Visualization of native chromatin-associated complexes to elucidate chromatin regulatory mechanisms - Project Summary Chromatin organizes into distinct functional domains and regulates a wide range of biological processes, including gene expression, genome integrity maintenance, and DNA accessibility. Chromatin dynamically adapts its functional states by altering protein composition or introducing chemical modifications in response to various biological contexts, such as cell differentiation, the cell cycle, environmental stimuli, and disease states. To understand chromatin regulatory mechanisms, it is critical to elucidate how chromatin-associated complexes modulate their structures and functions in response to these alterations. However, traditional biochemical and structural approaches, which rely on in vitro reconstituted complexes of known chromatin proteins and modifications, are often inadequate for studying these dynamic regulatory processes, as they involve numerous biological context-dependent factors and modifications that have yet to be identified. My lab will address this gap by determining the structures of native chromatin-associated complexes within their physiological chromatin context, aiming to elucidate the structural mechanisms underlying key chromosomal processes. Leveraging the unique cryo-EM methods I have developed in conjunction with the distinctive capabilities of the Xenopus egg extract system—which can recapitulate complex biological processes such as cell cycle progression—my lab will focus on two primary research objectives over the next five years. [Project 1]: The first project aims to elucidate the structure and function of the stably bound FACT-nucleosome complex. FACT plays a critical role in various chromosomal processes, including transcription and replication. Although substantial amounts of the FACT-nucleosome complex formed in cellular or Xenopus egg extract chromatin, the purified FACT complex does not bind to an intact nucleosome in vitro. Consequently, the structure and function of the stably bound FACT-nucleosome complex were not well characterized. The cryo- EM structure of the native FACT-nucleosome complex will reveal novel protein-protein, protein-DNA, and protein-RNA interactions within the complex. These insights will facilitate the design of follow-up functional experiments in egg extract that will identify the structural features critical to their functions. [Project 2]: The second project will focus on methodological development to enable the visualization of telomeric chromatin. We will explore various approaches, including telomeric chromatin assembly in Xenopus egg extracts. By achieving direct visualization of telomeric chromatin, we aim to address fundamental questions such as: How do shelterin complexes and columnar nucleosomes cooperatively assemble to form telomeric chromatin? How does telomeric structure protect chromosome ends from DNA repair machinery while regulating telomerase activity? Together, these projects will establish a robust foundation for our long-term goal of bridging the knowledge gap between dynamic chromatin regulation and its structural mechanisms by enabling the visualization of native chromatin-associated complexes within their physiological chromatin context.
