BETA2 activates transcription from the upstream glucokinase gene promoter in islet beta-cells and gut endocrine cells.

Glucokinase (GK) gene transcription initiates in the islet (beta-cell), gut, and brain from promoter sequences residing approximately 35 kbp upstream from those used in liver. Expression of betaGK is controlled in beta-cells by cell-enriched (i.e. pancreatic duodenal homeobox 1 [PDX-1]) and ubiquitously (i.e., Pal) distributed factors that bind to and activate from conserved sequence motifs within the upstream promoter region (termed betaGK). Here, we show that a conserved E-box element also contributes to control in the islet and gut. betaGK promoter-driven reporter gene activity was diminished by mutating the specific sequences involved in E-box-mediated basic helix-loop-helix factor activator binding in islet beta-cells and enteroendocrine cells. Gel shift assays demonstrated that the betaGK and insulin gene E-box elements formed the same cell-enriched (BETA2:E47) and generally distributed (upstream stimulatory factor [USF]) protein-DNA complexes. betaGK E-box-driven activity was stimulated in cotransfection assays performed in baby hamster kidney (BHK) cells with BETA2 and E47, but not USF. Chromatin immunoprecipitation assays performed with BETA2 antisera showed that BETA2 occupies the upstream promoter region of the endogenous betaGK gene in beta-cells. We propose that BETA2 (also termed NeuroD1) regulates betaGK promoter activity.

Transcription factor occupancy of the insulin gene in vivo. Evidence for direct regulation by Nkx2.2.

Consensus-binding sites for many transcription factors are relatively non-selective and found at high frequency within the genome. This raises the possibility that factors that are capable of binding to a cis-acting element in vitro and regulating transcription from a transiently transfected plasmid, which would not have higher order chromatin structure, may not occupy this site within the endogenous gene. Closed chromatin structure and competition from another DNA-binding protein with similar nucleotide specificity are two possible mechanisms by which a transcription factor may be excluded from a potential binding site in vivo. Multiple transcription factors, including Pdx-1, BETA-2, and Pax6, have been implicated in expression of the insulin gene in pancreatic beta cells. In this study, the chromatin immunoprecipitation assay has been used to show that these factors do, in fact, bind to insulin control region sequences in intact beta cells. In addition, another key islet-enriched transcription factor, Nkx2.2, was found to occupy this region using the chromatin immunoprecipitation assay. In vitro DNA-binding and transient transfection assays defined how Nkx2.2 affected insulin gene expression. Pdx-1 was also shown to bind within a region of the endogenous islet amyloid polypeptide, pax-4, and glucokinase genes that were associated with control in vitro. Because Pdx-1 does not regulate gene transcription in isolation, these sequences were examined for occupancy by the other insulin transcriptional regulators. BETA-2, Pax6, and Nkx2.2 were also found to bind to amyloid polypeptide, glucokinase, and pax-4 control sequences in vivo. These studies reveal the broad application of the Pdx-1, BETA-2, Pax6, and Nkx2.2 transcription factors in regulating expression of genes selectively expressed in islet beta cells.

Conserved sequences in a tissue-specific regulatory region of the pdx-1 gene mediate transcription in Pancreatic beta cells: role for hepatocyte nuclear factor 3 beta and Pax6.

Pancreas duodenum homeobox 1 (PDX-1) is absolutely required for pancreas development and the maintenance of islet beta-cell function. Temporal and cell-type-specific transcription of the pdx-1 gene is controlled by factors acting upon sequences found within its 5'-flanking region. Critical cis-acting transcriptional control elements are located within a nuclease hypersensitive site that contains three conserved subdomains, termed areas I, II, and III. We show that area II acts as a tissue-specific regulatory region of the pdx-1 gene, directing transgene expression to a subpopulation of islet cells. Mutation of the area II hepatocyte nuclear factor 3 (HNF3) binding element in the larger area I- and area II- containing PstBst fragment also decreases PB(hsplacZ) transgene penetrance. These two results indicate possible ontogenetic and/or functional heterogeneity of the beta-cell population. Several other potential positive- and negative-acting control elements were identified in area II after mutation of the highly conserved sequence blocks within this subdomain. Pax6, a factor essential for islet alpha-cell development and islet hormone gene expression, was shown to bind in area II in vitro. Pax6 and HNF3 beta were also found to bind to this region in vivo by using the chromatin immunoprecipitation assay. Collectively, these data suggest an important role for both HNF3 beta and Pax6 in regulating pdx-1 expression in beta cells.