Bioinformatics & identification of cis elements for dopamine gene expression

描述(申请人提供)：多巴胺是中枢神经系统中一种重要的神经递质，对各种运动和情绪行为有深远的影响。多巴胺能功能障碍与几种主要的神经精神障碍有关，从药物成瘾到精神分裂症再到帕金森病。对多巴胺功能的分子研究揭示了一组多巴胺调节基因(DRGs)，它们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。这一提议的中心假设是，这些DRG受到一组共同但复杂的顺式元件和转录因子的共同调控。我们的主要目标是使用整合的分子和生物信息学方法识别和验证DRG表达的顺式元件簇(CEDRG)。 具体目标1：我们将使用一套生物信息学和分子分析工具来尽可能全面地描述DRG的基因组组织，特别是努力确定DRG的TSS。我们将把DRGs的所有基因组信息整合到一个网络可访问的数据库--多巴胺数据库中，该数据库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2：在对人和鼠DRG进行系统发育分析以揭示其启动子内进化保守的区域之后，我们将使用一系列基于统计模型的算法(CLOVER[1]和GLAM[2])来识别统计上过度代表的多巴胺调节基因表达(CEDRG)的顺式元件。我们将分别通过芯片分析和凝胶漂移分析，系统地评估预测的已知和新的顺式元件结合活性，在假定的近端DRG启动子中。具体目标3：我们将通过检测具有统计学意义的CEDRG簇，以及通过检测纹状体克隆细胞系(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“避风港”转基因策略来评估已识别的CEDRG在转基因小鼠体内的功能。最后，我们将检测缺乏与CEDRG对应的转录因子的小鼠的DRG表达，以最终确定它们参与了DRG的表达。分子和生物信息学分析被整合到整个项目中，以克服每种单独技术的主要限制。从DRGs的系统分析中获得的信息将提供对多巴胺功能的关键见解，并识别新的多巴胺调节的转录因子，从而极大地促进药物成瘾等多巴胺相关神经精神障碍的新治疗策略的发展。

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.