title: Quantitative assessment of gene targeting in vitro and in vivo by the pancreatic transcription factor, Pdx1. Importance of chromatin structure in directing promoter binding.
abstract: The transcription factor Pdx1 is expressed in the pancreatic beta-cell, where it is believed to regulate several beta-cell-specific genes. Whereas binding by Pdx1 to elements of beta-cell genes has been demonstrated in vitro, almost none of these genes has been demonstrated to be a direct binding target for Pdx1 within cells (where complex chromatin structure exists). To determine which beta-cell promoters are bound by Pdx1 in vivo, we performed chromatin immunoprecipitation assays using Pdx1 antiserum and chromatin from beta-TC3 cells and Pdx1-transfected NIH3T3 cells and subsequently quantitated co-immunoprecipitated promoters using real-time PCR. We compared these in vivo findings to parallel immunoprecipitations in which Pdx1 was allowed to bind to promoter fragments in in vitro reactions. Our results show that in all cells Pdx1 binds strongly to the insulin, islet amyloid polypeptide, glucagon, Pdx1, and Pax4 promoters, whereas it does not bind to either the glucose transporter type 2 or albumin promoters. In addition, no binding by Pdx1 to the glucokinase promoter was observed in beta-cells. In contrast, in in vitro immunoprecipitations, Pdx1 bound all promoters to an extent approximately proportional to the number of Pdx1 binding sites. Our findings suggest a critical role for chromatin structure in directing the promoter binding selectivity of Pdx1 in beta-cells and non-beta-cells.
authors.affiliation: Department of Medicine, Division of Endocrinology and Metabolism, University of Virginia Health Sciences Center, 1300 Jefferson Park Avenue, Charlottesville, VA 22908, USA.
authors.forename: Swarup K
authors.initials: SK
authors.lastname: Chakrabarti
authors.forename: Joshua C
authors.initials: JC
authors.lastname: James
authors.forename: Raghavendra G
authors.initials: RG
authors.lastname: Mirmira
mesh_terms: D000818:Animals; D002460:Cell Line; D002843:Chromatin / Q000737:chemistry / Q000502:physiology*; D004247:DNA; D018390:Gene Targeting; D018398:Homeodomain Proteins; D007515:Islets of Langerhans / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011233:Precipitin Tests; D011401:Promoter Regions, Genetic; D011487:Protein Conformation; D015534:Trans-Activators / Q000235:genetics / Q000502:physiology*
chemical_list: D002843:Chromatin; D018398:Homeodomain Proteins; D015534:Trans-Activators; C084010:pancreatic and duodenal homeobox 1 protein; D004247:DNA