title: Transcription factor occupancy of the insulin gene in vivo. Evidence for direct regulation by Nkx2.2.
abstract: 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.
authors.affiliation: Department of Molecular Physiology and Biophysics, Vanderbilt University Medical School, Nashville, Tennessee 37232, USA.
authors.forename: Michelle A
authors.initials: MA
authors.lastname: Cissell
authors.forename: Li
authors.initials: L
authors.lastname: Zhao
authors.forename: Lori
authors.initials: L
authors.lastname: Sussel
authors.forename: Eva
authors.initials: E
authors.lastname: Henderson
authors.forename: Roland
authors.initials: R
authors.lastname: Stein
mesh_terms: D000682:Amyloid / Q000235:genetics; D000818:Animals; D051792:Basic Helix-Loop-Helix Proteins; D001665:Binding Sites; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000502:physiology*; D005136:Eye Proteins; D005786:Gene Expression Regulation; D051273:Glucose Transporter Type 2; D000090322:Homeobox Protein Nkx-2.2; D018398:Homeodomain Proteins / Q000502:physiology*; D007328:Insulin / Q000235:genetics*; D058228:Islet Amyloid Polypeptide; D007515:Islets of Langerhans / Q000378:metabolism; D051379:Mice; D009004:Monosaccharide Transport Proteins / Q000235:genetics; D000071841:PAX6 Transcription Factor; D051761:Paired Box Transcription Factors; D051381:Rats; D012097:Repressor Proteins; D015534:Trans-Activators / Q000502:physiology*; D014157:Transcription Factors / Q000502:physiology*; D029961:Zebrafish Proteins
chemical_list: D000682:Amyloid; D051792:Basic Helix-Loop-Helix Proteins; D004268:DNA-Binding Proteins; D005136:Eye Proteins; D051273:Glucose Transporter Type 2; D000090322:Homeobox Protein Nkx-2.2; D018398:Homeodomain Proteins; D007328:Insulin; D058228:Islet Amyloid Polypeptide; D009004:Monosaccharide Transport Proteins; D000071841:PAX6 Transcription Factor; D051761:Paired Box Transcription Factors; D012097:Repressor Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D029961:Zebrafish Proteins; C501008:Neurod1 protein, mouse; C000715627:Nkx2-2 protein, mouse; C000715628:Nkx2-2 protein, rat; C000605732:Pax6 protein, mouse; C000605733:Pax6 protein, rat; C000715630:nkx2.2b protein, zebrafish; C084010:pancreatic and duodenal homeobox 1 protein