Pancreatic and duodenal homeobox factor 1 (Pdx1) has been demonstrated to play a crucial role in pancreas development and in maintenance of mature beta-cell function. However, it remains to be elucidated how Pdx1 gene expression is regulated in non-beta cells during pancreas development. Pdx1 and Ptf1 a are expressed in pancreatic progenitor cells, which give rise to all three types of pancreatic tissue. In addition, Ptf1a has been shown to bind the mammalian Suppressor of Hairless (RBP-J) within the PTF1 complex. Furthermore, loss-of-function approaches have revealed that all three factors are essential for early pancreas development. We therefore hypothesized that Ptf1a and RBP-J regulate expression of the Pdx1 gene in pancreatic precursors. Reporter gene analyses showed that Ptf1a transactivated Pdx1 promoter in pancreatic Panc-1 cells, which was enhanced by RBP-J. Deletion/mutation analyses of the Pdx1 promoter and electrophoretic gel-mobility shift assays identified the Ptf1 a binding site in the well-conserved regulatory sequence domain, termed Area III, which was also confirmed by the chromatin immunoprecipitation assay. Furthermore, adenovirus-mediated overexpression of Ptf1a, together with RBP-J, markedly increased Pdx1 protein levels in pancreatic AR42J-B13 cells. Our data suggest a novel transcriptional network, where Ptf1a and RBP-J cooperatively regulate Pdx1 gene expression through binding to Area III. (c) 2007 Elsevier Inc. All rights reserved.