PLATELET-DERIVED GROWTH-FACTOR A-CHAIN GENE-TRANSCRIPTION IS MEDIATEDBY POSITIVE AND NEGATIVE REGULATORY REGIONS IN THE PROMOTER

Platelet-derived growth factor (PDGF) is a disulphide-linked heterodim er 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 n umber of transformed and nontransformed cell lines and is inducible by a wide variety of growth factors, cytokines and other mitogenic agoni sts. To localize DNA elements that mediate basal transcription in the promoter regulatory region of the A-chain gene, we have employed 5'-en dpoint deletion mutagenesis and transient expression analysis in the r enal epithelial cell line BSC-1 (African green monkey). Studies conduc ted in this cell line, which expresses high concentrations of PDGF A-c hain mRNA, reveal a positive regulatory element (PRE) in a GC-rich str etch of the A-chain promoter between -82 and -40, relative to the tran scription start site. Two discrete regions of the promoter were identi fied as negative regulatory elements (NREs), located between -1029 and -880 (NRE1) and between -1800 and -1029 (NRE2). The -1800 to -812 reg ion, which contains both NREs, functions as a potent NRE when relocate d in either orientation adjacent to the herpes simplex virus thymidine kinase promoter, reducing transcription activity by 60% in the positi ve orientation and 85% in the negative orientation. Comparison of BSC- 1 cells and Saos-2 cells (human osteogenic sarcoma), which do not expr ess significant quantities of PDGF A-chain mRNA or protein, indicates that basal transcription of the gene is determined by enhancer activit y 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 demonstr ate a requirement for the presence of an Spl-like core sequence (GGCGG G) but not Egr-1/Krox-24 [GCG(G/T)GGGCG] for the formation of specific DNA-protein complexes. Our observations suggest that basal transcript ion of the A-chain gene in renal epithelial cells is achieved through active enhancement, mediated by a GC-rich PRE and nuclear proteins tha t bind to Sp-1-like consensus DNA sequences.