Central Laboratory, Shenzhen PKU-HKUST Medical Center, Peking University Shenzhen Hospital, China.
Acta Biochim Biophys Sin (Shanghai). 2011 Jun;43(6):455-64. doi: 10.1093/abbs/gmr033.
We previously demonstrated that the region -87/+134 of the human ezrin gene (VIL2) exhibited promoter activity in human esophageal carcinoma EC109 cells, and a further upstream region -1324/-890 positively regulated transcription. In this study, to identify the transcriptional regulatory regions upstream of the VIL2 promoter, we cloned VIL2 -1541/-706 segment containing the -1324/-890, and investigated its transcriptional regulatory properties via luciferase assays in transiently transfected cells. In EC109 cells, it was found that VIL2 -1541/-706 possessed promoter and enhancer activities. We also localized transcriptional regulatory regions by fusing 5'- or 3'-deletion segments of VIL2 -1541/-706 to a luciferase reporter. We found that there were three positive and one negative transcriptional regulatory regions within VIL2 -1541/-706 in EC109 cells. When these regions were separately located upstream of the luciferase gene without promoter, or located upstream of the VIL2 promoter or SV40 promoter directing the luciferase gene, only VIL2 -1297/-1186 exhibited considerable promoter and enhancer activities, which were lower than those of -1541/-706. In addition, transient expression of Sp1 increased ezrin expression and the transcriptional activation of VIL2 -1297/-1186. Other three regions, although exhibiting significantly positive or negative transcriptional regulation in deletion experiments, showed a weaker or absent regulation. These data suggested that more than one region upstream of the VIL2 promoter participated in VIL2 transcription, and the VIL2 -1297/-1186, probably as a key transcriptional regulatory region, regulated VIL2 transcription in company with other potential regulatory regions.
我们之前证明，人 ezrin 基因（VIL2）的 -87/+134 区域在人食管癌细胞 EC109 中具有启动子活性，并且更远的上游区域 -1324/-890 正向调节转录。在这项研究中，为了鉴定 VIL2 启动子上游的转录调节区域，我们克隆了包含 -1324/-890 的 VIL2-1541/-706 片段，并通过瞬时转染细胞中的荧光素酶测定研究了其转录调节特性。在 EC109 细胞中，发现 VIL2-1541/-706 具有启动子和增强子活性。我们还通过将 VIL2-1541/-706 的 5'-或 3'-缺失片段融合到荧光素酶报告基因上来定位转录调节区域。我们发现 VIL2-1541/-706 中有三个阳性和一个阴性转录调节区域。当这些区域分别位于没有启动子的荧光素酶基因上游或位于 VIL2 启动子或 SV40 启动子上游时，只有 VIL2-1297/-1186 表现出相当大的启动子和增强子活性，其活性低于 -1541/-706。此外，瞬时表达 Sp1 增加了 ezrin 的表达并激活了 VIL2-1297/-1186 的转录。另外三个区域虽然在缺失实验中表现出明显的正或负转录调控，但调控作用较弱或不存在。这些数据表明，VIL2 启动子上游的多个区域参与了 VIL2 转录，并且 VIL2-1297/-1186 可能作为一个关键的转录调节区，与其他潜在的调节区共同调节 VIL2 转录。
