Title: Structure-function analysis of the essential islet regulatory factor Nkx2.2
Description: The specification and differentiation of the pancreatic beta cell lineage requires guidance by spatiotemporally regulated signaling cues and a highly orchestrated set of transcription factors. Defining the factors and their regulatory functions that are required for proper beta cell development will enhance our ability to recapitulate these developmental events in vitro to generate beta cells from alternate cell sources. The homeodomain transcription factor Nkx2.2 is essential for pancreatic endocrine cell development; Nkx2.2-/- mice lack all beta cells and have reductions in alpha and pancreatic polypeptide (PP) cells. In place of these cell populations, the Nkx2.2-/- null islet is replete with ghrelin-producing epsilon cells. An Nkx2.2-repressor fusion protein derivative (Pdx1:Nkx2.2-EnR) expressed in the Nkx2.2-/- background can fully rescue the alpha cell population, but can only specify a few immature beta cells, suggesting that Nkx2.2 must contain both repressor and activator functions to properly guide beta cell development.

Accordingly, Nkx2.2 has been shown to be an activator of several beta-cell targets. It has also been demonstrated that the corepressor Grg3 is expressed in the endocrine population and can physically interact with Nkx2.2, which points toward a mechanism by which Nkx2.2 confers transcriptional repression; however, the genes targeted by Nkx2.2/Grg3 are unknown. Additionally, how Nkx2.2 can both repress and activate genes in the same cellular context, and differentially regulate the same gene in different cellular contexts, is not understood. In this dissertation, I sought to determine the regulatory role of Nkx2.2 in the developing pancreas and its dependence on Grg interactions, and to elucidate whether post-translational modifications play a role in modulating Nkx2.2 regulatory activities. By analyzing mice carrying knock-in mutations in the Nkx2.2 Grg-interaction domain (Nkx2.2TNmut/TNmut), I show that the interaction between Nkx2.2 and Grg protein is required at two developmental stages of beta cell development: 1) Grg-mediated Nkx2.2 repression is necessary for correct beta-cell specification, and 2) the recruitment of Grg by Nkx2.2 is required to repress Arx in the beta cells to prevent beta-to-alpha cell reprogramming.

Additionally, by analyzing the Nkx2.2TNmut/TNmut and Nkx2.2TNmut/TNmut;Ins:Cre;Arxfl/fl mice, I have identified several additional genes that may be regulated by Grg-mediated Nkx2.2 repression. Finally, I also present data to suggest that Nkx2.2 protein is phosphorylated, and that the phosphorylation state determines whether Nkx2.2 functions as an activator or a repressor in a promoter-specific context. These studies have begun to elucidate the complex regulatory roles that Nkx2.2 plays in specifying and maintaining the beta-cell lineage. Future analyses will help us to better understand the spatiotemporal regulatory activities that are required to make and maintain functional beta cells.