The lung enriched transcription factor TTF-1 and the ubiquitously expressed proteins Sp1 and Sp3 interact with elements located in the minimal promoter of the rat Clara cell secretory protein gene
The lung enriched transcription factor TTF-1 and the ubiquitously expressed proteins Sp1 and Sp3 interact with elements located in the minimal promoter of the rat Clara cell secretory protein gene
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DOI：
10.1042/bj3160467
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发表时间：
1996-06-01
影响因子：
4.1
通讯作者：
Bingle, CD
中科院分区：
文献类型：
--
作者：
Toonen, RFG;Gowan, S;Bingle, CD
Clara细胞分泌蛋白（CCSP）基因在肺细支气管上皮细胞中的表达机制尚不清楚。已有的研究表明，大鼠CCSP基因转录起始位点-132 ~-76bp处的一个重要功能区（Region 1）结合了许多蛋白质。随后，我们表明，虽然区域1是CCSP基因表达的重要正调节因子，但该区域的3'序列（-75至+38）足以赋予报告基因的组织特异性表达。在本研究中，我们已经使用了瞬时转染与CCSP-CAT报告质粒（其中CAT是氯霉素乙酰转移酶）和凝胶迁移率变动分析与一系列重叠的寡核苷酸覆盖整个最小的启动子区域内研究蛋白质-DNA的相互作用在这个区域内的缺失系列。这些研究已经鉴定了肺和甲状腺富集同源结构域转录因子TTF-1的保守功能结合位点，其位于转录起始位点的位置-51和-42之间。含有该区域的CCSP-CAT嵌合报告基因在共转染试验中被TTF-1特异性激活，并且来自表达TTF-1的细胞的核提取物与该区域结合，体外翻译的大鼠TTF-1也是如此。鉴定了三个另外的保守区，并且在用跨越紧邻TTF-1位点5'的保守区的寡核苷酸进行的进一步凝胶迁移率变动研究中，我们鉴定了普遍表达的含锌指蛋白Spl和Sp3的结合位点。这些研究表明，细胞类型的限制和无处不在的核蛋白可能发挥联合作用，在细支气管上皮细胞内的CCSP基因的调节与最小的启动子区相互作用。
The mechanisms that direct expression of the Clara cell secretory protein (CCSP) gene to the bronchiolar epithelial cells of the lung remain to be elucidated, Previous studies have identified a number of proteins which bind to a functionally important region (Region 1) located -132 to -76 bp from the transcription start site in the rat CCSP gene. Subsequently we have shown that while Region 1 is an important positive regulator of CCSP gene expression, sequences 3' of this region (-75 to +38) are sufficient to confer tissue-specific expression of a reporter gene. In the present study we have used transient transfections with a deletion series of CCSP-CAT reporter plasmids (where CAT is chloramphenicol acetyltransferase) and gel mobility shift assays with a series of overlapping oligonucleotides covering the whole minimal promoter region to study protein-DNA interactions within this region. These studies have identified a conserved functional binding site for the lung and thyroid enriched homeodomain transcription factor TTF-1, located between positions -51 and -42 from the transcription start site. CCSP-CAT chimaeric reporters containing this region are specifically activated by TTF-1 in co-transfection assays, and nuclear extracts from cells which express TTF-I bind to this region, as does in vitro translated rat TTF-1. Three additional conserved regions were identified, and in further gel mobility shift studies with an oligonucleotide spanning the conserved region immediately 5' to the TTF-1 site we identified a binding site for the ubiquitously expressed zinc-finger-containing proteins Spl and Sp3. These studies suggest that cell-type-restricted and ubiquitous nuclear proteins may play a combined role in the regulation of the CCSP gene within the bronchiolar epithelium by interacting with the minimal promoter region.