Characterization of a Type 2 Diabetes–Associated Islet-Specific Enhancer Cluster in STARD10 by Genome Editing of EndoC-ßH1 Cells

Abstract

Genome-wide association studies have identified >100 genetic loci associated with type 2 diabetes. We recently analysed one locus on chromosome 11q and mapped 5 causal variants in an islet-specific enhancer cluster within the STARD10 gene (Carrat, et al., Am J Hum Gen, 2017). Here, we aimed to understand the function of this cluster in beta cells and how causal variants influence enhancer activity. Using published ATAC-seq data (Varshney A, et al., PNAS, 2017) we analysed the transcriptional activity of 10 open chromatin regions (0.2-2 kb) using promoter-luciferase assay in EndoC-βH1 cells. Of these regions, HS1b displayed a 6.25-fold increase compared with empty vector [luciferase/renilla ratio: 6.25 ± 0.14; Student’s t-test, p<0.001; n=3]. Deletion of HS1b using CRISPR/Cas9 technology led to a significant reduction of glucose-stimulated insulin secretion (GSIS) (15/0.5 mM glucose, fold change: Control, 4.23 ± 0.41 vs. HS1b-del 2.21 ± 0.06; p<0.05; n=3) and ∼50% reduction of STARD10 gene expression (Fold: control 1 vs. HS1b-del, 0.486 ± 0.04; p<0.001, n=3). Chromosome conformation capture (4C and 3C) identified two enhancer regions, termed HS1 and HS5, as interacting with the causal variants including rs140130268. Both regions contain multiple defined CTCF binding sites. To test whether these CTCF binding sites create a chromatin loop defining an enhancer complex, we mutated four of the CTCF binding sites one by one. This resulted in a significant (p<0.05; n=3) impairment in GSIS assays [Control, 4.± 0.46, vs. HS5-1-Mu, 2.75 ± 0.07; HS5-2-Mu, 2.66 ± 0.13; HS1-1-Mu, 3.49 ± 0.02 and HS1-2-Mu, 3.47 ± 0.57]. Taken together, we show that the modification of enhancer elements at the STARD10 locus affects beta cell function. The causal variants, which are physically associated with both HS5 and HS1, are likely to exert their effects through the formation and activity of an enhancer-cluster complex which affects the expression of STARD10 and possibly other genes. Disclosure M. Hu: None. P. Gadue: None. G.A. Rutter: Research Support; Self; Servier.