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Supplemental Data Epigenetic Modulation of Β-Cells By SUPPLEMENTAL DATA EPIGENETIC MODULATION OF β-CELLS BY INTERFERON-α VIA PNPT1-miR26a-TET2 TRIGGERS AUTOIMMUNE DIABETES Mihaela Stefan-Lifshitz1, Esra Karakose2, Lingguang Cui1, Abora Ettela1, Zhengzi Yi3, Weijia Zhang3, and Yaron Tomer1 1Division of Endocrinology and the Fleischer Institute for Diabetes and Metabolism, Albert Einstein College of Medicine, Bronx, NY, 10461; 2Diabetes, Obesity, and Metabolism Institute and 3Department of Medicine Bioinformatics Core, Icahn School of Medicine at Mount Sinai, New York, NY 10029 To whom correspondence should be addressed: Mihaela Stefan-Lifshitz, PhD, Albert Einstein College of Medicine, Forchheimer 702 1300 Morris Park Ave, Bronx, NY, 10461 Tel: 718-430-8530 E-mail: [email protected] KEYWORDS DNA methylation, beta cells, interferon alpha, type 1 diabetes SUPPLEMENTAL RESULTS SI Results: Confirmation of the DNAm array results To confirm the DNAm array results we preformed bisulfite DNA sequencing of four selected genes: the top ranked hypo- and hyper-methylated genes, GGA3 and SLC25A31 respectively, and two genes associated with T1D pathogenesis: IRF7(1) and ERAP1(2). Nine CpG sites from the GGA3 promoter and 45 CpG sites form the SLC25A31 5’UTR region overlapping the oligonucleotide probes cg19184818 and cg09796800 respectively, were analyzed in islet samples treated with IFNα for 24h and 120h and compared with CpG methylation status of untreated samples. The direction of methylation-change at analyzed CpGs was in agreement with the DNAm array analysis: 6.6% more CpG sites in the GGA3 promoter were methylated and 6.2% more CpG sites in the SLC25A31 5’UTR region were un-methylated after 24h of IFNα treatment while 10.8% more sites were methylated in GGA2 and 9.1% sites were un-methylated in SLC25A31 after 120h of IFNα treatment (Fig. S1A). Similarly, confirming the array results, 6.4% more of the 17 CpG sites analyzed in the IRF7 5’UTR region (cg09703963) and 3.1% more of the nine CpGs analyzed in the ERAP1 gene (cg16492584) were un-methylated after 24h of IFNα treatment (Fig. S1B). Genomic distribution of hyper- and hypo-methylation CpG sites We analyzed genomic functional distribution of the hyper- and hypo-methylated CpG sites relative to six gene regions annotated on the 450k array: regions located 1.5kb and 0.2kb from the TSS (TSS1500 and TSS200 respectively), regions within the gene 5’UTR (5’UTR), 1st exon, gene body, and regions within the gene 3’ UTR (3’UTR) (Fig. S2A). There were no significant differences between the distributions of the hyper- and hypo-methylated sites across these regions. Both hypo- and hyper-methylated sites were more prevalent (62%) in the gene body regions followed by regions up-stream to the TSS (14% hypo- and 18% hyper- methylated CpGs) in IFNα treated vs. untreated islets (Fig. S2A). We also assessed the distribution of differentially methylated CpG sites relative to CpG islands, shelfs and shores (3). Both hyper- and hypo- methylated sites were equally distributed relative to the CpG islands and genomic neighborhood regions: shores and shelfs (Fig. S2B). 1 miR-22, miR-29b miR-101 and miR-125 expression in human islets treated with IFNα We measured the expression levels of miR-22, miR-29b, miR-101 and miR-125b in 10 - 22 human islets treated with IFNα and compared with untreated islets. miR-22 and miR-29b expression was significantly downregulated in IFNα treated islets relative to untreated islets: miR-22 expression level was reduced 0.8-fold (P ≤ 0.02), and miR-29b expression was reduced 0.8-fold (P ≤ 0.05) (Fig. S9A). In contrast, miR-101 and miR- 125b expression remained unchanged or slightly increased upon IFNα treatment: miR-101 expression was 1.08- fold (P ≤ 0.3) and miR-125b expression was 1.3-fold (P ≤ 0.2) in IFNα treated compared to untreated islets (Fig. S9A). Because miR-29b and miR-22 levels showed modest but significant downregulation in islets treated with IFNα, we assessed the expression of their primary miRNA transcripts. miR-29b has two primary transcripts, miR- 29-b1 and miR-29b-2 that are encoded by different genes located in chromosome 7q32.3 and 1q32.2, respectively (4). The expression level of miR-29b-1 increased 1.6-fold (P ≤ 0.03) and that of miR-29b-2 increased 1.5-fold (P ≤ 0.005) (Fig. S9B). miR-22 is encoded by an exon of a long non-coding RNA gene, the miR-22 host gene (MIR-22HG) (5). A similar analysis for pri-miR-22 showed that its expression level in IFNα treated relative to untreated islets was non-significantly increased at 1.5-fold (P = 0.1) (Fig. S9B). 2 REFERENCES 1. Heinig M, et al. (2010) A trans-acting locus regulates an anti-viral expression network and type 1 diabetes risk. Nature 467(7314):460-464. 