The PDX-1 activation domain provides specific functions necessary for transcriptional stimulation in pancreatic beta-cells

PDX-1 is a homeodomain transcription faster whose targeted disruption resul ts in a failure of the pancreas to develop. Mutations in the human pdx-1 ge ne are linked to an early onset form of non-insulin-dependent diabetes mell itus. PDX-1 binds to and transactivates the promoters of several physiologi cally relevant genes within the beta -cell, including insulin, glucose tran sporter 2, glucokinase, and islet amyloid polypeptide. This study focuses o n the mechanisms by which PDX-1 activates insulin gene transcription. To ev aluate the role of PDX-1 in transcription of the insulin gene, a chloramphe nicol 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-ter minal transactivation domain sequences, this GAL4-substituted insulin const ruct was active in PDX-1-expressing beta -cells and not non-beta -cells. PD X-1 activation was mediated through three highly conserved segments of the transactivation domain. In addition, when cotransfected together with the G AL4-substituted insulin enhancer reporter gene in glucose-responsive MIN-6 p-cells, glucose-induced activation is observed with GAL4:PDX-1 but not wit h fusions of the heterologous activation domains from herpes virus VP16 or adenovirus-5 E1A proteins. Using A3 element-substituted GAL4 insulin enhanc er reporter constructs containing mutations in two additional key control e lements, E1 and C1, we also show that full activation requires cooperative interactions between other enhancer-bound factors, particularly the E1 elem ent activators. In contrast, the activity of the VP16 activation factor was not dependent on the activators of either the E1 or C1 sites. These result s suggest that the PDX-1 transactivation domain is specifically required fo r appropriate regulation of insulin enhancer function in beta -cells.