Dissecting the roles of type 2 diabetes-associated variants and effector genes in islet endoplasmic reticulum stress response

项目总结 2型糖尿病(T2D)是一种由遗传因素和环境因素共同引起的复杂疾病。全基因组 关联研究已在243个基因座(T2D变种)确定了403个增加T2D的关联信号 遗传风险。功能性(Epi)基因组分析强烈表明，非编码T2D变体改变了转录 胰岛的调控和靶基因表达，但只有20%的T2D变异体引起胰岛改变 在稳态条件下，顺式调节元件(Cre)的使用或基因表达。环境因素，如 内质网(ER)应激与胰岛功能障碍有关。然而，到目前为止的研究已经 未评估T2D变体是否或如何调节胰岛对内质网应激的反应。我假设这些T2D 变异体改变胰岛ER应激反应Cre的使用或活性和靶基因表达 T2D患者胰岛β细胞功能障碍或死亡。在我的初步数据分析中，我已经确定了内质网应激反应 在胰岛中重叠407个T2D变异体的CRES，并将这些CRE连接到22个可能的靶基因启动子 利用胰岛启动子捕获Hi-C图谱。在目标1中，我将全面评估T2D变体对 利用染色质可及性数量性状基因座(CaQTL)利用或激活内质网应激反应基因 和大规模平行报告分析(MPRA)。在目标2中，我将确定推定的CRE- 在人类胰岛细胞中，靶基因是其内质网应激反应和生存所必需的，通过改变其 使用CRISPR/Cas9表观基因组编辑平台进行表达。成功完成这些目标将产生 一组具有功能特征的T2D变异体和一组经过验证的下游T2D靶基因 调节胰岛功能和存活以应对中枢(病理)生理性应激源。更广泛地说，我 我预计我在这个机械化的从变量到功能的项目中采用的原则和框架可能是 应用于表征T2D变异在其他环境应激源和代谢背景下的影响 纸巾。重要的是，这个项目的完成将帮助我掌握当前的概念和最先进的技术 遗传学和功能基因组学技术，并通过以下途径提高我的科学交流技能 有广泛的机会介绍和发表我的研究成果。最后，我在康涅狄格州大学攻读医学博士的职位 健康和杰克逊基因组医学实验室不仅让我在这个世界上得到指导- 班级，高度协作的环境，但它将为我提供继续磨练我的临床的机会 在我的研究阶段中和之后，我获得了技能和专业经验。完成我的培训和发展 计划将是我未来职业生涯的关键一步，我将成为一名研究遗传学和(Epi)基因组的内科科学家。 患者中疾病相关变异的机制。

PROJECT SUMMARY Type 2 Diabetes (T2D) is a complex disease caused by both genetic and environmental factors. Genome-wide association studies (GWAS) have identified 403 association signals at 243 loci (T2D variants) that increase T2D genetic risk. Functional (epi)genomic analyses strongly suggest that non-coding T2D variants alter transcriptional regulation and target gene expression in pancreatic islets, but only ~20% of T2D variants elicit changes in islet cis-regulatory element (CRE) use or gene expression under steady state conditions. Environmental factors such as endoplasmic reticulum (ER) stress have been implicated in islet dysfunction. However, studies to date have not assessed if or how T2D variants modulate the response of islets to ER stress. I hypothesize that these T2D variants alter islet ER stress-responsive CRE use or activity and target gene expression to contribute to islet β cell dysfunction or death in T2D. In my preliminary data analysis, I have identified ER stress-responsive CREs that overlap 407 T2D variants in islets and connected these CREs to 22 putative target gene promoters using islet promoter capture Hi-C maps. In Aim 1, I will comprehensively assess the effects of T2D variants on the use or activity of ER stress-responsive CREs using chromatin accessibility quantitative trait locus (caQTL) and massively parallel reporter assays (MPRA), respectively. In Aim 2, I will determine if the putative CRE- targeted genes are required in human islet cells for their ER stress response and survival by altering their expression using CRISPR/Cas9 epigenomic editing platforms. Successful completion of these Aims will yield a functionally characterized set of T2D variants and a validated set of downstream 'T2D target genes’ that modulate islet function and survival in response to a central (patho)physiologic stressor. More broadly, I anticipate that the principles and framework I employ in this mechanistic variant-to-function project could be applied to characterize the effects of T2D variants in the context of other environmental stressors and metabolic tissues. Importantly, completion of this project will help me master current concepts and state-of-the-art techniques in genetics and functional genomics and increase my scientific communication skills through extensive opportunities to present and publish my studies. Finally, my position as an MD/PhD student at UConn Health and The Jackson Laboratory for Genomic Medicine will not only allow me to be mentored in a world- class, highly collaborative environment, but it will provide me with opportunities to continue honing my clinical skills and gain specialized experience during and after my research phase. Fulfilling my training and development plan will be a crucial step toward my future career as a physician-scientist studying the genetic and (epi)genomic mechanisms of disease-associated variants in patients.