PROJECT SUMMARY Posttranslational modification (PTM) of nucleosome-associated histone proteins, along with histone variant incorporation, influences transcriptional competence and their dysregulation has been identified in numerous pathological states. Histone H3 N-terminal acetylation, methylation, and phosphorylation are common PTMs; the precise combination of these PTMs can modulate chromatin architecture and genome organization, leading to changes in gene expression. Despite extensive efforts to characterize H3 PTMs and H3 variants, their mechanistic and functional interplay, and their ability to influence biological output, it remains unclear how histones and many histone PTMs integrate cues from upstream signaling cascades to regulate gene expression. The overarching objective for my laboratory is to define mechanisms that regulate histone PTM patterns and unmask how they influence transcriptional output. Over the next five years, we propose a combinatorial approach leveraging genetic, molecular, cellular, biochemical and computational methods to define novel mechanisms by which a poorly understood histone H3 PTM, H3 threonine 45 phosphorylation (pH3T45), relays cellular signals from upstream kinases to impact gene expression, and leverage this approach to delineate how novel pathogenic histone H3 variants dysregulate the epigenome to alter cellular function. Our preliminary data suggest that H3T45 phosphorylation status (1) modulates H3K4 methylation by directing specific H3K4-modifying complexes to chromatin; (2) disrupts H3K36me3 via competing histone acetylation/deacetylation; (3) regulates RNA processing through differential association with splicing factors and the RNA exosome complex. We will dissect how H3K4-methyltransferase complexes differ in structure, function, and biological output when associated with pH3T45 or unmodified H3T45. We will delineate how pH3T45 impacts the dynamics of H3K36 acetylation and H3K36 methylation in the context of DNA repair. We will then define how pH3T45 governs the production of mature RNA by examining the role of unmodified H3T45 and pH3T45 throughout RNA processing. Lastly, we have identified a series of cancer-associated H3 variants in which an amino acid is changed to a lysine, termed “H3 X to K” variants. Our preliminary data demonstrates that H3 X to K variants dysregulate proximal H3 PTMs to uniquely modulate gene expression. We will leverage our experience studying pH3T45 to mechanistically define how H3 X to K variants reprogram the epigenome to produce transcriptional and functional cellular changes. This research will address the regulation and effects of histone PTM patterns and H3 variant expression, which will inform our view of how H3 PTMs and variant expression underlies human disease.