Genome-wide DNA Methylation Differences Research Articles

Epigenome-wide differences in pathology-free regions of multiple sclerosis-affected brains.

Using the Illumina 450K array and a stringent statistical analysis with age and gender correction, we report genome-wide differences in DNA methylation between pathology-free regions derived from human multiple sclerosis-affected and control brains. Differences were subtle, but widespread and reproducible in an independent validation cohort. The transcriptional consequences of differential DNA methylation were further defined by genome-wide RNA-sequencing analysis and validated in two independent cohorts. Genes regulating oligodendrocyte survival, such as BCL2L2 and NDRG1, were hypermethylated and expressed at lower levels in multiple sclerosis-affected brains than in controls, while genes related to proteolytic processing (for example, LGMN, CTSZ) were hypomethylated and expressed at higher levels. These results were not due to differences in cellular composition between multiple sclerosis and controls. Thus, epigenomic changes in genes affecting oligodendrocyte susceptibility to damage are detected in pathology-free areas of multiple sclerosis-affected brains.

Abstract B24: Ras-mediated epigenetic reprogramming: NF-κB-dependent and independent effects

Abstract In addition to many genetic alterations, cancer cells also harbor global epigenetic abnormalities, a fundamental hallmark of cancer. K-Ras, the most commonly mutated oncogene, has been shown to induce aberrant epigenetic changes, including the methylation and subsequent silencing of key tumor suppressors. Furthermore, in the majority of K-Ras-driven tumors, the NF-κB pathway is constitutively active and contributes to cellular transformation. Specifically, our lab has shown that knocking out the p65 subunit of NF-κB significantly reduces tumor development in a mouse model of K-Ras-induced lung cancer. NF-κB subunits have been shown to associate with HDAC1 and to promote DNMT1 recruitment to chromatin, suggesting the involvement of NF-κB in epigenetic regulation. However, the role of the NF-κB pathway in Ras-mediated epigenetic changes remains unclear. Based on the above, I hypothesize that oncogenic K-Ras cooperates with the NF-κB pathway to alter the epigenetic landscape and promote transformation. Using the Infinium Methylation Assay, we have uncovered genome-wide differences in DNA methylation in lung epithelial cells expressing K-RasG12V (SAKRAS) compared to wild-type (SALEB) cells. Expression of oncogenic K-RasG12V was accompanied by hypermethylation of many tumor suppressors and demethylation of established oncogenes. Notably, many of these changes in methylation correlated with changes in gene expression. Inhibition of the NF-κB pathway in SAKRAS cells also led to significant changes in the methylation profile of a subset of genes. Together, these preliminary results support a role for the K-Ras and NF-κB pathways in the epigenetic reprogramming observed in K-Ras-driven cancers. Citation Format: Joel Durand, Albert Baldwin. Ras-mediated epigenetic reprogramming: NF-κB-dependent and independent effects. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Jun 19-22, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2013;73(13 Suppl):Abstract nr B24.

BRAF mutation-specific promoter methylation of FOX genes in colorectal cancer

BackgroundCancer-specific hypermethylation of (promoter) CpG islands is common during the tumorigenesis of colon cancer. Although associations between certain genetic aberrations, such as BRAF mutation and microsatellite instability, and the CpG island methylator phenotype (CIMP), have been found, the mechanisms by which these associations are established are still unclear. We studied genome-wide DNA methylation differences between colorectal tumors carrying a BRAF mutation and BRAF wildtype tumors.ResultsUsing differential methylation hybridization on oligonucleotide microarrays representing 32,171 CpG-rich regions, we identified 1,770 regions with differential methylation between colorectal tumor and paired normal colon. Next, we compared the tumor/normal methylation ratios between different groups of patients. Related to CIMP, we identified 749 differentially methylated regions, of which 86% had a higher tumor/normal methylation ratio in the CIMP-positive group. We identified 758 regions with a BRAF mutation-specific methylation change, of which 96% had a higher tumor/normal methylation ratio in the BRAF mutant group. Among the genes affected by BRAF mutation-specific methylation changes, we found enrichment of several cancer-related pathways, including the PI3 kinase and Wnt signaling pathways. To focus on genes that are silenced in a tumor-specific rather than a lineage-specific manner, we used information on the epigenetic silencing mark H3K27me3 in embryonic stem (ES) cells. Among the genes showing BRAF mutation-specific promoter methylation but no H3K27me3 mark in ES cells were forkhead box (FOX) transcription factors associated with the PI3 kinase pathway, as well as MLH1 and SMO. Repression of FOXD3 gene expression in tumors could be related to its promoter hypermethylation.ConclusionsWe identified new BRAF mutation-specific methylation changes in colorectal cancer. Epigenetic downregulation of these targets may contribute to mutationally active BRAF-driven tumorigenesis, explaining its association with aberrant DNA methylation.

