Abstract
Maturity onset diabetes of the young, subtype 1 (MODY1), is associated with defective glucose-dependent insulin secretion from pancreatic beta cells. MODY1 is caused by mutation in the transcription factor hepatocyte nuclear factor 4 alpha (HNF4 alpha). To understand better the MODY1 phenotype, we tested whether HNF4 alpha was able to modulate directly the insulin gene promoter. Transfection of cultured 293T cells with an HNF4 alpha expression vector led to 10-fold activation of a cotransfected reporter plasmid containing the rat insulin I gene promoter. Computer analysis revealed a potential HNF4 alpha-binding site between nucleotides -57 and -69 of the promoter; mutation of this sequence led to reduced ability of HNF4 alpha to activate the promoter. The ability of HNF4 alpha to bind this sequence was confirmed using gel shift analysis. In transfected INS-1 beta cells, mutation of either the HNF1 alpha site or the HNF4 alpha site in the insulin gene promoter led to 50-75% reduction in reporter gene activity; expression of dominant negative HNF4 alpha led to significant reduction in the activity of wild type and both mutated promoters. Thus, in addition to the previously described indirect action of HNF4 alpha on insulin gene expression mediated through elevated HNF1 alpha levels, HNF4 alpha also activates the insulin gene directly, through a previously unrecognized cis element.
Highlights
Maturity onset diabetes of the young, subtype 1 (MODY1), is associated with defective glucose-dependent insulin secretion from pancreatic beta cells
HNF4␣ Can Activate the Insulin Gene Promoter—Because HNF4␣ was previously shown to affect insulin mRNA levels in beta cells [27], its involvement in the direct transcriptional regulation of the insulin gene was examined. 293T cells were transfected with a reporter plasmid encoding the luciferase gene controlled by the rat insulin I gene promoter (Ϫ410 to Ϫ1) in the presence or absence of an HNF4␣ expression plasmid
Because the MODY phenotype involves a primary defect in beta cells, the defective function of these transcription factors must be manifested in these cells
Summary
293T, HeLa S3 Tet-Off (CLONTECH PT3001–1), and COS7 cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum, penicillin (200 IU/ml), and streptomycin (100 g/ml). Reporter Plasmids—pGL3.Synt.LUC, a plasmid containing the luciferase (LUC) gene controlled by the rat insulin I gene promoter, was generated by replacement of the SV40 promoter of pGL3-promoter vector (Promega) with the insulin promoter fragment (Ϫ410 to Ϫ1). PGL3.SV40.LUC is the plasmid pGL3-promoter vector (Promega), containing the luciferase gene controlled by the SV40 early promoter. RSV.LUC contains the luciferase gene controlled by the RSV promoter [31]. The reporter plasmids Synt 5 LUC, Synt 6 LUC, Synt 7 LUC, and Synt 8 LUC contain the luciferase gene controlled by Synt 5– 8 block replacement mutants of the rat insulin I promoter [5]. LUC was generated by subcloning of the mutated insulin promoter from Ϫ410MFInsLUC to pGL3-promoter vector (Promega). For induction of DN-HNF4␣, cells were incubated with 1 g/ml doxycycline for 24 h prior to transfection
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