Inter-individual DNA methylation variations were frequently hypothesized to alter individual susceptibility to Type 2 Diabetes Mellitus (T2DM). Sequence-influenced methylations were described in T2DM-associated genomic regions, but evidence for direct, sequence-independent association with disease risk is missing. Here, we explore disease-contributing DNA methylation through a stepwise study design: first, a pool-based, genome-scale screen among 1169 case and control individuals revealed an excess of differentially methylated sites in genomic regions that were previously associated with T2DM through genetic studies. Next, in-depth analyses were performed at selected top-ranking regions. A CpG site in the first intron of the FTO gene showed small (3.35%) but significant (P = 0.000021) hypomethylation of cases relative to controls. The effect was independent of the sequence polymorphism in the region and persists among individuals carrying the sequence-risk alleles. The odds of belonging to the T2DM group increased by 6.1% for every 1% decrease in methylation (OR = 1.061, 95% CI: 1.032-1.090), the odds ratio for decrease of 1 standard deviation of methylation (adjusted to gender) was 1.5856 (95% CI: 1.2824-1.9606) and the sensitivity (area under the curve = 0.638, 95% CI: 0.586-0.690; males = 0.675, females = 0.609) was better than that of the strongest known sequence variant. Furthermore, a prospective study in an independent population cohort revealed significant hypomethylation of young individuals that later progressed to T2DM, relative to the individuals who stayed healthy. Further genomic analysis revealed co-localization with gene enhancers and with binding sites for methylation-sensitive transcriptional regulators. The data showed that low methylation level at the analyzed sites is an early marker of T2DM and suggests a novel mechanism by which early-onset, inter-individual methylation variation at isolated non-promoter genomic sites predisposes to T2DM.

Understanding how the epigenetic blueprint of the genome shapes human phenotypes requires systematic evaluation of the complex interplay between gene activity and the different layers of the epigenome. Utilizing microarray-based techniques, we explored the relationships between DNA methylation, DNA replication timing and gene expression levels across a variety of human tissues and cell lines. The analyses revealed unequal methylation levels among early- and late-replicating fractions of the genome: late-replicating DNA was hypomethylated compared with early-replicating DNA. Moreover, late-replicating regions were gradually demethylated with cell divisions, whereas the methylation of early-replicating regions was better maintained. As active genes concentrate at early-replicating regions, they are overall hypermethylated relative to inactive genes. Accordingly, we show that the previously reported positive correlation between gene-body methylation (methylation of the transcribed portion of genes) and gene expression is restricted to proliferative tissues and cell lines, whereas in tissues containing few proliferating cells, active and inactive genes have similar methylation levels. We further show that active gene bodies are hypermethylated not only compared with inactive gene bodies, but also compared with their flanking sequences. This specific hypermethylation of the active gene bodies is severely disrupted in cells of an immunodeficiency, centromeric region instability, facial anomalies (ICF) syndrome patient bearing mutated DNA methyltransferase 3B (DNMT3B). Our data show that a high methylation level is preferentially maintained in active gene bodies through independent cellular processes. Rather than serving as a distinctive mark between active and inactive genes, gene-body methylation appears to serve a vital, currently unknown function in active genes.

MicroRNAs (miRs) regulate a variety of cellular processes, and their impaired expression is involved in cancer. Silencing of tumorsuppressive miRs in cancer can occur through epigenetic modifications, including DNA methylation and histone deacetylation. We performed comparative miR profiling on cultured lung cancer cells before and after treatment with 5′aza-deoxycytidine plus Trichostatin A to reverse DNA methylation and histone deacetylation, respectively. Several tens of miRs were strongly induced by such 'epigenetic therapy'. Two representatives, miR-512-5p (miR-512) and miR-373, were selected for further analysis. Both miRs were secreted in exosomes. Re-expression of both miRs augmented cisplatin-induced apoptosis and inhibited cell migration; miR-512 also reduced cell proliferation. TEAD4 mRNA was confirmed as a direct target of miR-512; likewise, miR-373 was found to target RelA and PIK3CA mRNA directly. Our results imply that miR-512 and miR-373 exert cell-autonomous and non-autonomous tumor-suppressive effects in lung cancer cells, where their re-expression may benefit epigenetic cancer therapy.

BackgroundType 2 diabetes mellitus (T2D) is highly prevalent in Middle-Eastern and North African Arab populations, but the molecular basis for this susceptibility is unknown. Altered DNA methylation levels were reported in insulin-secreting and responding tissues, but whether methylation in accessible tissues such as peripheral blood is associated with T2D risk remains an open question. Age-related alteration of DNA methylation level was reported in certain methylation sites, but no association with T2D has been shown. Here we report on a population-based study of 929 men and women representing the East Jerusalem Palestinian (EJP) Arab population and compare with the findings among Israeli Ashkenazi Jews. This is the first reported epigenetic study of an Arab population with a characteristic high prevalence of T2D.ResultsWe found that DNA methylation of a prespecified regulatory site in peripheral blood leukocytes (PBLs) is associated with impaired glucose metabolism and T2D independent of sex, body mass index, and white blood cell composition. This CpG site (Chr16: 53,809,231-2; hg19) is located in a region within an intron of the FTO gene, suspected to serve as a tissue-specific enhancer. The association between PBL hypomethylation and T2D varied by age, revealing differential patterns of methylation aging in healthy and diabetic individuals and between ethnic groups: T2D patients displayed prematurely low methylation levels, and this hypomethylation was greater and occurred earlier in life among Palestinian Arabs than Ashkenazi Jews.ConclusionsOur study suggests that premature DNA methylation aging is associated with increased risk of T2D. These findings should stimulate the search for more such sites and may pave the way to improved T2D risk prediction within and between human populations.Electronic supplementary materialThe online version of this article (doi:10.1186/s13148-015-0069-1) contains supplementary material, which is available to authorized users.

Cancers often display gene expression profiles resembling those of undifferentiated cells. The mechanisms controlling these expression programs have yet to be identified. Exploring transcriptional enhancers throughout hematopoietic cell development and derived cancers, we uncovered a novel class of regulatory epigenetic mutations. These epimutations are particularly enriched in a group of enhancers, designated ES-specific enhancers (ESSEs) of the hematopoietic cell lineage. We found that hematopoietic ESSEs are prone to DNA methylation changes, indicative of their chromatin activity states. Strikingly, ESSE methylation is associated with gene transcriptional activity in cancer. Methylated ESSEs are hypermethylated in cancer relative to normal somatic cells and co-localized with silenced genes, whereas unmethylated ESSEs tend to be hypomethylated in cancer and associated with reactivated genes. Constitutive or hematopoietic stem cell-specific enhancers do not show these trends, suggesting selective reactivation of ESSEs in cancer. Further analyses of a hypomethylated ESSE downstream to the VEGFA gene revealed a novel regulatory circuit affecting VEGFA transcript levels across cancers and patients. We suggest that the discovered enhancer sites provide a framework for reactivation of ES genes in cancer.