Page 1 of 33 Diabetes The dysregulation of the DLK1-MEG3 locus in islets from type 2 diabetics is mimicked by targeted epimutation of its promoter with TALE-DNMT constructs Vasumathi Kameswaran1, Maria Golson1, Mireia Ramis Rodriguez2,3,4, Kristy Ou1, Yue J. Wang1, Jia Zhang1, Lorenzo Pasquali2,3,4, Klaus H. Kaestner1* 1 University of Pennsylvania, Department of Genetics and Institute for Diabetes, Obesity, and Metabolism, Philadelphia, PA, USA 2Program of Predictive and Personalized Medicine of Cancer (PMPPC), Department of Endocrinology, Germans Trias i Pujol University Hospital and Research Institute, Badalona, Spain 3Josep Carreras Leukaemia Research Institute, Badalona, Spain, 4CIBER de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Barcelona, Spain. *Corresponding author. University of Pennsylvania, Department of Genetics and Institute for Diabetes, Obesity, and Metabolism, 12-126 Translational Research Center, 3400 Civic Center Blvd., Philadelphia, PA 19104-6145, USA. Tel.: +1-215-898-8759. E-mail: [email protected] 1 Diabetes Publish Ahead of Print, published online July 3, 2018 Diabetes Page 2 of 33 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 down-regulated in human T2D islets. Here, 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. 2 Page 3 of 33 Diabetes Diabetes mellitus refers to a group of metabolic diseases characterized by an insufficient insulin response to high blood glucose levels. Pancreatic β-cells are critical regulators of glucose homeostasis as they produce, store and secrete insulin to regulate glucose uptake by peripheral tissues. Their autoimmune destruction or functional decline can lead to Type 1 and Type 2 diabetes mellitus (T2DM), respectively. Thus, understanding the molecular mechanisms underlying β-cell physiology is fundamental to improving current diabetes treatment strategies. We previously demonstrated that the imprinted DLK1-MEG3 locus is mis-regulated in islets from T2DM donors (1). This locus consists of the paternally-active DLK1, RTL1 and DIO3 genes, as well as maternally-expressed long non-coding RNAs, MEG3, RTL1as and MEG8, a large miRNA cluster, and several snoRNAs (2; 3). The genes in this locus are active in human β, but not α-cells, at very high levels (1; 4) and repressed in islets from type 2 diabetics (1). This decreased expression correlates with hyper-methylation at the MEG3 promoter. Consistent with these human studies, Meg3 expression is decreased in mouse models of Type 1 and Type 2 diabetes (5). Little is known about the mechanism by which imprinting at the DLK1-MEG3 locus is regulated, particularly in human islets and β-cells. Mono-allelic expression at this locus is established and maintained through specific methylation patterns at two differentially methylated regions (DMRs), the intergenic IG-DMR and MEG3-DMR, overlapping the promoter of the maternal transcript (6; 7). These DMRs are paternally-methylated. While MEG3 promoter hyper- methylation and a concomitant decrease in expression have been reported in several human diseases (8-14), a causal relationship between these observations has not been established. Here, we demonstrate that hyper-methylation of this DMR using targeted DNA methylation in mouse βTC6 β-cells causes decreased transcription of Meg3, and that this repressed expression exacerbates β-cell death, consistent with our observation in islets from human T2DM donors (1). Furthermore, we identified a putative enhancer within an intron of the human MEG3 3 Diabetes Page 4 of 33 gene that is bound by transcription factors that are critical for islet function. We demonstrate that this sequence functions indeed as an active enhancer, and physically interacts with the MEG3 promoter more than 16 kilobases upstream. Intriguingly, this enhancer is bound by islet transcription factors in an allele-specific manner in human islets. Overall, our results suggest an important regulatory function for this newly characterized MEG3 enhancer and provide insights into the mechanism of imprinting at the DLK1-MEG3 locus in β-cells. Research Design and Methods Methods Human Islets Human islets and relevant donor information including age, gender, diabetes status, and BMI were obtained from the Islet Cell Resource Center of the University of Pennsylvania, the NIDDK-supported Integrated Islet Distribution Program (iidp.coh.org) and the National Disease Research Interchange. The donor’s diabetes status was defined by the patient’s medical record, and, when available, the hemoglobin A1c (Supplemental Table 1). TALE experiments TALEs targeting the mouse Meg3-DMR (mm10 chr12:109,540,635-109,540,653) were designed using an online resource and as described before (1). 