Foxa2 and MafA regulate islet-specific glucose-6-phosphatase catalytic subunit-related protein gene expression

Abstract

Islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP/G6PC2) is a major autoantigen in both mouse and human type 1 diabetes. IGRP is selectively expressed in islet b cells and polymorphisms in the IGRP gene have recently been associated with variations in fasting blood glucose levels and cardiovascular-associated mortality in humans. Chromatin immunoprecipitation (ChIP) assays have shown that the IGRP promoter binds the islet-enriched transcription factors Pax-6 and BETA2. We show here, again using ChIP assays, that the IGRP promoter also binds the islet-enriched transcription factors MafA and Foxa2. Single binding sites for these factors were identified in the proximal IGRP promoter, mutation of which resulted in decreased IGRP fusion gene expression in bTC-3, Hamster insulinoma tumor (HIT), and Min6 cells. ChiP assays have shown that the islet-enriched transcription factor Pdx-1 also binds the IGRP promoter, but mutational analysis of four Pdx-1 binding sites in the proximal IGRP promoter revealed surprisingly little effect of Pdx-1 binding on IGRP fusion gene expression in bTC-3 cells. In contrast, in both HIT and Min6 cells mutation of these four Pdx-1 binding sites resulted in a w50% reduction in fusion gene expression. These data suggest that the same group of islet-enriched transcription factors, namely Pdx-1, Pax-6, MafA, BETA2, and Foxa2, directly or indirectly regulate expression of the two major autoantigens in type 1 diabetes.

Multiple functional polymorphisms in the G6PC2 gene contribute to the association with higher fasting plasma glucose levels

“…Hamster insulinoma tumor (HIT), βTC-3 and Min6 cells were cultured and transfected with various G6PC2-luciferase fusion genes in the pGL3 MOD vector (2 µg) and an expression vector encoding Renilla luciferase (0.5 µg) using lipofectamine as previously described [3, 25]. Following transfection, cells were incubated for 18–20 hr in serum-containing medium.…”

“…Following transfection, cells were incubated for 18–20 hr in serum-containing medium. The cells were then harvested and firefly and Renilla luciferase activity were assayed as previously described [25]. …”

A Single-Nucleotide Polymorphism in a Methylatable Foxa2 Binding Site of the G6PC2 Promoter Is Associated With Insulin Secretion In Vivo and Increased Promoter Activity In Vitro

“…Our functional analysis of the rs573225 variant in ␤-cell lines found that the G allele was able to bind Foxa2 factor more efficiently than the A allele and was associated with a higher G6PC2 promoter activity. Previous studies have found that several mutated transcription factor sites located in G6PC2 promoter could modify its expression (6,17). Studies reviewed in Martin et al (6) support a positive role for Foxa2 in G6PC2 expression and a negative impact on insulin secretion in vitro (18 -20).…”

“…Previous studies have found that several mutated transcription factor sites located in G6PC2 promoter could modify its expression (6,17). Studies reviewed in Martin et al (6) support a positive role for Foxa2 in G6PC2 expression and a negative impact on insulin secretion in vitro (18 -20). G6PC2 appears to negatively affect stimulus-secretion coupling (3).…”

“…The G6PC2 promoter is inactive in HepG2 cells but has 150-fold more activity in HIT-T15 ␤-cells, due to the region located between Ϫ306 and ϩ3 (5). This proximal promoter region regulates G6PC2 expression in transiently transfected ␤TC-3, HIT-T15, and Min6 cells through the binding of Foxa2 and MafA transcription factors (6). We found three single-nucleotide polymorphisms (SNPs) in the G6PC2 promoter in public banks, among which we selected rs573225, a G/A variant located at position Ϫ231.…”