Genetic mechanisms of type 1 diabetes risk in stress-induced pancreatic islets

项目总结/摘要 1型糖尿病（T1 D）的特征是胰腺中产生胰岛素的β细胞的自身免疫性破坏。 小岛虽然T1 D风险机制的研究主要集中在免疫细胞功能上，但最近的证据表明， 表明β细胞本身积极参与疾病过程。β细胞暴露于不同的 T1 D发展过程中的环境刺激和应激源，如促炎细胞因子， 高血糖症可导致β细胞应激和死亡。然而，T1 D风险变异的程度 影响β细胞表观基因组，并且响应于这些外部信号的基因调控是未知的。获得 更深入地了解影响β细胞功能和T1 D生存的变异、基因和途径 在病理生理学中，在T1 D相关免疫调节的背景下绘制β细胞基因调控的变化是至关重要的。 和代谢应激源。我们已经从原发性胰岛样本中生成了染色质可及性图谱 暴露于T1 D相关的细胞因子，并确定了数千个精氨酸反应位点和转录 因素将这些数据与T1 D遗传精细图谱相结合，然后揭示了具有细胞因子的T1 D风险变体。 对胰岛染色质可及性的依赖性影响。拟议的项目将在这些研究结果的基础上， 人类遗传学，胰岛表观基因组学和基因组工程，以绘制影响β细胞的T1 D风险变体 染色质在体外暴露于多种T1 D相关的应激源，并确定应激诱导的靶基因 T1 D变体影响β细胞ER应激和存活。为了实现这一点，在目标1中，我们将生成 β细胞染色质可及性和转录因子结合的变化的综合图， 暴露于多种与T1 D相关的应激源。使用这些数据，我们将精细映射T1 D风险变体， 利用QTL定位对β细胞染色质胁迫诱导效应并利用报告基因验证其等位基因效应 测定。在目标2中，我们将通过产生和分析应激诱导的T1 D变体来鉴定靶基因。 暴露于相同应激源后β细胞基因表达和3D染色质结构的变化，以及 然后使用CRISPRi调控筛选验证胁迫诱导位点的靶基因。最后，在目标3中， 鉴定直接调节β细胞ER应激和存活表型的T1 D风险变体的靶基因 使用全基因组CRISPR介导的功能丧失筛选。这些基因的细胞表型将 在hiPSC衍生的β细胞中使用CRISPR介导的基因缺失进行验证。我们的研究结果将 为β细胞在T1 D病理生理学中的内在作用提供新的见解，并为治疗提供信息。 通过靶向发现参与β细胞应激反应和存活的T1 D风险基因进行干预。

PROJECT SUMMARY/ABSTRACT Type 1 diabetes (T1D) is characterized by autoimmune destruction of insulin-producing beta cells in pancreatic islets. While studies of T1D risk mechanisms have largely focused on immune cell function, recent evidence suggests the beta cells themselves actively contribute to the disease process. Beta cells are exposed to different environmental stimuli and stressors in the course of T1D development, such as pro-inflammatory cytokines and hyperglycemia which can contribute to beta cell stress and death. However, the extent to which T1D risk variants affect the beta cell epigenome and gene regulation in response to these external signals is unknown. To gain a deeper understanding of the variants, genes, and pathways that impact beta cell function and survival in T1D pathophysiology, it is critical to map changes in beta cell gene regulation the context of T1D-relevant immune and metabolic stressors. We have generated chromatin accessibility maps from primary pancreatic islet samples exposed to T1D-relevant cytokines and identified thousands of cytokine-responsive sites and transcription factors. Integrating these data with T1D genetic fine-mapping then revealed T1D risk variants with cytokine- dependent effects on islet chromatin accessibility. The proposed project will build on these findings in combining human genetics, islet epigenomics, and genome engineering to map T1D risk variants that affect beta cell chromatin upon in vitro exposure to multiple T1D-relevant stressors and identify target genes of stress-induced T1D variant effects that impact beta cell ER stress and survival. To accomplish this, in Aim 1 we will generate comprehensive maps of changes in beta cell chromatin accessibility and transcription factor binding upon exposure to multiple T1D-relevant stressors. Using these data, we will then fine-map T1D risk variants with stress-induced effects on beta cell chromatin using QTL mapping and validate their allelic effects using reporter assays. In Aim 2, we will identify target genes of stress-induced T1D variants by generating and analyzing changes in beta cell gene expression and 3D chromatin architecture upon exposure to the same stressors, and then validate target genes of stress-induced sites using a CRISPRi regulatory screen. Finally, in Aim 3 we will identify target genes of T1D risk variants that directly modulate beta cell ER stress and survival phenotypes using genome-wide CRISPR-mediated loss-of-function screens. The cellular phenotype of these genes will then be validated using CRISPR-mediated gene deletions in hiPSC-derived beta cells. Together our findings will provide novel insight into the intrinsic role of beta cells in T1D pathophysiology and inform therapeutic intervention through target discovery of T1D risk genes involved in beta cell stress response and survival.