Abstract
BackgroundGenome-wide association studies (GWAS) have identified more than 100 genetic loci associated with type 2 diabetes (T2D). However, the underlying biological mechanisms for many of these associations remain unknown. GWAS signals close to the glucokinase regulatory protein gene (GCKR) have been reported for lipid and glucose metabolism traits and the risk of T2D. We investigated the regulatory function of an intronic locus at GCKR represented by the lead single nucleotide polymorphism (SNP) rs780094.MethodsWe used ENCODE project histone modification and transcription factor binding data to determine the regulatory features of a GCKR intronic locus formed by the high linkage disequilibrium rs780094(C/T), rs780095(G/A), and rs780096(G/C) SNPs. Characterization of the transcriptional activity of this region was assessed by luciferase reporter assays in HepG2 cells and mouse primary hepatocytes. ChIP-qPCR was used to determine the levels of haplotype specific transcription factor binding and histone marks. A CRISPR-dCas9 transcriptional activator system and qPCR were used to activate the locus and measure GCKR expression, respectively. Differential haplotype expression was measured from human liver biopsies.ResultsThe ENCODE data suggest the existence of a liver-specific intragenic enhancer at the locus represented by s780094. We observed that FOXA2 increased the transcriptional activity of this region in a haplotype specific way (CGG > TAC; rs780094, rs780095, and rs780096). In addition, the CGG haplotype showed higher binding to FOXA2 and higher levels of the H3K27Ac histone mark. The epigenetic activation of this locus increased the expression of endogenous GCKR in HepG2 cells, confirming that GCKR is the direct target gene of the enhancer. Finally, we confirmed that the CGG haplotype exhibits higher levels of transcription in human liver.ConclusionsOur results demonstrate the existence of a liver-specific FOXA2-regulated transcriptional enhancer at an intronic T2D locus represented by rs780094, rs780095, and rs780096 SNPs that increases GCKR expression. Differential haplotype regulation suggests the existence of cis regulatory effects that may contribute to the associated traits at this locus.
Highlights
Genome-wide association studies (GWAS) have identified more than 100 genetic loci associated with type 2 diabetes (T2D)
Genomic and regulatory features of the rs780094 locus We investigated the chromatin features of the region containing rs780094, rs780095, and rs780096 by exploring the ENCODE Project data for histone modifications and transcription factor (TF) binding [22]
ChIP-seq data for TFs showed the binding of Forkhead box protein A2 (FOXA2), RXRA, and MAFK TFs to this locus, which are bound to the GCKR promoter (Fig. 1a)
Summary
Genome-wide association studies (GWAS) have identified more than 100 genetic loci associated with type 2 diabetes (T2D). GWAS signals close to the glucokinase regulatory protein gene (GCKR) have been reported for lipid and glucose metabolism traits and the risk of T2D. Genome-wide association studies (GWAS) have identified more than 100 common single nucleotide polymorphisms (SNPs) associated with T2D and glycemic traits [1]. GKRP inhibits GCK by a direct effect on enzyme kinetics or by sequestering GCK in the nucleus, where GCK is unable to facilitate glycolysis [2]. This inhibitory mechanism operates as a negative feedback loop in the control of glucose disposal by the liver. Given the major role of hepatic GCK in glucose metabolism in the liver, disturbances in GKRP–GCK regulation at the transcriptional or post-transcriptional level may directly affect glucose metabolism and produce subsequent changes in other metabolic traits
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