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• W2012332098 abstract "Genome-wide association studies (GWASs) have identified more than 70 loci associated with type 2 diabetes (T2D), but for most, the underlying causal variants, associated genes, and functional mechanisms remain unknown. At a T2D- and fasting-proinsulin-associated locus on 11q13.4, we have identified a functional regulatory DNA variant, a candidate target gene, and a plausible underlying molecular mechanism. Fine mapping, conditional analyses, and exome array genotyping in 8,635 individuals from the Metabolic Syndrome in Men study confirmed a single major association signal between fasting proinsulin and noncoding variants (p = 7.4 × 10−50). Measurement of allele-specific mRNA levels in human pancreatic islet samples heterozygous for rs11603334 showed that the T2D-risk and proinsulin-decreasing allele (C) is associated with increased ARAP1 expression (p < 0.02). We evaluated four candidate functional SNPs for allelic effects on transcriptional activity by performing reporter assays in rodent pancreatic beta cell lines. The C allele of rs11603334, located near one of the ARAP1 promoters, exhibited 2-fold higher transcriptional activity than did the T allele (p < 0.0001); three other candidate SNPs showed no allelic differences. Electrophoretic mobility shift assays demonstrated decreased binding of pancreatic beta cell transcriptional regulators PAX6 and PAX4 to the rs11603334 C allele. Collectively, these data suggest that the T2D-risk allele of rs11603334 could abrogate binding of a complex containing PAX6 and PAX4 and thus lead to increased promoter activity and ARAP1 expression in human pancreatic islets. This work suggests that increased ARAP1 expression might contribute to T2D susceptibility at this GWAS locus. Genome-wide association studies (GWASs) have identified more than 70 loci associated with type 2 diabetes (T2D), but for most, the underlying causal variants, associated genes, and functional mechanisms remain unknown. At a T2D- and fasting-proinsulin-associated locus on 11q13.4, we have identified a functional regulatory DNA variant, a candidate target gene, and a plausible underlying molecular mechanism. Fine mapping, conditional analyses, and exome array genotyping in 8,635 individuals from the Metabolic Syndrome in Men study confirmed a single major association signal between fasting proinsulin and noncoding variants (p = 7.4 × 10−50). Measurement of allele-specific mRNA levels in human pancreatic islet samples heterozygous for rs11603334 showed that the T2D-risk and proinsulin-decreasing allele (C) is associated with increased ARAP1 expression (p < 0.02). We evaluated four candidate functional SNPs for allelic effects on transcriptional activity by performing reporter assays in rodent pancreatic beta cell lines. The C allele of rs11603334, located near one of the ARAP1 promoters, exhibited 2-fold higher transcriptional activity than did the T allele (p < 0.0001); three other candidate SNPs showed no allelic differences. Electrophoretic mobility shift assays demonstrated decreased binding of pancreatic beta cell transcriptional regulators PAX6 and PAX4 to the rs11603334 C allele. Collectively, these data suggest that the T2D-risk allele of rs11603334 could abrogate binding of a complex containing PAX6 and PAX4 and thus lead to increased promoter activity and ARAP1 expression in human pancreatic islets. This work suggests that increased ARAP1 expression might contribute to T2D susceptibility at this GWAS locus. Genome-wide association studies (GWASs) have identified more than 70 loci associated with type 2 diabetes (T2D [MIM 125853])1Voight B.F. Scott L.J. Steinthorsdottir V. Morris A.P. Dina C. Welch R.P. Zeggini E. Huth C. Aulchenko Y.S. Thorleifsson G. et al.MAGIC investigatorsGIANT ConsortiumTwelve type 2 diabetes susceptibility loci identified through large-scale association analysis.Nat. Genet. 