Genome-Wide DNA Methylation Changes between the Superficial and Deep Backfat Tissues of the Pig

Adipose tissue is not only a storage organ involved in fuel metabolism, but also an endocrine organ involved in the regulation of insulin sensitivity, thermogenesis, immunity, and inflammation. There are anatomical, cellular, molecular and physiological differences among adipose tissues deposited in different body sites. However, current understanding of the intrinsic differences between the sub-compartments of the subcutaneous adipose tissue remains rudimentary. Here, we analyzed the genome-wide DNA methylation differences between the porcine superficial and deep backfat tissues using methylated DNA immunoprecipitation combined with high-throughput sequencing. We show that the genes with differentially methylated regions in their promoter are mainly involved in the processes of “lipid metabolism” and “regulation of immune-related cytokines”. Compared with the deep backfat tissue, the promoters of genes related to the ‘positive regulation of cytokine production’ were significantly hypermethylated in the superficial backfat tissue, which reflects the intrinsic functional and metabolic differences between the sub-compartments of the subcutaneous adipose tissue. This study provides epigenetic evidence for functionally relevant methylation differences between different layers of porcine backfat tissues.

Genome-Wide DNA Methylation Differences Between Late-Onset Alzheimer's Disease and Cognitively Normal Controls in Human Frontal Cortex

Evidence supports a role for epigenetic mechanisms in the pathogenesis of late-onset Alzheimer's disease (LOAD), but little has been done on a genome-wide scale to identify potential sites involved in disease. This study investigates human postmortem frontal cortex genome-wide DNA methylation profiles between 12 LOAD and 12 cognitively normal age- and gender-matched subjects. Quantitative DNA methylation is determined at 27,578 CpG sites spanning 14,475 genes via the Illumina Infinium HumanMethylation27 BeadArray. Data are analyzed using parallel linear models adjusting for age and gender with empirical Bayes standard error methods. Gene-specific technical and functional validation is performed on an additional 13 matched pair samples, encompassing a wider age range. Analysis reveals 948 CpG sites representing 918 unique genes as potentially associated with LOAD disease status pending confirmation in additional study populations. Across these 948 sites the subtle mean methylation difference between cases and controls is 2.9%. The CpG site with a minimum false discovery rate located in the promoter of the gene Transmembrane Protein 59 (TMEM59) is 7.3% hypomethylated in cases. Methylation at this site is functionally associated with tissue RNA and protein levels of the TMEM59 gene product. The TMEM59 gene identified from our discovery approach was recently implicated in amyloid-β protein precursor post-translational processing, supporting a role for epigenetic change in LOAD pathology. This study demonstrates widespread, modest discordant DNA methylation in LOAD-diseased tissue independent from DNA methylation changes with age. Identification of epigenetic biomarkers of LOAD risk may allow for the development of novel diagnostic and therapeutic targets.

DNA Methylation and Gene Expression Changes in Monozygotic Twins Discordant for Psoriasis: Identification of Epigenetically Dysregulated Genes

Monozygotic (MZ) twins do not show complete concordance for many complex diseases; for example, discordance rates for autoimmune diseases are 20%–80%. MZ discordance indicates a role for epigenetic or environmental factors in disease. We used MZ twins discordant for psoriasis to search for genome-wide differences in DNA methylation and gene expression in CD4+ and CD8+ cells using Illumina's HumanMethylation27 and HT-12 expression assays, respectively. Analysis of these data revealed no differentially methylated or expressed genes between co-twins when analyzed separately, although we observed a substantial amount of small differences. However, combined analysis of DNA methylation and gene expression identified genes where differences in DNA methylation between unaffected and affected twins were correlated with differences in gene expression. Several of the top-ranked genes according to significance of the correlation in CD4+ cells are known to be associated with psoriasis. Further, gene ontology (GO) analysis revealed enrichment of biological processes associated with the immune response and clustering of genes in a biological pathway comprising cytokines and chemokines. These data suggest that DNA methylation is involved in an epigenetic dysregulation of biological pathways involved in the pathogenesis of psoriasis. This is the first study based on data from MZ twins discordant for psoriasis to detect epigenetic alterations that potentially contribute to development of the disease.

