Systematic Mapping of the Functional Common Noncoding Variants in the TNFAIP3 Locus

项目摘要/摘要 诱导自身免疫的机制仍然是个谜，部分原因是许多未经证实的活动。 自身免疫相关的非编码常见遗传变异。来剖析所有这些问题的具体机制 常见的和疾病相关的非编码变异调节基因表达，我建议使用可伸缩的 三种细胞类型的检测将评估2708个非编码常见变异体的基因调制功能 包含负调控基因TNFAIP3/A20的重要自身免疫相关基因。一次化验就能 检测~2700个突变体在刺激前后对报告基因表达的影响。第二次化验将评估 基因组中约43,000个系统性基因座范围的缺失，以确定调节区(和潜在的活跃区域 内部变体)在细胞类型中。然后我将从这些检测中将活跃的疾病相关变体映射到开放的 自身免疫患者细胞类型的染色质，以创建那些可能在疾病中起作用的细胞的排序列表。这就做 然后通过遗传方法确定这些活性的、开放的染色质局部变异体的作用机制 使用CRISPR-Cas9同源定向修复和执行生化操作的工程风险和非风险等位基因 化验。该方法在今后的工作中可以扩展到多个基因座的研究，这将有助于我们对基因座的认识 疾病基因网络、准确的动物疾病模型和更有效的治疗方法。

Project Summary/Abstract The mechanisms for induction of autoimmunity remain enigmatic, in part due to the unconfirmed activity of many autoimmune-associated noncoding common genetic variants. To dissect the specific mechanisms of how all common and disease-associated noncoding variation modulates gene expression, I propose to employ scalable assays in three cell types that will assess 2708 noncoding common variants for gene-modulatory function in an important autoimmune-associated locus containing the negative regulatory gene TNFAIP3/A20. One assay will test ~2700 variants for their effects on reporter expression pre- and post-stimulation. A second assay will evaluate ~43,000 systematic locus-wide deletions in the genome to determine regulatory regions (and potentially active variants within) in cell types. I will then map the active disease-associated variants from these assays to the open chromatin of autoimmune patient cell types to create a ranked list of those that putatively operate in disease. I will then determine the mechanism of action for these active, open chromatin localized variants through genetically engineering risk and non-risk alleles using CRISPR-Cas9 homology directed repair and performing biochemical assays. This method is scalable to study many loci in future work, which will contribute to our knowledge of disease gene networks, accurate animal disease models, and more efficacious therapeutics.