Tasnim Dayeh

A six months exercise intervention influences the genome-wide DNA methylation pattern in human adipose tissue.

Epigenetic mechanisms are implicated in gene regulation and the development of different diseases. The epigenome differs between cell types and has until now only been characterized for a few human tissues. Environmental factors potentially alter the epigenome. Here we describe the genome-wide pattern of DNA methylation in human adipose tissue from 23 healthy men, with a previous low level of physical activity, before and after a six months exercise intervention. We also investigate the differences in adipose tissue DNA methylation between 31 individuals with or without a family history of type 2 diabetes. DNA methylation was analyzed using Infinium HumanMethylation450 BeadChip, an array containing 485,577 probes covering 99% RefSeq genes. Global DNA methylation changed and 17,975 individual CpG sites in 7,663 unique genes showed altered levels of DNA methylation after the exercise intervention (q<0.05). Differential mRNA expression was present in 1/3 of gene regions with altered DNA methylation, including RALBP1, HDAC4 and NCOR2 (q<0.05). Using a luciferase assay, we could show that increased DNA methylation in vitro of the RALBP1 promoter suppressed the transcriptional activity (p = 0.03). Moreover, 18 obesity and 21 type 2 diabetes candidate genes had CpG sites with differences in adipose tissue DNA methylation in response to exercise (q<0.05), including TCF7L2 (6 CpG sites) and KCNQ1 (10 CpG sites). A simultaneous change in mRNA expression was seen for 6 of those genes. To understand if genes that exhibit differential DNA methylation and mRNA expression in human adipose tissue in vivo affect adipocyte metabolism, we silenced Hdac4 and Ncor2 respectively in 3T3-L1 adipocytes, which resulted in increased lipogenesis both in the basal and insulin stimulated state. In conclusion, exercise induces genome-wide changes in DNA methylation in human adipose tissue, potentially affecting adipocyte metabolism.

Genome-Wide DNA Methylation Analysis of Human Pancreatic Islets from Type 2 Diabetic and Non-Diabetic Donors Identifies Candidate Genes That Influence Insulin Secretion

Impaired insulin secretion is a hallmark of type 2 diabetes (T2D). Epigenetics may affect disease susceptibility. To describe the human methylome in pancreatic islets and determine the epigenetic basis of T2D, we analyzed DNA methylation of 479,927 CpG sites and the transcriptome in pancreatic islets from T2D and non-diabetic donors. We provide a detailed map of the global DNA methylation pattern in human islets, β- and α-cells. Genomic regions close to the transcription start site showed low degrees of methylation and regions further away from the transcription start site such as the gene body, 3′UTR and intergenic regions showed a higher degree of methylation. While CpG islands were hypomethylated, the surrounding 2 kb shores showed an intermediate degree of methylation, whereas regions further away (shelves and open sea) were hypermethylated in human islets, β- and α-cells. We identified 1,649 CpG sites and 853 genes, including TCF7L2, FTO and KCNQ1, with differential DNA methylation in T2D islets after correction for multiple testing. The majority of the differentially methylated CpG sites had an intermediate degree of methylation and were underrepresented in CpG islands (∼7%) and overrepresented in the open sea (∼60%). 102 of the differentially methylated genes, including CDKN1A, PDE7B, SEPT9 and EXOC3L2, were differentially expressed in T2D islets. Methylation of CDKN1A and PDE7B promoters in vitro suppressed their transcriptional activity. Functional analyses demonstrated that identified candidate genes affect pancreatic β- and α-cells as Exoc3l silencing reduced exocytosis and overexpression of Cdkn1a, Pde7b and Sept9 perturbed insulin and glucagon secretion in clonal β- and α-cells, respectively. Together, our data can serve as a reference methylome in human islets. We provide new target genes with altered DNA methylation and expression in human T2D islets that contribute to perturbed insulin and glucagon secretion. These results highlight the importance of epigenetics in the pathogenesis of T2D.

