Project Summary Covalent modifications of histones, such as acetylation, methylation, phosphorylation, and ubiquitylation, are essential for regulating chromatin structure and function and defective histone modifications have causal roles in numerous developmental disorders and diseases. Our long-term goal is to use fission yeast heterochromatin assembly as an experimental model to study how diverse histone modification activities are coordinated to initiate different gene expression states and how these states are epigenetically inherited. Heterochromatin preferentially assembles at repetitive DNA elements, and it is essential for regulating gene expression and maintaining genome integrity. The formation of heterochromatin is critically dependent on the methylation of H3 lysine 9 (H3K9me). Interestingly, the activities of H3K9 methyltransferases are stimulated by the ubiquitylation of H3 lysine 14 (H3K14ub), although the mechanism is poorly understood. We will examine how H3K14ub stimulates H3K9 methyltransferases and how H3K14ub is regulated in vivo to aid heterochromatin assembly. Heterochromatin is also a great model to study chromatin-based mechanisms of epigenetic inheritance. An important unresolved question is how parental histones carrying modifications are deposited onto newly synthesized DNA during the S phase of the cell cycle to direct the duplication of these modifications on daughter chromatids. We developed eSPAN (enrichment of Sequencing Protein-Associated Nascent DNA) in fission yeast, which allows us to measure the segregation of parental histones on daughter DNA strands. We will integrate this new genomic tool and traditional genetic approaches to examine mechanisms that regulate the segregation of parental histones and how they contribute to epigenetic inheritance. Together, these studies will address fundamental questions about how histone modifications are regulated and inherited, and how their dysregulation contributes to human diseases.