Clone history shapes Populus drought responses.

Just as animal monozygotic twins can experience different environmental conditions by being reared apart, individual genetically identical trees of the genus Populus can also be exposed to contrasting environmental conditions by being grown in different locations. As such, clonally propagated Populus trees provide an opportunity to interrogate the impact of individual environmental history on current response to environmental stimuli. To test the hypothesis that current responses to an environmental stimulus, drought, are contingent on environmental history, the transcriptome- level drought responses of three economically important hybrid genotypes-DN34 (Populus deltoides × Populus nigra), Walker [P. deltoides var. occidentalis × (Populus laurifolia × P. nigra)], and Okanese [Walker × (P. laurifolia × P. nigra)]-derived from two different locations were compared. Strikingly, differences in transcript abundance patterns in response to drought were based on differences in geographic origin of clones for two of the three genotypes. This observation was most pronounced for the genotypes with the longest time since establishment and last common propagation. Differences in genome-wide DNA methylation paralleled the transcriptome level trends, whereby the clones with the most divergent transcriptomes and clone history had the most marked differences in the extent of total DNA methylation, suggesting an epigenomic basis for the clone history-dependent transcriptome divergence. The data provide insights into the interplay between genotype and environment in the ecologically and economically important Populus genus, with implications for the industrial application of Populus trees and the evolution and persistence of these important tree species and their associated hybrids.

Abstract LB-133: Genome-wide differences in DNA methylation between tumor and normal adjacent tissue mapped by high-throughput sequencing of methyl-CPG binding domain based affinity-purified genomic fragments

Abstract Aberrant DNA methylation is characteristic of many cancers and differences in methylation have been observed in a wide variety of genomic contexts; for example, both within “classic” promoter-associated CpG islands and also in distal, non-CpG island regions. Establishing a method to broadly and efficiently survey the methylation patterns genome-wide in matched tumor and normal genomic DNA and to elucidate differences in these “methylomes” is the objective of the work presented. The method combines the power of methyl-CpG binding domain (MBD) proteins to sensitively and selectively bind methylated DNA sequences with the coverage, precision, and accuracy provided by high-throughput sequencing. Notably, MBD-affinity capture can also be used to sub-fractionate genomic DNA based on its average methyl-CpG content. In parallel, patient-derived matched samples of fragmented genomic DNA from a breast tumor and normal adjacent tissue were enriched and fractionated based on their methyl-CpG content with a commercial MBD-based affinity reagent (see figure) and high-throughput sequencing libraries were prepared from each fraction. The libraries were sequenced at 50 bp read-length on a SOLiD 3 Plus system and mapped to the human reference genome. Peak analysis of the distribution of mapped reads permitted the discovery of hundreds of putative differentially methylation regions (DMRs). Differential methylation at a number of these positions was confirmed by bisulfite-sequencing. We conclude that such enrichment and fractionation, when coupled to high-throughput sequencing, can be used to efficiently survey the majority of DNA methylation marks within samples of genomic DNA and to discover genomic loci of differential methylation. We conclude that such enrichment and fractionation, when coupled to high-throughput sequencing, can be used to efficiently survey a large fraction of all DMRs between samples of genomic DNA and to discover genomic loci of differential methylation. These datasets can then be compared with ChIP-seq and/or RNA-seq data sets to begin to decipher the functional genomics and epigenomics of cancer. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-133.

Reproductive and epigenetic outcomes associated with aging mouse oocytes

Female aging entails a decline in fertility in mammals, manifested by reduced oocyte reserves and poor oocyte quality accompanied by chromosomal anomalies and reduced litter size. In addition to compromised genetic integrity, recent studies suggest that epigenetic mechanisms may be altered in aging oocytes, with age affecting the expression of DNA methyltransferases, which catalyze the important epigenetic modification, DNA methylation. Loss of DNA methylation patterns, most notably for imprinted genes, is lethal to mouse embryos. To investigate how maternal age affects embryonic development and underlying DNA methylation patterns, young and aged C57BL/6 females were mated with C57BL/6 or C57BL/6(CAST7) males to allow for the identification of parental alleles; resulting blastocysts and mid-gestation embryos and placentas were evaluated. Although pregnancy, ovulation and implantation rates were similar between age groups, an age-related increase in resorption sites, morphological abnormalities and delayed development was found. Interestingly, placental morphology was also perturbed by aging, with elevated numbers of trophoblast giant cells in aged pregnancies. Normal monoallelic expression of the imprinted genes H19 and Snrpn was unaltered in blastocysts from aged females. We failed to observe any age-related changes in methylation of the differentially methylated regions of imprinted genes Snrpn, Kcnq1ot1, U2af1-rs1, Peg1, Igf2r and H19. Restriction Landmark Genome Scanning showed no significant differences in genome-wide DNA methylation in embryos and placentas, regardless of maternal age. Our findings demonstrate that maternal age affects post-implantation embryo and placental development; however embryos capable of developing to mid-gestation appear to undergo normal acquisition and maintenance of DNA methylation patterning.