Impact of an Exercise Intervention on DNA Methylation in Skeletal Muscle From First-Degree Relatives of Patients With Type 2 Diabetes

To identify epigenetic patterns, which may predispose to type 2 diabetes (T2D) due to a family history (FH) of the disease, we analyzed DNA methylation genome-wide in skeletal muscle from individuals with (FH+) or without (FH−) an FH of T2D. We found differential DNA methylation of genes in biological pathways including mitogen-activated protein kinase (MAPK), insulin, and calcium signaling (P ≤ 0.007) and of individual genes with known function in muscle, including MAPK1, MYO18B, HOXC6, and the AMP-activated protein kinase subunit PRKAB1 in skeletal muscle of FH+ compared with FH− men. We further validated our findings from FH+ men in monozygotic twin pairs discordant for T2D, and 40% of 65 analyzed genes exhibited differential DNA methylation in muscle of both FH+ men and diabetic twins. We further examined if a 6-month exercise intervention modifies the genome-wide DNA methylation pattern in skeletal muscle of the FH+ and FH− individuals. DNA methylation of genes in retinol metabolism and calcium signaling pathways (P < 3 × 10−6) and with known functions in muscle and T2D including MEF2A, RUNX1, NDUFC2, and THADA decreased after exercise. Methylation of these human promoter regions suppressed reporter gene expression in vitro. In addition, both expression and methylation of several genes, i.e., ADIPOR1, BDKRB2, and TRIB1, changed after exercise. These findings provide new insights into how genetic background and environment can alter the human epigenome.

Increased DNA methylation and decreased expression of PDX-1 in pancreatic islets from patients with type 2 diabetes.

Mutations in pancreatic duodenal homeobox 1 (PDX-1) can cause a monogenic form of diabetes (maturity onset diabetes of the young 4) in humans, and silencing Pdx-1 in pancreatic β-cells of mice causes diabetes. However, it is not established whether epigenetic alterations of PDX-1 influence type 2 diabetes (T2D) in humans. Here we analyzed mRNA expression and DNA methylation of PDX-1 in human pancreatic islets from 55 nondiabetic donors and nine patients with T2D. We further studied epigenetic regulation of PDX-1 in clonal β-cells. PDX-1 expression was decreased in pancreatic islets from patients with T2D compared with nondiabetic donors (P = 0.0002) and correlated positively with insulin expression (rho = 0.59, P = 0.000001) and glucose-stimulated insulin secretion (rho = 0.41, P = 0.005) in the human islets. Ten CpG sites in the distal PDX-1 promoter and enhancer regions exhibited significantly increased DNA methylation in islets from patients with T2D compared with nondiabetic donors. DNA methylation of PDX-1 correlated negatively with its gene expression in the human islets (rho = -0.64, P = 0.0000029). Moreover, methylation of the human PDX-1 promoter and enhancer regions suppressed reporter gene expression in clonal β-cells (P = 0.04). Our data further indicate that hyperglycemia decreases gene expression and increases DNA methylation of PDX-1 because glycosylated hemoglobin (HbA1c) correlates negatively with mRNA expression (rho = -0.50, P = 0.0004) and positively with DNA methylation (rho = 0.54, P = 0.00024) of PDX-1 in the human islets. Furthermore, while Pdx-1 expression decreased, Pdx-1 methylation and Dnmt1 expression increased in clonal β-cells exposed to high glucose. Overall, epigenetic modifications of PDX-1 may play a role in the development of T2D, given that pancreatic islets from patients with T2D and β-cells exposed to hyperglycemia exhibited increased DNA methylation and decreased expression of PDX-1. The expression levels of PDX-1 were further associated with insulin secretion in the human islets.

Identification of CpG-SNPs associated with type 2 diabetes and differential DNA methylation in human pancreatic islets

Aims/hypothesisTo date, the molecular function of most of the reported type 2 diabetes-associated loci remains unknown. The introduction or removal of cytosine–phosphate–guanine (CpG) dinucleotides, which are possible sites of DNA methylation, has been suggested as a potential mechanism through which single-nucleotide polymorphisms (SNPs) can affect gene function via epigenetics. The aim of this study was to examine if any of 40 SNPs previously associated with type 2 diabetes introduce or remove a CpG site and if these CpG-SNPs are associated with differential DNA methylation in pancreatic islets of 84 human donors.MethodsDNA methylation was analysed using pyrosequencing.ResultsWe found that 19 of 40 (48%) type 2 diabetes-associated SNPs introduce or remove a CpG site. Successful DNA methylation data were generated for 16 of these 19 CpG-SNP loci, representing the candidate genes TCF7L2, KCNQ1, PPARG, HHEX, CDKN2A, SLC30A8, DUSP9, CDKAL1, ADCY5, SRR, WFS1, IRS1, DUSP8, HMGA2, TSPAN8 and CHCHD9. All analysed CpG-SNPs were associated with differential DNA methylation of the CpG-SNP site in human islets. Moreover, six CpG-SNPs, representing TCF7L2, KCNQ1, CDKN2A, ADCY5, WFS1 and HMGA2, were also associated with DNA methylation of surrounding CpG sites. Some of the type 2 diabetes CpG-SNP sites that exhibit differential DNA methylation were further associated with gene expression, alternative splicing events determined by splice index, and hormone secretion in the human islets. The 19 type 2 diabetes-associated CpG-SNPs are in strong linkage disequilibrium (r2 > 0.8) with a total of 295 SNPs, including 91 CpG-SNPs.Conclusions/interpretationOur results suggest that the introduction or removal of a CpG site may be a molecular mechanism through which some of the type 2 diabetes SNPs affect gene function via differential DNA methylation and consequently contributes to the phenotype of the disease.

