ABSTRACT Diffuse intrinsic pontine glioma (DIPG) is an aggressive tumor of young children that infiltrates the brainstem, and although radiation therapy can slow tumor growth, most children with DIPG die within two years of diagnosis. To develop more effective therapies, it is necessary to understand the molecular and cellular mechanisms that drive DIPG. Approximately 80% of DIPG tumors carry a point mutation in one of the histone 3 (H3) genes that causes a substitution of lysine 27 for methionine (H3K27M). Expression of H3K27M mutant protein is an oncogenic driver, increasing cell proliferation, and causes aberrant gene expression. Chromatin is a dynamic polymer of DNA and histone proteins, and the trimethylation of H3 lysine 27 (H3K27-me3) correlates with transcriptional repression. H3K27-me3 is catalyzed by EZH2, and the H3K27M mutant histone protein inhibits EZH2 activity, causing widespread loss of H3K27-me3 across the genome. However, this loss of H3K27-me3 does not closely correlate with transcriptional de-repression. For example, in cells expressing the H3K27M mutation, only about 4% of the genes that lose H3K27-me3 increase their expression. Does H3K27M instead affect gene expression by altering “higher-order” aspects of genome organization, such as the interaction of chromatin with specific nuclear “compartments?” The lamina of the inner nuclear membrane is a transcriptionally repressive nuclear compartment. Lamina associated domains (LADs) are large, specific genomic regions located at the nuclear periphery, and genes within LADs exhibit low transcriptional activity. H3K27-me3 is highly enriched at the “borders” between LADs and non-LAD genomic regions, suggesting a role for this chromatin modification in regulating this aspect of higher-order genome organization. In Preliminary Studies, we have mapped LADs in DIPG cells that carry the H3K27M mutation, finding that the LAD architecture is uniquely disrupted as compared to other types of cancer and normal cells. Given our biochemical understanding of H3K27M mutant protein, the function of EZH2, and our knowledge of LAD border structure, we propose experiments that test the following hypothesis: By inhibiting EZH2 and causing genome-wide changes in H3K27-me3 levels, the H3K27M mutant protein disrupts normal LAD structures, and that such disruption of nuclear compartment-associated genome organization underlies the abnormal gene expression of DIPG. This work challenges the current understanding of H3K27M oncogenic effects. Instead of building upon the current model that explains H3K27M oncogenic function at the level of local chromatin state changes, results obtained test a new paradigm, that H3K27M causes a much larger-scale change to the organization of the genome in the nucleus. Mutations in histone proteins such as H3K27M represent an emerging class of oncogenic drivers in a wide diversity of cancer, both rare and prevalent. Thus, understanding how H3K27M affects nuclear compartment-a...