Mapping Technology Development

测绘技术发展

• 批准号: 9149216
• 负责人: Bing Ren
• 金额: $ 67.43万
• 依托单位: LUDWIG INSTITUTE FOR CANCER RES LTD
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/9149216
• 关键词: 3-Dimensional; Address; Algorithms; Architecture; Binding; Biochemistry; Biological Assay; Biological Models; Cell Count; Cell Nucleus; Cells; Chromatin; Chromatin Structure; DNA; DNA Sequence; DNA biosynthesis; Data; Data Analyses; Data Set; Detection; Digestion; Enhancers; Environment; Eukaryotic Cell; Experimental Models; Face; Formaldehyde; Frequencies; Gene Expression; Genetic; Genome; Genomics; Goals; Gold; In Situ; Lead; Ligation; Mammalian Cell; Maps; Measures; Methodology; Methods; Microscopy; Modification; Mus; Nuclear; Population; Process; Protocols documentation; Reading; Research; Resolution; Resources; Structure; System; Techniques; Technology; Variant; cell fixing; chromosome conformation capture; cost; crosslink; embryonic stem cell; genome editing; genome-wide; genome-wide analysis; improved; innovation; nanometer; novel; promoter; restriction enzyme; technology development; tool

PROJECT SUMMARY/ABSTRACT The 3-dimensional organization of our genome has emerged as an important regulator of diverse nuclear processes, ranging from gene expression to DNA replication. A wide variety of tools have been useful for the genome-wide study of the 3D genome organization, and these assays generally resolve chromatin topology by detecting the frequency of ligation between proximal genomic fragments in the formaldehyde fixed cells. While these techniques have uncovered general features of chromatin organization in eukaryotic cells, they also produced widely different results that have clouded our view of chromatin organization. In addition, the modest resolution and the biases introduced by restriction digestion and ligation complicate the data interpretation. Here, we propose innovative solutions to address each of these barriers. In aim 1, we will combine genetics, biochemistry and microscopy to develop a gold standard dataset for evaluating and optimizing technologies mapping chromatin topology. Specifically, we will assess long-range chromatin interactions at ~100 pairs of enhancer/promoter loci in an experimental model cell system, with the use of genome editing tools, locus- specific 4C and 3D-FISH, to establish a set of positive and negative controls. In aim 2, we will optimize and refine existing genome wide approaches for assessing high-resolution chromatin structure, guided by the gold standard data generated in aim 1. In aim 3, we will develop and refine a complementary method, termed Genome Architecture Mapping (GAM), that can probe chromatin structure genome-wide without the need for restriction and ligation. This method avoids the potential bias of previous methodologies and also offers the opportunity for analysis of chromatin structure in single nuclei. If successful, the tools and resources developed through this component will transform our ability to study chromatin topology in mammalian cells.

项目摘要/摘要 我们基因组的三维组织已成为多种核的重要调节者 从基因表达到DNA复制的过程。多种工具对 3D基因组组织的全基因组研究，这些测定通常通过 检测甲醛固定细胞中近端基因组碎片之间的连接频率。尽管 这些技术在真核细胞中发现了染色质组织的一般特征，它们也 产生的结果很大，使我们对染色质组织的看法蒙上了困难。另外，谦虚 限制消化和连接引入的分辨率和偏见使数据解释变得复杂。 在这里，我们提出了创新的解决方案来解决这些障碍。在AIM 1中，我们将结合遗传学， 生物化学和显微镜开发用于评估和优化技术的黄金标准数据集 映射染色质拓扑。具体而言，我们将评估〜100对的远程染色质相互作用 实验模型细胞系统中的增强子/启动子基因座，使用基因组编辑工具，位置 - 特定的4C和3D-FISH，以建立一组阳性和阴性对照。在AIM 2中，我们将优化和 优化现有的基因组广泛的方法来评估高分辨率染色质结构，在黄金的指导下 在AIM 1中生成的标准数据。在AIM 3中，我们将开发和完善一种互补方法，称为 基因组结构映射（GAM），可以探测整个基因组的染色质结构而无需 限制和结扎。此方法避免了以前方法论的潜在偏见，还提供了 分析单核中染色质结构的机会。如果成功，工具和资源开发了 通过该成分将改变我们研究哺乳动物细胞中染色质拓扑的能力。