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.