Friday, May 23, 2025
5/23/2025
The Role of Dynamics in Regulating Multi-Activity Protein Complexes and Chromatin - PROPOSAL SUMMARY Many protein machines require access to genomic DNA to carry out the essential processes of transcription, DNA repair, and DNA replication. The balance between nucleosome assembly and disassembly within chromatin controls this DNA access in eukaryotic cells. Nucleosomes use an octamer of histone proteins to wrap 146 bp of DNA. They assemble throughout the genome and can form nucleosome-nucleosome contacts that further restrict DNA access. Cells have evolved several mechanisms to regulate transitions between active and inactive chromatin states and enable the turnover of nucleosomes at the right time and place. These mechanisms work together in an elaborate network that responds to changes in the cellular environment. Disruptions in this network cause developmental disorders and cancers with altered transcriptional profiles and genomic instability. Our overall vision is to understand the molecular details of crosstalk between various chromatin regulators and how they influence chromatin structure and dynamics. We will continue to employ an integrated structural and biophysical approach that revolves around the dynamics of protein complexes as measured by bottom-up hydrogen-deuterium exchange coupled with mass spectrometry (HDX-MS). HDX-MS is a highly specialized approach for studying the protein conformational ensemble in solution. It can quantify changes in this ensemble due to stimuli such as ligand binding, allostery, post-translational modification, mutation, and additives, to name but a few. Our bottom-up approach can localize changes with peptide-level resolution, allowing us to quantify each interface's occupancy or thermodynamic contribution in a multivalent complex. Multi- valency is particularly applicable to the multi-domain and multi-protein chromatin regulators we study. In the next five years, we will use HDX-MS to study histones, mono-nucleosomes, and multi-nucleosome arrays. For arrays, we will develop novel isotope-based strategies that distinguish each nucleosome. We will establish a high-throughput approach to enable rapid testing of many regulatory factors alone and in combination. We will also study regulatory complexes with various chromatin-related activities. Proteins within these complexes contain large regions of disorder that are refractory to traditional structural approaches. We will cover diverse regulatory mechanisms, including histone variants, histone post-translational modifications, histone chaperones, ATP-dependent nucleosome remodelers, and nucleosome-binding proteins. Our immediate focus is on H2A.Z and H3.3, histone acetylation, the deacetylase module of the nucleosome remodeling and deacetylase complex, and the yeast acetyltransferase complex with Rtt109, Asf1, and Vps75. We expect to contribute to the mechanistic understanding of these regulators and how they influence chromatin dynamics.
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