title: Platelet-derived growth factor A-chain gene transcription is mediated by positive and negative regulatory regions in the promoter.
abstract: Platelet-derived growth factor (PDGF) is a disulphide-linked heterodimer of two polypeptide chains, the A and B chains, which are encoded by genes on separate chromosomes. The A-chain gene is transcribed in a number of transformed and non-transformed cell lines and is inducible by a wide variety of growth factors, cytokines and other mitogenic agonists. To localize DNA elements that mediate basal transcription in the promoter regulatory region of the A-chain gene, we have employed 5'-endpoint deletion mutagenesis and transient expression analysis in the renal epithelial cell line BSC-1 (African green monkey). Studies conducted in this cell line, which expresses high concentrations of PDGF A-chain mRNA, reveal a positive regulatory element (PRE) in a GC-rich stretch of the A-chain promoter between -82 and -40, relative to the transcription start site. Two discrete regions of the promoter were identified as negative regulatory elements (NREs), located between -1029 and -880 (NRE1) and between -1800 and -1029 (NRE2). The -1800 to -812 region, which contains both NREs, functions as a potent NRE when relocated in either orientation adjacent to the herpes simplex virus thymidine kinase promoter, reducing transcription activity by 60% in the positive orientation and 85% in the negative orientation. Comparison of BSC-1 cells and Saos-2 cells (human osteogenic sarcoma), which do not express significant quantities of PDGF A-chain mRNA or protein, indicates that basal transcription of the gene is determined by enhancer activity mediated by the GC-rich region rather than through de-repression of the upstream NREs. Electrophoretic gel mobility shift assays reveal a complex pattern of nuclear protein binding to the GC-rich PRE (-73 to -46). Competition studies conducted with mutant oligonucleotides that alternately disrupt consensus binding sites for Sp-1 or Egr-1 demonstrate a requirement for the presence of an Sp1-like core sequence (GGCGGG) but not Egr-1/Krox-24 [GCG(G/T)-GGGCG] for the formation of specific DNA-protein complexes. Our observations suggest that basal transcription of the A-chain gene in renal epithelial cells is achieved through active enhancement, mediated by a GC-rich PRE and nuclear proteins that bind to Sp-1-like consensus DNA sequences.
authors.affiliation: Department of Pharmacology, University of Kentucky Medical Center, Lexington 40536.
authors.forename: D M
authors.initials: DM
authors.lastname: Kaetzel
authors.forename: R S
authors.initials: RS
authors.lastname: Maul
authors.forename: B
authors.initials: B
authors.lastname: Liu
authors.forename: D
authors.initials: D
authors.lastname: Bonthron
authors.forename: R A
authors.initials: RA
authors.lastname: Fenstermaker
authors.forename: D W
authors.initials: DW
authors.lastname: Coyne
mesh_terms: D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D015152:Blotting, Northern; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D002522:Chlorocebus aethiops; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D018014:Gene Transfer Techniques; D006801:Humans; D007668:Kidney; D008969:Molecular Sequence Data; D016296:Mutagenesis; D012516:Osteosarcoma; D010982:Platelet-Derived Growth Factor / Q000235:genetics*; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000378:metabolism; D011993:Recombinant Fusion Proteins; D012045:Regulatory Sequences, Nucleic Acid; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
chemical_list: D010982:Platelet-Derived Growth Factor; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D004247:DNA; D015500:Chloramphenicol O-Acetyltransferase