Long-range gene regulation links genomic type 2 diabetes and obesity risk regions to HHEX, SOX4, and IRX3

Abstract

Genome-wide association studies identified noncoding SNPs associated with type 2 diabetes and obesity in linkage disequilibrium (LD) blocks encompassing HHEX-IDE and introns of CDKAL1 and FTO [Sladek R, et al. (2007) Nature 445:881-885; Steinthorsdottir V, et al. (2007) Nat. Genet 39:770-775; Frayling TM, et al. (2007) Science 316:889-894]. We show that these LD blocks contain highly conserved noncoding elements and overlap with the genomic regulatory blocks of the transcription factor genes HHEX, SOX4, and IRX3. We report that human highly conserved noncoding elements in LD with the risk SNPs drive expression in endoderm or pancreas in transgenic mice and zebrafish. Both HHEX and SOX4 have recently been implicated in pancreas development and the regulation of insulin secretion, but IRX3 had no prior association with pancreatic function or development. Knockdown of its orthologue in zebrafish, irx3a, increased the number of pancreatic ghrelin-producing epsilon cells and decreased the number of insulin-producing beta-cells and glucagon-producing alpha-cells, thereby suggesting a direct link of pancreatic IRX3 function to both obesity and type 2 diabetes.

Fig. 1.

Disease-associated SNPs (Top) and tested elements (elements 1–6) in the context of vertebrate GRBs around HHEX (A), SOX4 (B), and IRX3 (C). GRBs were defined by minimal synteny blocks to zebrafish (tan shading) or stickleback (light blue shading). Hapmap recombination hotspots (blue bars) mark the edges of LD blocks (blocks of interest shaded red). HCNE density plots for pairwise comparisons are colored yellow, orange, and red, based on the minimum percentage identity of the elements ≥50 bp in size used to derive them: human vs. mouse (mm: ≥95%, ≥98%, 100% identity), chicken (gg: ≥90, ≥95, ≥98), Xenopus (xt: ≥70, ≥80, ≥90) and zebrafish (dr: ≥70, ≥80, ≥90). HCNE densities were calculated as number of bases in HCNEs in sliding windows of 300 kb, and curves for different species are drawn to the same scale. HCNEs (black bars) are shown at the lowest threshold for each pairwise comparison. All features, including the suggested regulatory target gene (red) and bystander genes (gray) are shown in human coordinates. Gene annotations were adapted from the UCSC Known Genes set.

Fig. 2.

Reporter expression patterns directed by noncoding elements from T2D risk regions. (A and B) Live dorsal image (A) and cryosection (B) of a 72-hpf transgenic zebrafish showing GFP-reporter expression driven by the Xenopus element equivalent to the rs1111875-containing human HCNE downstream of HHEX (element 1). Red arrowheads point to the pancreatic anlagen. The horizontal line in (A) shows the level of section in B. The cryosection (B) was stained for DNA using DAPI (blue), for muscle actin using rhodamine-phalloidin (red) and for GFP using anti-GFP (green). (C) Sections through the pancreas of a 14.5-d mouse transgenic for rs1111875-containing human HCNE downstream of HHEX (element 1). LacZ staining is seen in the pancreatic anlagen. (D) Live dorsal image of a 48-hpf transgenic zebrafish showing GFP-reporter expression driven by the human HCNE closest to rs7754840 in CDKAL1 (element 2). Arrowheads point to expression in the hindbrain (red) and the primordium of the swim bladder (green). (E and F). Live lateral images of 48-hpf transgenic zebrafish showing GFP-reporter expression driven by the most deeply conserved HCNEs in the obesity-associated LD block of FTO. Element 3 (E) drives expression in the pronephric duct (red arrowhead), whereas element 4 (F) drives expression in the notochord (green arrowheads) and in hindbrain rhombomeres (red arrowhead). (G and H) Live dorsal images of 48-hpf transgenic zebrafish showing GFP-reporter expression driven by the rs1421085-containing HCNE and the rs9939609- containing HCNE in the FTO intron 1. Red arrowheads point to expression in the pancreatic area.

Fig. 3.

Pancreatic buds of 48-hpf zebrafish embryos showing a reduction in the number of insulin-expressing cells and an increase of ghrelin-expressing cells in irx3a morphants. In situ hybridization with insulin (red) and ghrelin (blue) probes on control (A) and irx3a morphant embryos (B). Statistical support for the observation is given in Table 3. (C and D) irx3a morphants display a reduction in pancreatic α-cells. In situ hybridization with glucagon (red in C and D), somatostatin (red in E and F), and ghrelin (blue in C–F) probes on control (C and E) and irx3a morphant embryos (D and F). showing reduction in the number of α-cells (D) and increase of ghrelin-expressing cells (D and F) in the pancreatic bud of 48-hpf irx3a morphant embryos. The number of δ-cells is unaffected. Embryos are in ventral view. Statistical support for the observation is given in Table 4.