Twyman R M, Jones E A
Department of Biological Sciences, University of Warwick, Coventry, UK.
J Mol Neurosci. 1997 Feb;8(1):63-73. doi: 10.1007/BF02736864.
We investigated the regulation of the rat neuron-specific enolase gene using a transient transfection approach. Recent transgenic mouse studies have shown that a 1.8-kb segment of the rat NSE gene 5' flanking region, including the first (noncoding) exon but not the first intron, is able to drive expression of a reporter gene in parallel with endogenous NSE. These data suggest that cis-acting elements responsible for the spatial and temporal pattern of NSE gene expression are located within the proximal 1.8 kb of the 5' flanking sequence. To further investigate this region, we joined the 1.8-kb regulatory cassette to the cat reporter gene and generated a number of constructs in which the flanking sequence was progressively deleted from the 5' end. These constructs were tested by transient transfection into neuronal and nonneuronal cells, followed by an assay for CAT activity. We found that as little as 255 bp of 5' flanking sequence was able to confer cell type-specificity on the reporter gene. Further truncation to 120 bp of 5' sequence resulted in a sharp downregulation of reporter activity in PC12 cells but a significant rise in both Neuro-2A neuroblastoma cells and nonneuronal Ltk- cells, indicating that cis-acting elements controlling the regulation of NSE in Ltk-, Neuro-2A, and PC12 cells may lie within the 135 bp region covered by this deletion. This region contains an AP-2 site and an element similar in sequence and position to a motif identified in the proximal promoter region of the neuron-specific peripherin gene. Reduction to 95 bp of 5' sequence resulted in a slight downregulation of CAT activity in all cell lines tested, and further truncation to 65 bp of 5' sequence caused a universal reduction to background levels of CAT activity, concomitant with the disruption of the basal NSE promoter. Our results show that the 5' flanking region of the NSE gene is capable of conferring cell type-specificity on a heterologous gene in transfected cells and that elements responsible for this are located within the proximal 255 bp.
我们采用瞬时转染方法研究了大鼠神经元特异性烯醇化酶基因的调控。最近的转基因小鼠研究表明，大鼠NSE基因5'侧翼区的一个1.8kb片段，包括第一个（非编码）外显子但不包括第一个内含子，能够驱动报告基因与内源性NSE平行表达。这些数据表明，负责NSE基因表达时空模式的顺式作用元件位于5'侧翼序列的近端1.8kb内。为了进一步研究该区域，我们将1.8kb调控盒与猫报告基因连接，并构建了一些构建体，其中侧翼序列从5'端逐渐缺失。通过将这些构建体瞬时转染到神经元和非神经元细胞中，然后检测CAT活性来进行测试。我们发现，仅5'侧翼序列的255bp就能赋予报告基因细胞类型特异性。将5'序列进一步截短至120bp导致PC12细胞中报告基因活性急剧下调，但在Neuro-2A神经母细胞瘤细胞和非神经元Ltk-细胞中均显著升高，表明控制Ltk-、Neuro-2A和PC12细胞中NSE调控的顺式作用元件可能位于该缺失覆盖的135bp区域内。该区域包含一个AP-2位点和一个在序列和位置上与在神经元特异性外周蛋白基因近端启动子区域鉴定的基序相似的元件。将5'序列减少到95bp导致所有测试细胞系中CAT活性略有下调，进一步将5'序列截短到65bp导致CAT活性普遍降低到背景水平，同时基础NSE启动子被破坏。我们的结果表明，NSE基因的5'侧翼区能够在转染细胞中赋予异源基因细胞类型特异性，并且负责此功能的元件位于近端255bp内。
