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，与转录因子一起指导细胞- 发育、组织动态平衡和再生过程中的特定类型转录程序。计划 例如ENCODE项目，揭示了数万个基于线性接近的假定增强剂，使用 标准如染色质可及性、转铁蛋白结合和组蛋白修饰，如H3K27ac。然而，一个主要的 发现功能增强子并将它们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 染色体构象捕捉分析，我们最近捕捉到了3D增强子网络的动态重连 在小鼠体细胞重新编程期间，发现了多连接的增强子，我们将其命名为“3D 增强型中心“。在这里，我们将3D映射方法扩展到人类原发胰岛，并比较 健康和2型糖尿病(T2D)捐赠者组装4D图谱以捕捉3D增强子的重新连接 网络在疾病进展中的作用。同时，我们计划将成人胰岛中的增强子网络与 通过使用人类多能干细胞(HPSC)产生早期β细胞和 它们的发育前体。利用这些4D基因组数据，我们将计算命名为核心β细胞 与β细胞发育、功能和T2D相关的特定增强子，然后询问这些假定的 利用hPSC-β细胞通过大规模CRISPRI介导的干扰筛选获得增强子。增强剂 从筛选工作中确定的将在已建立的人类β细胞系和原代细胞中进一步验证 人胰岛β细胞。这项提议解决了4DN倡议中的一个关键差距，即如何翻译3D 将基因组学数据转化为与人类健康背景下的基因表达有关的功能数据。成功 我们的目标的完成将建立一个发现和询问功能增强剂的范例 指示特定于感兴趣的细胞类型的转录程序，揭示其独特的见解 行动机制，并确定与人类发展和疾病相关的促进剂。为 例如，发现功能增强子可以帮助识别所识别的非编码因果变体 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.