Wang Z, Melmed S
Cedars-Sinai Research Institute, UCLA School of Medicine, Los Angeles, California 90048, USA.
J Biol Chem. 1998 Oct 2;273(40):26069-77. doi: 10.1074/jbc.273.40.26069.
We recently reported a placenta-specific enhancer in the human leukemia inhibitory factor receptor (LIFR) gene and now show detailed characterization of the 226-base pair enhancer (-4625/-4400 nucleotides). Four of twenty-two mutants in linker analysis showed reduced promoter activities to 45, 30, 10, and 10%, respectively. Specific binding of region A (-4617/-4602) with nuclear extract was competed by a known Oct-1 oligo and supershifted by Oct-1 antibody. Specific binding of region B (-4549/-4535) was competed by a GATA oligo, but could not be supershifted by four GATA antibodies. Nevertheless, mutagenesis showed that critical bases in region B were identical to the GATA core motif, indicating that region B may bind to a novel GATA family transcription factor. The other two adjacent regions designated as region C (-4464/-4445) showed no known consensus binding sites, and their specific placental JEG-3 nuclear extract binding was not evident in nonplacental nuclear extracts and was not competed by a trophoblast specific element (TSE), indicating that region C is a novel placenta-specific element (PSE, CATTTCCTGAACTAGTTTTT). Footprinting localized the binding boundary of PSE-binding protein (PSEB), and three Gs were found to be important for specific PSE binding. UV cross-linking showed that PSEB had a molecular mass of approximately 160 kDa, substituting the PSE with two previously reported placenta elements TSE or chorionic somatomammotropin enhancer factor 1 (CSEF-1) motifs resulted in markedly different promoter activities, indicating that PSEB is indeed different from TSE binding protein or CSEF-1. These results are the first demonstration that a novel PSE is the major element for placenta-specific enhancer activity in human LIFR gene.
我们最近报道了人类白血病抑制因子受体（LIFR）基因中的一个胎盘特异性增强子，现在展示了对226个碱基对增强子（-4625/-4400核苷酸）的详细特征分析。连接子分析中的22个突变体中有4个分别显示启动子活性降低至45%、30%、10%和10%。区域A（-4617/-4602）与核提取物的特异性结合被已知的Oct-1寡核苷酸竞争，并被Oct-1抗体超迁移。区域B（-4549/-4535）的特异性结合被GATA寡核苷酸竞争，但不能被四种GATA抗体超迁移。然而，诱变表明区域B中的关键碱基与GATA核心基序相同，表明区域B可能与一种新型GATA家族转录因子结合。另外两个相邻区域被指定为区域C（-4464/-4445），未显示已知的共有结合位点，它们与胎盘JEG-3核提取物的特异性结合在非胎盘核提取物中不明显，也不被滋养层特异性元件（TSE）竞争，表明区域C是一个新型胎盘特异性元件（PSE，CATTTCCTGAACTAGTTTTT）。足迹分析确定了PSE结合蛋白（PSEB）的结合边界，发现三个鸟嘌呤对于特异性PSE结合很重要。紫外线交联显示PSEB的分子量约为160 kDa，用两个先前报道的胎盘元件TSE或绒毛膜生长催乳素增强子因子1（CSEF-1）基序替代PSE导致明显不同的启动子活性，表明PSEB确实不同于TSE结合蛋白或CSEF-1。这些结果首次证明了一种新型PSE是人类LIFR基因中胎盘特异性增强子活性的主要元件。
