Ohmori M, Shimura H, Shimura Y, Ikuyama S, Kohn L D
Section on Cell Regulation, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892.
Endocrinology. 1995 Jan;136(1):269-82. doi: 10.1210/endo.136.1.7828540.
A thyroid transcription factor-1 (TTF-1)-binding element in the rat TSH receptor (TSHR) promoter, between -189 and -175 basepairs (bp), is important for both thyroid-specific expression and thyroid-specific TSH/cAMP autoregulation of the TSHR. The identification of an up-stream TTF-1-binding site and its relationship to the function of the down-stream TTF-1 element are the subjects of this report. Sequence analysis identifies a potential TTF-1 site at -878 bp; deoxyribonuclease-I footprinting shows that the -881 to -866 bp region is protected by recombinant TTF-1 protein and by nuclear extracts from FRTL-5 thyroid cells that contain TTF-1, but not by extracts from nonfunctioning FRT thyroid or Buffalo rat liver (BRL) cells, which have no TTF-1, or by Pax-8. FRTL-5, but not FRT or BRL cell nuclear extracts, form a specific protein-DNA complex with this region in gel mobility shift analyses; its formation is prevented by TTF-1-binding elements from the thyroglobulin promoter. The upstream TTF-1 site acts as an enhancer when coupled to a heterologous simian virus-40 promoter-chloramphenicol acetyltransferase (CAT) chimera and transfected into FRTL-5 thyroid cells. There is a greater increase, 3-vs. 2-fold (P < 0.05), when TSHR promoter-CAT chimeras, which contain the up-stream TTF-1 element, pTRCAT5'-907 or pTRCAT5'-886, as opposed to those in which it is deleted, pTRCAT5'-907 delta USTTF-1, are transfected into FRTL-5 cells or cotransfected with a TTF-1 expression vector into BRL cells, which have no endogenous TTF-1. The TTF-1-dependent activity of pTRCAT5'-907 delta USTTF-1 is the same as that of the minimal promoter, -220 to -39 bp, containing only the down-stream TTF-1 site in both cells. Transfection of chimeric TSHR-CAT plasmids with the down- and/or up-stream TTF-1 site deleted reveals that the down-stream TTF-1 element functions in the absence of the up-stream element, but function of the up-stream site requires the down-stream TTF-1 element. Like the down-stream TSHR TTF-1-binding site, the up-stream TTF-1 site is different from TTF-1 sites in the thyroglobulin and thyroid peroxidase promoter, in that it does not interact with Pax-8.(ABSTRACT TRUNCATED AT 400 WORDS)
大鼠促甲状腺激素受体（TSHR）启动子中位于-189至-175碱基对（bp）之间的甲状腺转录因子-1（TTF-1）结合元件，对于TSHR的甲状腺特异性表达以及甲状腺特异性促甲状腺激素/环磷酸腺苷（TSH/cAMP）自身调节都很重要。本报告的主题是鉴定一个上游TTF-1结合位点及其与下游TTF-1元件功能的关系。序列分析在-878 bp处鉴定出一个潜在的TTF-1位点；脱氧核糖核酸酶I足迹分析表明，-881至-866 bp区域受到重组TTF-1蛋白以及来自含有TTF-1的FRTL-5甲状腺细胞的核提取物的保护，但不受来自无功能的FRT甲状腺或布法罗大鼠肝（BRL）细胞（不含TTF-1）的提取物或Pax-8的保护。在凝胶迁移率变动分析中，FRTL-5细胞（而非FRT或BRL细胞核提取物）与该区域形成特异性蛋白质-DNA复合物；甲状腺球蛋白启动子的TTF-1结合元件可阻止其形成。当与异源猿猴病毒40启动子-氯霉素乙酰转移酶（CAT）嵌合体偶联并转染到FRTL-5甲状腺细胞中时，上游TTF-1位点起增强子作用。当含有上游TTF-1元件的TSHR启动子-CAT嵌合体pTRCAT5'-907或pTRCAT5'-886，与缺失该元件的pTRCAT5'-907 delta USTTF-1相比，转染到FRTL-5细胞中或与TTF-1表达载体共转染到无内源性TTF-1的BRL细胞中时，前者的增加幅度更大，为3倍而非2倍（P<0.05）。pTRCAT5'-907 delta USTTF-1的TTF-1依赖性活性与仅含有下游TTF-1位点的最小启动子-220至-39 bp在两种细胞中的活性相同。缺失下游和/或上游TTF-1位点的嵌合TSHR-CAT质粒转染实验表明，下游TTF-1元件在没有上游元件时仍起作用，但上游位点的功能需要下游TTF-1元件。与下游TSHR TTF-1结合位点一样，上游TTF-1位点与甲状腺球蛋白和甲状腺过氧化物酶启动子中的TTF-1位点不同，因为它不与Pax-8相互作用。（摘要截短于400字）