2010; 42: 579-589Crossref PubMed Scopus (1435) Google Scholar, 2Cho Y.S. Chen C.-H. Hu C. Long J. Ong R.T.H. Sim X. Takeuchi F. Wu Y. Go M.J. Yamauchi T. et al.DIAGRAM ConsortiumMuTHER ConsortiumMeta-analysis of genome-wide association studies identifies eight new loci for type 2 diabetes in east Asians.Nat. Genet. 2012; 44: 67-72Crossref Scopus (477) Google Scholar, 3Cho Y.S. Lee J.-Y. Park K.S. Nho C.W. Genetics of type 2 diabetes in East Asian populations.Curr. Diab. Rep. 2012; 12: 686-696Crossref PubMed Scopus (48) Google Scholar, 4Imamura M. Maeda S. Yamauchi T. Hara K. Yasuda K. Morizono T. Takahashi A. Horikoshi M. Nakamura M. Fujita H. et al.Diabetes Genetics Replication and Meta-analysis (DIAGRAM) ConsortiumA single-nucleotide polymorphism in ANK1 is associated with susceptibility to type 2 diabetes in Japanese populations.Hum. Mol. Genet. 2012; 21: 3042-3049Crossref PubMed Scopus (81) Google Scholar, 5Perry J.R.B. Voight B.F. Yengo L. Amin N. Dupuis J. Ganser M. Grallert H. Navarro P. Li M. Qi L. et al.MAGICDIAGRAM ConsortiumGIANT ConsortiumStratifying type 2 diabetes cases by BMI identifies genetic risk variants in LAMA1 and enrichment for risk variants in lean compared to obese cases.PLoS Genet. 2012; 8: e1002741Crossref PubMed Scopus (157) Google Scholar, 6Morris A.P. Voight B.F. Teslovich T.M. Ferreira T. Segrè A.V. Steinthorsdottir V. Strawbridge R.J. Khan H. Grallert H. Mahajan A. et al.Wellcome Trust Case Control ConsortiumMeta-Analyses of Glucose and Insulin-related traits Consortium (MAGIC) InvestigatorsGenetic Investigation of ANthropometric Traits (GIANT) ConsortiumAsian Genetic Epidemiology Network–Type 2 Diabetes (AGEN-T2D) ConsortiumSouth Asian Type 2 Diabetes (SAT2D) ConsortiumDIAbetes Genetics Replication And Meta-analysis (DIAGRAM) ConsortiumLarge-scale association analysis provides insights into the genetic architecture and pathophysiology of type 2 diabetes.Nat. Genet. 2012; 44: 981-990Crossref PubMed Scopus (1409) Google Scholar, 7Li H. Gan W. Lu L. Dong X. Han X. Hu C. Yang Z. Sun L. Bao W. Li P. et al.DIAGRAM ConsortiumAGEN-T2D ConsortiumA genome-wide association study identifies GRK5 and RASGRP1 as type 2 diabetes loci in Chinese Hans.Diabetes. 2013; 62: 291-298Crossref PubMed Scopus (142) Google Scholar, 8Tabassum R. Chauhan G. Dwivedi O.P. Mahajan A. Jaiswal A. Kaur I. Bandesh K. Singh T. Mathai B.J. Pandey Y. et al.DIAGRAMINDICOGenome-wide association study for type 2 diabetes in Indians identifies a new susceptibility locus at 2q21.Diabetes. 2013; 62: 977-986Crossref PubMed Scopus (129) Google Scholar, 9Saxena R. Saleheen D. Been L.F. Garavito M.L. Braun T. Bjonnes A. Young R. Ho W.K. Rasheed A. Frossard P. et al.DIAGRAMMuTHERAGENGenome-wide association study identifies a novel locus contributing to type 2 diabetes susceptibility in Sikhs of Punjabi origin from India.Diabetes. 2013; 62: 1746-1755Crossref PubMed Scopus (126) Google Scholar and ten loci associated with fasting proinsulin levels.10Strawbridge R.J. Dupuis J. Prokopenko I. Barker A. Ahlqvist E. Rybin D. Petrie J.R. Travers M.E. Bouatia-Naji N. Dimas A.S. et al.DIAGRAM ConsortiumGIANT ConsortiumMuTHER ConsortiumCARDIoGRAM ConsortiumC4D ConsortiumGenome-wide association identifies nine common variants associated with fasting proinsulin levels and provides new insights into the pathophysiology of type 2 diabetes.Diabetes. 2011; 60: 2624-2634Crossref PubMed Scopus (269) Google Scholar, 11Huyghe J.R. Jackson A.U. Fogarty M.P. Buchkovich M.L. Stančáková A. Stringham H.M. Sim X. Yang L. Fuchsberger C. Cederberg H. et al.Exome array analysis identifies new loci and low-frequency variants influencing insulin processing and secretion.Nat. Genet. 