title: Comparative analysis of type 2 diabetes-associated SNP alleles identifies allele-specific DNA-binding proteins for the KCNQ1 locus.
abstract: Although recent genome-wide association studies (GWAS) have been extremely successful, it remains a big challenge to functionally annotate disease‑associated single nucleotide polymorphisms (SNPs), as the majority of these SNPs are located in non‑coding regions of the genome. In this study, we described a novel strategy for identifying the proteins that bind to the SNP‑containing locus in an allele‑specific manner and successfully applied this method to SNPs in the type 2 diabetes mellitus susceptibility gene, potassium voltage‑gated channel, KQT‑like subfamily Q, member 1 (KCNQ1). DNA fragments encompassing SNPs, and risk or non‑risk alleles were immobilized onto the novel nanobeads and DNA‑binding proteins were purified from the nuclear extracts of pancreatic β cells using these DNA‑immobilized nanobeads. Comparative analysis of the allele-specific DNA-binding proteins indicated that the affinities of several proteins for the examined SNPs differed between the alleles. Nuclear transcription factor Y (NF‑Y) specifically bound the non‑risk allele of the SNP rs2074196 region and stimulated the transcriptional activity of an artificial promoter containing SNP rs2074196 in an allele‑specific manner. These results suggest that SNP rs2074196 modulates the affinity of the locus for NF‑Y and possibly induces subsequent changes in gene expression. The findings of this study indicate that our comparative method using novel nanobeads is effective for the identification of allele‑specific DNA‑binding proteins, which may provide important clues for the functional impact of disease‑associated non‑coding SNPs.
authors.affiliation: Department of Metabolic Disorder, Diabetes Research Center, Research Institute, National Center for Global Health and Medicine, Tokyo 162‑8655, Japan.
authors.forename: Masaki
authors.initials: M
authors.lastname: Hiramoto
authors.affiliation: Department of Metabolic Disorder, Diabetes Research Center, Research Institute, National Center for Global Health and Medicine, Tokyo 162‑8655, Japan.
authors.forename: Haruhide
authors.initials: H
authors.lastname: Udagawa
authors.affiliation: Laboratory of Research Advancement, Research Institute, National Center for Geriatrics and Gerontology, Obu, Aichi 474‑8511, Japan.
authors.forename: Atsushi
authors.initials: A
authors.lastname: Watanabe
authors.affiliation: Department of Biochemistry, Tokyo Medical University, Tokyo 160‑8402, Japan.
authors.forename: Keisuke
authors.initials: K
authors.lastname: Miyazawa
authors.affiliation: Department of Metabolic Disorder, Diabetes Research Center, Research Institute, National Center for Global Health and Medicine, Tokyo 162‑8655, Japan.
authors.forename: Naoko
authors.initials: N
authors.lastname: Ishibashi
authors.affiliation: Department of Metabolic Disorder, Diabetes Research Center, Research Institute, National Center for Global Health and Medicine, Tokyo 162‑8655, Japan.
authors.forename: Miho
authors.initials: M
authors.lastname: Kawaguchi
authors.affiliation: Department of Metabolic Disorder, Diabetes Research Center, Research Institute, National Center for Global Health and Medicine, Tokyo 162‑8655, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Uebanso
authors.affiliation: Department of Metabolic Disorder, Diabetes Research Center, Research Institute, National Center for Global Health and Medicine, Tokyo 162‑8655, Japan.
authors.forename: Wataru
authors.initials: W
authors.lastname: Nishimura
authors.affiliation: Department of Metabolic Disorder, Diabetes Research Center, Research Institute, National Center for Global Health and Medicine, Tokyo 162‑8655, Japan.
authors.forename: Takao
authors.initials: T
authors.lastname: Nammo
authors.affiliation: Department of Metabolic Disorder, Diabetes Research Center, Research Institute, National Center for Global Health and Medicine, Tokyo 162‑8655, Japan.
authors.forename: Kazuki
authors.initials: K
authors.lastname: Yasuda
mesh_terms: D000818:Animals; D023081:CCAAT-Binding Factor / Q000378:metabolism*; D045744:Cell Line, Tumor; D004268:DNA-Binding Proteins / Q000378:metabolism*; D003924:Diabetes Mellitus, Type 2 / Q000235:genetics*; D020022:Genetic Predisposition to Disease; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D050417:Insulin-Secreting Cells / Q000378:metabolism; D051657:KCNQ1 Potassium Channel / Q000235:genetics*; D058185:Magnetite Nanoparticles; D020641:Polymorphism, Single Nucleotide; D011401:Promoter Regions, Genetic / Q000235:genetics; D051381:Rats; D015533:Transcriptional Activation / Q000235:genetics
chemical_list: D023081:CCAAT-Binding Factor; D004268:DNA-Binding Proteins; D051657:KCNQ1 Potassium Channel; C496909:KCNQ1 protein, human; D058185:Magnetite Nanoparticles