Pax-6 and c-Maf functionally interact with the alpha-cell-specific DNA element G1 in vivo to promote glucagon gene expression

Abstract

Specific expression of the glucagon gene in the rat pancreas requires the presence of the G1 element localized at -100/-49 base pairs on the promoter. Although it is known that multiple transcription factors such as Pax-6, Cdx-2/3, c-Maf, Maf-B, and Brain-4 can activate the glucagon gene promoter through G1, their relative importance in vivo is unknown. We first studied the expression of Maf-B, c-Maf, and Cdx-2/3 in the developing and adult mouse pancreas. Although Maf-B was detectable in a progressively increasing number of alpha-cells throughout development and in adulthood, c-Maf and Cdx-2/3 were expressed at low and very low levels, respectively. However, c-Maf but not Cdx-2/3 was detectable in adult islets by Western blot analyses. We then demonstrated the in vivo interactions of Pax-6, Cdx-2/3, Maf-B, and c-Maf but not Brain-4 with the glucagon gene promoter in glucagon-producing cells. Although Pax-6, Cdx-2/3, Maf-B, and c-Maf were all able to bind G1 by themselves, we showed that Pax-6 could interact with Maf-B, c-Maf, and Cdx-2/3 and activate transcription of the glucagon gene promoter. Overexpression of dominant negative forms of Cdx-2/3 and Mafs in alpha-cell lines indicated that Cdx-2/3 and the Maf proteins interact on an overlapping site within G1 and that this binding site is critical in the activation of the glucagon gene promoter. Finally, we show that specific inhibition of Pax-6 and c-Maf but not Cdx-2/3 or Maf-B led to decreases in endogenous glucagon gene expression and that c-Maf binds the glucagon gene promoter in mouse islets. We conclude that Pax-6 and c-Maf interact with G1 to activate basal expression of the glucagon gene.