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.
