PLATELET-DERIVED GROWTH-FACTOR A-CHAIN GENE-TRANSCRIPTION IS MEDIATED BY POSITIVE AND NEGATIVE REGULATORY REGIONS IN THE PROMOTER
PLATELET-DERIVED GROWTH-FACTOR A-CHAIN GENE-TRANSCRIPTION IS MEDIATED BY POSITIVE AND NEGATIVE REGULATORY REGIONS IN THE PROMOTER
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DOI：
10.1042/bj3010321
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发表时间：
1994-07-15
影响因子：
4.1
通讯作者：
COYNE, DW
中科院分区：
文献类型：
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作者：
KAETZEL, DM;MAUL, RS;COYNE, DW
血小板衍生生长因子（PDGF）是由两条多肽链（A和B链）的二硫键连接的异源二聚体，所述两条多肽链由不同染色体上的基因编码。A链基因在许多转化和非转化细胞系中转录，并且可由多种生长因子、细胞因子和其他促有丝分裂激动剂诱导。为了定位在A链基因的启动子调控区中介导基础转录的DNA元件，我们在肾上皮细胞系BSC-1（非洲绿色猴）中采用了5 '-末端缺失诱变和瞬时表达分析。在表达高浓度PDGF A链mRNA的该细胞系中进行的研究揭示了相对于转录起始位点，在A链启动子的富含GC的区段中在-82和-40之间的正调控元件（PRE）。启动子的两个离散区域被鉴定为负调控元件（NRE），位于-1029和-880（NRE 1）之间和-1800和-1029（NRE 2）之间。包含两个NRE的-1800至-812区域，当以邻近单纯疱疹病毒胸苷激酶启动子的任一方向重新定位时，作为有效的NRE发挥功能，在正方向上降低转录活性60%，在负方向上降低转录活性85%。BSC-1细胞和Saos-2细胞（人成骨肉瘤），不表达大量的PDGF A链mRNA或蛋白质的比较表明，基因的基础转录是由富含GC的区域介导的增强子活性决定的，而不是通过上游NRE的去抑制。电泳凝胶迁移率变动分析揭示了与富含GC的PRE（-73至-46）结合的核蛋白的复杂模式。用交替破坏Sp-1或Egr-1的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Sl样核心序列（GGCGGG）而不是Egr-1/Krox-24 [GCG（G/T）GGGCG]。我们的观察结果表明，在肾上皮细胞中的A链基因的基础转录是通过主动增强，介导的GC丰富的PRE和核蛋白结合到SP-1样的共识DNA序列。
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 nontransformed 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 Spl-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.