Genome-Wide Associations between Genetic and Epigenetic Variation Influence mRNA Expression and Insulin Secretion in Human Pancreatic Islets

Genetic and epigenetic mechanisms may interact and together affect biological processes and disease development. However, most previous studies have investigated genetic and epigenetic mechanisms independently, and studies examining their interactions throughout the human genome are lacking. To identify genetic loci that interact with the epigenome, we performed the first genome-wide DNA methylation quantitative trait locus (mQTL) analysis in human pancreatic islets. We related 574,553 single nucleotide polymorphisms (SNPs) with genome-wide DNA methylation data of 468,787 CpG sites targeting 99% of RefSeq genes in islets from 89 donors. We identified 67,438 SNP-CpG pairs in cis, corresponding to 36,783 SNPs (6.4% of tested SNPs) and 11,735 CpG sites (2.5% of tested CpGs), and 2,562 significant SNP-CpG pairs in trans, corresponding to 1,465 SNPs (0.3% of tested SNPs) and 383 CpG sites (0.08% of tested CpGs), showing significant associations after correction for multiple testing. These include reported diabetes loci, e.g. ADCY5, KCNJ11, HLA-DQA1, INS, PDX1 and GRB10. CpGs of significant cis-mQTLs were overrepresented in the gene body and outside of CpG islands. Follow-up analyses further identified mQTLs associated with gene expression and insulin secretion in human islets. Causal inference test (CIT) identified SNP-CpG pairs where DNA methylation in human islets is the potential mediator of the genetic association with gene expression or insulin secretion. Functional analyses further demonstrated that identified candidate genes (GPX7, GSTT1 and SNX19) directly affect key biological processes such as proliferation and apoptosis in pancreatic β-cells. Finally, we found direct correlations between DNA methylation of 22,773 (4.9%) CpGs with mRNA expression of 4,876 genes, where 90% of the correlations were negative when CpGs were located in the region surrounding transcription start site. Our study demonstrates for the first time how genome-wide genetic and epigenetic variation interacts to influence gene expression, islet function and potential diabetes risk in humans.

A Central Role for GRB10 in Regulation of Islet Function in Man

Variants in the growth factor receptor-bound protein 10 (GRB10) gene were in a GWAS meta-analysis associated with reduced glucose-stimulated insulin secretion and increased risk of type 2 diabetes (T2D) if inherited from the father, but inexplicably reduced fasting glucose when inherited from the mother. GRB10 is a negative regulator of insulin signaling and imprinted in a parent-of-origin fashion in different tissues. GRB10 knock-down in human pancreatic islets showed reduced insulin and glucagon secretion, which together with changes in insulin sensitivity may explain the paradoxical reduction of glucose despite a decrease in insulin secretion. Together, these findings suggest that tissue-specific methylation and possibly imprinting of GRB10 can influence glucose metabolism and contribute to T2D pathogenesis. The data also emphasize the need in genetic studies to consider whether risk alleles are inherited from the mother or the father.