2013; 45: 197-201Crossref PubMed Scopus (206) Google Scholar A 11q13.4 locus near ARAP1 (MIM 606646), PDE2A (MIM 602658), STARD10, ATG16L2, and FCHSD2 is strongly associated with T2D (rs1552224, p = 1.4 × 10−22),1Voight B.F. Scott L.J. Steinthorsdottir V. Morris A.P. Dina C. Welch R.P. Zeggini E. Huth C. Aulchenko Y.S. Thorleifsson G. et al.MAGIC investigatorsGIANT ConsortiumTwelve type 2 diabetes susceptibility loci identified through large-scale association analysis.Nat. Genet. 2010; 42: 579-589Crossref PubMed Scopus (1435) Google Scholar fasting proinsulin (rs11603334, p = 3.2 × 10−102),10Strawbridge R.J. Dupuis J. Prokopenko I. Barker A. Ahlqvist E. Rybin D. Petrie J.R. Travers M.E. Bouatia-Naji N. Dimas A.S. et al.DIAGRAM ConsortiumGIANT ConsortiumMuTHER ConsortiumCARDIoGRAM ConsortiumC4D ConsortiumGenome-wide association identifies nine common variants associated with fasting proinsulin levels and provides new insights into the pathophysiology of type 2 diabetes.Diabetes. 2011; 60: 2624-2634Crossref PubMed Scopus (269) Google Scholar and 32,33-split proinsulin (rs11603334, p = 1.2 × 10−25).10Strawbridge R.J. Dupuis J. Prokopenko I. Barker A. Ahlqvist E. Rybin D. Petrie J.R. Travers M.E. Bouatia-Naji N. Dimas A.S. et al.DIAGRAM ConsortiumGIANT ConsortiumMuTHER ConsortiumCARDIoGRAM ConsortiumC4D ConsortiumGenome-wide association identifies nine common variants associated with fasting proinsulin levels and provides new insights into the pathophysiology of type 2 diabetes.Diabetes. 2011; 60: 2624-2634Crossref PubMed Scopus (269) Google Scholar This locus is also nominally associated with the insulinogenic index (rs1552224, p = 2 × 10−6) and both insulin (p = 0.001) and glucose (p = 2 × 10−5) levels at 30 min during an oral glucose tolerance test.12Nielsen T. Sparsø T. Grarup N. Jørgensen T. Pisinger C. Witte D.R. Hansen T. Pedersen O. Diabetes Genetics Replication and Meta-analysis (DIAGRAM) ConsortiumType 2 diabetes risk allele near CENTD2 is associated with decreased glucose-stimulated insulin release.Diabetologia. 2011; 54: 1052-1056Crossref PubMed Scopus (30) Google Scholar The clustering of multiple phenotypic associations related to proinsulin processing and insulin secretory response during an oral glucose challenge suggests that the affected target gene(s) might play a role in pancreatic beta cell function. Currently, the functional DNA variant(s), the affected gene(s), and the underlying molecular genetic mechanism(s) contributing to these associations are unknown. SNPs rs1552224 and rs11603334 are in perfect linkage disequilibrium (LD) with each other (r2 = 1, 1000 Genomes European ancestry [EUR]) and are located within the first exon and 5′ UTR of ARAP1 RefSeq isoforms NM_015242.4 and NM_001135190.1 (rs1552224, +305 nt from the transcription start site [TSS]; rs11603334, +418 nt from the TSS) at 72.11 Mb (hg18, UCSC Genome Browser) on chromosome 11. A third ARAP1 RefSeq isoform, NM_001040118.2, is expressed from an alternative promoter and TSS located at 72.14 Mb. We designate the promoter at 72.11 Mb as P1 and the promoter at 72.14 Mb as P2. We hypothesized that functional variant(s) at this locus are in high LD (r2 ≥ 0.8) with rs1552224 and rs11603334. None of the variants in high LD with these SNPs are within gene coding regions where they could alter protein function, suggesting that functional common SNP(s) at this locus might influence gene regulation. Genes within the LD region containing rs1552224 and rs11603334 have reported expression in human pancreas, islets, and flow-sorted beta cells;1Voight B.F. Scott L.J. Steinthorsdottir V. Morris A.P. Dina C. Welch R.P. Zeggini E. Huth C. Aulchenko Y.S. Thorleifsson G. et al.MAGIC investigatorsGIANT ConsortiumTwelve type 2 diabetes susceptibility loci identified through large-scale association analysis.Nat. Genet. 