Project Summary NANOG Structure and Function in Stem Cell Pluripotency NANOG directs access to stem cell pluripotency. There are considerable gaps in our molecular understanding of how NANOG coordinates this process. This is also compounded by the fact that NANOG (in particular, human NANOG) has been a challenging system to work with. The investigators have successfully applied innovative fluorescence techniques to characterize NANOG’s key structure and functional properties. NANOG is an intrinsically disordered protein (IDP), with a prion-like domain critical for NANOG self-assembly and for establishing contacts in pluripotency specific regions. NANOG is a ‘molecular hub’, reported to interact with >100 transcription factors (TFs) and other proteins to form cooperative hub condensates and coordinate activation of pluripotency genes and repression of differentiation genes. The investigators will investigate and deconvolute how NANOG dose-sensitivity play a role in chromatin looping and recruitment of TFs, co-activators, and epigenetic regulators. They will use an intensive holistic combination of standard and innovative structural, molecular, genomic and cell biology approaches. The first aim will elucidate NANOG’s dose-sensitivity mechanism in establishing pluripotency by assessing chromatin reorganization and recruitment of cofactors using Hi-C 3.0 and single molecule fluorescence techniques. The second aim will probe the molecular elements (i.e., OCT4-SOX2-NANOG triad cooperativity and KLF4 biomolecular condensation) involved in activating NANOG expression. In addition, the aim will investigate the molecular basis for NANOG autorepression. The last aim will focus on inhibition of NANOG stabilization and oligomerization with dominant negative NANOG-based peptides. These studies will provide unprecedented insights into how NANOG orchestrates establishment of stem cell pluripotency and reversal of developmental aging.