Control and function of repressive chromatin domains

Project Summary/Abstract The long-term goals of this project are to understand the composition, assembly, and function of specialized chromatin domains. Polycomb Group (PcG) proteins interact to form enzyme complexes that modify chromatin and assemble repressed chromatin states in animals, plants, and some fungi. Polycomb Repressive Complex 2 (PRC2) is a highly conserved histone methyltransferase complex that is essential for multicellular development and plays key roles in maintenance of stem cell identify, X-chromosome inactivation, and gene regulation. Mutations that alter PRC2 function are responsible for human Weaver syndrome and multiple cancers. Despite the importance of PcG proteins in human health and nearly 80 years of investigation, important mechanistic details regarding the establishment and epigenetic inheritance of PcG repressed chromatin remain unknown. Our studies with the model fungus Neurospora crassa have begun to address these critical gaps in knowledge. N. crassa is a powerful experimental system that shares key features of the PcG system with higher eukaryotes. Importantly, components of PRC2 are structurally and functionally conserved, in contrast to major yeast models Saccharomyces cerevisiae and Schizosaccharomyces pombe. Our recent efforts have uncovered new genes required for PcG repression and have begun to generate mechanistic insights into their functions. We will apply hypothesis-driven genetic, molecular, proteomic, and genomic approaches to define functions of newly identified genes, and we will perform discovery-driven genetic screens to identify new regulators and components of Polycomb repressed chromatin domains. Major questions will be addressed during the grant period include. 1) What are the mechanisms that control de novo establishment, spreading, and epigenetic inheritance of H3K27me3? 2) How does ISWI, a nucleosome sliding ATP-dependent chromatin remodeling enzyme control to assembly and maintenance of PcG repressed domains? 3) How are H3K27me3 levels re- established following DNA replication? 4) What other factors control and comprise PcG-repressed chromatin domains? Successful completion of the proposed aims will uncover general principles and mechanisms governing assembly and function of PcG-repressed chromatin domains. Findings from these studies will serve as a framework for future studies in higher eukaryotes, including humans. In addition, because PcG proteins are linked to cancer and other genetic diseases, understanding the regulation and function of these key chromatin regulators will ultimately improve diagnosis and treatment of human disease.