The Dysregulation of the DLK1- MEG3 Locus in Islets From Patients With Type 2 Diabetes Is Mimicked by Targeted Epimutation of Its Promoter With TALE-DNMT Constructs

Abstract

Type 2 diabetes mellitus (T2DM) is characterized by the inability of the insulin-producing β-cells to overcome insulin resistance. We previously identified an imprinted region on chromosome 14, the DLK1-MEG3 locus, as being downregulated in islets from humans with T2DM. In this study, using targeted epigenetic modifiers, we prove that increased methylation at the promoter of Meg3 in mouse βTC6 β-cells results in decreased transcription of the maternal transcripts associated with this locus. As a result, the sensitivity of β-cells to cytokine-mediated oxidative stress was increased. Additionally, we demonstrate that an evolutionarily conserved intronic region at the MEG3 locus can function as an enhancer in βTC6 β-cells. Using circular chromosome conformation capture followed by high-throughput sequencing, we demonstrate that the promoter of MEG3 physically interacts with this novel enhancer and other putative regulatory elements in this imprinted region in human islets. Remarkably, this enhancer is bound in an allele-specific manner by the transcription factors FOXA2, PDX1, and NKX2.2. Overall, these data suggest that the intronic MEG3 enhancer plays an important role in the regulation of allele-specific expression at the imprinted DLK1-MEG3 locus in human β-cells, which in turn impacts the sensitivity of β-cells to cytokine-mediated oxidative stress.

Figure 1

Targeted methylation of the Meg3-DMR results in decreased expression and increased β-cell death. βTC6 mouse insulinoma cells were transfected with TALE molecules specific to the mouse Meg3-DMR fused to either a WT or mutant DNMT catalytic domain. Transfected cells were sorted by GFP expression. A: Schema of mouse Meg3-DMR spanning ∼2 kb, depicting TALE-DNMT (mm10 chr12:109,540,635-109,540,653) binding positions relative to the Meg3 TSS. Regions assayed for methylation levels by pyrosequencing are depicted by blue boxes. Percent methylation levels determined by pyrosequencing of CpGs in the Meg3-DMR (mm10 chr12:109,540,014-109,542,041) in untransfected βTC6 (n = 3), WT TALE-DNMT (n = 3), or mutant TALE-DNMT–transfected (n = 2) cells (B) and Dlk1 promoter region (mm10 chr12:109452986-109453847) in untransfected βTC6 cells (n = 2), WT TALE-DNMT GFP⁺ (n = 3), and mutant TALE-DNMT GFP⁺ cells (n = 1) (C). *P < 0.05; **P < 0.01. D: Relative expression of Meg3 and Dlk1 measured by quantitative RT-PCR between WT (n = 8) and mutant (n = 7) TALE-DNMT–transfected βTC6 cells, sorted by GFP levels. Data are represented as mean ± SEM. **P < 0.01. E: Percent of WT or mutant TALE-DNMT GFP⁺ βTC6 cells labeled by CellROX fluorescence, a marker of oxidative stress, following cytokine treatment for 48 h (n = 4). Data are represented as mean ± SEM and P < 0.02.

Figure 2

Characteristics of a novel intronic enhancer human islet. A: The chromatin landscape for a putative enhancer in an intron of MEG3 is shown with human islet ChIP sequencing tracks for histone modification marks associated with enhancers (H3K4me1 and H3K27ac) and active promoters (H3K4me3). Occupancy of islet transcription factors PDX1, NKX2.2, and FOXA2 at the putative enhancer is also shown. Data retrieved from Pasquali et al. (15). B: Activity of the MEG3 enhancer was validated by luciferase reporter assays. pGL3 vectors with the MEG3-DMR and the enhancer sequence in either its native (For) or reverse (Rev) orientation were transfected into βTC6 cells. Data are represented as mean ± SEM. n = 3. P values calculated by Student t test. **P < 0.1 × 10⁻³; ***P < 0.1 × 10⁻⁵.

Figure 3

The MEG3 promoter and enhancer interact with other putative enhancers within the DLK1-MEG3–imprinted region. Genome browser image of the selective interactions of the MEG3 promoter and enhancer within 350 kb of the MEG3 promoter. 4C-Seq was performed using the MEG3 promoter and enhancer, respectively, as viewpoints (indicated with red triangles) using three human islet donors (represented by different shaded tracks) per viewpoint. Bars under each track represent the significant interaction sites (P < 1 × 10⁻⁸). The MEG3 promoter and enhancer make frequent contact with putative enhancers within the imprinted locus. ChIP sequencing for the histone modification marks and the putative enhancer identification was performed by Pasquali et al. (15).

Figure 4

Allele-specific transcription factor occupancy at the MEG3 enhancer. A: Schematic representation of the allele-specific ChIP experimental design. Transcription factor ChIP is performed on islets from donors heterozygous for the SNP rs3783355. Following amplification using primers surrounding the SNP, the PCR products are sequenced to quantitatively determine the relative representation of the two alleles. B: Relative amplification (percent sequencing reads) of the rs3783355 alleles as determined by high-throughput sequencing of input, NKX2.2, FOXA2, and PDX1 ChIP DNA from islet donors heterozygous for rs3783355 (G/A). C: The minor allele of rs3783355 alters the recognition sequence of a restriction enzyme, BanII. Following ChIP-PCR to determine FOXA2 occupancy at the MEG3 enhancer, the PCR products were digested with BanII to qualitatively assess the allelic representation of rs3783355. A representative gel of BanII-digested input and FOXA2 ChIP-PCR products from islets from two donors heterozygous for rs3783355 is shown. Data are represented as mean ± SEM. P values calculated using Student t test. ***P < 0.001.