Department of Neurophysiology & Neural Repair, Gunma University Graduate School of Medicine, Maebashi, Gunma, 371-8511, Japan.
Department of Ophthalmology, Gunma University Graduate School of Medicine, Maebashi, Gunma, 371-8511, Japan.
Cerebellum. 2017 Dec;16(5-6):913-922. doi: 10.1007/s12311-017-0866-5.
We investigated the neuron-specific enolase (NSE) promoter in terms of its promoter strength and neuronal specificity in the cerebellum in vivo. The 1.8 kb rat NSE promoter was divided into three regions, A (0.8 kb), B (0.7 kb), and C (0.3 kb), starting from the 5' side. Then, we made various deletion constructs and assessed them by virally expressing GFP under the control of one of the deleted promoters. Removing region A reduced GFP expression to ~6% of that of the original 1.8 kb promoter. Further deletion of region B (presence of region C alone) did not influence the promoter strength, but removing region B from the original 1.8 kb promoter reduced the GFP expression to ~6% of the original level, similar to the level observed after deletion of region A. Immunohistochemistry showed robust GFP expression in Purkinje cells and modest expression in interneurons by the original promoter. Removing region A and/or region B abolished the GFP expression in Purkinje cells in most cerebellar lobules, with the expression in interneurons almost unchanged. These results suggest that region C, which is a proximal 0.3 kb sequence, contains cis-acting elements that drive transcription predominantly in interneurons. The addition of either region A or B onto region C does not alter the promoter properties; however, the addition of both regions A and B to region C drastically enhanced the promoter activity in Purkinje cells, suggesting the synergistic action of cis-acting regulatory elements in regions A and B for strong activation in Purkinje cells.
我们研究了神经元特异性烯醇化酶（NSE）启动子在体内小脑的启动子强度和神经元特异性。从 5'侧开始，将 1.8kb 大鼠 NSE 启动子分为三个区域，A（0.8kb）、B（0.7kb）和 C（0.3kb）。然后，我们构建了各种缺失构建体，并通过病毒表达 GFP 来评估它们，GFP 由其中一个缺失启动子控制。去除区域 A 将 GFP 表达降低到原始 1.8kb 启动子的约 6%。进一步删除区域 B（仅存在区域 C）不会影响启动子强度，但从原始 1.8kb 启动子中删除区域 B 将 GFP 表达降低至原始水平的约 6%，与删除区域 A 后观察到的水平相似。免疫组织化学显示，原始启动子在浦肯野细胞中表现出强烈的 GFP 表达，在中间神经元中表现出适度的表达。去除区域 A 和/或区域 B 会使大多数小脑叶的浦肯野细胞中的 GFP 表达消失，而中间神经元的表达几乎不变。这些结果表明，近端 0.3kb 序列的区域 C 包含顺式作用元件，主要驱动中间神经元的转录。将区域 A 或 B 添加到区域 C 上不会改变启动子特性；然而，将区域 A 和 B 两者都添加到区域 C 上会极大地增强浦肯野细胞中的启动子活性，这表明区域 A 和 B 中的顺式作用调节元件在浦肯野细胞中具有强烈激活的协同作用。
