Advisory Committee Chair
Chad S Hunter
Advisory Committee Members
Hubert M Tse
Kirk M Habegger
Adam R Wende
Bradley K Yoder
Document Type
Dissertation
Date of Award
2020
Degree Name by School
Doctor of Philosophy (PhD) Heersink School of Medicine
Abstract
The Ldb1:Isl1 LIM transcriptional complex is critical for endocrine pancreas development as well as b-cell terminal differentiation. These complexes have been shown to interact with various proteins to elicit transcriptional regulation in other tissues, yet few interactors have been identified in b-cells. Therefore, the objective of this dissertation was to identify interactors of Ldb1 and Isl1 and assess their contributions to endocrine pancreas development and function. Using a reversible crosslink immunoprecipitation (ReCLIP) and mass spectrometry (MS) strategy to isolate endogenous Ldb1 and/or Isl1 interacting protiens, SSBP3, Rnf20, and Rnf40 (E3 ubiquitin ligases) coregulators were identified. SSBP3 was found to interact with both Ldb1 and Isl1 in mouse b-cell lines and in mouse and human islets. Immunofluorescence demonstrated coexpression of SSBP3 with Ldb1 and Isl1 in human and mouse islets. b-cell knockdown of SSBP3 imparted similar mRNA deficiencies exhibited in Ldb1 knockdowns. Rnf20 and Rnf40 however, were found to only interact with Isl1 in mouse bcells and in human islets. Knockdown of Rnf20 or Rnf40 led to reductions in monoubiquitination of histone H2b (H2bub1) and histone H3 lysine 4 trimethylation (H3K4me3). Surprisingly, reductions of H2bub1 and H3K4me3 marks were also exhibited in Isl1 and SSBP3 knockdowns, suggesting they have novel epigenetic roles. Furthermore, glucose stimulated insulin secretion was significantly reduced in Rnf20-, Rnf40-, SSBP3-, and Isl1-deficient mouse b-cells. The observation that Ldb1 possessed Isl1-independent effects led me to assess if other LIM homeodomain transcription factors (LIM-HD) were interacting in the endocrine pancreas to impact b-cell development and function. LIM homebox 1 (Lhx1), an Isl1-related LIM-HD transcription factor appeared to be expressed in the developing and the adult pancreas. Lhx1 knockdown and chromatin immunoprecipitation (ChIP) assays in a mouse b-cell line demonstrated shared regulation of Ldb1 and Isl1 target gene, Glp1r, which encodes the glucagon like peptide 1 receptor. I next determined the in vivo contributions of Lhx1, by generating a pancreas wide knockout model, Lhx1ΔPanc. Lhx1ΔPanc mice exhibited altered glucose tolerance, elevated α-cell mRNAs, as well as reduced Glp1R. My findings identify new interactors of the Ldb1:Isl1 LIM transcriptional complex that impact b-cell development and function.
Recommended Citation
Bethea, Maigen, "Lim Transcriptional Complexes That Impact Endocrine Pancreas Development And Function" (2020). All ETDs from UAB. 733.
https://digitalcommons.library.uab.edu/etd-collection/733