MECHANISMS OF EPIDERMAL HOMEOSTASIS PROJECT SUMMARY Biomolecular cues that mediate homeostasis are not fully elucidated. During the most recent funding cycle, AR043799 found that intracellular glucose increases during differentiation of diverse cell types where it controls multimerization of nucleic acid binding proteins, independent of its role in energetics. This new role for glucose is analogous to action as a second messenger. In epidermis, glucose directly bound specific RNA binding proteins (RBPs), including DDX21, as well as specific DNA binding protein (DBP) transcription factors (TFs), including IRF6, to alter their dimerization in ways essential for differentiation. To understand principles of glucose action in homeostasis, this competing renewal will define the function of glucose binding to additional RBPs and TFs. For RBPs, we found that DEAD-box DDX RBPs were among the most enriched glucose binding proteins essential for epidermal differentiation. Glucose binding dissociated DDX21 dimers, propelling DDX21 monomers out of the nucleolar rRNA production machinery into nuclear complexes controlling splicing of essential pro-differentiation mRNAs. In contrast, glucose binding altered DDX50 association with an entirely different set of interactors and had no effect on RNA splicing, indicating that glucose binding engages a diversity of pro-differentiation mechanisms for specific DDX RBPs. Aim I will characterize glucose-enabled DDX50 pro-differentiation functions as well as the impacts of glucose binding on the subcellular localization, protein interactions, RNA interactions, mRNA splicing, and RNA-dependent protein assemblies of 3 other glucose-binding RNA helicases essential for epidermal homeostasis, namely DDX1, DDX17, and DDX18. For TFs, we found that glucose binding to IRF6 – in contrast to its dissociating effects on both DDX21 and DDX50 RBP dimers - induced IRF6 homodimerization, along with IRF6 DNA binding, genomic targeting, and differentiation gene transcription. This raised the question as to how glucose impacts other glucose-binding TFs essential for epidermal homeostasis. Among the latter is the pro- differentiation TF, TFAP2A, which binds DNA as a dimer with other AP-2 subunits. Aim II will characterize the effects of glucose binding on TFAP2A dimerization and target gene induction. Epidermal differentiation is enabled by other known factors, including calcium, specific adhesion proteins, and dominant differentiation-driving TFs. Aim II will therefore also determine the interplay between physiologic elevation of intracellular glucose and differentiation enabled by representative important contextual factors in epidermal cells. This effort will define the function of glucose-binding regulators in epidermal differentiation to expand insight into the mechanistic actions of newly identified biomolecular cues in homeostasis.