Discovery of diabetes-relevant ò cell enhancers through 4D enhancer mapping, integrative analysis, and large-scale CRISPRi perturbation screens

摘要 增强子是重要的调控元件，与转录因子（TF）一起指导细胞- 在发育、组织稳态和再生过程中的类型特异性转录程序。举措 例如ENCODE项目，揭示了数万个基于线性邻近的推定增强子， 标准如染色质可及性、TF结合和组蛋白修饰如H3 K27 ac。然而，主要 发现功能增强子并将其分配给靶基因的挑战在于， 3D染色质组织，这可以影响增强子的特异性和活性。使用高级 染色体构象捕获分析，我们最近捕获了3D增强子网络的动态重新布线 在小鼠体细胞重编程过程中发现了多连接的增强子，我们将其命名为“3D 增强器集线器”。在这里，我们将3D映射方法扩展到人类初级胰岛，并比较来自 健康和2型糖尿病（T2 D）供体组装4D图谱，以捕获3D增强子的重新布线 疾病进展的网络。同时，我们计划将成人胰岛中的增强子网络与 通过使用人多能干细胞（hPSC）产生早期β细胞， 他们的发展先驱。利用这些4D基因组数据，我们将计算提名核心β细胞 与β细胞发育、功能和T2 D相关的特异性增强子，然后询问这些推定的增强子。 通过使用hPSC-β细胞的大规模CRISPRi介导的扰动筛选来筛选增强子。增强剂 将在已建立的人β细胞系和原代细胞系中进一步验证从筛选工作中鉴定的 人胰岛β细胞该提案解决了4DN计划中的一个关键差距，即如何将3D 基因组学数据转化为人类健康背景下基因表达的功能数据。成功 我们目标的完成将为功能增强剂的发现和研究建立一个范例 指导特定于感兴趣细胞类型的转录程序，揭示了对其独特的见解 作用机制，并确定与人类发育和疾病相关的增强剂。为 例如，发现功能增强子可以帮助识别非编码的因果变异， 在全基因组关联研究中。

ABSTRACT Enhancers are essential regulatory elements that together with transcription factors (TFs) instruct cell- type specific transcriptional programs during development, tissue homeostasis and regeneration. Initiatives such as the ENCODE project, revealed tens of thousands putative enhancers based on linear proximity, using criteria like chromatin accessibility, TF binding, and histone modifications such as H3K27ac. However, a main challenge of uncovering functional enhancers and assigning them to target genes lies in the complexity of the 3D chromatin organization, which can influence enhancer specificity and activity. Using an advanced chromosome conformation capture assay, we recently captured the dynamic rewiring of 3D enhancer networks during mouse somatic cell reprogramming and discovered multi-connected enhancers that we named “3D enhancer hubs”. Here we extend the 3D mapping approach to human primary islets, and compare islets from healthy and type 2 diabetes (T2D) donors to assemble a 4D atlas to capture the rewiring of 3D enhancer network in disease progression. At the same time, we plan to compare the enhancer network in adult islets to earlier stages of development by using human pluripotent stem cells (hPSCs) to generate early β cells and their developmental precursors. Utilizing these 4D genomic data, we will computationally nominate core β-cell specific enhancers relevant to β cell development, function, and T2D, and then interrogate these putative enhancers through large-scale CRISPRi mediated perturbation screens using hPSC-β cells. Enhancers identified from the screening effort will be further validated in an established human β cell line and primary human islet β cells. This proposal addresses a critical gap in the 4DN initiative, that is how to translate 3D genomics data into functional data with respect to gene expression in the context of human health. Successful completion of our aims will establish a paradigm for the discovery and interrogation of functional enhancers that instruct transcriptional programs specific to a cell type of interest, reveal unique insights into their mechanisms of action, and identify enhancers with relevance to human development and disease. For instance, uncovering functional enhancers could assist the identification of noncoding causal variants identified in genome-wide association studies.