2010; 42: 579-589Crossref PubMed Scopus (1435) Google Scholar however, islet expression of ATG16L2, FCHSD2, and PDE2A might not be above background.13Wu C. Orozco C. Boyer J. Leglise M. Goodale J. Batalov S. Hodge C.L. Haase J. Janes J. Huss 3rd, J.W. Su A.I. BioGPS: an extensible and customizable portal for querying and organizing gene annotation resources.Genome Biol. 2009; 10: R130Crossref PubMed Scopus (1093) Google Scholar, 14Su A.I. Wiltshire T. Batalov S. Lapp H. Ching K.A. Block D. Zhang J. Soden R. Hayakawa M. Kreiman G. et al.A gene atlas of the mouse and human protein-encoding transcriptomes.Proc. Natl. Acad. Sci. USA. 2004; 101: 6062-6067Crossref PubMed Scopus (2848) Google Scholar, 15Parker S.C.J. Stitzel M.L. Taylor D.L. Orozco J.M. Erdos M.R. Akiyama J.A. van Bueren K.L. Chines P.S. Narisu N. Black B.L. et al.NISC Comparative Sequencing ProgramNational Institutes of Health Intramural Sequencing Center Comparative Sequencing Program AuthorsNISC Comparative Sequencing Program AuthorsChromatin stretch enhancer states drive cell-specific gene regulation and harbor human disease risk variants.Proc. Natl. Acad. Sci. USA. 2013; 110: 17921-17926Crossref PubMed Scopus (462) Google Scholar None of these genes have been demonstrated to play roles in insulin processing or secretion. ARAP1 activates Arf and Rho GTPases, which regulate membrane trafficking and actin cytoskeleton reorganization.16Miura K. Jacques K.M. Stauffer S. Kubosaki A. Zhu K. Hirsch D.S. Resau J. Zheng Y. Randazzo P.A. ARAP1: a point of convergence for Arf and Rho signaling.Mol. Cell. 2002; 9: 109-119Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar STARD10 binds and transfers membrane phospholipids.17Olayioye M.A. Vehring S. Müller P. Herrmann A. Schiller J. Thiele C. Lindeman G.J. Visvader J.E. Pomorski T. StarD10, a START domain protein overexpressed in breast cancer, functions as a phospholipid transfer protein.J. Biol. Chem. 2005; 280: 27436-27442Crossref PubMed Scopus (72) Google Scholar PDE2A is a cyclic nucleotide phosphodiesterase that degrades second messengers cGMP and cAMP.18Rosman G.J. Martins T.J. Sonnenburg W.K. Beavo J.A. Ferguson K. Loughney K. Isolation and characterization of human cDNAs encoding a cGMP-stimulated 3′,5′-cyclic nucleotide phosphodiesterase.Gene. 1997; 191: 89-95Crossref PubMed Scopus (114) Google Scholar ATG16L2 shares sequence homology with ATG16, a protein required for autophagy in yeast.19Matsushita M. Suzuki N.N. Obara K. Fujioka Y. Ohsumi Y. Inagaki F. Structure of Atg5.Atg16, a complex essential for autophagy.J. Biol. Chem. 2007; 282: 6763-6772Crossref PubMed Scopus (183) Google Scholar FCHSD2 is named for its FCH and SH3 protein domains.20Katoh M. Katoh M. Identification and characterization of human FCHSD1 and FCHSD2 genes in silico.Int. J. Mol. Med. 2004; 13: 749-754PubMed Google Scholar Here, we show data supporting rs11603334 as a functional variant regulating ARAP1 expression. Dense fine-mapping data and conditional analyses support a single association signal. We demonstrate that the T2D-risk allele (C) of rs11603334 is associated with increased ARAP1 transcript levels in primary human pancreatic islets, disrupts binding of a protein complex containing transcriptional regulators, and increases transcriptional activity at the ARAP1 P1 promoter. These data suggest that increased ARAP1 P1 promoter activity and ARAP1 expression might be molecular consequences of the T2D-associated variants in this region. In the proinsulin association analyses, we included 8,635 Finnish men without diabetes and not taking diabetes medication (mean age = 57.2 years [range = 45–74 years]; mean body mass index [BMI] = 26.8 kg/m2 [range = 16.2–51.6 kg/m2]; fasting plasma glucose levels < 7 mmol/l; and plasma glucose levels after 2 hr oral glucose tolerance test < 11.1 mmol/l) from the population-based Metabolic Syndrome in Men (METSIM) study.21Stancáková A. Javorský M. Kuulasmaa T. Haffner S.M. Kuusisto J. Laakso M. Changes in insulin sensitivity and insulin release in relation to glycemia and glucose tolerance in 6,414 Finnish men.Diabetes. 