Jin H M, Robinson D F, Liang Y, Fahl W E
McArdle Laboratory for Cancer Research, University of Wisconsin Medical School, Madison, 53706.
J Biol Chem. 1994 Nov 18;269(46):28648-54.
Platelet-derived growth factor BB, encoded by the SIS/PDGF-B gene, is a potent mitogen for cells of mesenchymal origin, and the SIS/PDGF-B gene is expressed in a large percentage of human mesenchymal tumor cells establishing a growth-promoting, autocrine growth circuit. A 4-kb fragment, containing the SIS/PDGF-B promoter, was isolated from a human genomic library, and a series of 5'-nested deletions and linker-scanning mutants were used to identify positive regulatory elements that are necessary for the constitutive expression of this gene in human U2-OS osteosarcoma cells. A 250-bp fragment, lying immediately 5' to the SIS/PDGF-B mRNA initiation site (+1), retained full promoter activity, and positive regulatory elements at -228 to -219, -97 to -88 (SIS distal element) and -58 to -39 (SIS proximal element, SPE) were identified. Insertion of the 20-bp SPE into a heterologous, minimal promoter resulted in >5-fold transcriptional activation which was ablated by mutations to the SPE. High resolution mutagenesis within the 20-bp SPE, indicated the necessity of a CACCC motif for activity. Gel shift analysis of SPE-binding proteins in U2-OS nuclear extracts identified Sp1 and two additional binding factors that could be competed away from SPE binding by adding excess consensus Sp1 or CACC oligonucleotides. The individual and aggregate roles of the SPE and two weaker positive regulatory elements in regulating SIS/PDGF-B transcription in these tumor cells is considered.
由SIS/PDGF - B基因编码的血小板衍生生长因子BB是一种对间充质来源细胞具有强大作用的有丝分裂原，并且SIS/PDGF - B基因在很大比例的人间充质肿瘤细胞中表达，从而建立了一个促进生长的自分泌生长回路。从人基因组文库中分离出一个包含SIS/PDGF - B启动子的4 kb片段，并使用一系列5'端嵌套缺失和接头扫描突变体来鉴定该基因在人U2 - OS骨肉瘤细胞中组成型表达所必需的正调控元件。一个位于SIS/PDGF - B mRNA起始位点（+1）紧邻5'端的250 bp片段保留了完整的启动子活性，并且鉴定出了位于 - 228至 - 219、 - 97至 - 88（SIS远端元件）和 - 58至 - 39（SIS近端元件，SPE）的正调控元件。将20 bp的SPE插入异源最小启动子中导致转录激活增加5倍以上，而对SPE的突变消除了这种激活。在20 bp的SPE内进行高分辨率诱变表明，活性需要一个CACCC基序。对U2 - OS核提取物中SPE结合蛋白的凝胶迁移分析鉴定出Sp1和另外两个结合因子，通过添加过量的共有Sp1或CACC寡核苷酸可以使它们从SPE结合中被竞争掉。本文考虑了SPE和另外两个较弱的正调控元件在调节这些肿瘤细胞中SIS/PDGF - B转录方面的个体和总体作用。