12x106 βTC6 cells in a 10cm dish were transfected with 12µg of either TALE WT or mutant plasmids using FuGENE HD transfection reagent (Promega). Cells were FACS-sorted for GFP+ cells after 72 hours on Diva 206 (Penn Flow Cytometry and Cell Sorting Facility). After sorting, the cells were pooled for total RNA and genomic DNA extraction using an Allprep DNA/RNA mini kit (QIAGEN). DNA Methylation analysis 4 Page 5 of 33 Diabetes Genomic DNA was isolated with using All Prep DNA/RNA kit (QIAGEN). 325 nanograms of extracted DNA or unsonicated chromatin input were bisulfite treated with the EpiTect Bisulfite kit (QIAGEN) and eluted in 20µl of Buffer EB. PCR and sequencing primers were designed using the PyroMark assay design software version 2.0 (QIAGEN, sequences listed in Supplemental Table 2) to cover CpGs throughout the Meg3-Dlk1 locus. Bisulfite-converted DNA was amplified by PCR using the PyroMark PCR kit (QIAGEN) at 95°C for 15 mins followed by 45 cycles at 95°C for 15s, 57°C for 30s and 72°C for 15s. Biotinylated PCR products were immobilized onto streptavidin-coated sepharose beads (GE Healthcare) and DNA strands were separated using PyroMark denaturation solution (QIAGEN), washed and then neutralized using a vacuum prep station (QIAGEN PyroMark Q96 workstation). After annealing the sequencing primer to the immobilized strand, pyrosequencing was performed on the PyroMark Q96 MD (QIAGEN) using PyroMark Gold CDT kit (QIAGEN) according to the manufacturer’s instructions. Data were analyzed using the Pyro Q-CpG software program (QIAGEN). Gene expression by RT-qPCR Primers used for to quantify gene expression of Meg3 and Dlk1 in TALE experiments are listed in Supplemental table 3. Cell Death Assays βTC6 cells were seeded in six-well plates at a density of 1x10^6 cells per well. The next day, cells were transfected with 1 mg plasmid and 4 mL Lipofectamine 2000 per well. After 72 hours, cells were treated with 20 ng/mL mouse TNFa, 5 ng/mL mouse IL1b, and 10 ng/mL mouse IFNg. 48 hours later, cells were treated with CellROX Deep Red (ThermoFisher) according to manufacturer’s instructions and analyzed for GFP and CellROX fluorescence using a BD LSRII. Dual luciferase reporter assay – enhancer activity 5 Diabetes Page 6 of 33 The human MEG3-DMR (hg19 - chr14: 100,824,307-100,826,452) and MEG3 enhancer (chr14:101,308,419-101,309,405) were subcloned into pGL3-Basic or pGL4.23[luc2/minP] luciferase reporter (Promega). 50,000 βTC6 cells were seeded per well of a 24-well plate and transfected with 500ng of plasmid DNA and 10ng of pRL-SV40 (Promega). Cells were harvested 24 hours post-transfection and processed for luciferase readout. Experiments were performed in triplicates with four technical replicates per experiment. Allele-specific ChIP-PCR Allele-specific ChIP was performed according to the schema in figure 4A. 30ng of genomic DNA from islet donors was used to identify donors heterozygous for the SNP rs3783355. SNP genotyping was performed using Taqman SNP genotyping Assay (ThermoFisher Scientific C_1259770_10, Cat #4351379) and Taqman genotyping Master Mix (ThermoFisher Scientific Cat# 4371353) on a Stratagene Mx3000P thermocycler. Chromatin was extracted from non- diabetic donors’ islets as previously described (2). The ChIP antibodies and conditions used for this experiment were described by (3). The primers used for this experiment are listed in Supplemental Table 4. Following PCR of the input and ChIP DNA, libraries were prepared using the NuGEN Mondrian SP+ system and sequenced on an Illumina MiSeq to obtain approximately 200,000 reads per library (sequence read count ranged from 24,568 to 916096 reads). For the qualitative assay, 300ng of PCR amplified input or FOXA2 ChIP DNA was digested using the restriction enzyme BanII. Uncut PCR product (150 bp) and BanII digested PCR products (113 bp + 37 bp fragments) were run on a 3% gel to visualize differences. Circular chromosome conformation capture (4C-Seq) 4C-Seq was performed on ~10,000 IEQ of human islets using the enzymes DpnII (first enzyme) and NlaIII (second enzyme) according to (4). The libraries were prepared using the BiS-PCR2 protocol 6 Page 7 of 33 Diabetes (1).