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

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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.