Genome-wide DNA methylation analysis of human pancreatic islets from type 2 diabetic and non-diabetic donors identifies candidate genes that influence insulin secretion - PubMed (original) (raw)

. 2014 Mar 6;10(3):e1004160.

doi: 10.1371/journal.pgen.1004160. eCollection 2014 Mar.

Petr Volkov 1, Sofia Salö 1, Elin Hall 1, Emma Nilsson 1, Anders H Olsson 1, Clare L Kirkpatrick 2, Claes B Wollheim 2, Lena Eliasson 3, Tina Rönn 1, Karl Bacos 1, Charlotte Ling 1

Affiliations

Genome-wide DNA methylation analysis of human pancreatic islets from type 2 diabetic and non-diabetic donors identifies candidate genes that influence insulin secretion

Tasnim Dayeh et al. PLoS Genet. 2014.

Abstract

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.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1

Figure 1. The human methylome in pancreatic islets from 15 T2D and 34 non-diabetic donors.

(A) All analyzed DNA methylation sites on the Infinium HumanMethylation450 BeadChip are mapped to gene regions based on their functional genome distribution and CpG island regions based on CpG content and neighbourhood context . TSS: proximal promoter, defined as 200 bp or 1500 bp upstream of the transcription start site. UTR: untranslated region. CpG island: 200 bp (or more) stretch of DNA with a C+G content of >50% and an observed CpG/expected CpG in excess of 0.6. Shore: the flanking region of CpG islands, 0–2000 bp. Shelf: regions flanking island shores, i.e., covering 2000–4000 bp distant from the CpG island . Global DNA methylation in human pancreatic islets of T2D and non-diabetic donors is shown for (B) each gene region and (C) CpG island regions. Global DNA methylation is calculated as average DNA methylation based on all CpG sites in each annotated region on the chip. (D) The absolute difference in DNA methylation of 3,116 individual sites, including 2,988 sites with decreased and 128 sites with increased DNA methylation in T2D compared with non-diabetic human islets with q<0.05 based on a FDR analysis. 1,649 sites with an absolute difference in DNA methylation ≥5% are represented by grey bars. (E) Pie chart describing the number of sites that exhibit increased or decreased DNA methylation in T2D compared with non-diabetic human islets with an absolute difference in methylation ≥5% and q<0.05. The degree of DNA methylation is shown for (F) the 1,596 CpG sites with decreased DNA methylation and (G) the 53 sites with increased DNA methylation in T2D vs. non-diabetic islets in comparison to the degree of methylation of all analyzed CpG sites in the human islets using the Infinium HumanMethylation450 BeadChip.

Figure 2

Figure 2. Distribution of individual sites that exhibit differential DNA methylation in human islets from 15 T2D versus 34 non-diabetic donors.

(A) The chromosomal location of the 1,649 sites that exhibit differential DNA methylation in islets of T2D versus non-diabetic donors in comparison to all analyzed sites on the Infinium HumanMethylation450 BeadChip. (B) Distribution of significant CpG sites in relation to nearest gene regions. (C) Distribution of significant CpG sites in relation to CpG island regions. *The distribution of the significant sites compared with all analyzed sites on the Infinium HumanMethylation450 BeadChip is significantly different from what is expected by chance based on Chi2 tests. _P_-values have been corrected for multiple testing using Bonferroni correction. (D) The average degree of DNA methylation of analyzed non-CpG sites in human pancreatic islets. Genes with previous known function in (E) pancreatic islets, (F) the exocytotic process and (G) apoptosis that exhibit differential DNA methylation in T2D versus non-diabetic human islets with an absolute difference in methylation ≥5%. * q<0.05. Data are mean ± SEM.

Figure 3

Figure 3. Relation between DNA methylation and gene expression in human pancreatic islets.

(A) The number of genes that exhibit both differential DNA methylation and gene expression in pancreatic islets from 15 T2D versus 34 non-diabetic donors. One gene (SLC44A4) was counted twice in the figure because it showed increased expression in T2D islets and was associated with probes that showed both increased and decreased DNA methylation. Distribution of differentially methylated CpG sites located in/near genes that also exhibit differential expression in T2D islets with an inverse relationship or a positive relationship in relation to their (B) functional genome distribution and in relation to (C) CpG island regions. Decreased DNA methylation and increased mRNA expression of (D) CDKN1A and (E) PDE7B in pancreatic islets of T2D versus non-diabetic donors. (F) A diagram of the two luciferase reporter plasmids used to test the effect of DNA methylation on CDKN1A and PDE7B promoter activity is shown. The two plasmids contain 1500 bp of either the CDKN1A or the PDE7B promoter regions inserted into a pCpGL-basic vector. Methylated (grey and black bars) or mock-methylated (white bars) promoter constructs were transfected into clonal β-cells for 48 hours prior to luciferase assay. The data were normalized with co-transfected renilla luciferase control vector and are the average of three separate experiments with five replicates each. In each experiment, cells were transfected with an empty pCpGL-basic vector as a background control. * P<0.05. Data are mean ± SEM.

