Central Laboratory, Peking University Shenzhen Hospital, Shenzhen PKUHKUST Medical Center, Peoples Republic of China.
Cell Biol Int. 2011 Oct;35(10):967-71. doi: 10.1042/CBI20100854.
We previously demonstrated that the VIL2 -87/+134 region exhibited promoter activity in some human cells, and a region further upstream of this promoter might contain an enhancer. However, the properties and location of this VIL2 enhancer remain unclear. In this study, we cloned the VIL2 -1541/-706 segment and investigated its transcriptional regulatory properties via luciferase assays in transiently transfected HEK-293 cells (human embryonic kidney cells). The VIL2 -1541/-706 was found to exhibit promoter activity. Furthermore, when this segment was located upstream of the VIL2 or SV40 (simian virus 40) promoters in the forward orientation, the expression levels of luciferase were dramatically enhanced. However, this transcriptional enhancement disappeared when this segment was located upstream of the promoter in the reverse orientation or downstream of the reporter gene in the forward or reverse orientation. In deletion experiments, we found several potential regulatory regions within the VIL2 -1541/-706. When these regions were separately located upstream of the VIL2 or SV40 promoters, only the -1297/-1186 considerably enhanced the activity of these promoters. Although the other regulatory regions exhibited significant transcriptional regulation in deletion experiments, they weakly enhanced VIL2 promoter activity and/or did not regulate SV40 promoter activity. These results suggest that the DNA sequence upstream of the VIL2 promoter functions as an enhancer in a position- and orientation-dependent manner, and the VIL2 -1297/-1186, which acts as a key enhancer, probably regulates VIL2 transcription in combination with other potential regulatory regions located upstream of the VIL2 promoter.
我们之前证明 VIL2-87/+134 区域在一些人体细胞中具有启动子活性，并且该启动子的上游区域可能含有增强子。然而，这个 VIL2 增强子的性质和位置仍不清楚。在这项研究中，我们克隆了 VIL2-1541/-706 片段，并通过瞬时转染 HEK-293 细胞（人胚肾细胞）中的荧光素酶测定研究了其转录调控特性。发现 VIL2-1541/-706 具有启动子活性。此外，当该片段在正向方向位于 VIL2 或 SV40（猿猴病毒 40）启动子的上游时，荧光素酶的表达水平显著增强。然而，当该片段在反向方向位于启动子的上游或正向或反向方向位于报告基因的下游时，这种转录增强消失了。在缺失实验中，我们在 VIL2-1541/-706 内发现了几个潜在的调控区域。当这些区域分别位于 VIL2 或 SV40 启动子的上游时，只有-1297/-1186 显著增强了这些启动子的活性。尽管其他调控区域在缺失实验中表现出显著的转录调控，但它们微弱地增强了 VIL2 启动子的活性，并且/或者不调节 SV40 启动子的活性。这些结果表明，VIL2 启动子上游的 DNA 序列以位置和方向依赖的方式作为增强子发挥作用，而作为关键增强子的 VIL2-1297/-1186 可能与位于 VIL2 启动子上游的其他潜在调控区域一起调节 VIL2 转录。
