PROJECT SUMMARY Signaling proteins, including G-protein-coupled receptors (GPCRs), impact genome modulators, including ATP-dependent chromatin remodelers (ADCRs), to control gene expression in homeostasis and disease. The signaling and chromatin regulators operative in epidermis, however, have not been fully characterized, in part due to the hundreds of genes that comprise each of these classes of proteins. This cycle, AR049737 knocked out all 101 GPCR and 116 ADCR genes expressed in epidermis to identify essential roles for the ADGRL2 adhesion GPCR as well as for ncBAF and SRCAP ADCRs in controlling epidermal differentiation. This revised competing renewal will define the mechanistic basis for these new essential actions, sustaining the long-term focus of AR049737 in characterizing signaling and genome regulators of epidermal homeostasis. First, we will test a working model in which the ADGRL2 GPCR is activated by FLRT3 and/or TENM2 from adjacent cells to induce pro-differentiation signals, which are modulated by specific proteins that AR049737 recently found associated with the ADGRL2 cytoplasmic domain in differentiating keratinocytes. These proteins include the RapGEF2 guanine nucleotide exchange factor and the PPP2R2A phosphatase, which are posited to enable ADGRL2-pro-differentiation signaling, and the RASAL2 GTPase-activating protein, which is postulated to oppose it. Aim I will test key features of this model using genetic approaches in tissue to illuminate the mechanistic basis for the actions of the ADGRL2 GPCR in epidermal differentiation. Second, we will study the premise that the diverse impacts of ADCRs are enabled by the specific ADCR-associated proteins that target their actions across the genome. These efforts focus on ncBAF, which AR049737 found to be essential for induction of epidermal differentiation genes and SRCAP, which AR049737 found to be essential to suppress ectopic differentiation in keratinocyte progenitors. We will define the impact of DNA sequence-specific transcription factors (TFs) on ncBAF and SRCAP targeting and activity in epidermal homeostasis, including IRF6, which co- regulates nBAF differentiation genes, and SNAI2, which co-suppresses SRCAP differentiation targets. Aim II will thus use genomics approaches to define the molecular mechanisms responsible for the opposite impacts of ncBAF and SRCAP ADCRs in epidermal differentiation. At the end of proposed funding, this effort will have defined mechanistic features of newly identified essential roles for GPCRs and ADCRs in the control of epidermal homeostasis.