THYROID-SPECIFIC ENHANCER-BINDING PROTEIN (T/EBP) - CDNA CLONING, FUNCTIONAL-CHARACTERIZATION, AND STRUCTURAL IDENTITY WITH THYROID TRANSCRIPTION FACTOR TTF-1
THYROID-SPECIFIC ENHANCER-BINDING PROTEIN (T/EBP) - CDNA CLONING, FUNCTIONAL-CHARACTERIZATION, AND STRUCTURAL IDENTITY WITH THYROID TRANSCRIPTION FACTOR TTF-1
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DOI：
10.1128/mcb.11.10.4927
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发表时间：
1991-10-01
影响因子：
5.3
通讯作者：
KIMURA, S
中科院分区：
文献类型：
--
作者：
MIZUNO, K;GONZALEZ, FJ;KIMURA, S
利用双链寡核苷酸探针从大鼠甲状腺源性FRTL-5细胞lambda-gt 11表达文库中分离到甲状腺特异性增强子结合蛋白（T/EBP）的cDNA克隆。该寡核苷酸先前被证明在位于人甲状腺过氧化物酶基因转录起始位点上游5.5 kbp的甲状腺特异性增强子区域的三个顺式作用DNA元件之间具有最强的结合亲和力。cDNA的核苷酸和推导出的氨基酸序列显示，T/EBP与先前报道的甲状腺特异性转录因子1 （TTF-1）相同，TTF-1结合大鼠甲状腺球蛋白基因的启动子并控制其甲状腺特异性表达。当将含有6.3 kbp人甲状腺过氧化物酶上游序列的载体与荧光素酶报告基因连接时，在人巨细胞病毒主要立即早期基因启动子控制下表达的T/EBP cDNA，可使非允许的人肝癌HepG2细胞具有高达26倍的甲状腺特异性增强子活性。利用牛痘病毒表达系统进一步在HepG2细胞中表达T/EBP。表达的蛋白通过序列特异性亲和柱层析得到部分纯化，并通过凝胶迁移实验进一步证明，可以特异性结合增强子衍生的双链寡核苷酸。这些结果清楚地表明，T/EBP （TTF-1）与特定的顺式作用增强因子的结合是甲状腺特异性增强因子活性的主要原因。
A cDNA clone encoding a thyroid-specific enhancer-binding protein (T/EBP) was isolated from a rat thyroid-derived FRTL-5 cell lambda-gt 11 expression library, using a double-stranded oligonucleotide probe. This oligonucleotide was previously demonstrated to have the strongest binding affinity among three cis-acting DNA elements within the thyroid-specific enhancer region located 5.5 kbp upstream of the human thyroid peroxidase gene transcription start site. Nucleotide and deduced amino acid sequences of the cDNA revealed that T/EBP is identical to the previously reported thyroid-specific transcription factor 1 (TTF-1), which binds to the promoter of the rat thyroglobulin gene and controls its thyroid-specific expression. Expression of the T/EBP cDNA under control of the human cytomegalovirus major immediate-early gene promoter conferred thyroid-specific enhancer activity of as high as 26-fold to nonpermissive human hepatoma HepG2 cells when cotransfected with a vector containing 6.3 kbp of upstream sequence of the human thyroid peroxidase gene connected to a luciferase reporter gene. T/EBP was further expressed in HepG2 cells by using the vaccinia virus expression system. The expressed protein was partially purified by using sequence-specific affinity column chromatography and was further shown, by gel mobility shift experiments, to specifically bind to the enhancer-derived double-stranded oligonucleotide. These results clearly indicate that the binding of T/EBP (TTF-1) to the specific cis-acting enhancer element is largely responsible for thyroid-specific enhancer activity.