ATP-dependent chromatin remodelers catalyze nucleosome changes to regulate essential DNA-related processes, such as replication, transcription, and repair. Mutations of protein subunits within these remodeling complexes have been linked to various diseases including cancer and developmental disorders; however, the precise underlying mechanisms remain unclear. The ACF remodeling complex has served as a model system for understanding molecular mechanisms used in nucleosome remodeling. ACF is composed of the SNF2h ATPase and the Acf1 accessory subunit and generates evenly spaced nucleosome arrays that are important for forming silenced chromatin. Recent NMR and cryo-EM studies indicate SNF2h alone is able to deform histones within a nucleosome to carry out its functions, suggesting remarkable nucleosome plasticity. However, the exact molecular nature of the histone conformational changes remains unresolved. Additionally, the structure and function of the Acf1 subunit of ACF remains poorly characterized, and how ACF remodels nucleosomes in the presence of other heterochromatin-associated proteins remains unclear. The ability to address these questions using single-particle cryo-EM has faltered due to the difficulty in preparing cryo-EM sample grids of remodeling complexes bound to nucleosome substrate. We have now developed a new method to routinely prepare cryo-EM grids with intact remodeler-nucleosome complexes without the use of chemical fixatives. This technology will facilitate our ability to use an interdisciplinary approach combining single-particle cryo-EM, biochemical assays, and single-molecule fluorescence spectroscopy to elucidate mechanisms of ACF function as delineated in the research strategy. Specifically, I will: 1. determine the structural basis for ACF-mediated chromatin remodeling using single-particle cryo-EM; 2. define the interplay between ACF and linker histone H1 variants using biochemical assays, fluorescence-based assays, and single- particle cryo-EM; and 3. elucidate the connection between ACF and heterochromatin protein 1? using interaction assays and single-particle cryo-EM. In the long-term, we envision that the methods applied here will be generally applicable to study the molecular mechanisms of other chromatin-related enzymes and to understand why mutations of these enzymes lead to disease. The Principal Investigator (Un Seng Chio) will carry out the proposed experiments under the guidance of both Dr. Yifan Cheng (Sponsor), an expert in cryo-EM methodology, and Dr. Geeta Narlikar (Co- Sponsor), an expert in chromatin biochemistry, at the University of California, San Francisco (UCSF). UCSF is a well-established research institution with abundant resources for single-particle cryoEM and chromatin biochemistry both intellectually and equipment-wise, making it an ideal location for the PI to receive postdoctoral training while performing the proposed research. UCSF also offers many resources for the PI to develop career- wise as he prepares for the next stage of his career as an independent investigator.
ATP-dependent chromatin remodelers play important roles in organizing chromatin for all essential DNA- related processes, such as replication, transcription, and repair. Mutations of protein subunits within these chromatin remodeling complexes have been linked to various diseases including cancer and developmental defects. The research proposed here will further our ability to determine structures of chromatin remodeling complexes using single-particle cryo-EM to understand functional and disease mechanisms at the molecular level, enabling the development of therapeutic drugs targeting these enzyme machines.
