The PDX-1 transcription factor plays a key role in pancreatic development and in the regulation of the insulin gene in the adult ␤ cell. As its functions appear to be similar in humans and mice, we analyzed the functional conservation of homologous sequences important for the maintenance and the cell-specific regulation of the pdx-1 gene. Apart from the proximal promoter region, three highly homologous (PH1 to PH3) sequences were apparent in the human and mouse 5 flanking regions of the gene. By transient transfections in ␤ and non-␤ cells, we show that mainly PH1 and PH2 preferentially confer ␤-cell-specific activation on a heterologous promoter. DNase I footprinting and binding analyses revealed that both bind to and are transactivated by hepatocyte nuclear factor 3␤ (HNF-3␤). Furthermore, the PH1 enhancer element also binds the PDX-1 transcription factor itself, which acts cooperatively with adjacent HNF-3␤ to regulate its transcriptional potency. This finding suggests a possible autoregulatory loop as a mechanism for PDX-1 to control its own expression.The mammalian pancreas develops from the endoderm in the upper duodenal part of the foregut as dorsal and ventral buds which fuse together to form the organ. Recently, considerable progress has been made in identifying genes that control the embryonic development of the islet. Most are transcription factors such as isl-1, PDX-1, BETA2 (also called NeuroD), Nkx 2.2, Pax4, and Pax6 (for reviews, see references 8, 9, 14, 15, 26, 30, and 31). The first molecular marker identified that specifies the early pancreatic epithelium was the homeodomain-containing transcription factor PDX-1 (1, 2, 13). PDX-1 function appears to be well conserved. Its absence in both humans and mice leads to agenesis of the pancreas, while in the adult pancreatic islet, its expression is restricted to the ␤ cell, where it acts as the glucose-sensitive transcription factor of the insulin gene (19).Initiation of transcription appears to be the major level of regulation for many cell type-specific genes. To elucidate the regulation of pdx-1 gene expression, a 6.5-kb fragment upstream of the transcription start site of the rat pdx-1 gene (also called stf-1) (28) and a fragment extending from the region from kb Ϫ4.5 to ϩ8.2 of the mouse pdx-1 gene (33) were shown to direct the expression of the ␤-galactosidase (␤-Gal) reporter gene to pancreatic islet cells in transgenic mice. Using transiently transfected ␤ cells, it was shown that the expression of the rat pdx-1 gene, apart from a proximal E box, depended on a distal enhancer element that was activated by the cooperative action of the endodermal transcription factors hepatocyte nuclear factor 3␤ (HNF-3␤) and BETA2 (27). A study of the mouse pdx-1 promoter revealed that the region involved in regulating ␤-cell-specific transcription and directing appropriate developmental and adult-specific expression in transgenic animals contained an HNF-3-like element (33). Thus, these initial studies with the rat and the mouse enhancer-promoter regions su...

The PDX-1 transcription factor plays a key role in pancreas development. Although expressed in all cells at the early stages, in the adult it is mainly restricted to the beta-cell. To characterize the regulatory elements and potential transcription factors necessary for human PDX-1 gene expression in beta-cells, we constructed a series of 5' and 3' deletion fragments of the 5'-flanking region of the gene, fused to the luciferase reporter gene. In this report, we identify by transient transfections in beta- and non-beta-cells a novel beta-cell-specific distal enhancer element located between -3.7 and -3.45 kilobases. DNase I footprinting analysis revealed two protected regions, one binding the transcription factors SP1 and SP3 and the other hepatocyte nuclear factor 3beta (HNF-3beta) and HNF-1alpha. Cotransfection experiments suggest that HNF-3beta, HNF-1alpha, and SP1 are positive regulators of the herein-described human PDX-1 enhancer element. Furthermore, mutations within each motif abolished the binding of the corresponding factor(s) and dramatically impaired the enhancer activity, therefore suggesting cooperativity between these factors.

Intestinal metaplasia (IM) is a preneoplastic lesion of the stomach in which there is transdifferentiation of the gastric mucosa to an intestinal phenotype. The caudal-related homeobox gene CDX2 encodes an intestine-specific transcription factor crucial for the regulation of proliferation and differentiation of intestinal cells. In addition, CDX2 is involved in the induction of IM in the stomach. The aim of this study was to access the putative involvement of OCT-1 in the induction of CDX2 expression de novo in gastric mucosa leading to the onset of IM. OCT-1 protein expression was evaluated by immunohistochemistry in 31 biopsies with chronic gastritis, 15 biopsies with foci of IM and adjacent gastric mucosa and 42 gastric carcinomas. Furthermore, we evaluated OCT-1 binding by electrophoretic mobility shift assay and activation of the CDX2 promoter by co-transfecting a CDX2 promoter/reporter construct with an OCT-1 expression vector in two gastric carcinoma cell lines, GP220 and MKN45. Our results show that OCT-1 is expressed in chronic gastritis, particularly when it is adjacent to IM and is expressed in 87% of IM foci. Furthermore, 74% of the gastric carcinomas were positive for OCT-1 and a strong association was observed between OCT-1 expression and intestinal-type carcinoma. We identified that OCT-1 binds to the CDX2 promoter, although we could not see a transactivation effect in gastric carcinoma cell lines. In conclusion, we observed increased OCT-1 expression in IM and in intestinal gastric carcinomas and identified the capacity of OCT-1 to bind to the CDX2 promoter, although we could not demonstrate a direct effect of OCT-1 in the transactivation of CDX2.

