Pancreatic β cell-specific transcription of the pdx-1 gene -: The role of conserved upstream control regions and their hepatic nuclear factor 3β 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 critical cis-regulatory elements. The sequences associated with hypersensitive site 1 (HSS1) represented the principal area of homology within which three conserved 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 identities 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 chicken. 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 of pdx-1-driven reporter activity in transfected beta- and non-beta cells. Mutational and functional analyses of conserved hepatic nuclear factor 3 (HNF3)-like sites located within area I and area II demonstrated that activation by these regions was mediated by HNF3 beta, To determine if a similar regulatory relationship might exist within the context of the endogenous gene, pdx-1 expression was measured in embryonic stem cells in which one or both alleles of HNF3 beta were inactivated, pdx-1 mRNA levels induced upon differentiation to embryoid bodies were down-regulated in homozygous null HNF3 beta cells. Together, these results suggest that the conserved sequences represented by areas I and II define the binding sites for factors such as HNF3 beta, which control islet beta cell-selective expression of the pdx-1 gene.