2. Fung EY, et al. (2009) Analysis of 17 autoimmune disease-associated variants in type 1 diabetes identifies 6q23/TNFAIP3 as a susceptibility locus. Genes Immun 10(2):188-191. 3. Portela A & Esteller M (2010) Epigenetic modifications and human disease. Nat Biotechnol 28(10):1057- 1068. 4. Kriegel AJ, Liu Y, Fang Y, Ding X, & Liang M (2012) The miR-29 family: genomics, cell biology, and relevance to renal and cardiovascular injury. Physiol Genomics 44(4):237-244. 5. Huang ZP & Wang DZ (2014) miR-22 in cardiac remodeling and disease. Trends Cardiovasc Med 24(7):267-272. 3 SUPPLEMENTAL FIGURE LEGENDS Figure S1: Confirmation of CpG methylation status for individual genes in human islets treated and untreated with IFNα. (A) CpG methylation status of top ranked hypo- and hyper-methylated genes in IFNα treated vs. un-treated samples. DNAm of the GGA3 gene (Golgi-Localized, Gamma Ear-Containing, ARF- Binding Protein 3) that showed increased DNAm (Log2Rat 1.5) in the promoter region (Illumina ID: cg19184818) and of the SLC25A31 gene (Solute Carrier Family 25 Member 31) that showed decreased DNAm (Log2Rat - 0.9) in regions 0.2 Kb to the TSS (Illumina ID: cg09796800) was analyzed by direct sequencing of bisulfite DNA. Nine CpG sites in the GGA2 promoter and 45 CpG sites in the SLC25A31 5’UTR region were analyzed in islets treated with IFNα for 24h and 120h and compared with CpG methylation status of untreated samples. (B) CpG methylation status of genes showing IFNα induced mRNA expression and hypo-methylated CpG sites: IRF7 (Interferon regulatory factor 7) and ERAP1 (Endoplasmic reticulum aminopeptidase 1). 17 CpG sites from the IRF7 5’ UTR region (Illumina ID: cg09703963) and nine CpGs from the ERAP1 gene (Illumina ID: cg16492584) were analyzed by direct sequencing of bisulfite DNA in islets treated with IFNα for 24h and compared with untreated samples. Figure S2: Genomic distribution and classification of the hypo- and hyper-methylated CpG sites in IFNα treated compared with un-treated islets. (A) Distribution of the hyper- and hypo-methylated CpG sites across six annotation gene sets relative to TSS: first exon, 5’ UTR, 3’ UTR, gene body, 1500 kb and 200 kb downstream the TSS; (B) Distribution of hyper- and hypo-methylated CpG sites according to the CpG content of the DNA region and genomic neighborhood context: regions annotated as islands (regions with at least 500 bp, and > 55% GC content), shores (regions 2 kb either side of an island), shelfs (regions 2 kb outside the shores). N represents North, indicating the region upstream from a CpG island; S represents South indicating, the region downstream from a CpG island. Figure S3: Gene interaction networks of differentially-expressed genes in human islets treated with IFNα. Functional gene interaction networks were identified by Ingenuity Pathway Analysis (IPA). Red color indicates up-regulated gene expression; color intensity is indicative of fold-change level with deep red indicating 4 the highest detected level of expression for genes in the network. Legend insert: each gene was assigned a shape and function by IPA. Figure S4: Confirmation of RNA-seq data by qRT-PCR. Up-regulation of mRNA gene expression was confirmed in islet samples after 24h of IFNα treatment and compared with untreated samples for OAS1, SP100, IFIH1, CD40, TLR3 and IRS1 genes. Results are presented as fold change of expression level in IFNα treated samples relative to untreated samples. Figure S5: IFNα induces up-regulation of Tet1/2/3 mRNA expression in NIT-1 cells. qRT-PCR analysis of Tet1/2/3 gene expression in NIT-1 cells treated with IFNα for 24h and 48h. Results represents fold change of gene expression level in IFNα treated samples relative to untreated samples and are presented as mean fold change of quadruplicated experiments ± SD; *, p<0.05. Figure S6: DNMT3A and DNMT3B expression in human pancreatic islets treated with IFNα. mRNA levels of DNMT3A and DNMT3B was analyzed by qRT-PCR . Results represents fold change of gene expression level in IFNα treated samples relative to untreated samples. The average fold expression level in IFNα treated vs. untreated islets for DNMT1A was 1.28 (p = 0.003) and for DNMT3B was 1.10 (p = 0.11). Each diamond represents an islet sample; dotted horizontal lines represent the median fold level. Figure S7: Quantification of TET hydroxylase activity and 5hmC content in IFNα treated vs. untreated human islets. (A) Quantification of TET hydroxylase activity in nuclear extracts from seven islets treated and un-treated with IFNα. (B) Quantification of 5-hydroxymethylcytosine (5hmC) in genomic DNA from islets treated with IFNα for 24h and compared with untreated islets. (C) 5hmC quantification in islets treated with IFNα for 24h and 48h. White circles, samples untreated with IFNα, black circles, samples treated with IFNα; dotted squares individual pair samples, treated and untreated with IFNα.
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