Molecular mechanisms of heterochromatin spreading at the leading edge

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Molecular mechanisms of heterochromatin spreading at the leading edgeThis project will uncover molecular details of how genes are stably turned off to produce different types of cells. Stable gene silencing can distinguish single-celled organisms and it also helps the human body use the same genome to create very different cell types that form organs. One major determinant of stable gene silencing is how far along a chromosome the wave of silencing spreads. Understanding how spreading occurs and how it is stopped are the primary focuses of this project. Achieving the goals of this project will help us understand variation across genetically identical cells that underlies development and environmental effects on gene regulation. The project will be undertaken by a team of scientists lead by the PI and including postdoctoral fellows, graduate students, and undergraduates. Summer research experiences for high school students and their teachers from a local school with a high minority population will enhance the training impact of the project. Together, the team will contribute to the research, combining their resources and skill sets while mentoring each other across levels. The results from this project will be broadly shared with the research community and the public and the resources generated will be made available to the scientific field.Heterochromatin is a genomic structure that represses transcription in a manner that is epigenetically maintained through cell division, contributing to stable cell differentiation in all eukaryotes. Establishment of a heterochromatin domain involves site-specific initiation and subsequent spreading across large regions of the genome, incorporating repressor proteins that stably associate with chromatin. Spreading is coupled to the conversion of histone modifications at the “leading edge” and eventual transition to the stable heterochromatin state. This mechanism requires two main modes of action for the heterochromatin machinery: dynamic spreading and stable repressive association with chromatin. While much is known of the components of heterochromatin in multiple systems, we do not understand at molecular resolution how the machinery coordinates the transitions through the steps of the mechanism. This project harnesses a purified budding yeast heterochromatin system that fully recapitulates the dynamic conversion of histone modifications and the formation of a stable repressive heterochromatin state. Previous work from the research group using this system has generated models for many steps in the mechanism that will now be tested at a new level of molecular resolution. Advanced cross-linking mass spectrometry will be used, combined with “designer” chromatin to capture the heterochromatin machinery in the multiple modes of action that drive formation of an epigenetic repressive genomic structure. The wealth of genetic and biochemical tools available will allow for identification of the most important molecular interactions for heterochromatin assembly, generating a new level of mechanistic understanding of this fundamental genomic regulatory system.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.