Summary
Reductions in ß-cell mass underlie the pathogenesis of all forms of diabetes, raising the relevance of
understanding the mechanisms controlling postnatal ß-cell growth. Transcriptional networks regulate the
development, differentiation, and expansion of ß 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 ß-cell differentiation, proliferation, and function. Despite the
importance of Pdx1 for ß-cell growth, knowledge of the topological and biophysical properties of Pdx1 that
regulate ß-cell mass are unclear. Our preliminary data reveal that ß 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 ß-cell death, suggesting that dynamic regulation of
expression is required for effective ß-cell expansion and maintenance of functional ß-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 ß-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 ß-cell growth and glycemic control. Results of our proposed studies will inform therapeutic
efforts to optimize ß-cell mass expansion ex vivo for cell based therapies and in vivo to treat patients with
diabetes.