Project Summary The hallmark of pluripotent stem cells (PSCs) is their capability to self-renew and differentiate, which is governed by the core pluripotency circuitry consisting of pluripotency factors OCT4, SOX2, and NANOG. Enhancers are fundamental in regulating the spatial and temporal expression of pluripotency genes and lineage specific genes during cellular differentiation and embryogenesis. Enhancer-regulating epigenetic modifiers play critical roles in normal physiological processes and human pathogenesis. Epigenetic marks such as H3K4me1 and H3K27ac are widely believed to regulate the activity and higher-order chromatin structure of enhancers by directly remodeling local chromatin and/or recruiting reader proteins. However, recent discoveries of catalytic- independent functions of multiple histone modifiers suggest that these enzymes govern enhancers and stem cell differentiation via a non-catalytic manner. Despite the importance of epigenetic modifiers in mammalian development and human diseases, how they sustain stem cell identity and impact human health is poorly understood. I previously demonstrated that while enhancer activation does not require the catalytic activity of histone methyltransferase MLL4 in PSCs, it is regulated by the functional antagonism between MLL4 and histone demethylase LSD1. Using state-of-the-art unbiased approaches, my lab recently unveiled novel mechanisms underlying the role of enhancer-regulating epigenetic modifiers in PSCs, providing insight into elucidating gene regulation and cell fate transition. Here, I propose to build two research areas in my laboratory focused on enhancer-regulating epigenetic modifiers. The first research area will focus on identifying catalytic-independent functions of LSD1 in governing gene expression and cellular differentiation. The second research area will focus on determining how MLL4 and its interactors modulate enhancer activity and cell fate transition. I anticipate that accomplishing the proposed studies will reveal novel mechanisms underlying enhancer regulation, decipher how stem cell self-renewal and differentiation are governed, and pave the way for understanding the pathogenesis of diseases driven by enhancer malfunction.