CRCNS-Bioinformatics & ident.:cis-elements: DA receptors

描述（由申请人提供）：多巴胺是中枢神经系统中的一种重要神经递质，对各种运动和情绪行为有深刻的影响。多巴胺能功能障碍与几种主要的神经精神疾病有关，从药物成瘾到精神分裂症再到帕金森病。多巴胺功能的分子研究揭示了一组多巴胺调节基因（DRGs），这些基因对纹状体独特的神经化学和行为特性起着至关重要的作用。这个建议的中心假设是，这些DRG是共同的，但复杂的一套顺式元件和转录因子的共同调节。我们的主要目标是确定和验证集群的顺式元件的DRG表达（CEDRG）使用集成的分子和生物信息学方法。 具体目标1：我们将采用一套生物信息学和分子分析工具，尽可能充分地表征DRG的基因组组织，特别是努力确定DRG的TSS。我们将把DRG的所有基因组信息整合到一个可通过网络访问的数据库DopamineDB中，这将为多巴胺神经生物学的研究提供一个独特的资源。 具体目标二：在对人类和小鼠DRG进行系统发育分析以揭示其启动子内的进化保守区域之后，我们将采用一系列基于统计模型的算法（三叶草[1]和Glam [2]）来鉴定统计学上过度代表的多巴胺调节基因表达（CEDRG）顺式元件。我们将系统地评估预测已知的和新的顺式元件结合活性的ChIP芯片分析和凝胶迁移试验，分别在推定的近端DRG启动子。具体目标3：我们将通过检测统计学上显著的CEDRG簇，并通过测定纹状体克隆细胞系（ST14A）中CEDRG簇的转录活性来确定CEDRG的功能相互作用。此外，我们将采用“安全港”转基因策略来评估转基因小鼠中鉴定的CEDRG的体内功能。最后，我们将检查DRG表达在小鼠缺乏相应的CEDRG转录因子，最终确定他们参与DRG表达。分子和生物信息学分析被整合在整个项目中，以克服每种技术的主要局限性。来自DRG的系统分析的信息将提供关键的见解多巴胺的功能，并确定新的多巴胺调节的转录因子，从而大大促进多巴胺相关的神经精神疾病，如药物成瘾的新的治疗策略的发展。

DESCRIPTION (provided by applicant): Dopamine is an important neurotransmitter in CNS and contributes profoundly to a variety of motor and emotional behaviors. Dopaminergic dysfunction has been associated with several major neuropsychiatric disorders, ranging from drug addiction to schizophrenia to Parkinson's disease. Molecular studies of dopamine function have revealed a set of Dopamine-Regulated Genes (DRGs), which contribute critically to the unique neurochemical and behavioral properties of the striatum. The central hypothesis of this proposal is that these DRGs are co-regulated by a common but complex set of cis-elements and transcription factors. Our primary goal is to identify and validate the clusters of cis-elements for DRG expression (CEDRG) using integrated molecular and bioinformatics approaches. Specific Aim 1: We will employ a set of bioinformatics and molecular analysis tools to characterize as fully as possible the genomic organization of the DRGs, with particular effort to determine the TSSs of DRG. We will integrate all genomic information for DRGs into a Web-accessible database, DopamineDB, which will provide a unique resource for investigation of dopamine neurobiology. Specific Aim 2: Following phylogenetic analysis of human and mouse DRGs to reveal evolutionally conserved regions within their promoters, we will employ a range of statistical model-based algorithms (Clover [1]) and Glam [2]) to identify statistically over-represented cis-Elements for Dopamine-Regulated Gene expression (CEDRG). We will systematically evaluate the predicted known and novel cis-element binding activity by ChIP-chip analysis and gel shift assay, respectively, in the putative proximal DRG promoters. Specific Aim 3: We will determine functional interactions of CEDRGs by detecting statistically significant CEDRG clusters, and by assaying transcription activity of CEDRG clusters in a striatal cloned cell line (ST14A). Furthermore, we will employ a "safe-haven" transgenic strategy to evaluate in vivo function of identified CEDRG in transgenic mice. Finally, we will examine DRG expression in mice deficient in transcription factors corresponding to the CEDRG to conclusively determine their involvement in DRG expression. The molecular and bioinformatics analyses are integrated throughout the project to overcome major limitations of each individual technique. The information derived from systematic analyses of DRGs will provide critical insights into dopamine functions and identify novel dopamine-regulated transcription factors and thus greatly facilitate the development of novel treatment strategies for dopamine-associated neuropsychiatric disorders such as drug addiction.