The pancreatic duodenal homeodomain transcription factor (PDX-1) is an essential regulator of pancreatic endocrine cell development and adult islet ␤-cell function. PDX-1 function appears to be well conserved, as its absence in humans and mice leads to agenesis of the pancreas (1,2). The gene is expressed both in endocrine and exocrine cells of the developing pancreas; however, in the adult pancreatic islet, its expression is restricted to the ␤-cells, where it acts as the glucose-sensitive transcription factor of the insulin gene (3,4). In mice, ␤-cell-selective disruption of pdx-1 leads to diabetes associated with reduced insulin and GLUT2 expression (5). To identify key components that direct the expression of the pdx-1 gene to ␤-cells, we performed deletion analysis of 7 kb of the 5Ј flanking region of the human pdx-1. We identified a novel ␤-cell-specific distal enhancer element that showed no sequence homology with the mouse pdx-1 gene.Because it appears that PDX-1 function is similar in humans and rodents, we deduced that conserved sequences located within the 5Ј flanking region of the gene would be of importance for its expression. Comparison of 4.5-kb upstream regions of the human and mouse pdx-1 genes revealed that, in addition to the proximal promoter region described by Sharma et al. (6), only three relatively short sequences designated PH1, PH2, and PH3 (for pdx-1 homology 1-3) were highly conserved between human and mouse. Using transient transfection experiments, we showed that PH1 and PH2 preferentially drive ␤-cell-specific expression of a reporter gene. Using DNAse I footprinting analyses and electrophoretic mobility shift assays, we demonstrate that PH1 element binds the transcription factor PDX-1 adjacent to the endodermal factor hepatocyte nuclear factor (HNF)-3␤, where they act cooperatively to transactivate a PH1-driven reporter construct. Similar studies reveal that HNF-3␤ mediates the transcriptional activity of the PH2 domain. Our results suggest a possible feedback mechanism where PDX-1 could regulate its own expression. RESULTSHuman pdx-1 sequences involved in ␤-cell-specific transcriptional activity. To delineate the putative DNA sequences controlling pdx-1 gene expression, we performed a deletion analysis of the 5Ј flanking region of human pdx-1 extending from -7 to +0.117 kb. The fragment spanning 3.7 kb of the upstream region conferred strong ␤-cell-specific expression to the reporter gene, suggesting the presence of a strong positive regulatory element(s). The human pdx-1 sequence -3.7 to -3.4 kb acts as a ␤-cell-specific enhancer element. Detailed deletion analysis of 3.7-kb fragment allowed us to delineate a regulatory element spanning the region from -3.7 to -3.4 kb that exhibits no homology with the mouse pdx-1 sequence. This element had the characteristics of an enhancer and strongly activated the minimal pdx-1 promoter in a tissue-specific manner. ␤-Cell expression of conserved sequences in the human pdx-1 gene. Sequence comparison of 4.5 kb upstream of the human and mouse ...

␤-cell-restricted expression of the insulin gene relies on the interaction of 5Ј flanking sequence motifs in the promoter region with a number of ubiquitous and islet-specific transcription factors. These interactions determine the temporal expression of the gene and its inducibility by physiological stimuli. Most of the studies on the regulation of insulin gene expression were conducted with rat insulin 1 and 2 and the human genes. They share a number of conserved cis elements in their 5Ј flanking region and are probably regulated by similar trans-acting factors. Among such elements, the E and A boxes appear to be the major determinants of ␤-cell-specific insulin gene expression. The E boxes (E1 and E2), with the consensus CANNTG, bind transcription factors of the basic helix-loop-helix family. The A boxes (A1-A5) containing AT-rich sequences bind factors that belong to the homeodomain-containing protein family. The most conserved A1 and A3 boxes in the insulin promoter bind the pancreatic duodenal homeobox transcription factor (PDX-1), the mammalian homolog of the Xenopus laevis XlHbox8, previously described as insulin promoter factor-1, somatostatin transcription factor-1 (STF-1), islet duodenal homeobox-1 (IDX-1), glucose-sensitive factor (GSF), and insulin upstream factor-1 (IUF-1). Gene disruption experiments in mice showed that PDX-1 is needed for pancreas formation. Thus, tissue-specific expression of the insulin gene appears to depend on a combination of transcription factors, some of which are restricted to the ␤-cell.Because of its central role in metabolic control, tight regulation of insulin production and release is critical. Inducible transcription may depend on the very same sequences in the promoter/enhancer regions that control the cell-type specificity of the insulin gene. In pancreatic ␤-cells, glucose is the major regulator of insulin biosynthesis and secretion. Experiments with rat and human islets, as well as ␤-cell lines, indicated that glucose regulates insulin gene expression by simultaneously stimulating transcription and inhibiting proinsulin mRNA degradation.The glucose-responsiveness of the insulin gene is similarly mediated by cis-acting elements within the 5Ј flanking region relative to the transcription start site. We have previously demonstrated the presence of an islet-specific GSF, whose DNA binding to the A3 motif of the rat I and human insulin promoters is modulated by extracellular glucose. A single mutation in the GSF binding site of the human insulin promoter abolishes the stimulation by high glucose in normal islets. The sequence of the purified protein was shown to correspond to PDX-1. Mapping of GSF/PDX-1 functional domains in normal islet cells showed that its transactivation domain is located within the NH 2 -terminal region of the protein and is also regulated by extracellular glucose levels. Thus, in addition to its essential roles in the development and differentiation of pancreatic islets and in ␤-cell-specific gene expression, PDX-1 protein functions as a mediat...