Significant advances in cellular reprogramming have facilitated the directed differentiation of islet beta cells from a variety of pluripotent cell populations. Furthermore, discovery of the previously unappreciated plasticity in adult islet cell populations has led to many studies demonstrating transdifferentiation/reprogramming of mature non-beta islet cell populations into beta cells, and vice versa. However, the downside of this innate islet cell plasticity is that these cells also display inherent phenotypic instability. As such, there is growing evidence in rodent and human islets for spontaneous reprogramming of beta cells in pathophysiological conditions that can lead to loss of cellular identity, phenotypic dysfunction and ultimately diabetes. In light of these advances and discoveries, the questions have shifted from “can we generate new sources of beta cells?” to “can a functional beta cell phenotype be maintained?” and “what are the transcriptional mechanisms and epigenetic landscapes that distinguish beta cells from the other islet populations?”. Our studies have demonstrated that Nkx2.2 is a critical regulator of the specification and differentiation of several defined islet cell populations in mice and humans. We also determined that Nkx2.2 is critical for the maintenance of a functional beta cell phenotype; deletion of Nkx2.2 in the beta cell causes reprogramming of beta cells to alpha, delta and PP cells. Importantly, Nkx2.2 function is required continuously throughout life to directly activate key beta cell genes, such as MafA and NeuroD1, and directly repress non-beta cell master regulator genes, such as Arx and MafB (alpha cells) and Hhex (delta cells). Nkx2.2 repressor functions are at least partially mediated through interactions with the corepressor protein Grg3/TLE3, the predominant groucho family member expressed in the islet. Since Nkx2.2 and Grg3 are also highly expressed in alpha cells, where they presumably do not repress alpha cell genes, comparative analysis of Nkx2.2/Grg activity in the two islet cell populations will provide insight into the regulation of the alpha cell versus beta cell transcriptional and epigenetic programs. We hypothesize that Nkx2.2 recruits distinct transcriptional complexes – with and without Grg3 - to the same genes in alpha versus beta cells. The goal of this application is to further dissect the Nkx2.2-mediated regulatory and epigenetic events that are essential for alpha versus beta cell identities and functions. This study will provide important new knowledge about the molecular mechanisms that regulate islet cell differentiation and identity that will 1) inform the directed differentiation of alpha versus beta cells; 2) identify the transcriptional programs that are important in the reprogramming of alpha cells to beta cells; and 3) determine the epigenetic landscapes that must be established to ensure the maintenance of distinct islet cell phenotypes in pathophysiological conditions.