A pancreatic beta -cell-specific enhancer in the human PDX-1 gene is regulated by hepatocyte nuclear factor 3beta (HNF-3beta ), HNF-1alpha, and SPs transcription factors.

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.

Regulatory elements involved in human pdx-1 gene expression.

PDX-1 was shown to be expressed early during development in cells of both exocrine and endocrine origin; later it becomes restricted primarily to beta-cells where it regulates the expression of beta-cell-specific genes and mediates the glucose effect on insulin gene transcription. Therefore, it was important to identify the molecular mechanisms that specifically govern the expression of pdx-1 in the mature beta-cell. To address this question, we analyzed 7 kb of the 5' flanking region of the human pdx-1 gene. By transient transfections of beta- and non-beta-cell lines with different 5' and 3' deletions of that region, a strong beta-cell-specific enhancer element located between -3.71 and 3.46 kb was revealed. We also sequenced about 4.5 kb of the human 5' flanking region and compared it with that of the mouse pdx-1 gene. This comparison revealed three short conserved regions, designated PH1, PH2, and PH3. We showed that HNF-3beta can bind and stimulate the activity of the human PH1 and PH2 elements in non-beta-cells. Results reported by Wu et al. (7) and Sharma et al. (6) also indicate that expression of the mouse pdx-1 is controlled by an HNF-3-like element. Thus, it can be stated that at least some aspects of pdx-1 expression rely on the transcription factor HNF-3beta. Because HNF-3beta is not restricted to beta-cells, the selective transcription of pdx-1 is likely to rely on additional factors. Our findings that the PH1 enhancer element binds both HNF-3beta and PDX-1 and that mutations in each individual site dramatically impair its transcriptional activity suggest that these factors cooperate with one another. We therefore propose that a possible feedback mechanism might control the expression of pdx-1 at different stages during development.

Functional conservation of regulatory elements in the pdx-1 gene: PDX-1 and hepatocyte nuclear factor 3beta transcription factors mediate beta-cell-specific expression.

The PDX-1 transcription factor plays a key role in pancreatic development and in the regulation of the insulin gene in the adult beta 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 beta and non-beta cells, we show that mainly PH1 and PH2 preferentially confer beta-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 3beta (HNF-3beta). Furthermore, the PH1 enhancer element also binds the PDX-1 transcription factor itself, which acts cooperatively with adjacent HNF-3beta to regulate its transcriptional potency. This finding suggests a possible autoregulatory loop as a mechanism for PDX-1 to control its own expression.