2009; 58: 1212-1221Crossref PubMed Scopus (254) Google Scholar Fasting plasma-specific proinsulin (Human Proinsulin RIA Kit, Linco Research; no cross-reaction with insulin or C-peptide) and fasting insulin (ADVIA Centaur Insulin IRI, 02230141, Siemens Medical Solutions Diagnostics; minimal cross-reaction with proinsulin or C-peptide) were measured by immunoassay. For the T2D association analyses, 1,389 T2D cases and 5,748 normoglycemic controls were included. The study was approved by the ethics committee of the University of Kuopio and Kuopio University Hospital, and informed consent was obtained from all study participants. Samples were genotyped with the Illumina HumanOmniExpress BeadChip. In sum, 681,789 autosomal SNPs passed quality control (Hardy-Weinberg equilibrium [HWE] p ≥ 1 × 10−6 in the total sample; call frequency ≥ 0.97). The same samples were previously genotyped with the Illumina HumanExome BeadChip,11Huyghe J.R. Jackson A.U. Fogarty M.P. Buchkovich M.L. Stančáková A. Stringham H.M. Sim X. Yang L. Fuchsberger C. Cederberg H. et al.Exome array analysis identifies new loci and low-frequency variants influencing insulin processing and secretion.Nat. Genet. 2013; 45: 197-201Crossref PubMed Scopus (206) Google Scholar which focuses on protein-altering variants selected from >12,000 exome and genome sequences. We used a two-step imputation strategy wherein samples were pre-phased with ShapeIT version 222Delaneau O. Zagury J.-F. Marchini J. Improved whole-chromosome phasing for disease and population genetic studies.Nat. Methods. 2013; 10: 5-6Crossref PubMed Scopus (866) Google Scholar before genotypes were imputed with Minimac.23Howie B. Fuchsberger C. Stephens M. Marchini J. Abecasis G.R. Fast and accurate genotype imputation in genome-wide association studies through pre-phasing.Nat. Genet. 2012; 44: 955-959Crossref PubMed Scopus (1205) Google Scholar To increase imputation quality, we used 5,474 haplotypes from the 2,737 central-northern European samples sequenced within the Genetics of Type 2 Diabetes (GoT2D) study as a reference panel (C.F., J. Flannick, K. Gaulton, H. Kang, and the GoT2D Consortium, unpublished data). Assuming an additive genetic model and adjusting for age, BMI, and log-transformed fasting plasma insulin, we tested SNP associations with log-transformed fasting plasma proinsulin levels by using a linear mixed model with an empirical kinship matrix to account for relatedness as implemented in EMMAX.24Kang H.M. Sul J.H. Service S.K. Zaitlen N.A. Kong S.-Y. Freimer N.B. Sabatti C. Eskin E. Variance component model to account for sample structure in genome-wide association studies.Nat. Genet. 2010; 42: 348-354Crossref PubMed Scopus (1619) Google Scholar We analyzed both raw residuals and rank-based inverse-normal-transformed residuals to assess robustness of rare-variant associations with outliers. Genotyped variants with minor allele count ≥ 5 (minor allele frequency [MAF] ∼ 0.03%) and HWE p ≥ 1 × 10−6 and imputed variants with imputation quality score R2 > 0.3 and MAF ≥ 0.03% were included in the analyses. To identify additional independent signals in the region, we performed conditional analyses on previously reported lead SNP rs11603334 or on our fine-mapped lead SNP rs7109575 by using allele count (genotyped variants) or allelic dosage (imputed variants) as a covariate in the model. SNP associations with T2D were tested similarly but with adjustment for age only. Kang et al.24Kang H.M. Sul J.H. Service S.K. Zaitlen N.A. Kong S.