Hahm S H, Eiden L E
Section on Molecular Neuroscience, National Institutes of Health, Bethesda, Maryland 20892, USA.
Ann N Y Acad Sci. 1998 Dec 11;865:10-26. doi: 10.1111/j.1749-6632.1998.tb11158.x.
The cis-acting elements of the VIP gene important for basal and stimulated transcription have been studied by transfection of VIP-reporter gene constructs into distinct human neuroblastoma cell lines in which VIP transcription is constitutively high, or can be induced to high levels by protein kinase stimulation. The 5.2 kb flanking sequence of the VIP gene conferring correct basal and inducible VIP gene expression onto a reporter gene in these cell lines was systematically deleted to define its minimal components. A 425-bp fragment (-4656 to -4231) fused to the proximal 1.55 kb of the VIP promoter-enhancer was absolutely required for cell-specific basal and inducible transcription. Four additional components of the VIP gene were required for full cell-specific expression driven by the 425 bp TSE (region A). Sequences from -1.55 to -1.37 (region B), -1.37 to -1.28 (region C), -1.28 to -.094 (region D), and the CRE-containing proximal 94 bp (region E) were deleted in various combinations to demonstrate the specific contributions of each region to correct basal and inducible VIP gene expression. Deletion of region B, or mutational inactivation of the CRE in region E, resulted in constructs with low transcriptional activity in VIP-expressing cell lines. Deletion of regions B and C together resulted in a gain of transcriptional activity, but without cell specificity. All five domains of the VIP gene were also required for cell-specific induction of VIP gene expression with phorbol ester. Gelshift analysis of putative regulatory sequences in regions A-D suggests that both ubiquitous and neuron-specific trans-acting proteins participate in VIP gene regulation.
通过将VIP报告基因构建体转染到不同的人神经母细胞瘤细胞系中，对VIP基因中对基础转录和刺激转录很重要的顺式作用元件进行了研究。在这些细胞系中，VIP转录本底水平较高，或者可通过蛋白激酶刺激诱导至高水平。系统地删除了VIP基因的5.2 kb侧翼序列，该序列可在这些细胞系中赋予报告基因正确的基础和可诱导的VIP基因表达，以确定其最小组成部分。一个425 bp的片段（-4656至-4231）与VIP启动子增强子的近端1.55 kb融合，是细胞特异性基础转录和诱导转录绝对必需的。由425 bp TSE（区域A）驱动的全细胞特异性表达还需要VIP基因的另外四个组成部分。以各种组合删除了从-1.55至-1.37（区域B）、-1.37至-1.28（区域C）、-1.28至-0.94（区域D）以及含CRE的近端94 bp（区域E）的序列，以证明每个区域对正确的基础和可诱导VIP基因表达的具体贡献。删除区域B或使区域E中的CRE发生突变失活，导致构建体在表达VIP的细胞系中具有低转录活性。一起删除区域B和C导致转录活性增加，但没有细胞特异性。VIP基因的所有五个结构域对于佛波酯对VIP基因表达的细胞特异性诱导也是必需的。对区域A-D中假定调控序列的凝胶迁移分析表明，普遍存在的和神经元特异性的反式作用蛋白都参与了VIP基因的调控。
