Wang Z, Melmed S
Cedars Sinai Research Institute-UCLA School of Medicine, Los Angeles, California 90048, USA.
J Biol Chem. 1997 Oct 31;272(44):27957-65. doi: 10.1074/jbc.272.44.27957.
Knockout of the leukemia inhibitory factor receptor (LIFR) gene results in disrupted placental architecture, imbalanced bone development, and losses of functional neurons. We here report the identification of an enhancer in a functional human LIFR gene promoter and alternative promoter usage by this gene. A single transcription start site was identified in placental JEG-3 cells and a genomic clone containing 4876-nucleotide upstream sequences was found to have promoter activity in JEG-3 cells. However, in osteogenic sarcoma U-2 OS cell, Northern blot using a probe of the first exon detected in JEG-3 cells failed to detect LIFR transcripts. 5'-Rapid amplification of cDNA ends (RACE) revealed an alternative first exon and a 0.6-kilobase pair (kb) 5'-flanking region possessed promoter activity in U-2 OS cells. For the 4.8-kb promoter active in placental cells, a minimal promoter was localized within -162 nucleotides. Three regions increased and one inhibited promoter activity. Subcloning of an activation region (-4876 to -3453 nucleotides) into SV40 promoter either upstream or downstream in either orientation to the luciferase reporter resulted in 10-35-fold luciferase induction, demonstrating the characteristics of an enhancer. Transfections into nine cell lines of different tissue origin indicated that the cloned promoter and enhancer in the 4.8-kb fragment was placental tissue-specific. A 226-base pair fragment (-4625 to -4400 nucleotides) was further localized as the minimal enhancer region, in which deletion of either element A (-4625 to -4581 nucleotides) or element B (-4418 to -4400 nucleotides) resulted in the loss of enhancer activity. Electrophoretic mobility shift assay confirmed that these two elements bind to specific nuclear proteins individually. In the middle region between element A and B, disruption of enhancer integrity also led to a loss of enhancer activity, although two SP1 and three NF-kappaB/c-Rel binding sites did not contribute to enhancer function. These results demonstrate a complex regulation of the human LIFR gene, including alternative promoter usage and tissue-specific elements at the transcription level.
白血病抑制因子受体（LIFR）基因的敲除会导致胎盘结构破坏、骨骼发育失衡以及功能性神经元丧失。我们在此报告在功能性人类LIFR基因启动子中鉴定出一个增强子以及该基因的替代启动子使用情况。在胎盘JEG - 3细胞中鉴定出一个单一转录起始位点，并且发现一个包含4876个核苷酸上游序列的基因组克隆在JEG - 3细胞中具有启动子活性。然而，在骨肉瘤U - 2 OS细胞中，使用在JEG - 3细胞中检测到的第一个外显子探针进行Northern印迹未能检测到LIFR转录本。5' - cDNA末端快速扩增（RACE）揭示了一个替代的第一个外显子以及一个0.6千碱基对（kb）的5'侧翼区域在U - 2 OS细胞中具有启动子活性。对于在胎盘细胞中具有活性的4.8 kb启动子，最小启动子定位于 - 162个核苷酸范围内。三个区域增强而一个区域抑制启动子活性。将一个激活区域（ - 4876至 - 3453个核苷酸）亚克隆到SV40启动子中，无论其相对于荧光素酶报告基因是上游还是下游、何种方向，均导致荧光素酶诱导10 - 35倍，证明其具有增强子的特性。转染到九种不同组织来源的细胞系表明，4.8 kb片段中的克隆启动子和增强子具有胎盘组织特异性。一个226碱基对片段（ - 4625至 - 4400个核苷酸）被进一步定位为最小增强子区域，其中元件A（ - 4625至 - 4581个核苷酸）或元件B（ - 4418至 - 4400个核苷酸）的缺失均导致增强子活性丧失。电泳迁移率变动分析证实这两个元件分别与特定核蛋白结合。在元件A和B之间的中间区域，增强子完整性的破坏也导致增强子活性丧失，尽管两个SP1和三个NF - κB/c - Rel结合位点对增强子功能没有贡献。这些结果证明了人类LIFR基因的复杂调控，包括替代启动子使用以及转录水平上的组织特异性元件。
