Dataset Information

Promoter region of the human platelet-derived growth factor A-chain gene.

ABSTRACT: The platelet-derived growth factor (PDGF) A- and B-chain genes are widely expressed in mammalian tissues and their homodimeric gene products appear to regulate the autocrine growth of both normal and transformed cells. In this study, we analyzed the 5' flanking sequences of the human PDGF A-chain gene to seek elements important to regulating its transcription. The promoter region was exceptionally G + C-rich and contained a "TATA box" but no "CAAT box." The transcription start site was identified 845 base pairs 5' to the translation initiation site by S1 nuclease mapping and by primer extension. Both in vitro transcription and transient expression of the chloramphenicol acetyltransferase gene linked to the PDGF A-chain 5' flanking sequences established that the putative promoter region was active, and RNase H mapping established that the three characteristic mRNAs (1.9, 2.3, and 2.8 kilobases) used the same transcription start site, which was used in normal endothelial cells and in two human tumor cell lines that express high levels of A-chain transcripts. The results established an exceptionally G + C-rich promoter region and a single transcription start site active for each of the three mRNAs of the PDGF A-chain gene. DNA sites of potential importance in mediating the activation of the PDGF A-chain gene in normal cells and in transformed cell lines expressing high levels of PDGF A chain were identified.

SUBMITTER: Takimoto Y

PROVIDER: S-EPMC51089 | biostudies-other | 1991 Mar

REPOSITORIES: biostudies-other

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Promoter region of the human platelet-derived growth factor A-chain gene.

Takimoto Y Y Wang Z Y ZY Kobler K K Deuel T F TF

Proceedings of the National Academy of Sciences of the United States of America 19910301 5

The platelet-derived growth factor (PDGF) A- and B-chain genes are widely expressed in mammalian tissues and their homodimeric gene products appear to regulate the autocrine growth of both normal and transformed cells. In this study, we analyzed the 5' flanking sequences of the human PDGF A-chain gene to seek elements important to regulating its transcription. The promoter region was exceptionally G + C-rich and contained a "TATA box" but no "CAAT box." The transcription start site was identifie ...[more]

PMID: 1848007

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Project description: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.

Dataset Information

Promoter region of the human platelet-derived growth factor A-chain gene.

Publications

Promoter region of the human platelet-derived growth factor A-chain gene.

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