Gene repression by Polycomb Group (PcG) proteins is a conserved mechanism across metazoans and most eukaryotic organisms. Polycomb Repressive Complex 2 (PRC2) maintains gene repression by establishing Histone H3 lysine 27 trimethylation (H3K27me3), a repressive histone post-translational modification (PTM), at target regions across the genome. The mechanisms that underly PRC2 recruitment to these regions remain incompletely understood across all model systems. In this dissertation, I used the model fungus Neurospora crassa to study the molecular factors that underly PRC2 localization and activity. The first half describes a model system I developed to assay the kinetics of de-novo facultative heterochromatin establishment by PRC2. The second half looks at the role of the conserved, replication-dependent histone chaperone, Chromatin Assembly Factor 1 (CAF-1), in maintaining gene silencing within facultative heterochromatin regions. Collectively, this work deepens our understanding of how PRC2 activity is targeted in fungi and elucidates the role of other chromatin modifying complexes in this process. The findings from these studies may provide insights into how PRC2 function is regulated across all eukaryotes and may be relevant to the development of therapeutics that target the Polycomb repression pathway to treat cancer and other diseases.
