Genome-wide sequencing technologies have allowed an unprecedented discovery of somatic mutations in epigenetic modifiers in human cancers, providing mechanistic links between cancer epigenomes and genetic alterations. The collective number of oncogenic activating mutations in epigenetic regulators has led to the emerging view of "driver mutations" underlying cancer epigenomes. Nowhere is this better illustrated than with recent findings of high-frequency missense mutations in core histones, such as histone H3 lysine 27 to methionine (H3K27M) and glycine 34 to arginine/valine (G34R/V) mutations in pediatric gliomas, and H3K36M mutations in pediatric chondroblastomas, particularly aggressive cancers that remain poorly understood and for which there are no effective therapies. Our biochemical studies suggest that the 'K-to-M' mutant histones can inhibit the enzymatic activity of responsible histone methyltransferases (HMTs), such as Ezh2 for H3K27 methylation, and SETD2 for H3K36 methylation. Oddly, histone mutations and HMT mutations are never found in the same type of cancer. These observations lead us to hypothesize that H3 'K-to-M' mutations play distinct functions beyond just inactivation of HMTs, which may be key to the lineage-specific pathogenesis of the respective cancers. Here, we propose multidisciplinary and integrative approaches, using genetics (cell line and mouse models), epigenetics (ChIP-seq and RNA-seq), proteomics (quantitative mass spectrometry) and chemical biology ("designer chromatin") to gain mechanistic insights into how a "mutated" histone code functions towards disrupting epigenetic landscapes that, in turn, lead to cancer progression. A world-class team of experts in cancer, chromatin and chemical biology are assembled to explore novel approaches to these devastating childhood cancers. The single goal of our Program is to illuminate the molecular mechanisms underlying "oncohistone" mutations to advance the diagnosis and exploration of therapeutic avenues for the associated pediatric cancers. Specifically, we will: i) investigate how histone mutations affect the cross-talk between other histone and DNA modifications; ii) identify the changes in chromatin landscape by histone mutations using cell-based systems, animal models and patient tumor samples; iii) characterize misregulated developmental programs that help establish tumorigenesis; and iv) specifically engineer chemically-defined chromatin templates for use in in vitro biochemical reactions aimed at a detailed mechanistic dissection of how histone mutations alter HMT activities. These studies will provide guidance for the development of therapeutic strategies designed to ameliorate the pathogenic effects of histone mutations and HMTs in these childhood cancers. Also, novel immunological reagents will be generated for much-needed immunohistochemistry (IHC) diagnosis of tumor samples.