Identification and characterization of a FOXA2-regulated transcriptional enhancer at a type 2 diabetes intronic locus that controls GCKR expression in liver cells

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.Electronic supplementary materialThe online version of this article (doi:10.1186/s13073-017-0453-x) contains supplementary material, which is available to authorized users.

Functional Variant in the GCKR Gene Affects Lactate Levels Differentially in the Fasting State and During Hyperglycemia

“…This may generate a cytoplasmic pool of free GCK that is suggested to induce elevated rate of glycolysis in the fasting state. Thus, our current results, together with our previous work 17 and the characterization of the P446L variant 16 , allow us to propose a model for the combined effect of these two genetic variants on hepatic glucose metabolism (Fig. 4 ).…”

“…We recently demonstrated that a locus represented by rs780094 is a transcriptional enhancer that regulates GCKR expression 17 . We showed that the CGG haplotype, formed by the rs780094, rs780095 and rs780096 genetic variants, had higher transcriptional activity compared to the TAC haplotype, suggesting that differences in GCKR expression between the carriers of these two haplotypes may explain previously reported associations of this locus with metabolic parameters, including lactate.…”

“…Our previous characterization of an intronic locus including rs780094 demonstrated that a transcriptional enhancer regulates GCKR expression in response to FOXA2 in a haplotype specific way (CGG > TAC; rs780094-rs780095-rs780096) 17 . This suggests that FOXA2 activating signals may induce differential GCKR transcription through the allele-specific activation of the enhancer by FOXA2.…”

“…Functional studies of rs1260326 have shown that the minor T allele of a non-synonymous P446L substitution of GCKR leads to a weak GCK binding at low glucose concentrations, and impaired response to fructose-6 phosphate 15 , 16 . We recently demonstrated that the intronic locus including rs780094 is a transcriptional enhancer that regulates GCKR expression in a haplotype specific manner 17 .…”

CRISPR/Cas9-Based Engineering of the Epigenome

“…Moreover, the use of strong transactivators and repressors coupled to dCas9 has recently been demonstrated as a powerful tool to evaluate the role of candidate genes to model diseases in vitro , based on iPSC differentiation and culture of primary samples (Ho et al, 2017; Lopez Rodriguez et al, 2017). The next step is to apply epigenome engineering in vivo , and explore its potential for therapeutic applications.…”