1242-P: Defining the Nkx6.1 Interactome in Beta Cells Reveals a Novel Interaction with Pdx1

The beta cell transcription factor Nkx6.1 plays a critical role in pancreatic progenitor cell differentiation, mature beta cell proliferation and insulin secretion. The pathways regulated by Nkx6.1 that results in these phenotypes are still being defined. Given that transcription factors generally function in complexes, we sought to determine proteins that interact with Nkx6.1 and may be harnessed to expand functional beta cell mass. INS-1 832/13 beta cells were transduced with AdCMV-Nkx6.1-BioID, AdCMV-GFP-BioID or left untreated. Biotinylated proteins were isolated and the Nkx6.1 interactome was defined using liquid chromatography-tandem mass spectrometry (LC-MS/MS). We defined 176 biotinylated proteins in cells transduced with AdCMV-Nkx6.1-BioID that were not observed with AdCMV-GFP-BioID or naturally biotinylated proteins from untreated lysates (P≤0.05, n=9). These 176 proteins are enriched in terms of nucleotide binding (P=3.6E-5), ribosomal proteins (P=9.1E-4), and transcription factors (P=4.3E-4). Among the enriched transcription factors, we validated that Pdx1, Mef2d and Nr2c2 were biotinylated in response to transduction with AdCMV-Nkx6.1 using biotin-targeted immunoprecipitation and western blot. To examine if these transcription factors are likely to interact with Nkx6.1 on the genome, we analyzed the frequency of proximal binding sites for Nkx6.1 and our candidate genes. Nr2c2 and Pdx1 binding sites in close proximity to Nkx6.1 binding sites were statistically overrepresented in the genome (P≤0.01). Given the role of Pdx1 on functional beta cell mass, we further characterized the interaction of endogenous Nkx6.1 and Pdx1 by co-immunoprecipitation (Co-IP). Our data demonstrates a significant Pdx1 binding by Nkx6.1 Co-IP, as compared to Co-IP with the IgG control (P=0.015, n=3). These data define potential Nkx6.1 binding partners in INS-1 832/13 cells, as well as a novel interaction between two essential beta cell transcription factors, Nkx6.1 and Pdx1.