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The PDX-1 Activation Domain Provides Specific Functions Necessary for Transcriptional Stimulation in Pancreatic ß-Cells

Department of Molecular Physiology and Biophysics (M.P., M.A.C., E.H., R.S.) Vanderbilt University Medical School Nashville, Tennessee 37232
Hagedorn Research Institute (H.V.P.) DK-2820 Gentofte, Denmark

PDX-1 is a homeodomain transcription factor whose targeted disruption results in a failure of the pancreas to develop. Mutations in the human pdx-1 gene are linked to an early onset form of non-insulin-dependent diabetes mellitus. PDX-1 binds to and transactivates the promoters of several physiologically relevant genes within the ß-cell, including insulin, glucose transporter 2, glucokinase, and islet amyloid polypeptide. This study focuses on the mechanisms by which PDX-1 activates insulin gene transcription. To evaluate the role of PDX-1 in transcription of the insulin gene, a chloramphenicol acetyltransferase reporter construct was designed with a single yeast GAL4-DNA binding site in place of the A3/PDX-1 binding element in the rat insulin II enhancer. In the presence of GAL4:PDX chimeras containing N-terminal transactivation domain sequences, this GAL4-substituted insulin construct was active in PDX-1-expressing ß-cells and not non-ß-cells. PDX-1 activation was mediated through three highly conserved segments of the transactivation domain. In addition, when cotransfected together with the GAL4-substituted insulin enhancer reporter gene in glucose-responsive MIN-6 ß-cells, glucose-induced activation is observed with GAL4:PDX-1 but not with fusions of the heterologous activation domains from herpes virus VP16 or adenovirus-5 E1A proteins. Using A3 element-substituted GAL4 insulin enhancer reporter constructs containing mutations in two additional key control elements, E1 and C1, we also show that full activation requires cooperative interactions between other enhancer-bound factors, particularly the E1 element activators. In contrast, the activity of the VP16 activation factor was not dependent on the activators of either the E1 or C1 sites. These results suggest that the PDX-1 transactivation domain is specifically required for appropriate regulation of insulin enhancer function in ß-cells.

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