Structure dictates dynamic topology and function of pancreatic transcriptional regulators - Summary Reductions in β-cell mass underlie the pathogenesis of all forms of diabetes, raising the relevance of understanding the mechanisms controlling postnatal β-cell growth. Transcriptional networks regulate the development, differentiation, and expansion of β cells, operating through islet enhancers, super-enhancers and promoters forming 3-dimensional hubs. The homeodomain transcription factor and diabetes gene Pdx1 is a critical member of this network, playing roles in β-cell differentiation, proliferation, and function. Despite the importance of Pdx1 for β-cell growth, knowledge of the topological and biophysical properties of Pdx1 that regulate β-cell mass are unclear. Our preliminary data reveal that β cells exhibit altered subnuclear localization and reduced levels of Pdx1 protein as they advance through the cell cycle. Further, ectopically elevated levels of Pdx1 prevent cell cycle progression and increase β-cell death, suggesting that dynamic regulation of expression is required for effective β-cell expansion and maintenance of functional β-cell mass. We identify an intrinsically disordered protein region (IDPR) of unknown function in the Pdx1 C-terminus (aa 207-223). IDPRs, commonly found within transcription factors, lack fixed secondary structure and are amenable to flexible conformations and phase separation. IDPRs promote protein-protein interactions and transcriptional hub formation at super enhancers necessary for coordinated gene regulation. We previously identified the E3 ubiquitin ligase substrate adaptor protein SPOP as a PDX1 C-terminus protein partner (via aa224-238) that mediates ubiquitination and proteasomal degradation of PDX1. SPOP binds other IDPR partners in phase separated nuclear compartments critical for their function. Thus, we hypothesize that the IDPR and SPOP interaction domains within the Pdx1 C-terminus play critical, possibly interdependent, roles in Pdx1 protein localization, expression, and function crucial for β-cell gene expression and proliferation. This hypothesis will be tested in three Aims: (1) To investigate the dynamic subnuclear localization of PDX1 during the cell cycle and in response to glucose. (2) To determine the role of IDPR-regulated phase separation in the function and localization of PDX1. (3) To determine the role of SPOP in fine tuning PDX1 protein level and function. Our studies will determine a novel and cohesive role for unstudied structural features of the Pdx1 C-terminus and how they influence β-cell growth and glycemic control. Results of our proposed studies will inform therapeutic efforts to optimize β-cell mass expansion ex vivo for cell based therapies and in vivo to treat patients with diabetes.
