PROJECT SUMMARY Diabetes is characterized by the loss of functional pancreatic β-cell mass. With the prevalence of diabetes increasing across the globe, it is crucial that we develop a complete understanding of the gene regulatory mechanisms governing the maintenance of insulin-producing β-cell identity and function. In our previous work, we demonstrated that the Isl1 transcription factor and its histone-modifying co-regulators Rnf20 and Rnf40 are crucial for β-cell gene expression. Recently, we observed that in mice, β-cell-specific loss of Rnf20 (Rnf20Δβ-cell) prompts a disruption of glucose homeostasis, loss of β-cell identity gene expression, and an upregulation of cell cycle inhibitor mRNA, p19Arf. In aging human and mouse β-cells, cell cycle inhibitors encoded by Cdkn2a, p19Arf and p16Ink4a, are upregulated and impart a reduction in functional capacity known as senescence. While suppression of the age-associated upregulation in Cdkn2a expression is known to reverse β-cell senescence, the transcriptional mechanisms that repress Cdkn2a in healthy β-cells are understudied. In this proposal, I will investigate the regulatory relationship between Rnf20 and Cdkn2a to assess whether Rnf20 mediates β-cell senescence. My primary hypothesis is that Rnf20 protects against senescence-associated loss of β-cell functional identity through direct regulation of the Cdkn2a locus. I will address this hypothesis with two aims. In Aim 1, I will evaluate how the β-cell specific loss of Rnf20 affects the β-cell cycle and the appearance of the senescence-associated secretory phenotype. I will also perform systemic senolytic treatment to determine if targeted elimination of senescent cells rescues the glucose intolerance and reduced plasma insulin levels that occur in Rnf20Δβ-cell mice. In Aim 2, I will determine whether Rnf20 regulates the transcription of Cdkn2a. I will perform Chromatin Immunoprecipitation (ChIP) to assess if Rnf20 directly occupies the Cdkn2a promoter and ATAC-seq to see if loss of Rnf20 increases Cdkn2a chromatin accessibility. Our results will increase understanding of the transcriptional mechanisms regulating both the maintenance of β-cell identity and the process of β-cell senescence. Additionally, our outcomes may be used to inform future diabetes therapies through either the incorporation of Rnf20 into either β-cell differentiation protocols or as a biomarker for β-cell health. Furthermore, the proposed research plan provides a balanced training platform wherein I will expand both my critical thinking and technical skillset through my investigations of novel regulatory mechanisms of β- cell homeostasis. As a trainee at the University of Alabama at Birmingham (UAB) and in UAB’s Diabetes Research Center, I have access to a strong community of supportive faculty members to assist in the successful completion of the proposed study. Taken together, this research and training plan is a fundamental catalyst of my trajectory toward becoming an i...