing (bisulfite-seq) to comprehensively profile a primary human colorectal tumor and adjacent normal colon tissue at single-basepair resolution. Regions of focal hypermethylation in the tumor were located primarily at CpG islands and were concentrated within regions of long-range (>100 kb) hypomethylation. These hypomethylated domains covered nearly half of the genome and coincided with late replication and attachment to the nuclear lamina in human cell lines. We confirmed the confluence of hypermethylation and hypomethylation within these domains in 25 diverse colorectal tumors and matched adjacent tissue. We propose that widespread DNA methylation changes in cancer are linked to silencing programs orchestrated by the three-dimensional organization of chromatin within the nucleus.

Epigenomic Profiling of Multiple Myeloma Shows Widespread Stage Specific Alterations In DNA Methylation That Occur Early During Myelomagenesis

AbstractAbstract 784Gene expression is a tightly regulated process and is influenced by aberrant epigenetic changes that can lead to carcinogenesis. We used the HELP (HpaII tiny fragment Enrichment by Ligation-mediated PCR) assay to perform an unbiased genome-wide analysis of DNA methylation in 11 MGUS, 16 newly diagnosed myeloma, 17 relapsed myeloma, and 8 healthy control samples. The HELP assay uses differential methylation specific restriction digestion by HpaII and MspI followed by amplification, 2 color labeling, and co-hybridization to quantitatively determine individual promoter methylation of 25,626 loci. The methylome analysis was performed using CD 138+ sorted bone marrow plasma cells in all cases. We observed extensive DNA methylation changes in myeloma that were seen even in MGUS cases when compared to normal plasma cells. Unsupervised clustering of all samples showed that MGUS samples were distinct but epigenetically closer to normal plasma cells than to cases of newly diagnosed or relapsed Myeloma. MGUS cases were characterized predominantly by aberrant hypomethylation, with 456 hypomethylated probes versus 130 hypermethylated probes (cutoff criteria were defined as a difference of means > 1.5 and significance of this difference with p < 0.001, Figure 1) that affected pathways regulated by NF-kb transcription factor. Untreated newly diagnosed myeloma samples were also predominantly hypomethylated (461 hypomethylated probes, 83 hypermethylated probes) when compared to controls. In addition to NF-kB, the MAP kinase and PI3 kinase regulated pathways were affected by hypomethylated genes. In contrast, cases of relapsed myeloma showed predominantly hypermethylated loci (221 hypomethylated, 560 hypermethylated probes) when compared to controls and involved the TNF and retinoblastoma pathways. A large number of important genes including the tumor suppressors CDKN2A and CDKN2B were aberrantly hypermethylated in this cohort. [Display omitted] Analysis of our differentially methylated targets using the Molecular Signatures Database (MSigDB, Tamayo, et al. 2005, PNAS 102, 15545–15550), showed significant overlap for hypomethylated genes in MGUS and new multiple myeloma (MM). These gene sets contain genes with promoter regions around transcription start site containing motifs which match annotation for transcription factors SP1 and TCF3 as well as enrichment for genes with gene ontology annotations for plasma membrane proteins. Hypermethylated genes in new MM show significant overlap with genes in the neighborhood of RAD23A, CTBP1, G22P1 and SMC1, suggesting impairment of DNA repair, as well as enrichment for genes associated with apoptosis and programmed cell death.In conclusion, genome-wide DNA methylation analysis is able to clearly differentiate between normal bone marrow plasma cells, MGUS as well as new MM and relapsed MM cells. Correlation of significantly differentially methylated genes with published gene sets reveals enrichment for genes involved in DNA repair, cell-cell signaling, cell death, apoptosis and cell cycle regulation. Disclosures:Mehta:Celgene: Consultancy, Speakers Bureau; Takeda/Millennium: Speakers Bureau; Onyx: Research Funding. Singhal:Takeda/Millennium: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Onyx: Research Funding; Celgene: Speakers Bureau.

Extent and pattern of DNA methylation alteration in rice lines derived from introgressive hybridization of rice and Zizania latifolia Griseb

We have reported previously that introgression by Zizania latifolia resulted in extensive DNA methylation changes in the recipient rice genome, as detected by a set of pre-selected DNA segments. In this study, using the methylation-sensitive amplified polymorphism (MSAP) method, we globally assessed the extent and pattern of cytosine methylation alterations in three typical introgression lines relative to their rice parent at approximately 2,700 unbiased genomic loci each representing a recognition site cleaved by one or both of the isoschizomers, HpaII/MspI. Based on differential digestion by the isoschizomers, it is estimated that 15.9% of CCGG sites are either fully methylated at the internal Cs and/or hemi-methylated at the external Cs in the rice parental cultivar Matsumae. In comparison, a statistically significant increase in the overall level of both methylation types was detected in all three studied introgression lines (19.2, 18.6, 19.6%, respectively). Based on comparisons of MSAP profiles between the isoschizomers within the rice parent and between parent and the introgression lines, four major groups of MSAP banding patterns are recognized, which can be further divided into various subgroups as a result of inheritance of, or variation in, parental methylation patterns. The altered methylation patterns include hyper- and hypomethylation changes, as well as inter-conversion of hemi- to full-methylation, or vice versa, at the relevant CCGG site(s). Most alterations revealed by MSAP in low-copy loci can be validated by DNA gel blot analysis. The changed methylation patterns are uniform among randomly selected individuals for a given introgression line within or among selfed generations. Sequencing on 31 isolated fragments that showed different changing patterns in the introgression line(s) allowed their mapping onto variable regions on one or more of the 12 rice chromosomes. These segments include protein-coding genes, transposon/retrotransposons and sequences with no homology. Possible causes for the introgression-induced methylation changes and their implications for genome evolution and crop breeding are discussed.