Functional Conservation of Regulatory Elements in the pdx-1 Gene: PDX-1 and Hepatocyte Nuclear Factor 3β Transcription Factors Mediate β-Cell-Specific Expression

Abstract

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

ACKNOWLEDGMENTS

We thank Roland Stein (Vanderbilt University, Nashville, Tenn.) for the mouse pdx-1 sequence and Alan M. Permutt (Washington University) for the human pdx-1 gene. We are infinitely grateful to Robert Costa (University of Illinois at Chicago) for the gifts of HNF-3α and HNF-3β expression vectors and antibodies and to Peter Carlsson (Göteborg University, Göteborg, Sweden) for FREAC-1, FREAC-2, and FREAC-4 expression vectors. Our sincere thanks go to Rahel Oron for excellent technical help.

This work was supported by grants from the Juvenile Diabetes Foundation International, BIOMED 2 (BMH4-CT98-3448), and the Israel Science Foundation.