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The PDX-1 homeodomain transcription factor regulates pancreatic development and adult islet beta cell function. Expression of the pdx-1 gene is almost exclusively localized to beta cells within the adult endocrine pancreas. Islet beta cell-selective transcription is controlled by evolutionarily conserved subdomain sequences (termed Areas I (-2839 to -2520 base pairs (bp)), II (-2252 to -2023 bp), and III (-1939 to -1664 bp)) found within the 5'-flanking region of the pdx-1 gene. Areas I and II are independently capable of directing beta cell-selective reporter gene activity in transfection assays, with Area I-mediated stimulation dependent upon binding of hepatic nuclear factor 3 beta (HNF3 beta), a key regulator of islet beta cell function. To identify other transactivators of Area I, highly conserved sequence segments within this subdomain were mutagenized, and their effect on activation was determined. Several of the sensitive sites were found by transcription factor data base analysis to potentially bind endodermally expressed transcription factors, including HNF1 alpha (-2758 to -2746 bp, Segment 2), HNF4 (-2742 to -2730 bp, Segment 4; -2683 to -2671 bp, Segment 7-8), and HNF6 (-2727 to -2715 bp, Segment 5). HNF1 alpha, but not HNF4 and HNF6, binds specifically to Area I sequences in vitro. HNF1 alpha was also shown to specifically activate Area I-driven transcription through Segment 2. In addition, PDX-1 itself was found to stimulate Area I activation. The chromatin immunoprecipitation assay performed with PDX-1 antisera also demonstrated that this factor bound to Area I within the endogenous pdx-1 gene in beta cells. Our results indicate that regulatory factors binding to Area I conserved sequences contribute to the selective transcription pattern of the pdx-1 gene and that control is mediated by endodermal regulators like HNF1 alpha, HNF3 beta, and PDX-1.