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

通过 4D 增强子图谱、综合分析和大规模 CRISPRi 扰动筛选发现糖尿病相关的 β 细胞增强子

• 批准号: 10665641
• 负责人: Effie Apostolou
• 金额: $ 69.81万
• 依托单位: SLOAN-KETTERING INST CAN RESEARCH
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-15 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10665641
• 关键词: 3-Dimensional; ATAC-seq; Address; Adult; Affect; Atlases; Beta Cell; Binding; Biological Assay; Biology; CRISPR interference; Cell Line; Cell Reprogramming; Cells; Chromatin; Clinical Data; Complex; Computational Biology; Computing Methodologies; Data; Development; Diabetes Mellitus; Disease; Disease Progression; Embryonic Development; Enhancers; Ensure; Gene Expression; Genes; Genetic Transcription; Genome; Genomic approach; Genomics; Goals; Health; Hi-C; Homeostasis; Human; Human Development; Human Genetics; Islet Cell; Islets of Langerhans; Knowledge; Link; Malignant Neoplasms; Maps; Mediating; Mus; Mutation; Names; Natural regeneration; Non-Insulin-Dependent Diabetes Mellitus; Nucleic Acid Regulatory Sequences; Pathogenicity; Pathologic; Population; Publishing; Regulatory Element; Research Personnel; Resolution; Somatic Cell; Specificity; Time; Tissues; Translating; Untranslated RNA; Update; Validation; Variant; Work; causal variant; cell type; chromatin immunoprecipitation; chromosome conformation capture; clinical application; diabetes pathogenesis; diabetic; directed differentiation; endocrine pancreas development; gene function; gene regulatory network; genome wide association study; genomic data; histone modification; human disease; human pluripotent stem cell; improved; innovation; innovative technologies; insight; interest; islet; machine learning model; multidisciplinary; novel; precursor cell; programs; promoter; screening; tool; transcription factor

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.

抽象的 增强子是必不可少的调节元素，与转录因子（TFS）一起指示细胞 - 在开发，组织稳态和再生过程中键入特定的转录程序。倡议 例如编码项目，基于线性接近性，揭示了数以万计的推定增强剂 诸如染色质可及性，TF结合和组蛋白修饰等标准（例如H3K27AC）。但是，一个主要 发现功能增强子并将其分配给靶基因的挑战在于复杂性 3D染色质组织，可以影响增强子的特异性和活动。使用高级 染色体会议捕获测定法，我们最近捕获了3D增强器网络的动态重新布线 在鼠标体细胞细胞重编程和发现的多连接增强器中，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主要胰岛，并比较 健康和2型糖尿病（T2D）供体组装4D地图集以捕获3D增强剂的重新布线 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细胞和 他们的发展前体。利用这些4D基因组数据，我们将在计算中提名核心β细胞 与β细胞开发，功能和T2D相关的特定增强剂，然后询问这些推定的 通过大规模CRISPRI的增强子使用HPSC-β细胞介导的扰动筛选。增强剂 从筛选工作中确定的将在已建立的人β细胞系和原发性中进一步验证 人类胰岛β细胞。该提案解决了4DN计划中的一个关键差距，这是翻译3D的方法 基因组学数据在人类健康中有关基因表达的功能数据。成功的 我们的目标的完成将建立一个范式，以发现和询问功能增强器 该指导特定于单元格的转录程序，揭示其独特的见解 作用机制，并确定与人类发展和疾病相关的增强子。为了 实例，发现功能增强器可以帮助识别已识别的非编码因果变体 在全基因组关联研究中。