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，对患者和社会造成巨大负担。生物学因素有很强的牵连，大规模的全基因组关联研究现在已经集中在几个位点，这表明精神分裂症的第一个有效基因已经被确定。可以说，最特殊的经验证据是TCF 4（转录因子4），这是一个基因，在迄今为止最大的精神分裂症遗传研究中一直排在最前面。至关重要的是，我们的研究小组最近在一个大的以家庭为基础的样本中复制了TCF 4相关性（6，298个个体，包括3，286个病例，来自1，811个核心家庭）。基于家族的设计排除了由于人群分层而产生伪影的可能性。此外，TCF 4与精神分裂症内表型相关，与其他已知的精神分裂症基因在生物学上相互作用，并表现出迷人的生物相容性。 现在是时候进一步描述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TCF 4编码的蛋白质是碱性螺旋-环-螺旋转录因子，已知其识别Ephrussi盒（“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TCF 4的结合位点，并且还没有实证研究在全基因组水平上绘制TCF 4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，在一个称为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与对感兴趣的蛋白质特异性的抗体共沉淀，纯化并在NGS仪器上测序。数以百万计的序列标签被生成并映射回参考基因组，从而允许确定与蛋白质结合的基因组区域。由于ChIP-seq在过去5年中的快速发展，用于定位转录因子结合位点的实验室和计算方法得到了很好的发展。 一旦我们获得了TCF 4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，以验证我们的实验结果。然后，我们将通过检查11，185例病例和10，768例对照的GWAS荟萃分析数据，以及我们的全甲基化病例对照研究（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，来测试TCF 4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TCF 4进行彻底的调查，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。所有生成的数据将存放在适当的公共存储库中。 公共卫生相关性：大规模遗传学研究最终集中在精神分裂症的特定风险基因上，而TCF 4可以说是显示出最好支持证据的基因。TCF 4参与了其他基因的调控，因此在这里我们提出了尖端的DNA测序技术来发现TCF 4控制下的基因网络。然后，我们将在精神分裂症病例和对照组的多个生物数据集中研究这种遗传网络，以促进我们对TCF 4如何影响这种疾病风险的理解。

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. PUBLIC HEALTH RELEVANCE: Large-scale genetics studies are finally converging on specific risk genes for schizophrenia, and TCF4 is arguably the gene displaying the best supporting evidence of all. TCF4 is involved in the regulation of other genes, so here we propose cutting- edge DNA sequencing technologies to discover the network of genes under TCF4 control. We will then study this genetic network in multiple biological datasets of schizophrenia cases and controls, in order to advance our understanding of how TCF4 may affect risk for the disorder.