PROJECT ABSTRACT/SUMMARY Normal development requires tightly controlled gene expression to precisely regulate tissue patterning and failure to properly control the expression profiles can lead to disease. Post-translational modifications to the histone proteins, which package the DNA genome, can regulate expression of the underlying genes by making DNA more or less available to transcriptional machinery. Polycomb repressive complex 2 (PRC2), a highly conserved histone methyltransferase, maintains gene repression during development by facilitating chromatin compaction. PRC2 catalyzes the methylation of lysine 27 on histone H3 (H3K27me3) and loss of this epigenetic mark is associated with a number of developmental diseases. Diffuse intrinsic pontine glioma (DIPG), a deadly pediatric brain cancer, is caused by a lysine-to-methionine mutation on histone H3 (H3 K27M) that inhibits PRC2, resulting in a near-total loss of H3K27me3. Similarly, inhibition of PRC2 by the aberrant expression of EZHIP results in another pediatric glioma, posterior fossa ependymoma type A (PFA). By contrast, loss-of-function mutations in two core PRC2 subunits result in Weaver syndrome, a congenital disorder marked by somatic overgrowth and cognitive delays, but not cancer. The disparate clinical phenotypes caused by these disease alleles may be the result of different modes of PRC2 inhibition and the tissue-specific developmental context in which they are expressed. This proposal seeks to leverage a combination of genetic and genomic approaches to define the importance of distinct modes of PRC2 inhibition and developmental context to clinical phenotypes. Because PRC2 is highly conserved across species, Drosophila melanogaster will be used to test the relative contributions of each of these features to disease using an array of experimental approaches. The effects of disease alleles on tissue patterning, PRC2 function, and gene regulation will be assessed in a dynamic, developmental context. Genes that modulate the effects of disease alleles, including potential therapeutic targets, will be systematically tested. These approaches bridge the expertise of the Harrison lab (gene expression and development) and the Lewis lab (chromatin regulation and cancer) to address fundamental mechanistic questions about these disease alleles. The abundant resources at the University of Wisconsin School of Medicine and Public Health and the UW Carbone Cancer Center provide an ideal location to carry out the proposed work. Together this work will define mechanistic differences between PRC2-disease alleles and will reveal underlying differences that may lead to their distinct, devastating clinical phenotypes. Completion of this research will ensure development of experimental, mentorship, clinical, and communication skills that will enable successful transition to a career as a physician-scientist.