-Y. Freimer N.B. Sabatti C. Eskin E. Variance component model to account for sample structure in genome-wide association studies.Nat. Genet. 2010; 42: 348-354Crossref PubMed Scopus (1619) Google Scholar describe and motivate the use of a linear mixed model for analysis of a binary outcome. Human islets from nondiabetic organ donors were obtained from the National Disease Research Interchange and the Islet Cell Resource Center Integrated Islet Distribution Program. DNA and RNA were obtained from 87 primary human pancreatic islet samples with the use of the PureGene (DNA) or RNAeasy (RNA) kits (QIAGEN). Reverse transcription of the RNA was performed with the Superscript III First-Strand Synthesis System (Life Technologies). Common, transcribed SNPs (MAF ≥ 0.1) with the highest LD values with previously reported lead SNPs rs11603334 and rs1552224 were selected for testing allelic expression of ARAP1, STARD10, PDE2A, and FCHSD2. LD plots including the transcribed SNPs were created with the use of the HapMap Genome Browser (Figure S1, available online).25Thorisson G.A. Smith A.V. Krishnan L. Stein L.D. The International HapMap Project Web site.Genome Res. 2005; 15: 1592-1593Crossref PubMed Scopus (463) Google Scholar High-quality genomic DNA (gDNA) and mRNA were available for five islet samples heterozygous for rs11603334 and rs1552224; one additional high-quality heterozygous sample each was available for mRNA only or gDNA only. For each transcribed SNP, the relative proportions of each allele comprising the gDNA and cDNA were quantified with Sequenom iPLEX MALDI-TOF mass-spectrometry-based genotyping (Sequenom). Primers were designed with MassARRAY (Sequenom) (Table S1). We designed primers within a single exon to avoid amplicon size differences between gDNA and cDNA, except for rs2291289, which is located near an exon-intron boundary. To control for assay variation, we analyzed the proportions of each SNP allele measured in the cDNA relative to measurements taken in the gDNA. Data are reported as the percentage of total gDNA or cDNA containing a given transcribed SNP allele. The statistical significance of the differences in allelic representation was determined on the basis of LD scenarios as previously described.26Fogarty M.P. Xiao R. Prokunina-Olsson L. Scott L.J. Mohlke K.L. Allelic expression imbalance at high-density lipoprotein cholesterol locus MMAB-MVK.Hum. Mol. Genet. 2010; 19: 1921-1929Crossref PubMed Scopus (27) Google Scholar In brief, two-sided t tests were used when the transcribed SNP and lead SNPs were in perfect LD (D′ = 1.0, r2 = 1.0) or when the transcribed SNP and lead SNPs were in intermediate LD with low pairwise correlation (D′ ≈ 1.0, r2 < 0.2). For all t tests, F tests were first used for determining equal or unequal variance. Nonparametric Wilcoxon pairwise tests were used instead of t tests when gDNA or cDNA measurement data were not normally distributed. One-sided F tests were used when the transcribed SNP and lead SNPs were in low LD (D′ < 0.2, r2 < 0.2). All known variants in high LD (r2 ≥ 0.8, EUR) with rs11603334 and rs1552224 were identified with the use of Phase 1 data from the 1000 Genomes Project.27Abecasis G.R. Auton A. Brooks L.D. DePristo M.A. Durbin R.M. Handsaker R.E. Kang H.M. Marth G.T. McVean G.A. 1000 Genomes Project ConsortiumAn integrated map of genetic variation from 1,092 human genomes.Nature. 