Revealing the RNA foundations of chromatin-based epigenetics

PROJECT SUMMARY The goal of this proposal is to determine how the crosstalk between RNA and chromatin shapes epigenetic states in pluripotent and differentiated cells. My laboratory studies epigenetic memory, both at a mechanistic level, using biochemistry and functional genomics in mouse embryonic stem cells, and at an organismal level, using ants and flies as model systems. In the nine years since the lab opened, our mechanistic work has been recognized for our contributions to understanding the role of protein–RNA interactions in chromatin regulation and in particular the Polycomb pathway. We have developed technologies to map RNA-binding sites on specific protein complexes or the entire proteome using photocrosslinking and mass spectrometry, and used this information to dissect the functional contribution of RNA to epigenetic pathways. We also established an acute depletion system in embryonic stem cells that allow us to interrogate epigenetic dynamics during cell fate transitions. In the next five years, we will build on these studies and develop new systems and technologies to obtain a deeper understanding of the RNA foundations of chromatin regulation and epigenetic phenomena in mammalian cells. Using a directed differentiation system from embryonic stem cells to neurons combined with acute protein and RNA depletion technologies, we will determine the order of biochemical events that culminate in Polycomb-mediated silencing and the role of RNA at two critical steps: Polycomb target selection and gene repression via Polycomb body formation. Expanding on our protein–RNA studies with mass spectrometry, we will study more broadly the role of different classes of RNA and their chemical modifications in chromatin factor recruitment and design separation-of-function mutants using a newly developed high-throughput mutational screening method. Finally, we will follow up on some intriguing preliminary findings from a genome-wide knockout screen to explore a new direction, the molecular mechanism by which mobile RNAs are selected for incorporation into extracellular vesicles and transfer to recipient cells, where they might exert epigenetic functions. This work will add to our mechanistic understanding of RNA-mediated regulation of chromatin processes, which in turn will provide new opportunities to decode and engineer epigenetic states, with broad impact on research, biotechnology, and medicine.