PROJECT SUMMARY Medulloblastoma (MB) is the most common malignant primary brain tumor in children. MB is frequently induced by the alterations of cellular signaling pathways, such as sonic hedgehog and wingless pathways, which have been extensively characterized. Nevertheless, current treatment of MB causes severe life-long side effects and fails to cure many patients. Thus, there is an unmet need for a new mechanistic understanding that would be helpful for designing a mechanism-based approach for MB treatment. Epigenetic aberrations, which are heritable aberrations in gene expression or cellular phenotypes without accompanying changes in DNA sequences, are a major factor for tumorigenesis. Epigenetic modifiers often harbor DNA alterations, such as mutations and deletions, in human MB. However, the roles of epigenetic modifiers in MB development remain largely unknown. Histone lysine methylation, a type of histone posttranslational modification, is a hallmark of epigenetic and transcriptional regulation of gene expression and is reversibly modified by histone methyltransferases and demethylases. Of histone lysine methylation, methylations at histone H3 lysine 4 (H3K4) are key gene-activating epigenomic marks. For example, monomethyl H3K4 is a mark for enhancers, which activate genes by interacting with gene promoters. In addition, trimethyl H3K4 occupies as much as 75 % of all human gene-regulatory regions, and broad trimethyl H3K4 is a gene-activating signature that denotes tumor suppressor and cell identity genes. We have previously reported that the H3K4 methyltransferase KMT2D (also called MLL4, ALR, and MLL2; a transcriptional coactivator) is required for retinoic acid-induced neuronal differentiation of human neuron-lineage NT2/D1 stem cells. Notably, our other study showed that homozygous loss of Kmt2d in the mouse brain developed spontaneous MB in the cerebellum, a brain region that controls motor coordination and balance. Strikingly, our additional results showed that heterozygous loss (single-allelic) of Kmt2d highly promoted MB. Based on these compelling findings, our long-term goal is to define the oncogenic role of heterozygous loss of Kmt2d in MB pathogenesis. Our central hypothesis is that heterozygous loss of Kmt2d causes epigenomic alterations to downregulate tumor suppressor genes and thereby promotes MB. Here, we propose to study to 1) characterize the MB-promoting effect of heterozygous Kmt2d loss using genetically engineered mouse models; 2) define the molecular mechanism by which heterozygous Kmt2d loss promotes MB; and 3) determine how heterozygous Kmt2d loss causes epigenomic alterations. Because KMT2D is one of the most frequently mutated genes in MB and a majority of KMT2D mutations in MB are heterozygous and truncations, our proposed studies are significant and clinically relevant. In addition, our studies using genetically engineered mouse models will define an in vivo MB-promoting role for heterozygous Kmt2d loss. Fu...