Characterization of the c-specific promoter of the gene encoding human endothelin-converting enzyme-1 (ECE-1)
Characterization of the c-specific promoter of the gene encoding human endothelin-converting enzyme-1 (ECE-1)
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DOI：
10.1016/s0014-5793(00)01086-3
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发表时间：
2000-01-28
期刊：
影响因子：
3.5
通讯作者：
Orzechowski, HD
中科院分区：
文献类型：
--
作者：
Funke-Kaiser, H;Bolbrinker, J;Orzechowski, HD
人ECE-1有四种不同组织分布的异构体表达，在一定病理生理条件下，其mRNA和蛋白水平发生改变。为了研究ECE-1的转录调控，我们研究了ECE-1主要异构体ECE-1c的调控区域，获得了一个包含完整的人类ECE-1基因的基因组克隆，包括推定的ECE-1特异性启动子，在推定的c特异性翻译起始起始密码子上游968 bp处和几个序列缺失突变体被亚克隆到一个报告载体中，并转染到内皮细胞（BAEC， EA.hy926, ECV304）和上皮细胞(MDA MB435S，MCF7)细胞，与SV40启动子和之前描述的ECE-1a和1b启动子相比，显示出非常强的启动子活性。转染序列缺失突变体表明，启动子内有两个正调控区域（-142/-240和-240/490）可能与GATA和ETS转录因子结合有关，RNase保护试验（RPA）和5'-RACE显示，多个转录起始位点位于-110、-140和-350 bp左右，位点导向突变；证明了E2F顺式元件对基础ECE-1c启动子活性的关键作用，此外，我们发现了亚型特异性ECE-1 mRNA水平与相应的ECE-1a， 1b， 1c启动子活性之间的相关性。(C) 2000年欧洲生化学会联合会。
Human ECE-1 is expressed in four isoforms with different tissue distribution and its mRNA and protein levels are altered under certain pathophysiological conditions. To investigate the transcriptional regulation of ECE-1, we studied the regulatory region of ECE-1c, the major ECE-1 isoform, A genomic clone comprising the complete human ECE-1 gene including the putative ECE-1c-specific promoter was obtained, Up to 968 bp upstream of the putative c-specific translation initiation start codon and several serial deletion mutants were subcloned into a reporter vector and transfected into endothelial (BAEC, EA.hy926, ECV304) and epithelial (MDA MB435S, MCF7) cells, showing very strong promoter activity in comparison to the SV40 promoter and to the previously described ECE-1a and 1b promoters. Transfection of serial deletion mutants indicated two positive regulatory regions within the promoter (-142/-240 and -240/490) likely involved in binding GATA and ETS transcription factors, RNase protection assay (RPA) and 5'-RACE revealed multiple transcriptional start sites located at about -110, -140 and -350 bp, Site-directed mutagenesis; demonstrated a crucial role for the E2F cis-element for basal ECE-1c promoter activity, Additionally, we found a correlation between isoform-specific ECE-1 mRNA levels and corresponding ECE-1a, 1b, 1c promoter activities. (C) 2000 Federation of European Biochemical Societies.