Epidermal homeostasis requires a balance of growth and differentiation, a balance which is disrupted in skin cancer. We undertook exome and RNA-sequencing of squamous cell carcinomas (SCCs) to characterize regulators of epidermal homeostasis that are disrupted in skin cancer. We identified recurrent abnormalities in protein coding as well as non-coding genes. 4 major categories of protein-coding genes were recurrently mutated, including receptor tyrosine kinase (RTK)-Ras-MAPK pathway genes, Notch regulators, chromatin remodelers and Mediator genes. Parallel RNA-seq identified 11 new SCC Mis-Regulated Transcripts (SMRTs), among top aberrantly expressed genes in SCC, most of which are long non-coding RNAs (lncRNAs). This competing renewal will characterize the role of these genes in normal epidermal growth and differentiation and in early tumor progression. First, we will characterize the impact of protein-coding genes on homeostasis and early tumor progression, focusing on novel members of each category found to be mutated at high frequency in SCCs. Among these are the ROS1 RTK, the Notch pathway regulators GXYLT1 and DNER, the CHD3 and SMARCC1 chromatin regulators, and the MED12 Mediator. Human epidermal tissue with SCC-associated alterations in these genes will be generated and analyzed. Aim I will test a synergy model in which RTK-Ras-MAPK growth regulators interact with differentiation control by Notch pathway regulators, chromatin remodelers and Mediator transcriptional regulators to control homeostasis and early neoplastic progression. Second, we will characterize new SCC Mis-Regulated Transcripts (SMRTs). These entirely new, previously unannotated and hitherto unidentified SMRT genes encode RNAs that rank among the top dysregulated transcripts in SCC. The first functional studies of any SMRT indicate that SMRT-2 is required for epidermal differentiation. We will characterize the function of all 11 SMRT genes in epidermal homeostasis and early tumor progression. To begin mechanistic characterization of each SMRT that proves functional, we will then apply new high throughput methodologies designed to yield insight into lncRNA function by identifying the biomolecules that bind them. Aim II will test the hypothesis that multiple SMRT genes regulate epidermal homeostasis and early tumor progression and will provide initial insight into mechanism by elucidating the molecular interactome of individual SMRTs. At the end of proposed funding, we plan to have gained further insight into the control of skin homeostasis as a foundation for future strategies for cancer prevention and treatment.