The beta cell transcription factor Nkx6.1 is critical in pancreatic progenitor cells and mature beta cells. The pathways regulated by Nkx6.1 that results in these phenotypes are still being defined. We sought to define the transcriptional factors 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. 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. These potential Nkx6.1 interacting proteins are enriched in nucleotide binding, ribosomal proteins, and transcription factors. Among the enriched transcription factors, we validated that Pdx1 is biotinylated in response to transduction with AdCMV-Nkx6.1 using biotin-targeted immunoprecipitation and western blot. To examine if Pdx1 is likely to interact with Nkx6.1 on the genome, we analyzed the frequency of proximal binding sites for Nkx6.1 and Pdx1, and demonstrated that Pdx1 and Nkx6.1 binding sites in close proximity were statistically overrepresented in the genome. 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 significant Pdx1 binding by Nkx6.1 Co-IP and significant Nkx6.1 binding by Pdx1 Co-IP, as compared to Co-IP with the IgG control. Finally, we demonstrate coregulation of Unc3 expression by Nkx6.1 and Pdx1 and demonstrate that CRISPR mediated deletion of either Nkx6.1 or Pdx1 is sufficient to impair INS-1 832/13 mediated expression of Unc3. These data define potential Nkx6.1 binding partners in INS-1 832/13 cells, as well as novel interaction between Nkx6.1 and Pdx1 in the regulation of Unc3.


J.S.Tessem: None. C.C.Littlefield: None. J.T.Hill: None.

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