Identification of an enhancer of the human activating protein-2α gene that contains a critical Ets1 binding site
Identification of an enhancer of the human activating protein-2α gene that contains a critical Ets1 binding site
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DOI：
10.1210/jc.2002-021831
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发表时间：
2003-07-01
影响因子：
5.8
通讯作者：
Handwerger, S
中科院分区：
文献类型：
--
作者：
Cheng, YH;Handwerger, S
对激活蛋白-2 α（AP-2 α）基因的5 '侧翼区的缺失分析研究表明，近端152 bp是最小启动子活性所必需的，并且从核苷酸-1279到-1139的140 bp片段充当基础转录活性的增强子。将140-bp片段与最小AP-2 α启动子或异源猿猴病毒40启动子荧光素酶报告质粒连接，赋予滋养层细胞增强子活性。在脱氧核糖核酸酶I足迹研究中，滋养层细胞的核提取物保护了140 bp片段的两个区域，E2和E3。E2中Ets 1结合位点的定点突变显著抑制AP-2 α增强子活性，而E2上两个推定的多瘤病毒增强子激活因子-3结合位点和E3中胰岛素上游因子1结合位点的诱变对增强子活性没有影响。凝胶位移和supershift分析表明，Ets 1结合E2的Ets网站，和Ets 1在转染研究诱导AP-2 α启动子活性的过表达。由于转录因子Ets 1在滋养层细胞中含量丰富，这些研究强烈表明，在胎盘中AP-2 α基因的表达是由一个顺式作用元件在核苷酸-1279到-1139，包含一个关键的Ets 1结合位点。
Deletion analysis studies of the 5'-flanking region of the activating protein-2alpha (AP-2alpha) gene indicates that the proximal 152 bp are essential for minimal promoter activity and that a 140-bp fragment from nucleotides -1279 to -1139 acts as an enhancer of basal transcriptional activity. Ligation of the 140-bp fragment to a minimal AP-2alpha promoter or a heterologous simian virus 40 promoter luciferase reporter plasmid conferred enhancer activity in trophoblast cells. In deoxyribonuclease I footprint studies, nuclear extracts from trophoblast cells protected two regions of the 140-bp fragment, E2 and E3. Site-directed mutagenesis of an Ets1-binding site in E2 significantly inhibited AP-2alpha enhancer activity, whereas mutagenesis of two putative polyomavirus enhancer activator-3-binding sites on E2 and an insulin upstream factor 1-binding site in E3 had no effect on enhancer activity. Gel shift and supershift assays indicated that Ets1 binds to the Ets site in E2, and overexpression of Ets1 in transfection studies induced AP-2alpha promoter activity. As the transcription factor Ets1 is abundant in trophoblast cells, these investigations strongly suggest that AP-2alpha gene expression in the placenta is enhanced by a cis-acting element at nucleotides -1279 to -1139 that contains a critical Ets1-binding site.