The Rnf20 epigenetic modifier is required for pancreatic organogenesis and neonatal islet function

About This Grant

PROJECT SUMMARY Pancreatic islets are essential for glucagon and insulin secretion from - and -cells, respectively, to maintain glucose homeostasis. During diabetes mellitus, chronic dysglycemia results from autoimmune destruction (Type 1) or dysregulation (Type 2) of pancreatic β-cells. While treatments such as exogenous insulin manage disease symptoms, there is currently no cure. Future therapies may include the transplantation of stem cell-derived islet- like cells, but success requires a deeper understanding of pancreas development. Pancreatic organogenesis occurs in three phases – the primary, secondary, and tertiary transitions – to drive multipotent progenitor cell initiation, cellular differentiation, and islet maturation, respectively. Notably, many studies identified transcription factors (TFs) required for pancreatic organogenesis and differentiation. TF activities also require interacting transcriptional co-regulators, but the role of co-regulators in pancreas development is largely undescribed. We study the Islet-1 (Isl1) TF, which is a regulator of islet development and function. Previously, we found that Isl1 interacts with co-regulators Ring finger (Rnf)20 and Rnf40 in β-cells. RNF20 and, less robustly, Rnf40 are ubiquitin ligases that act as homo- or heterodimers required for the monoubiquitination of histone 2B, termed H2Bub1. H2Bub1 precedes H3K4 trimethylation to generally activate gene targets. Upon the deletion of Rnf20 in adult -cells, we observed -cell dysfunction, including glucose intolerance and fasting hyperglycemia. Because of the known roles for Isl1 in embryonic pancreas development and its interaction with Rnf20 in adult -cells, we tested for the requirement of Rnf20 and downstream H2Bub1 in the early embryonic mouse pancreas. Strikingly, we observed severe pancreatic dysfunction in pancreas-specific Rnf20-deficient neonates (Rnf20Flox;Pdx1-Cre - termed Rnf20panc) as indicated by hyperglycemia and loss of plasma insulin. Overall, these observations suggest a novel requirement for Rnf20 (and H2Bub1) in pancreatic organogenesis and neonatal islet function. For this training plan, I propose to investigate the role of Rnf20 during pancreas development and identify the genes directly or indirectly impacted by Rnf20. My overarching hypothesis is that Rnf20 promotes pancreatic development beginning after the initial budding stage and establishes the proliferative and morphological dynamics in the early secondary transition. I will assess pancreatic development and endocrine cell identity (e.g., insulin and glucagon) markers by immunofluorescence, as well as proliferation and apoptosis, and assess neonatal glucose homeostasis. In Aim 2, I will characterize Rnf20, Isl1, and H2Bub1 loci occupancy using CUT&RUN and assess gene expression changes impacted by Rnf20 loss via scRNA-seq. Together, I will evaluate the transcriptional consequences of pancreas-specific Rnf20 loss. The results of this research will increase our understanding of mammalian pancreas development and advance future diabetes treatments, like stem-cell therapy.