Kaetzel D M, Maul R S, Liu B, Bonthron D, Fenstermaker R A, Coyne D W
Department of Pharmacology, University of Kentucky Medical Center, Lexington 40536.
Biochem J. 1994 Jul 15;301 ( Pt 2)(Pt 2):321-7. doi: 10.1042/bj3010321.
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.
血小板衍生生长因子（PDGF）是由两条多肽链通过二硫键连接而成的异二聚体，即A链和B链，它们由位于不同染色体上的基因编码。A链基因在许多转化和未转化的细胞系中都有转录，并且可被多种生长因子、细胞因子和其他促有丝分裂激动剂诱导。为了定位介导A链基因启动子调控区域基础转录的DNA元件，我们在肾上皮细胞系BSC-1（非洲绿猴）中采用了5'末端缺失诱变和瞬时表达分析。在该细胞系中进行的研究表明，其表达高浓度的PDGF A链mRNA，在相对于转录起始位点的A链启动子富含GC的区域（-82至-40）中发现了一个正调控元件（PRE）。启动子的两个离散区域被确定为负调控元件（NREs），分别位于-1029至-880（NRE1）和-1800至-1029（NRE2）之间。包含两个NREs的-1800至-812区域，当以任何方向重新定位到单纯疱疹病毒胸苷激酶启动子附近时，都作为一个有效的NRE发挥作用，在正向时将转录活性降低60%，在负向时降低85%。对不表达大量PDGF A链mRNA或蛋白质的BSC-1细胞和Saos-2细胞（人骨肉瘤细胞）的比较表明，该基因的基础转录是由富含GC区域介导的增强子活性决定的，而不是通过上游NREs的去抑制作用。电泳凝胶迁移率变动分析揭示了核蛋白与富含GC的PRE（-73至-46）结合的复杂模式。用交替破坏Sp-1或Egr-1共有结合位点的突变寡核苷酸进行的竞争研究表明，形成特异性DNA-蛋白质复合物需要存在Sp1样核心序列（GGCGGG），而不需要Egr-1/Krox-24 [GCG(G/T)-GGGCG]。我们的观察结果表明，肾上皮细胞中A链基因的基础转录是通过由富含GC的PRE和与Sp1样共有DNA序列结合的核蛋白介导的活性增强来实现的。
