Feuerman M H, Godbout R, Ingram R S, Tilghman S M
Howard Hughes Medical Institute, Princeton University, New Jersey 08544.
Mol Cell Biol. 1989 Oct;9(10):4204-12. doi: 10.1128/mcb.9.10.4204-4212.1989.
Previous work identified four upstream cis-acting elements required for tissue-specific expression of the alpha-fetoprotein (AFP) gene: three distal enhancers and a promoter. To further define the role of the promoter in regulating AFP gene expression, segments of the region were tested for the ability to direct transcription of a reporter gene in transient expression assay. Experiments showed that the region within 250 base pairs of the start of transcription was sufficient to confer liver-specific transcription. DNase I footprinting and band shift assays indicated that the region between -130 and -100 was recognized by two factors, one of which was highly sequence specific and found only in hepatoma cells. Competition assays suggested that the liver-specific binding activity was HNF-1, previously identified by its binding to other liver-specific promoters. Mutation of the HNF-1 recognition site at -120 resulted in a significant reduction in transcription in transfection assays, suggesting a biological role for HNF-1 in the regulation of AFP expression.
先前的研究确定了甲胎蛋白（AFP）基因组织特异性表达所需的四个上游顺式作用元件：三个远端增强子和一个启动子。为了进一步明确启动子在调节AFP基因表达中的作用，在瞬时表达试验中对该区域的片段进行了报告基因转录指导能力的测试。实验表明，转录起始点250个碱基对内的区域足以赋予肝脏特异性转录。DNA酶I足迹法和凝胶迁移试验表明，-130至-100之间的区域被两个因子识别，其中一个因子具有高度序列特异性，且仅在肝癌细胞中发现。竞争试验表明，肝脏特异性结合活性是肝细胞核因子-1（HNF-1），它先前通过与其他肝脏特异性启动子结合而被鉴定。-120处HNF-1识别位点的突变导致转染试验中转录显著减少，表明HNF-1在AFP表达调节中具有生物学作用。
