Mapping of long-range INS promoter interactions reveals a role for calcium-activated chloride channel ANO1 in insulin secretion

Abstract

We used circular chromatin conformation capture (4C) to identify a physical contact in human pancreatic islets between the region near the insulin (INS) promoter and the ANO1 gene, lying 68 Mb away on human chromosome 11, which encodes a Ca(2+)-dependent chloride ion channel. In response to glucose, this contact was strengthened and ANO1 expression increased, whereas inhibition of INS gene transcription by INS promoter targeting siRNA decreased ANO1 expression, revealing a regulatory effect of INS promoter on ANO1 expression. Knockdown of ANO1 expression caused decreased insulin secretion in human islets, establishing a physical proximity-dependent feedback loop involving INS transcription, ANO1 expression, and insulin secretion. To explore a possible role of ANO1 in insulin metabolism, we carried out experiments in Ano1(+/-) mice. We observed reduced serum insulin levels and insulin-to-glucose ratios in high-fat diet-fed Ano1(+/-) mice relative to Ano1(+/+) mice fed the same diet. Our results show that determination of long-range contacts within the nucleus can be used to detect novel and physiologically relevant mechanisms. They also show that networks of long-range physical contacts are important to the regulation of insulin metabolism.

Keywords: chloride channel; diabetes; insulin secretion.

Fig. 1.

4C-Seq analysis reveals the physical association of the ANO1 gene locus with the INS promoter in human pancreatic islets. (A) 4C-Seq analysis of INS-associated loci in the IDDM4 locus in chromosome 11 (NCBI36/hg18) (Upper) and the ANO1 gene locus within the IDDM4 (Lower). The four Bgl II sites used in 3C-PCR for confirmation of 4C results are shown. (B) 3C-PCR analysis of the interactions of the INS promoter with the ANO1/FADD genes and the nearby FGF3 and PPF1A1 genes in human islets cultured in the basal islet media containing 5.5 mM glucose. (C) TaqMan quantitative 3C analysis of the INS–ANO1 interactions in islets, islet-derived hIPCs, and primary human fibroblasts. Data are mean ± SEM (n = 8). (D) TaqMan quantitative 3C analysis of the INS–ANO1 interactions in islets from two donors before and after treatment with 25 mM glucose for 1 h. Data are mean ± SEM (n = 8). (E) qRT-PCR analysis of ANO1 (orange bar) and FADD (green bar) gene expression in islets from three donors before and after treatment with 25 mM glucose for the indicated times. The RNA levels are normalized to those of HPRT1. Plotted are mRNA levels relative to those at t = 0. Data are mean ± SEM (n = 8).

Fig. 2.

The INS promoter positively regulates ANO1 gene expression in human islets. (A) qRT-PCR analysis of INS (red bar), ANO1 (orange bar), and FADD (black bar) gene expression in islets and islet-derived hIPCs from two donors. Plotted are mRNA levels in hIPCs relative to those in the corresponding islets. Data are mean ± SEM (n = 8). (B) qRT-PCR analysis of ANO1 (orange bar) and FADD (black bar) gene expression in islets from two donors treated with nontargeting control (Ctl) siRNA or one of the two siRNAs targeting to the INS promoter (INSP). The mRNA levels are plotted relative to the nontargeting control. (C) Promoter luciferase reporter assays in transfected MIN6 mouse β cells. The human ANO1 promoter luciferase constructs without (Top) or with insertion of human INS promoter in the forward (Middle) or reverse (Bottom) orientation was transfected into MIN6 cells. Plotted are the reporter luciferase activities relative to the ANO1 promoter luciferase construct without the INS promoter. Data are mean ± SEM (n = 12).

Fig. 3.

ANO1 is a positive regulator of insulin secretion in human islets. (A) qRT-PCR analysis of ANO1 (orange bar), FADD (black bar), and INS (red bar) gene expression in islets treated with nontargeting control siRNA or with one of the two ANO1-gene specific siRNAs. The mRNA levels are plotted relative to nontargeting control. (B) ELISA analysis of insulin levels in the medium for islets from two donors treated separately as in A. The insulin levels were normalized to the number of islets needed to produce 1 µg of total RNA. Data are mean ± SEM (n = 4). (C) ELISA analysis of insulin levels in the medium for islets from three donors treated with mock (DMSO, red bars), 1.5 or 3 μM ANO1 channel inhibitor T16Ainh–A01 (blue bars), or 2 μM ANO1 channel activator Ecat (green bar). Insulin levels were normalized to the amount of genomic DNA made from the corresponding islets. Plotted are insulin levels relative to those in islets without 25 mM glucose treatment (column 1). Data are mean ± SEM (n = 4).

Fig. 4.

Impaired insulin response to a glucose load in HFD-fed Ano1^+/− mice. Ano1^+/+ and Ano1^+/− mice fed either a regular diet (RD; n = 8–9) or an HFD (n = 9) were subjected to a GTT after 5 wk (A) or 13 wk (B) of challenge. After an overnight fast, mice were injected i.p. with 2 mg/g glucose. Blood glucose (Left) was measured before the injection (t0) and at 15, 30, 60, 120 min after the injection. Plasma insulin (Center) was measured at t0 and at 15 and 120 min after the injection. Values are presented with respect to time (Left and Center) and as a ratio (a.u., arbitrary unit) for the 15-min time point (Right). *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.005, t test.