Binding of HNF-3α in nuclear extracts of pancreatic islet α-cell line to the novel HNF-3 site-A in the glucagon gene

The pancreatic islet hormone glucagon stimulates hepatic glucose production and thus maintains blood glucose levels in the fasting state. Transcription factors of the hepatocyte nuclear factor-3 (HNF-3, also called Foxa) family are expressed in endoderm-derived tissues such as liver and pancreatic islets and, as part of their metabolic control, are required for cell-specific activation of the glucagon gene in pancreatic islet α-cells. However, their action at the glucagon gene is poorly understood. We have shown previously that the well characterized HNF-3 binding site in the G2 enhancer element of the rat glucagon gene is not conserved in humans and that the human G2 sequence lacks basal enhancer activity. However, a novel HNF-3 site (called site-A) has been identified that is conserved in rat, mouse, and humans and that mediates an activation of the glucagon gene by HNF-3 proteins expressed in an heterologous cell line. In the present study, a molecular characterization using protein-DNA binding and transient transfection assays revealed that the novel HNF-3 site-A confers cell-specific promoter activity in glucagon-producing pancreatic islet α-cell lines (InR1G9, αTC2). In contrast to other HNF-3 binding sites in the glucagon promoter that bind nuclear HNF-3β, the novel HNF-3 site-A was found to bind preferentially HNF-3α in nuclear extracts of a glucagon-producing pancreatic islet α-cell line. Mice with a targeted disruption of the HNF-3α gene have been shown to develop normally but exhibit neonatal growth retardation and die postnatally prior to 4 weeks of age, exhibiting hypoglycemia associated with inappropriately low levels of circulating glucagon. The results of the present study offer a mechanism that explains the decrease in glucagon gene expression in HNF-3α-deficient mice. The novel HNF-3 site-A is located just upstream of the TATA box (between -30 and -50) suggesting a role for HNF-3 proteins in addition to direct transcriptional activation