Molecular mechanisms of NKX2.2 function in adult human beta cells - PROJECT SUMMARY. Loss of pancreatic β cell function and/or mass is central to the development of type 2 diabetes (T2D). Understanding how β cell function is normally regulated in adult human islets will help elucidate mechanisms of dysfunction in T2D, which are not well understood. A large body of work in mouse models suggests that the islet-enriched transcription factor (TF) NKX2.2 is a critical regulator of β cell development and plays a role in the maintenance of adult β cell function. Further, patients with loss-of-function NKX2-2 mutations have neonatal diabetes, highlighting an important role of NKX2.2 in human islet development. However, the role of NKX2.2 in adult human β cells remain undefined. Interestingly, we found increased insulin secretion from primary human pseudoislets following global NKX2-2 knockdown, suggesting different roles of NKX2.2 across species and developmental stages. We hypothesize that, in adult human islets, NKX2.2 regulates insulin secretion via transcriptional repression of β cell-intrinsic pathways. To test this hypothesis, we will first determine the role of NKX2.2 in adult human islet function in a β cell-specific manner. Using florescence-activated cell sorting and CRISPR/Cas9 technology, we will perform targeted knockout of NKX2-2 in adult β cells in primary human pseudoislets. We will assess β cell intracellular Ca2+ signaling events and function in vitro using an integrated live cell imaging and microfluidic platform. To evaluate the impact of chronic loss of NKX2.2, we will examine pseudoislet function in vivo following transplantation into immunodeficient mice. Results of this aim will determine the impact of NKX2.2 on β cell-intrinsic pathways that lead to insulin secretion. Secondly, we will define molecular mechanisms of NKX2.2 function in adult human β cells using a single nucleus (sn)RNA-seq+ ATAC-seq multiome approach on the same nucleus. snRNA-seq will determine if NKX2.2 functions as a transcriptional repressor of insulin secretory machinery in β cells. In combination, snATAC-seq will reveal how NKX2.2 alters chromatin accessibility to regulate the β cell transcriptome. To study the impact of chronic NKX2-2 knockout on β cell phenotype and function, we will analyze harvested pseudoislet transplants for changes in proteins corresponding to top differentially expressed genes of interest. This aim will provide mechanistic insight into how NKX2.2 regulates adult human β cell gene transcription and function at the chromatin and transcript level. Overall, these studies will reveal molecular mechanisms of NKX2.2 function in adult human β cells, with implications for new therapeutic approaches to improve β cell function in T2D. Training under this fellowship will be enhanced by a rich environment, including a large community of islet biology investigators under the NIH-funded Vanderbilt Diabetes Research and Training Center, collaborations with experts in the field, and a variety of opportunities to promote career development, leadership, and scientific communication. Together, the proposed research, training plan, and environment will provide a strong foundation on which to base a career as a physician-scientist.