Figure 4

Figure 4. Impact of Cdkn1a, Pde7b, Sept9 and Exoc3l on clonal β- and α-cells.

Glucose-sensitive clonal INS-1 832/13 β-cells were used to study the impact of Cdkn1a, Pde7b, Sept9 and Exoc3l on insulin secretion and β-cell function. (A) Overexpression of Cdkn1a, Pde7b and Sept9 with pcDNA3.1 expression vectors in clonal β-cells resulted in elevated mRNA levels (black bars) compared with β-cells transfected with an empty pcDNA3.1 vector (white bars). * P<0.05. Data are mean ± SEM. Overexpression was also evident at the protein level as determined by immunoblot detection of the HA-tag situated on the c-terminal of the cloned cDNAs (rightmost panel) (B) Insulin secretion in response to 2.8 mM (white bars) and 16.7 mM (black bars) glucose in clonal β-cells overexpressing either Cdkn1a, Pde7b or Sept9 compared with control cells transfected with an empty pcDNA3.1 vector (n = 5). * P<0.05. Data are mean ± SEM (C) Decreased cell proliferation in clonal β-cells overexpressing Cdkn1a (black bar) compared with cells transfected with an empty pcDNA3.1 vector (white bar) (n = 4). * P<0.05. Data are mean ± SEM (D) Insulin secretion in response to 2.8 mM and 16.7 mM glucose (Gluc) or 16.7 mM glucose (Gluc) in combination with 100 µM IBMX in clonal β-cells overexpressing Pde7b (black bars) compared with cells transfected with an empty pcDNA3.1 vector (white bars) (n = 3). * P<0.05. NS = not significant. (E) Increased DNA methylation and decreased mRNA expression of EXOC3L2 in pancreatic islets of 15 T2D versus 34 non-diabetic donors. (F) Transfection of clonal β-cells with siRNA targeting Exoc3l resulted in decreased Exoc3l mRNA expression (siExo3l, black bar) when compared with clonal β-cells transfected with negative control siRNA (siNC, white bar) (n = 3). * P<0.05. Silencing of Exoc3l resulted in decreased Ca2+-dependent exocytosis; (G) Depolarizationevoked a decrease in membrane capacitance (ΔCm) in single INS1-832/13 β-cells where Exoc3l was silenced (black trace) compared with control β-cells (grey trace) treated with negative control siRNA. (H) Histogram of the summed increase in membrane capacitance evoked by the two first depolarizations (Σ1–2), the latter eight depolarizations (Σ3–10) or all depolarizations in the train (Σall). Data are mean ± SEM of n = 11 β-cells treated with control siRNA (white bars) and n = 4 β-cells treated with siRNA against Exoc3l (black bars). * P<0.05. (I) Depolarization-evoked increase in current (I) in single INS1-832/13 β-cells treated with control siRNA (grey trace) or siRNA targeting Exoc3l (black trace). Notice that the rapid Na+-current is markedly diminished in siExoc3l treated clonal β-cells. (J) Reduced expression of Exoc3l has no effect on the Ca2+ influx. Charge (Q)-voltage (Vm) relationship in β-cells treated with control (white squares) siRNA and siRNA against Exoc3l (black circles). The charge is representative of the Ca2+-influx into the cell through the voltage-dependent Ca2+ channels. (K) As in J, but the peak-current (Ipeak)-voltage (Vm)-relationship was estimated as a representation of the voltage-dependent Na+ current. The Na+ current is significantly reduced in siExoc3l cells (P<0.05) versus control siRNA cells except for the depolarization to −40 mV. Data in J and K are mean ± SEM of n = 11 β-cells treated with control siRNA and n = 7 siExoc3l treated β-cells. (L) αTC1-6 cells were used to determine the impact of Cdkn1a, Pde7b and Sept9 on glucagon secretion. Overexpression of Cdkn1a and Pde7b resulted in significantly increased glucagon secretion at 1 mM glucose (white bars) (n = 4, * P<0.05), while overexpression of Pde7b and Sept9 resulted in increased glucagon secretion at 16.7 mM glucose (black bars) (n = 4, ¤ P<0.05) compared with control cells transfected with an empty pcDNA3.1 vector.

Figure 5

Figure 5. The human methylome in FACS sorted human β- and α-cell fractions.

All analyzed DNA methylation sites on the Infinium HumanMethylation450 BeadChip are mapped to gene regions based on their functional genome distribution and CpG island regions based on CpG content and neighbourhood context . Global DNA methylation in whole human islets (n = 4) and purified human β- (n = 3) and α-cell (n = 2) fractions is shown for (A) each gene region and (B) CpG island regions. Global DNA methylation is calculated as average DNA methylation based on all CpG sites in each annotated region on the chip. Data are mean ± SEM.

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