Discovery of diabetes-relevant ò 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 α 细胞增强子

• 批准号: 10456285
• 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/10456285
• 关键词: 3-Dimensional; ATAC-seq; Address; Adult; Affect; Atlases; B-Cell Development; Beta Cell; Binding; Biological Assay; Biology; CRISPR interference; Cell Line; 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; base; causal variant; cell type; chromatin immunoprecipitation; chromosome conformation capture; clinical application; diabetes pathogenesis; diabetic; directed differentiation; gene function; gene regulatory network; genome wide association study; genomic data; histone modification; human disease; human pluripotent stem cell; improved; innovation; 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.

摘要