Identification and characterization of MAFA coregulators: MLL3/4 and its role in mouse and human islet β-cells

Abstract

CELL AND DEVELOPMENTAL BIOLOGY

Identification and characterization of MAFA coregulators: MLL3/4 and its role in mouse and human islet β-cells

David William Scoville

Dissertation under the direction of Professor Roland Stein Pancreatic islet β-cells are critical regulators of glycemic control, and their dysfunction or absence leads to Diabetes Mellitus. Several transcription factors play crucial roles in the development of functional β-cells, including the transcription factor MafA. MafA expression is restricted to islet β-cells in the pancreas where it regulates postnatal β-cell maturation and function in the mouse. However, little is known about the mechanisms through which MafA regulates its target genes. The goal of this dissertation work was to identify coregulators which interact with and contribute to the function of the MafA transcription factor. Utilizing an unbiased proteomics approach in a mouse β-cell line, I identified several potential coregulators of MafA that could control the many activities associated with this protein, including β-cell proliferation and glucose-stimulated insulin secretion. Among them were the mixed-lineage leukemia 3 and 4 (Mll3/4) complexes, known for their role in histone 3 lysine 4 (H3K4) methylation and gene activation. MafA was bound to the Mll3/4 complexes in experiments performed with size fractionated β-cell extracts and by immunoprecipitation analysis. Likewise, the MLL3 and MLL4 complexes bound human MAFB, which is closely related to MAFA, important to mouse β-cell development, and co-produced with MAFA in adult human islet β-cells. Knockdown of NCOA6, a core subunit of these methyltransferases, reduced expression of only a small subset of MAFA and MAFB target genes in mouse and human β-cell lines. In contrast, a more profound effect on MafA/MafB gene activation was observed upon NCoA6 deletion specifically in embryonic β-cells using rat insulin promoter-driven Cre. We propose that this broader impact is due to the coordinated recruitment of the Mll3/4 complexes by MafB during development and MafA postnatally. Future studies may elucidate the context-dependent mechanisms involved in regulating MAFA and MAFB coregulator binding. This may allow for development of therapeutic strategies for enhancing β-cell function and mass in the treatment of diabetes.