Sex differences in the genome-wide DNA methylation pattern and impact on gene expression, microRNA levels and insulin secretion in human pancreatic islets

BackgroundEpigenetic factors regulate tissue-specific expression and X-chromosome inactivation. Previous studies have identified epigenetic differences between sexes in some human tissues. However, it is unclear whether epigenetic modifications contribute to sex-specific differences in insulin secretion and metabolism. Here, we investigate the impact of sex on the genome-wide DNA methylation pattern in human pancreatic islets from 53 males and 34 females, and relate the methylome to changes in expression and insulin secretion.ResultsGlucose-stimulated insulin secretion is higher in female versus male islets. Genome-wide DNA methylation data in human islets clusters based on sex. While the chromosome-wide DNA methylation level on the X-chromosome is higher in female versus male islets, the autosomes do not display a global methylation difference between sexes. Methylation of 8,140 individual X-chromosome sites and 470 autosomal sites shows sex-specific differences in human islets. These include sites in/near AR, DUSP9, HNF4A, BCL11A and CDKN2B. 61 X-chromosome genes and 18 autosomal genes display sex-specific differences in both DNA methylation and expression. These include NKAP, SPESP1 and APLN, which exhibited lower expression in females. Functional analyses demonstrate that methylation of NKAP and SPESP1 promoters in vitro suppresses their transcriptional activity. Silencing of Nkap or Apln in clonal beta-cells results in increased insulin secretion. Differential methylation between sexes is associated with altered levels of microRNAs miR-660 and miR-532 and related target genes.ConclusionsChromosome-wide and gene-specific sex differences in DNA methylation associate with altered expression and insulin secretion in human islets. Our data demonstrate that epigenetics contribute to sex-specific metabolic phenotypes.

Blood-based biomarkers of age-associated epigenetic changes in human islets associate with insulin secretion and diabetes.

Aging associates with impaired pancreatic islet function and increased type 2 diabetes (T2D) risk. Here we examine whether age-related epigenetic changes affect human islet function and if blood-based epigenetic biomarkers reflect these changes and associate with future T2D. We analyse DNA methylation genome-wide in islets from 87 non-diabetic donors, aged 26–74 years. Aging associates with increased DNA methylation of 241 sites. These sites cover loci previously associated with T2D, for example, KLF14. Blood-based epigenetic biomarkers reflect age-related methylation changes in 83 genes identified in human islets (for example, KLF14, FHL2, ZNF518B and FAM123C) and some associate with insulin secretion and T2D. DNA methylation correlates with islet expression of multiple genes, including FHL2, ZNF518B, GNPNAT1 and HLTF. Silencing these genes in β-cells alter insulin secretion. Together, we demonstrate that blood-based epigenetic biomarkers reflect age-related DNA methylation changes in human islets, and associate with insulin secretion in vivo and T2D.

DNA methylation of the glucagon-like peptide 1 receptor (GLP1R) in human pancreatic islets

BackgroundInsulin secretion is enhanced upon the binding of Glucagon-like peptide-1 (GLP-1) to its receptor (GLP1R) in pancreatic β cells. Although a reduced expression of GLP1R in pancreatic islets from type 2 diabetic patients and hyperglycaemic rats has been established, it is still unknown if this is caused by differential DNA methylation of GLP1R in pancreatic islets of type 2 diabetic patients.MethodsIn this study, DNA methylation levels of 12 CpG sites close to the transcription start site of GLP1R were analysed in pancreatic islets from 55 non-diabetic and 10 type 2 diabetic human donors as well as in β and α cells isolated from human pancreatic islets. DNA methylation of GLP1R was related to GLP1R expression, HbA1c levels and BMI. Moreover, mRNA expression of MECP2, DNMT1, DNMT3A and DNMT3B was analysed in pancreatic islets of the non-diabetic and type 2 diabetic donors.ResultsOne CpG unit, at position +199 and +205 bp from the transcription start site, showed a small increase in DNA methylation in islets from donors with type 2 diabetes compared to non-diabetic donors (0.53%, p=0.02). Furthermore, DNA methylation levels of one CpG site located 376 bp upstream of the transcription start site of GLP1R correlated negatively with GLP1R expression (rho=−0.34, p=0.008) but positively with BMI and HbA1c (rho=0.30, p=0.02 and rho=0.30, p=0.03, respectively). This specific CpG site is located in an area with known SP1 and SP3 transcription factor binding sites. Moreover, when we compared the DNA methylation of the GLP1R promoter in isolated human β and α cells, we found that it was higher in α- compared with β-cells (p=0.009). Finally, there was a trend towards decreased DNMT3A expression (p=0.056) in type 2 diabetic compared with non-diabetic islets.ConclusionsTogether, our study shows that while BMI and HbA1c are positively associated with DNA methylation levels of GLP1R, its expression is negatively associated with DNA methylation of GLP1R in human pancreatic islets.