2012; 491: 56-65Crossref PubMed Scopus (5677) Google Scholar Variants were prioritized on the basis of location relative to regions of potential regulatory function, as indicated by the following: open chromatin in primary human pancreatic islets detected by FAIRE28Gaulton K.J. Nammo T. Pasquali L. Simon J.M. Giresi P.G. Fogarty M.P. Panhuis T.M. Mieczkowski P. Secchi A. Bosco D. et al.A map of open chromatin in human pancreatic islets.Nat. Genet. 2010; 42: 255-259Crossref PubMed Scopus (415) Google Scholar and DNase hypersensitivity,29Stitzel M.L. Sethupathy P. Pearson D.S. Chines P.S. Song L. Erdos M.R. Welch R. Parker S.C.J. Boyle A.P. Scott L.J. et al.NISC Comparative Sequencing ProgramGlobal epigenomic analysis of primary human pancreatic islets provides insights into type 2 diabetes susceptibility loci.Cell Metab. 2010; 12: 443-455Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar accessible chromatin in primary human pancreatic islets detected by chromatin immunoprecipitation sequencing (ChIP-seq) of active histone H3 lysine methylation or acetylation modifications (H3K4me3, H3K4me1, and H3K9ac) as reported in the Human Epigenome Atlas,30Bernstein B.E. Stamatoyannopoulos J.A. Costello J.F. Ren B. Milosavljevic A. Meissner A. Kellis M. Marra M.A. Beaudet A.L. Ecker J.R. et al.The NIH Roadmap Epigenomics Mapping Consortium.Nat. Biotechnol. 2010; 28: 1045-1048Crossref PubMed Scopus (1242) Google Scholar and transcription factor binding in ENCODE tissues or cell lines detected by ChIP-seq.31ENCODE Project ConsortiumA user’s guide to the encyclopedia of DNA elements (ENCODE).PLoS Biol. 2011; 9: e1001046Crossref PubMed Scopus (1114) Google Scholar The MIN6 mouse insulinoma beta cell line32Miyazaki J. Araki K. Yamato E. Ikegami H. Asano T. Shibasaki Y. Oka Y. Yamamura K. Establishment of a pancreatic beta cell line that retains glucose-inducible insulin secretion: special reference to expression of glucose transporter isoforms.Endocrinology. 1990; 127: 126-132Crossref PubMed Scopus (1053) Google Scholar was grown in Dulbecco’s modified Eagle’s medium (Sigma) supplemented with 10% fetal bovine serum (FBS), 1 mM sodium pyruvate, and 100 μM 2-mercaptoethanol. The INS-1-derived 832/13 rat insulinoma beta cell line33Hohmeier H.E. Mulder H. Chen G. Henkel-Rieger R. Prentki M. Newgard C.B. Isolation of INS-1-derived cell lines with robust ATP-sensitive K+ channel-dependent and -independent glucose-stimulated insulin secretion.Diabetes. 2000; 49: 424-430Crossref PubMed Scopus (706) Google Scholar (a gift from C.B. Newgard, Duke University) was grown in RPMI 1640 culture medium (Corning Cellgro) supplemented with 10% FBS, 2 mM L-glutamine, 1 mM sodium pyruvate, 10 mM HEPES, and 0.05 mM 2-mercaptoethanol. Both lines were maintained at 37°C and 5% CO2. One day prior to transfection, MIN6 cells were seeded at a density of 200,000 cells per well on 24-well plates. 832/13 cells were seeded at a density of 300,000 cells per well on 24-well plates or 150,000 cells per well on 48-well plates. Genomic regions including variant positions were PCR amplified from human gDNA with 5 PRIME Mastermix (5 PRIME) or the Expand High Fidelity PCR System (Roche) with the primers listed in Table S2. Amplified DNA was subcloned with restriction enzymes XhoI, KpnI, and/or NheI and T4 DNA Ligase (New England Biolabs) into the multiple cloning site upstream of the firefly luciferase gene in the pGL4.10 promoterless vector (Promega). Site-directed mutagenesis was performed with the QuikChange II XL Site-Directed Mutagenesis Kit (Agilent) and the primers listed in Table S2. For each construct, two to ten independent clones were selected. The fidelity and genotype of each gDNA construct were verified by sequencing. Equal amounts, between 600 and 800 ng, of each gDNA construct or empty pGL4.10 promoterless vector were cotransfected with 80 ng of Renilla luciferase vector into duplicate wells of MIN6 or 832/13 cells with