Project Summary Cancer epigenetics is an emerging field of high significance. It has led to the identification of novel therapeutic targets and biomarkers. Among chromatin remodeling complexes, driver mutations of the MLL family histone methyltransferases are commonly found in cancer. They lead to epigenetic aberrations that underlie cancer initiation, evolution, and metastasis. Understanding how the activity of the MLL family enzymes on chromatin is regulated and how its dysregulation leads to malignancy will shed light on disease mechanisms. We previously reconstituted a functionally active MLL1 core complex in vitro and identified four MLL1 core complex components, i.e. ASH2L, RbBP5, WDR5 and DPY30, which are essential for MLL1 enzymatic activity. Here we show that a novel function of ASH2L in MLL1 regulation and a previously uncharacterized mode of action. We will use biochemical, structural and epigenomic approaches to delineate how ASH2L confers new complexity to regulation of H3K4 methylation and gene expression. We expect to establish a new paradigm on how epigenetic heterogeneity is established by a well-defined MLL1 core complex, which also has general implications for other chromatin modifying enzymes. Our study will ultimately benefit discovery of novel therapeutic targets or biomarkers for cancer diagnosis and treatment. This project matches the mission of National Cancer Institute (NCI).