Determining the role of Nkx2.2 in the maintenance of pancreatic a cell identity - Similar to many other biological systems, maintenance of pancreatic islet cell types is governed by transcription factor function. NKX2.2 is one such transcription factor that is critical for all stages of islet cell development, including α and β cells8-12. Mice carrying Nkx2.2 null mutations have impaired differentiation of α and β cells and die shortly after birth9. In adult β cells, NKX2.2 promotes and actively maintains β cell identity by regulating several cell specific genes, including repressing the α cell master regulator Arx13. NKX2.2 is also expressed in the α cell population, but its role in maintaining α cell identity and function is not yet known9. To determine whether NKX2.2 is also important for α cell identity and function, I generated a constitutive α cell specific knockout (KO) of Nkx2.2. The preliminary in vivo data shows there is a reduction in α cell expression area that appears to be accompanied by a loss of α cell identity genes, with a concomitant gain of non-α islet cell genes. This suggests that in α cells, NKX2.2 is promoting the α cell transcriptional program, while repressing alternate endocrine cell genes. In preliminary studies to identify NKX2.2 α cell-specific DNA occupancy, an immortalized α cell line (αTC)14 was used to perform NKX2.2 chromatin immunoprecipitation coupled with next generation sequencing (ChIP-seq) and qPCR. Interestingly, this analysis identified a novel α cell-specific occupancy site in the promoter region of Arx – an α gene that is repressed by NKX2.2 in β cells. When combined with data from a pilot αTC RNA-seq of a Nkx2.2 knockdown in αTC cells that shows significant Arx downregulation, these data suggest Arx is a direct transcriptional target of NKX2.2 in α cells. Furthermore, comparisons between expression profiles of α cells versus β or δ cells shows that within α cells, NKX2.2 is binding to significantly more β or δ cell specific genes than α cell genes. This suggests that a major part of NKX2.2’s role in α cells is repressing alternate islet transcriptional programs. Lastly, NKX2.2 binds more frequently overall to promoter regions in α cells; whereas it predominantly occupies intergenic enhancer regions in β cells, suggesting global differences in NKX2.2 occupancy in α versus β cells13. Together, previous research and preliminary evidence supports the overarching hypothesis that in α cells, NKX2.2 is important for maintaining α cell identity by promoting α cell gene transcription and repressing β and δ cell genes. Aim 1 of this proposal assesses the role of NKX2.2 in the maintenance of α cell identity and function using morphometrics, gene expression analysis, and functional assays in α cell specific Nkx2.2 KO mice. Aim 2 determines the molecular mechanism of NKX2.2 identity regulation in α cells using Nkx2.2 ChIP-correlated RNA-seq to find direct targets, ChIP-seq to explore the functional epigenetics of NKX2.2 occupancy, and co-immunoprecipitation coupled with mass spectrometry (Co- IP-MS) to identify interacting factors. These experiments will further define the role of NKX2.2 in α cell maintenance and function, and NKX2.2’s molecular mechanism in α cells.
