Kikkawa F, Gonzalez F J, Kimura S
Laboratory of Molecular Carcinogenesis, National Cancer Institute, Bethesda, Maryland 20892.
Mol Cell Biol. 1990 Dec;10(12):6216-24. doi: 10.1128/mcb.10.12.6216-6224.1990.
A 6.3-kbp segment of DNA, upstream of the human thyroid peroxidase gene, and various deletions thereof were linked to a promoterless bacterial chloramphenicol acetyltransferase reporter gene. These constructs were analyzed by transfection and expression in rat FRTL-5 thyroid cells and in human hepatoma HepG2 cells to localize sequences that are important for thyroid cell-specific expression of the thyroid peroxidase gene. A thyroid-specific enhancer element, capable of activating enhancerless simian virus 40 promoter expression in FRTL-5 cells, was localized to a 230-bp region approximately 5.5 kbp upstream of the human thyroid peroxidase gene transcription start site. DNase I footprinting, using nuclear extracts prepared from FRTL-5 cells, revealed three regions within the 230-bp fragment; none of these regions were protected by nuclear extracts from HepG2 cells. Gel mobility shift assays, using double-stranded oligonucleotides corresponding to the three protected regions, further confirmed the existence of factors in FRTL-5 cells, but not HepG2 cells, able to specifically bind to the enhancer sequences. These results suggest the presence of three cis-acting DNA elements in the human thyroid peroxidase gene enhancer that interact with thyroid-specific trans-acting factors.
人甲状腺过氧化物酶基因上游一段6.3千碱基对的DNA片段及其各种缺失片段与无启动子的细菌氯霉素乙酰转移酶报告基因相连。通过在大鼠FRTL - 5甲状腺细胞和人肝癌HepG2细胞中进行转染和表达来分析这些构建体，以定位对甲状腺过氧化物酶基因甲状腺细胞特异性表达重要的序列。一个能够激活FRTL - 5细胞中无增强子的猿猴病毒40启动子表达的甲状腺特异性增强子元件，定位于人甲状腺过氧化物酶基因转录起始位点上游约5.5千碱基对处的一个230碱基对区域。使用从FRTL - 5细胞制备的核提取物进行的DNase I足迹分析，揭示了230碱基对片段内的三个区域；这些区域均未被HepG2细胞的核提取物保护。使用对应于三个受保护区域的双链寡核苷酸进行凝胶迁移率变动分析，进一步证实了FRTL - 5细胞中存在能够特异性结合增强子序列的因子，而HepG2细胞中不存在。这些结果表明人甲状腺过氧化物酶基因增强子中存在三个顺式作用DNA元件，它们与甲状腺特异性反式作用因子相互作用。
