PROJECT SUMMARY High grade glioma (HGG) is the most common malignant brain tumor in adults and is universally lethal. The standard of care for HGG has remained unchanged for over 30 years and median survival is devastatingly low at only 15 months post diagnosis. Epigenetic aberrations are a key molecular feature that contribute to disease pathogenesis. Therefore, the goal of this proposal is to define epigenomic dysregulations that promote HGG growth and determine the mechanisms through which these alterations drive tumorigenesis. HGG tumors with increased chromatin accessibility tend to have more favorable outcomes. We recently showed Sox9 inhibits histone deacetylases in HGG to increase H3K27 acetylation, which significantly extends median survival in mice. Analysis of Sox9 in human HGG revealed it interacts with numerous epigenetic modifying proteins, including Hp1bp3, a heterochromatin binding protein. Furthermore, whole genome sequencing of familial glioma patients and their families nominated Hp1bp3 as a potential tumor suppressor, with loss-of- function (LOF) mutations driving oncogenesis. Using an in-utero electroporation (IUE) mouse model of HGG, we found that Hp1bp3 loss-of-function (LOF) tumors are hyperproliferative with decreased median survival while HP1BP3 gain-of-function (GOF) stunts tumor growth and extends survival. Additionally, ATAC-Sequencing of LOF tumors revealed a global decrease in accessible chromatin. These preliminary results lead me to hypothesize that Hp1bp3 orchestrates chromatin remodeling to slow tumor growth in HGG. To understand the role of Hp1bp3 in regulating the epigenome and how this impacts HGG progression, I purpose three aims. In Aim 1, I will determine the effect of Hp1bp3 in our IUE and PDX models. In the second aim, I will determine the epigenomic alterations associated with Hp1bp3 by profiling H3Kme3 and H4K20me3 marks which appear to be altered by Hp1bp3 in HGG. Lastly, ChIP-Seq on Hp1bp3 in HGG mouse tumor revealed Hp1bp3 binds DNA at gene loci involved with methionine and one-carbon metabolism. Therefore, aim three will identify the metabolic changes in Hp1bp3 mutant tumors using metabolomics and determine how these alterations drive tumor growth. Overall, this research aims to expand our understanding of how epigenetic dysregulation in HGG drives disease progression. Importantly, this work will nominate new epigenetic candidates as potential drug targets in HGG.