Functional characterization of pathways regulated by schizophrenia gene TCF4

描述（由申请人提供）：精神分裂症是痛苦的主要原因，是患者和社会的巨大负担。生物因素与此密切相关，大规模的全基因组关联研究现在已经集中在几个位点上，这表明第一个有效的精神分裂症基因已经被确定。可以说，最特殊的经验证据是TCF4（转录因子4），这个基因在迄今为止最大规模的精神分裂症基因研究中一直名列前茅。至关重要的是，我们的团队最近在一个以家庭为基础的大型样本中复制了TCF4的关联（来自1811个核心家庭的6298个人，包括3286例病例）。基于家庭的设计排除了由于人口分层而产生人工制品的可能性。此外，TCF4与精神分裂症内表型相关，与其他已知的精神分裂症基因相互作用，并显示出令人着迷的生物学合理性。现在是时候进一步表征TCF的功能及其参与的途径，以更好地了解精神分裂症的发病机制。由TCF4编码的蛋白质是一种基本的螺旋-环-螺旋转录因子，已知可识别ephrus -box ('E-box') DNA结合位点（'CANNTG'）。该基序太小且非特异性，无法通过计算确定TCF4的结合位点，目前还没有实证研究在全基因组水平上绘制TCF4的结合位点。获得这张地图是我们的主要实验目标。研究蛋白质- dna结合的主要工具是染色质免疫沉淀（ChIP）。最近，该技术已与下一代测序（NGS）相结合，在称为ChIP-seq的程序中，在全基因组水平上发现蛋白质结合位点。在ChIP-seq中，蛋白质结合的DNA与感兴趣的蛋白质特异性抗体共沉淀，在NGS仪器上纯化和测序。生成数百万个序列标签并映射回参考基因组，从而确定与蛋白质结合的基因组区域。由于近5年来ChIP-seq的快速发展，用于转录因子结合位点定位的实验室和计算方法得到了很好的发展。一旦我们获得了tcf4结合位点的图谱，我们将在全基因组基因表达研究中寻找共调控的证据，以验证我们的实验发现。然后，我们将检验TCF4基因网络与精神分裂症的关联，通过检查GWAS荟萃分析数据，包括11185例病例和10768例对照，以及我们的甲基组范围病例对照研究（750例病例，750例对照），以及精神分裂症病例和对照脑组织的死后基因表达研究。我们的团队非常适合使用这些方法对TCF4进行彻底的调查，因为我们在精神病学遗传学和我们自己的NGS设备方面有创新的历史。所有生成的数据都将存储在适当的公共存储库中。

DESCRIPTION (provided by applicant): Schizophrenia is a major cause of suffering and an enormous burden to patients and society. Biological factors have been strongly implicated and large-scale genome-wide association studies have now converged on several loci, suggesting that the first valid genes for schizophrenia have been identified. Arguably, the most exceptional empirical evidence is for TCF4 (transcription factor 4), a gene that consistently ranked among the top findings in the largest schizophrenia genetic studies to date. Crucially, our group recentl replicated the TCF4 association in a large family-based sample (6,298 individuals, including 3,286 cases, from 1,811 nuclear families). Family-based designs preclude the possibility of artifacts due to population stratification. Additionally, TCF4 is associated with schizophrenia endophenotypes, interacts biologically with other known schizophrenia genes and demonstrates fascinating biological plausibility. The time is right to further characterize the function of TCF and the pathways in which it participates, to obtain better insight into schizophrenia pathogenesis. The protein encoded by TCF4 is a basic helix-loop-helix transcription factor, known to recognize an Ephrussi-box ('E-box') DNA binding site ('CANNTG'). This motif is too small and non-specific to identify the binding sites of TCF4 computationally, and no empirical study has yet mapped TCF4 binding sites on a genome-wide level. Obtaining this map is our primary experimental aim. The main tool for investigating protein-DNA binding is chromatin immunoprecipitation (ChIP). Recently, this technique has been coupled with next generation sequencing (NGS), in a procedure known as ChIP-seq, to discover protein binding sites on a genome-wide level. In ChIP-seq, protein-bound DNA is co-precipitated with an antibody specific to the protein of interest, purified and sequenced on an NGS instrument. Millions of sequence tags are generated and mapped back to a reference genome, allowing determination of the genomic regions bound to the protein. Owing to the rapid progress of in ChIP-seq over the last 5 years, the laboratory and computational methods used in mapping transcription factor binding sites are well worked out. Once we have obtained the map of TCF4-binding sites, we will look for evidence of co-regulation in genome-wide gene expression studies, to validate our experimental findings. We will then test for association of the TCF4 gene network with schizophrenia, by examining GWAS meta-analysis data on 11,185 cases plus 10,768 controls, in addition to our methylome-wide case-control study (750 cases, 750 controls), and post- mortem gene expression studies of brain tissue from schizophrenia cases and controls. Our group is ideally suited to perform a thorough investigation of TCF4 using these methods, having a history of innovation in psychiatric genetics and our own NGS equipment. All generated data will be deposited in the appropriate public repository.