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

项目摘要 基因组调控是控制基因精确时空表达的主要机制 这反过来又在多细胞生物的组织和器官中建立和维持细胞状态。在 在人类中，改变这种调节的基因组变化可能导致广泛的疾病，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， 有效疾病管理和促进健康的基础。稳态基因组调控 确保在适当绝缘的染色质结构中进行精确的时空染色体基因调节，在 顺式构型（相同染色体），并通过有丝分裂期间的忠实对称分离。形成鲜明 相反，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。 这使得绝缘交叉、反式调节和非对称继承成为可能。2021年癌症大名单 美国国家癌症研究所的挑战，兆碱基大小的环状ecDNA通常含有常见的 癌基因和调控元件存在于主要的人类癌症类型中，驱动大量癌基因 扩增和表达，使肿瘤内的异质性，并赋予耐药性和不良反应。 患者生存率。调控ecDNA表达、相互作用和 传播是未知的。尽管强大的生物化学、遗传学和基因组学方法已经奠定了基础， 基因组调控的现代理解的概念框架，主要是基于人口的，时间- 平均的，有时脱离上下文的测量不足以完全描述空间 在单个活细胞中的区室化的、时间动态的和生理相关的高阶相互作用。 根本的挑战来自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 监管程序。在这个项目中，我们计划通过从根本上解决这些原始挑战。 独特的技术发展，强大的，有效的，和多路复用的染色质标记策略，为活的， 细胞染色质生物学，这将使我们能够应用它们来解决ecDNA的基本调控， 癌症基因组我们将整合先进的成像技术，尖端的显微镜技术，现代合成基因组技术 工程和光学/遗传微扰，系统地研究ecDNA 协调大量的癌基因转录、广泛的染色体重塑和不对称分离 迄今为止，通过常规生物化学、遗传学或基因组学方法是难以解决的。这些削减的积极成果- 我们的提案中包含的边缘方法和高风险高回报的问题将产生变革性的影响。 对癌症基因组调控机制解剖的影响，并可能帮助我们发现一个致命弱点， 其靶向ecDNA驱动的癌症，从而对人类健康产生广泛的积极影响。

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.