ABSTRACT Insulin-secreting b-cells within the pancreatic islets of Langerhans are absolutely required for glucose homeostasis. Loss of b-cell survival or function are hallmarks of type 1 or type 2 diabetes mellitus, respectively, which affects millions of Americans, with numbers expected to greatly increase. This has created enormous economic and health care burdens. Improvements in diabetic therapies will require the deeper understanding of novel mouse and human b-cell regulators and/or pathways directly implicated in b-cell function, including glucose-stimulated insulin secretion (GSIS). The LIM-Homeodomain class transcription factor Islet-1 (Isl1) is an islet-enriched regulator of pancreatic islet cell development, maturation, and function. Despite this, little is known of the transcriptional mechanisms or protein interactors employed by Isl1 to elicit these functions in mouse or human b-cells. To identify components of Isl1 transcriptional complexes, we performed reverse-crosslinked immunoprecipitation and mass spectroscopy experiments using mouse b-cells. We found the novel Isl1 interactors Ring Finger (Rnf)20 and Rnf40, which are E3 ubiquitin ligases that act as a homo- or hetero-dimeric complex to promote transcription via specifically mono-ubiquitinating histone H2B (H2Bub1), a precursor to active histone 3 lysine 4 trimethylation (H34me3). In vitro assays with b-cell lines revealed that (at least) Rnf20 is required for the expression of many Isl1 target genes (e.g., Glut2, MafA, Ins1), as well as GSIS. Strikingly, we also found that reduction of Rnf20 or Isl1 in b-cell lines reduced H2Bub1 and H3K4me3 marks in vitro, thus linking Isl1:Rnf20 to b-cell epigenetics. Our preliminary mouse knockout data support that Rnf20 may act as a homodimer (i.e., without Rnf40) to drive Isl1-mediated target gene regulation in mouse b-cells. Therefore, in this proposal we will compare the functional and transcriptional impacts in mouse models of Isl1 or Rnf20 deficiency, as well as in human b-cells lacking ISL1, RNF20, or RNF40. Our central hypothesis is that b-cell formation and function requires deposition of the H2Bub1 modification by recruited Isl1:Rnf20. We designed three specific aims that will examine the relative functional and gene expression importance of Isl1 and Rnf20 (and thus H2Bub1) in embryonic and adult mouse b-cells, and also in adult human b-cells. Results reported from these studies will establish Rnf20 and the H2Bub1 epigenetic mark as fundamental gene regulatory effectors of Isl1, thus impacting mouse and human b-cell function. Concepts learned will yield new insight into development of novel diabetes drugs or enhance strategies to produce therapeutic b-like cells in vitro.