REGULATION OF NEURONAL NICOTINIC ACETYLCHOLINE RECEPTOR

我们的总体长期目标是理解转录 控制神经系统内细胞类型多样性的机制。至 为了实现这一目标，我们正在研究顺式和反式转录 神经烟碱受体编码基因的控制。这些基因编码 可以组装成各种不同功能的亚基 异构体兴奋性配体门控离子通道。的表达模式 这些基因表明不同的异构体在肾上腺中表达。 嗜铬细胞、外周神经节、视网膜和整个大脑。 我们研究的重点是一组nAchR基因，Beta4，alpha3和 编码组装成单个受体亚型的亚基的字母5 神经节，可能还有中枢神经元。推动我们的 研究是如何协调控制该集群的成员 为异构体产生必要的重叠表达模式 集合？共同表达和集群组织表明，这些 基因受共享的顺式元件的控制。但是，表达式 这些基因的模式并不完全一致，因此 该簇中的单个基因很可能由独立的 顺元素也是如此。我们已经确定了邻近的独立发起人 α3和β4基因以及潜在的增强子 β4/α3基因间隔区。我们现在的兴趣是调查这些 表达nAchR的PC12细胞中的顺式元件确定其功能 在转基因动物中确定它们在神经细胞中的作用 这些nAchR基因的特异性表达。我们还发现了反式- 调节α3和β4启动子活性的作用因子。锌- 手指蛋白Sp1或Sp1相关因子反式激活α3 通过位于Alpha3附近的富含G+A基序的启动子 转录起始位点区域。SP1属于一种差分 表达的基因家族，因此一个目标是鉴定Sp1家族 在PC12细胞中表达的成员并评估它们在 NAchR转录。对于这种煤，我们发现Sp1与 在PC12细胞中，SP4与Sp1共表达。因此，我们将 研究第二个锌指在脑内的表达和功能。 NAchR基因转录。我们还将把这些研究扩展到测试版4 启动子，以确定α3和β4是否协调 由这些蛋白质控制。我们发现了一个POU-域 转录因子SCIP/Tst-1/Oct-6是一种有效的、特异的激活剂 字母3。这代表了第一个被发现的细胞基因 受到SCIP的正向调制，这增加了SCIP 控制神经元的胆碱感受型。我们最近对PC12的研究 细胞表明，SCIP对Alpha3的调节是细胞类型特异性的，并且 表明激活是通过一种新的机制发生的。我们感兴趣的是 用PC12细胞作为神经模型研究其潜能 SCIP对α3和其他启动子作用的替代机制。 综合起来，拟议的工作将导致更好地理解如何 胆碱能递质系统的建立，更普遍地将有助于 提供神经元中基因表达控制的清晰视图。 最终，这些研究可能会为未来奠定基础 异常基因控制在特异体细胞中的作用研究 神经紊乱。

Our general long-term objective is to understand the transcriptional mechanisms that control cell-type diversity within the nervous system. To pursue this objective we are investigating cis and trans transcriptional control of genes encoding neuronal nicotinic receptors. These genes encode subunits that can be assembled into a variety of functionally distinct heteromeric excitatory ligand-gated ion channels. Expression patterns of these genes indicates that different heteromers are expressed in adrenal chromaffin cells, peripheral ganglia, retina, and throughout the brain. The focus of our research is a cluster of nAchR genes, beta4, alpha3, and alpha5 that encode subunits assembled into a single receptor subtype in ganglia and possibly central neurons. The basic question driving our research is how are members of this cluster coordinately controlled to generate requisite overlapping patterns of expression for heteromer assembly? Coexpression and the clustered organization suggest that these genes are subject to control by shared cis elements. However, expression patterns of these genes are not entirely concordant and therefore individual genes in the cluster are likely to be controlled by independent cis elements as well. We have identified independent promoters adjacent to the alpha3 and beta4 genes as well as a potential enhancer within the beta4/alpha3 intergenic region. Our interest now is to investigate these cis elements in nAchR expressing PC12 cells to define their functional properties and in transgenic animal to determine their role in neural- specific expression of these nAchR genes. We have also identified trans- acting factors that modulate alpha3 and beta4 promoter activity. The zinc- finger protein Sp1 or an Sp1-related factor transactivates the alpha3 promoter via a G+A-rich motif positioned adjacent to the alpha3 transcription start site region. Sp1 belongs to a differentially expressed gene family and therefore one goal is to identify Sp1 family members that are expressed in PC12 cells and to assess their function in nAchR transcription. Toward this coal we have found that the Sp1-related factor, Sp4, is coexpressed with Sp1 in PC12 cells. Thus we will investigate the expression and function of this second zinc-finger in nAchR gene transcription. We will also extend these studies to the beta4 promoter in order to determine whether alpha3 and beta4 are coordinately controlled by these proteins. We have discovered that a POU-domain transcription factor, SCIP/Tst-1/Oct-6, is a potent and specific activator of alpha3. This represents the first cellular gene identified that is positively modulated by SCIP and it raises the possibility that SCIP controls cholinoceptive phenotype in neurons. Our recent studies in PC12 cells indicate that alpha3 regulation by SCIP is cell-type specific and suggest that activation occurs via a novel mechanism. We are interested in using PC12 cells as a neural model to investigate the potential alternative mechanism of SCIP action on alpha3 and other promoters. Together the proposed work will lead to a better understanding of how cholinergic transmitter systems are built and more generally will help to provide a clear view of the control of gene expression in neurons. Ultimately, these studies are likely to establish a foundation for future investigations of the role of aberrant gene control in specific neurological disorders.