Lipofectamine 2000 (Life Technologies). Transfected cells were incubated at 37°C and 5% CO2 overnight, and the transfection media were replaced with fresh culture media after 24 hr. Forty-eight hours posttransfection, cell lysates were collected and assayed for luciferase activity with the Dual-Luciferase Reporter Assay System (Promega). We normalized firefly luciferase activity to that of Renilla luciferase to control for differences in transfection efficiency. Normalized data are reported as the fold change (±SD) in activity relative to that of the empty pGL4.10 promoterless vector control. Two-sided Student’s t tests were used for comparing luciferase activity between genotypes or haplotypes. F tests were used for determining equal or unequal variance. Two-way ANOVA was used for simultaneous comparisons of haplotypes resulting from two independently tested SNPs. Four sets of complementary 21-mer oligonucleotides centered on rs11603334 (C/T) or rs1552224 (T/G) were generated; each contained either of the two alternate alleles, both with and without biotin end labeling (Integrated DNA Technologies). We annealed each set to create double-stranded oligonucleotides by incubating 50 pmol of each single-stranded oligonucleotide in buffer containing 10 mM Tris, 1 mM EDTA, and 50 mM NaCl at 95°C for 5 min and then gradually cooled them to 4°C. Double-stranded oligonucleotide sequences are specified in Table S3. Nuclear protein lysates were extracted from MIN6 and 832/13 cell pellets with NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific), and protein concentrations were determined with the Pierce BCA Protein Assay (Thermo Scientific). Electrophoretic mobility shift assays (EMSAs) were carried out with the LightShift Chemiluminescent EMSA Kit (Thermo Scientific) according to the manufacturer’s instructions. Each protein-DNA binding reaction contained 1× binding buffer, 1 μg poly(dIdC), 4 μg nuclear protein lysates, and 100 fmol biotin-labeled double-stranded oligonucleotide in 20 μl total reaction volume. For reactions demonstrating DNA competition, 60-fold excess unlabeled double-stranded oligonucleotide was preincubated with nuclear protein lysates in the reaction mixture for 20 min before the addition of biotin-labeled oligonucleotide. For EMSA reactions with supershift, 4 μg antibody was added to the final reaction mixture and the samples were incubated for an additional 30 min. We used JASPAR,34Portales-Casamar E. Thongjuea S. Kwon A.T. Arenillas D. Zhao X. Valen E. Yusuf D. Lenhard B. Wasserman W.W. Sandelin A. JASPAR 2010: the greatly expanded open-access database of transcription factor binding profiles.Nucleic Acids Res. 2010; 38: D105-D110Crossref PubMed Scopus (471) Google Scholar TRANSFAC,35Matys V. Fricke E. Geffers R. Gössling E. Haubrock M. Hehl R. Hornischer K. Karas D. Kel A.E. Kel-Margoulis O.V. et al.TRANSFAC: transcriptional regulation, from patterns to profiles.Nucleic Acids Res. 2003; 31: 374-378Crossref PubMed Scopus (1657) Google Scholar CONSITE,36Sandelin A. Wasserman W.W. Lenhard B. ConSite: web-based prediction of regulatory elements using cross-species comparison.Nucleic Acids Res. 2004; 32: W249-W252Crossref PubMed Scopus (354) Google Scholar PWMSCAN,37Levy S. Hannenhalli S. Identification of transcription factor binding sites in the human genome sequence.Mamm. Genome. 2002; 13: 510-514Crossref PubMed Scopus (95) Google Scholar and Tfsitescan38Ghosh D. Object-oriented transcription factors database (ooTFD).Nucleic Acid" @default.
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• W2012332098 title "A Common Functional Regulatory Variant at a Type 2 Diabetes Locus Upregulates ARAP1 Expression in the Pancreatic Beta Cell" @default.
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