Pax-6 and c-Maf Functionally Interact with the α-Cell-specific DNA Element G1 in Vivo to Promote Glucagon Gene Expression

Yvan Gosmain,Isabelle Avril,Aline Mamin,Jacques Philippe

doi:10.1074/jbc.m702795200

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.

Highlights

We and others have proposed Pax-6, Cdx-2/3, large Mafs, Brain-4, and Foxa-2 as potential factors implicated in glucagon gene expression through the transactivation of the G1 element in glucagon-producing cells by transfection studies (8 –11)
Brain-4 is expressed in ␣-cells throughout development and in the adult and has been proposed as a candidate for ␣-cell specific expression of the glucagon gene [10, 23]; how
We show here by Chromatin Immunoprecipitation Assay (ChIP) analyses that Pax-6, Cdx-2/3, Maf-B, and c-Maf interact with the glucagon gene promoter in InR1G9 cells, whereas only Pax-6, Maf-B and c-Maf do in ␣-TC1 cells, suggesting that neither Cdx-2/3 nor Brain-4 are necessary for glucagon gene expression

Summary

EXPERIMENTAL PROCEDURES

Cell Culture—The glucagon-producing hamster InR1G9 [24] and mice ␣-TC1 cells [25] as well as the non-islet Syrian baby hamster kidney (BHK-21) cell lines were grown in RPMI 1640 (R-6504; Sigma) supplemented with 5% fetal bovine serum and 5% newborn calf serum, 2 mM glutamine, 100 units/ml of penicillin, and 100 ␮g/ml of streptomycin. Cdx-2/3 was of Pax-6, Cdx-2/3, and large Mafs on the glucagon gene pronot detected either by Western blot of adult islet nuclear moter, we performed transient transfections with the rat glucaextracts, whereas it was present in InR1G9 cells (data not gon gene promoter (Ϫ292/ϩ50), linked to the CAT gene with shown). DNA interactions, we performed EMSAs with the G1 element of the rat glucagon gene promoter and nuclear extracts from BHK-21 cells overexpressing the Pax-6, Cdx-2/3, or the large. We performed cotransfection experiments in BHK-21 cells using fragments of the 5Ј-flanking sequence of the rat glucagon gene promoter (Ϫ292/ϩ50) linked to the CAT reporter gene and SV40-driven expression vectors containing cDNAs encoding Pax-6, Cdx-2/3, Maf-B, or the dominant negative forms of Cdx2/3 and Maf-A. We conclude that Pax-6 and c-Maf are able to bind the glucagon gene promoter and are critical for basal glucagon gene expression

DISCUSSION

Indeed only overexpression studies