Live-Cell Chromatin Imaging and Biology: Application to Extrachromosomal DNA

活细胞染色质成像和生物学：在染色体外 DNA 中的应用

• 批准号: 10685017
• 负责人: Liangqi Xie
• 金额: $ 144.9万
• 依托单位: CLEVELAND CLINIC LERNER COM-CWRU
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-01 至 2026-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10685017
• 关键词: Address; Automobile Driving; Biochemical; Biology; Cells; Chromatin; Chromatin Structure; Chromosomes; Disease; Disease Management; Dissection; Drug resistance; Ensure; Foundations; Gene Expression; Gene Expression Regulation; Genetic; Genetic Transcription; Genome; Genome engineering; Genomic approach; Genomics; Health; Health Promotion; Human; Image; Label; Malignant Neoplasms; Measurement; Methods; Microscopy; Mitosis; Modernization; Molecular; Monitor; Names; National Cancer Institute; Oncogenes; Optics; Organ; Organism; Outcome; Patients; Physiological; Process; Regulation; Regulatory Element; Resolution; Time; Tissues; Visualization; cancer genome; cancer type; design; extrachromosomal DNA; genetic approach; high reward; high risk; improved; innovation; molecular dynamics; novel strategies; population based; programs; segregation; spatiotemporal; stem; technology development; tool; tumor heterogeneity

PROJECT SUMMARY Genome regulation is the prime mechanism that governs the precise spatiotemporal gene expression program that, in turn, establishes and maintains the cellular states in tissues and organs in multicellular organisms. In humans, genomic changes that alter this regulation can cause a wide range of diseases such as cancer. Demystifying genome regulation has become a central task of modern biology in the post-genomic era and is the foundation for effective disease management and promotion of health. Homeostatic genome regulation ensures precise spatiotemporal chromosomal gene regulation in properly insulated chromatin structures, in the cis-configuration (same chromosome), and through faithful symmetric segregation during mitosis. In stark contrast, these rules are broken in human cancers particularly in the form of extrachromosomal DNA (ecDNA) that enables insulation crossover, trans regulation, and asymmetric inheritance. Named as a 2021 Cancer Grand Challenge by the National Cancer Institute, megabase-sized circular ecDNA typically contains common oncogenes and regulatory elements present in major human cancer types, driving massive oncogene amplification and expression, enabling intra-tumoral heterogeneity, and conferring drug resistance and poor patient survival. The fundamental molecular mechanisms governing ecDNA expression, interaction, and propagation are largely unknown. Although powerful biochemical, genetic, and genomic approaches have laid the conceptual framework of modern understanding of genome regulation, the largely population-based, time- averaged, and sometimes out-of-context measurements are insufficient to fully describe the spatially compartmentalized, temporally dynamic, and physiologically relevant higher-order interactions in single live cells. The fundamental challenges stem from lack of chromatin tools to label the intrinsically heterogeneous chromatin, limited spatiotemporal resolution to monitor the highly concentrated and dynamic molecular transactions, and the paucity of quantitative and rigorous methods to extract fundamental physical rules underlying genome regulatory processes. In this project, we plan to overcome these primitive challenges by initiating a radically distinctive technological development of robust, efficient, and multiplexable chromatin labeling strategies for live- cell chromatin biology, which will allow us to apply them to address the fundamental regulation of ecDNA in the cancer genome. We will integrate advanced imaging, cutting-edge microscopy, modern synthetic genome engineering, and optical/genetic perturbation to systematically study the basic mechanisms by which ecDNA orchestrates massive oncogene transcription, extensive chromosomal remodeling, and asymmetric segregation hitherto intractable by conventional biochemical, genetic, or genomic methods. Positive outcome of these cutting- edge approaches and high-risk high-reward questions embedded in our proposal will have a transformative impact on mechanistic dissection of cancer genome regulation and may help us to uncover an Achilles' heel with which to target ecDNA-driven cancer, thereby affording wide-ranging positive influence on human health.

项目概要 基因组调控是控制精确时空基因表达程序的主要机制 反过来，它建立并维持多细胞生物组织和器官的细胞状态。在 对于人类来说，改变这种调节的基因组变化可能会导致多种疾病，例如癌症。 揭开基因组调控的神秘面纱已成为后基因组时代现代生物学的中心任务， 有效的疾病管理和促进健康的基础。稳态基因组调控 确保正确隔离的染色质结构中精确的时空染色体基因调控 顺式构型（相同染色体），并通过有丝分裂过程中忠实的对称分离。赤裸裸地 相比之下，这些规则在人类癌症中被打破，特别是以染色体外 DNA (ecDNA) 的形式 实现绝缘交叉、反式调节和不对称继承。被评为 2021 年癌症大奖 受到国家癌症研究所的挑战，兆碱基大小的环状 ecDNA 通常包含常见的 致癌基因和调控元件存在于主要人类癌症类型中，驱动大量致癌基因 扩增和表达，实现肿瘤内异质性，并赋予耐药性和不良 患者生存。控制 ecDNA 表达、相互作用和合成的基本分子机制 传播很大程度上是未知的。尽管强大的生化、遗传学和基因组方法已经奠定了 现代理解基因组调控的概念框架，主要是基于人群的、时间- 平均的，有时脱离上下文的测量不足以充分描述空间 单个活细胞中分隔的、时间动态的和生理相关的高阶相互作用。 根本挑战源于缺乏染色质工具来标记本质上异质的染色质， 有限的时空分辨率来监测高度集中和动态的分子交易，以及 缺乏定量和严格的方法来提取基因组的基本物理规则 监管流程。在这个项目中，我们计划通过发起一个彻底的解决方案来克服这些原始挑战。 稳健、高效、可多重染色质标记策略的独特技术开发，用于活体染色质标记 细胞染色质生物学，这将使我们能够应用它们来解决细胞中 ecDNA 的基本调控问题 癌症基因组。我们将整合先进的成像、尖端显微镜、现代合成基因组 工程和光学/遗传微扰系统地研究 ecDNA 的基本机制 协调大量癌基因转录、广泛的染色体重塑和不对称分离 迄今为止，传统的生化、遗传或基因组方法难以处理。这些削减的积极成果- 我们提案中嵌入的边缘方法和高风险高回报问题将具有变革性 对癌症基因组调控机制剖析的影响，可能有助于我们发现致命弱点 它针对 ecDNA 驱动的癌症，从而对人类健康产生广泛的积极影响。