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