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

项目摘要/摘要 1型糖尿病(T1D)的特征是胰腺中产生胰岛素的β细胞受到自身免疫破坏 小岛。虽然对T1D风险机制的研究主要集中在免疫细胞功能上，但最近有证据表明 这表明β细胞本身在疾病过程中起着积极的作用。β细胞暴露在不同的环境中 T1D发育过程中的环境刺激和应激源，如促炎细胞因子和 高血糖可导致β细胞应激和死亡。然而，T1D风险变异的程度 影响β细胞表观基因组和基因调控对这些外部信号的反应尚不清楚。为了获得一份 对T1D中影响β细胞功能和存活的变异、基因和途径有更深入的了解 病理生理学，将β细胞基因调控的变化映射到T1D相关免疫的背景下是至关重要的 和代谢应激源。我们已经从原始的胰岛样本中生成了染色质可及性图 暴露于与T1D相关的细胞因子，并鉴定了数千个细胞因子反应位点和转录 各种因素。将这些数据与T1D遗传精细图谱结合起来，揭示了带有细胞因子的T1D风险变异。 对胰岛染色质可及性的依赖效应。拟议的项目将以这些调查结果为基础，结合 人类遗传学、胰岛表观基因组学和基因组工程，以定位影响β细胞的T1D风险变异 染色质在体外暴露于多种T1D相关应激源及识别应激诱导的靶基因 影响β细胞内质网应激和存活的T1D变化效应。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变化的综合图谱 暴露于多种与T1D相关的压力源。使用这些数据，然后我们将精细映射T1D风险变量 应激诱导的β细胞染色质QTL定位及其等位基因效应验证 化验。在目标2中，我们将通过产生和分析来识别应激诱导的T1D变体的靶基因 暴露在相同应激源下的β细胞基因表达和3D染色质结构的变化 然后使用CRISPRi调节筛选来验证应激诱导部位的靶基因。最后，在目标3中，我们将 确定直接调节β细胞内质网应激和存活表型的T1D风险变异体的靶基因 使用全基因组CRISPR介导的功能丧失筛查。然后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。我们的发现将结合在一起 对β细胞在T1D病理生理学中的内在作用提供新的见解并提供治疗信息 通过靶向发现参与β细胞应激反应和存活的T1D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.