The RIPE3b1 activator of the insulin gene is composed of a protein(s) of approximately 43 kDa, whose DNA binding activity is inhibited by protein phosphatase treatment

Glucose-stimulated and pancreatic islet beta cell-specific expression of the insulin gene is mediated in part by the C1 DNA-element binding complex, termed RIPE3b1. In this report, we define the molecular weight range of the protein(s) that compose this beta cell-enriched activator complex and show that protein phosphatase treatment inhibits RIPE3b1 DNA binding activity. Fractionation of beta cell nuclear extracts by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that RIPE3b1 binding was mediated by a protein(s) within the 37-49-kDa ranges. Direct analysis of the proteins within the RIPE3b1 complex by ultraviolet light cross-linking analysis identified three binding species of approximately 51, 45, and 38 kDa. Incubating beta cell nuclear extracts with either calf alkaline phosphatase or a rat brain phosphatase preparation dramatically reduced RIPE3b1 DNA complex formation. Phosphatase inhibition of RIPE3b1 binding was prevented by sodium pyrophosphate, a general phosphatase inhibitor. We discuss how changes in the phosphorylation status of the RIPE3b1 activator may influence its DNA binding activity.     

Pancreatic beta cell-specific transcription of the pdx-1 gene. The role of conserved upstream control regions and their hepatic nuclear factor 3beta 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 HNF3beta. 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 HNF3beta were inactivated. pdx-1 mRNA levels induced upon differentiation to embryoid bodies were down-regulated in homozygous null HNF3beta cells. Together, these results suggest that the conserved sequences represented by areas I and II define the binding sites for factors such as HNF3beta, which control islet beta cell-selective expression of the pdx-1 gene.