PROJECT ABSTRACT UTX/KDM6A is an important tumor suppressor and developmental regulator, and is frequently mutated in human cancers and certain neurodevelopmental disorders including Kabuki syndrome. Though UTX is a histone H3K27 demethylase, the demethylase activity is often dispensable in mediating tumor suppression and developmental regulation, and the key molecular activity of UTX in these processes remains elusive. UTX is thought to regulate chromatin activity by associating with and coordinating the function of MLL3/KMT2C and MLL4/KMT2D, the major H3K4 mono-methylase at enhancers, and the p300 histone acetyltransferase, all of which are also frequently mutated in human cancers. In our preliminary studies, we found that the tumor suppressive activity of UTX requires its phase separation property conferred by its core Intrinsically Disordered Region (cIDR). Endogenous UTX forms dynamic condensates in mouse embryonic stem cells (ESCs) and its cIDR is important for ESC differentiation. UTX and MLL4 form co-condensates that enhance the H3K4 mono-methylation activity. We also found that UTY, the Y chromosome homolog of UTX, has weaker tumor suppressive activity that is associated with formation of less dynamic condensates. These results let us formulate our central hypothesis that UTX needs to be in condensates with appropriate biophysical properties to be active in regulating gene expression on chromatin in regulating tumorigenesis and stem cell differentiation. We proposal two Specific Aims. Aim 1. Determine the mechanisms by which UTX condensation regulates chromatin activity. We will determine how UTX condensation regulates chromatin modification activities by biochemical reconstitution, and also through integrative analyses of multiple levels of chromatin activity in both transduced cancer cells and endogenous Utx-edited ESCs. Aim 2. Determine how biophysical properties of the UTX condensates regulate its biological activities. We will study biophysical properties of UTX WT and variant condensates and its associated co-activators. We will determine impacts of condensate properties on biological activities at different levels in in both transduced cancer cells and endogenous Utx-edited ESCs. We will also examine effect of disease-associated missense mutations of UTX on condensate properties.