Enhancer-based Immune and Beta Cell Dysregulation Underlying T1D Risk

摘要 1型糖尿病(T1D)是一种器官特异性自身免疫性疾病，通过免疫细胞介导和 炎性细胞因子导致胰腺中产生胰岛素的β细胞的丧失。全基因组关联 研究已经确定了大约60个与T1D风险相关的基因组区域。GWA的绝大多数风险 与T1D相关的变异驻留在非编码区，特别是增强子，这表明基因 法规的变化在很大程度上导致了个体之间对T1D的易感性的差异。由我们的 T1D GWAS注释数据，强调了对免疫和β进行系统分析的重要性 T1D患者和对照的细胞系统，我们建议识别因果增强子变体和因果 使用当代计算方法和尖端全球基因组学来靶向基因。我们将表演我们的 PRO-CAP和PRO-SEQ检测以全面识别含有T1D相关变体的活性增强子 在来自T1D患者和对照组的T细胞、单核细胞和干细胞来源的β细胞(sc-β细胞)中，其次是 使用我们的eSTARR-seq分析验证含有T1D相关变体的活性增强子(目标1)。我们 将执行我们的三重分析，以前所未有的高分辨率描述增强子-启动子相互作用 来自T1D患者和对照组以及胰岛的原代T细胞、原代单核细胞和sc-β细胞 全面鉴定T1D相关变异体的靶基因，提炼T1D相关的靶基因 利用我们的耦合单细胞在每种细胞类型中具有T1D特异性改变的靶基因表达的变体 核(SN)RNA-SEQ和SnATAC-SEQ分析(目标2)。我们将验证T1D相关变体的目标 使用CRISPR/Cas9内源性增强子突变策略的T细胞、单核细胞和β细胞 每种细胞类型的内源性染色质景观。我们的分析和实验框架代表了 一种令人兴奋的研究T1D的新范式，以及基于我们的结果的后续临床研究具有 开发预防和治疗T1D策略的潜力。这些研究产生的数据集 将为T1D研究界提供重要但目前缺乏的资源。

Abstract Type 1 diabetes (T1D) is an organ-specific autoimmune disease, whereby immune cell-mediated and inflammatory cytokines lead to loss of the insulin-producing β cells in the pancreas. Genome-wide association studies (GWAS) have identified ~60 genomic regions associated with T1D risk. The vast majority of GWAS risk variants associated with T1D reside in non-coding regions, particularly enhancers, suggesting that gene regulatory changes substantially contribute to inter-individual differences in susceptibility to T1D. Driven by our T1D GWAS annotation data, highlighting the importance of a systematic analysis of both immune and β cell systems of both T1D patients and controls, we propose to identify causal enhancer variants and causal target genes using contemporary computational methods and cutting-edge global genomics. We will perform our PRO-cap and PRO-seq assays to comprehensively identify active enhancers harboring T1D-associated variants in T cells, monocytes and stem cell derived β cells (sc-β cells) from T1D patients and controls, followed by validation of active enhancers harboring T1D-associated variants using our eSTARR-seq assays (Aim 1). We will perform our Tri-HiC assays to profile the enhancer-promoter interactomes at unprecedented high resolution in primary T cells, primary monocytes and sc-β cells from T1D patients and controls as well as pancreatic islets to comprehensively identify target genes of T1D-associated variants, and refine targets of T1D-associated variants with T1D-sepcific alteration in target gene expression in each cell type using our coupled single cell nucleus (sn) RNA-seq and snATAC-seq assays (Aim 2). We will validate targets of T1D-associated variants in T cells, monocytes and β cells using CRISPR/Cas9 endogenous enhancer mutational strategies in the context of the endogenous chromatin landscape in each cell type. Our analytical and experimental framework represents an exciting new paradigm for studying T1D, and the subsequent clinical research based on our results has the potential to develop preventive and therapeutic strategies against T1D. The data sets generated by these studies will represent important, but currently lacking, resources for the T1D research community.