Pancreatic beta cell-specific transcription of the pdx-1 gene - The role of conserved upstream control regions and their hepatic nuclear factor 3 beta sites

To identify potential transactivators of pdx-1, we sequenced approximately 4.5 kilobases of the 5' promoter region of the human and chicken homologs, assuming that sequences conserved with the mouse gene would contain critica l cis-regulatory elements. The sequences associated with hypersensitive sit e 1 (HSS1) represented the principal area of homology within which three co nserved subdomains were apparent: area I (-2694 to -2561 base pairs (bp)), area II (-2139 to -1958 bp), and area III (-1879 to -1799 bp), The identiti es between the mouse and chicken/human genes are very high, ranging from 78 to 89%, although only areas I and III are present within this region in ch icken. Pancreatic beta cell-selective expression was shown to be controlled by mouse and human area I or area II, but not area III, from an analysis o f pdx-1-driven reporter activity in transfected beta- and non-beta cells. M utational and functional analyses of conserved hepatic nuclear factor 3 (HN F3)-like sites located within area I and area II demonstrated that activati on by these regions was mediated by HNF3 beta, To determine if a similar re gulatory relationship might exist within the context of the endogenous gene , pdx-1 expression was measured in embryonic stem cells in which one or bot h alleles of HNF3 beta were inactivated, pdx-1 mRNA levels induced upon dif ferentiation to embryoid bodies were down-regulated in homozygous null HNF3 beta cells. Together, these results suggest that the conserved sequences r epresented by areas I and II define the binding sites for factors such as H NF3 beta, which control islet beta cell-selective expression of the pdx-1 g ene.