Genetic vs. Epigenetic Disruption of the CEBPA Locus Yields Epigenomically and Biologically Distinct Leukemia Phenotypes.

Acute Myeloid Leukemia (AML) is a heterogeneous disease from the molecular and biological standpoints. In order to resolve some of this complexity, a recent microarray-based expression profiling study segregated cohorts of patients with common gene signatures. One of these signatures was associated with alterations of the CCAAT/enhancer-binding protein alpha (CEBPA) gene. Among these patients, a subset harbored CEBPA mutations, while the remainder failed to express CEBPA, which in a number of cases correlated with hypermethylation of its promoter. This latter subgroup of leukemias with silenced CEBPA presented with significant biological differences compared to CEBPA mutant patients, including expression of T-cell markers and activating mutations of NOTCH1 (Wouters BJ et. al., PMID:17671232). Since our preliminary data show that DNA methylation profiling is extremely accurate in identifying distinct biological phenotypes in AML and other tumors, we wondered whether genome-wide epigenetic analysis would identify the biological difference between these patients. In order to determine the DNA methylation profiles of these patients we performed HELP (HpaII tiny fragment Enrichment by Ligation-mediated PCR), a quantitative genome-wide DNA methylation method, using a 400,000 feature cutom-desinged microarray, representing 24,000 gene promoters with 50-mer oligonucleotides. We studied the complete previously identified cluster of AML cases presenting with a CEBPA expression signature, and compared and contrasted the DNA methylation profiles of cases carrying the CEBPA mutation and those presenting with CEBPA silencing. Remarkably, unsupervised (unbiased) clustering of DNA methylation profiles revealed that samples were readily segregated into two groups that overlapped perfectly with the presence or absence of the CEBPA mutation, indicating the presence of underlying genome-wide DNA methylation differences between these two groups. We next performed supervised analysis of the samples to compare the DNA methylation profiles of CEBPA mutated vs. CEBPA non-mutated samples. The analysis was performed using a moderated T test, and 291 genes promoters were identified as differentially methylated between the two groups at a significance level of p <0.001. Within this differentially methylated signature, we detected a clear predominance (90%) of hypermethylated genes in the CEBPA silenced group. The critical importance of CEBPA loss of function in mediating the phenotype of these tumors was underlined by the fact that multiple other members of the CEBPA network were likewise hypermethylated. Other than the CEBPA network, the two other most hypermethylated biological pathways involved p38MAPK signaling and PDGF/LCK signaling. Thus, we conclude i) that hypermethylation of the CEBPA promoter is not an isolated event, but rather part of a more widespread epigenetic regulation, and ii) that the original CEBPA signature group is composed of two distinct subgroups originating through two distinct mechanisms, a genetic one and an epigenetic one. The significance of these findings may be supported by the fact that the hypermethylated cohort of patients tended to show a worse prognosis than CEBPA mutant patients. These patients might be good candidates for DNA methyltransferase inhibitors in prospective clinical trials.

Differences in DNA methylation during oogenesis and spermatogenesis and their persistence during early embryogenesis in the mouse.

We have examined the relative methylation levels of several dispersed repeated and low-copy-number gene sequences during gametogenesis and early embryogenesis. Southern blot analyses revealed that L1, intercisternal A particle (IAP), and major urinary protein (MUP) sequences were undermethylated extensively at MspI sites in DNA from diplotene oocytes. In contrast, the same sequences were highly methylated in DNA from pachytene spermatocytes, round spermatids, and epididymal sperm. These results indicate that there are genome-wide DNA methylation differences between oogenesis and spermatogenesis. Repeated sequences in DNA from cleavage-stage embryos and inner cell masses (ICM) were methylated at intermediate levels, consistent with transient maintenance of gametic methylation levels during early embryogenesis. Gametic differences in DNA methylation observed here indicate that methylation could provide a mechanism for imprinting maternal and paternal genomes resulting in differential regulation of parental genomes during early development.