Project Summary MITF, particularly the melanocyte-restricted m-MITF isoform, is a key transcription factor that regulates melanocyte differentiation, survival, and pigmentation. Its expression is dynamically regulated by the MC1R receptor through the PKA signaling pathway. For decades, it was thought that CREB1 was the PKA-controlled transcription factor that drove m-MITF transcription. However, I have discovered that a non-canonical mechanism activates m-MITF independent of CREB1 and its redundant paralogs. This mechanism requires the transcriptional co-activator CRTC, which must be recruited to m-MITF promoter DNA through an unknown factor. We propose here to define this non-canonical mechanism, discover its unknown regulators, and characterize the precise molecular interactions required for m-MITF transcriptional activation. Specifically, we will 1) generate a toolkit of mutant cell lines designed to perturb select events in PKA signaling and dissect the differences between non-canonical and canonical regulation, 2) perform functional genomic screens to unveil new regulators of m- MITF using cellular proliferation and transcriptional reporter readouts, and 3) investigate physical interactions between identified regulators and the m-MITF promoter to develop a molecular model for the non-canonical mechanism. As a feature of each of these aims, we will apply our findings to genetically modified, iPSC-derived primary melanocytes and organotypic skin reconstitutions to address this mechanism's role in physiological tissue pigmentation. This work will deepen our understanding of the molecular biology of pigmentation and give insight into PKA signaling in other specialized cell types. It will lay the foundation for molecular approaches to manipulate m-MITF in a melanocyte-specific manner for the chemoprevention and treatment of numerous sun- and pigmentation-related conditions, including skin cancer and aging.