Functional characterization of pathways regulated by schizophrenia gene TCF4

描述(申请人提供)：精神分裂症是痛苦的主要原因，对患者和社会来说是一个巨大的负担。生物因素被强烈地牵连在一起，大规模的全基因组关联研究现在已经集中在几个基因位点上，这表明第一批有效的精神分裂症基因已经被识别出来。可以说，最特殊的经验证据是TCF4(转录因子4)，这个基因一直是迄今为止最大的精神分裂症基因研究中最重要的发现之一。至关重要的是，我们小组最近在一个以家庭为基础的大样本中复制了TCF4相关性(来自1,811个核心家庭的6,298个个体，包括3,286个病例)。以家庭为基础的设计排除了由于人口分层而产生文物的可能性。此外，TCF4与精神分裂症的内表型相关，与其他已知的精神分裂症基因在生物学上相互作用，并显示出迷人的生物学合理性。现在是进一步确定TCF的功能及其参与的途径，以更好地了解精神分裂症发病机制的时候了。TCF4编码的蛋白质是一个基本的螺旋-环-螺旋转录因子，已知识别E-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，而且还没有经验研究在全基因组水平上定位TCF4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，这项技术已经与下一代测序(NGS)相结合，在被称为芯片序列的程序中，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与目标蛋白质的特异性抗体共沉淀，纯化后在NGS仪器上进行测序。数以百万计的序列标签被生成并映射回参考基因组，从而能够确定与蛋白质结合的基因组区域。由于芯片内序列技术在过去5年中的快速发展，用于绘制转录因子结合位点的实验室和计算方法已经制定得很好了。一旦我们获得了TCF4结合位点的地图，我们将在全基因组基因表达研究中寻找协同调控的证据，以验证我们的实验发现。然后，我们将通过检查11,185例患者和10,768名对照的GWASMeta分析数据，以及我们的全基因组病例对照研究(750例，750名对照)，以及精神分裂症病例和对照的脑组织死后基因表达研究，来测试TCF4基因网络与精神分裂症的关联。我们的团队非常适合使用这些方法对TCF4进行彻底的调查，我们在精神病学遗传学和我们自己的NGS设备方面有着创新的历史。所有生成的数据都将存放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.