Human NEUROG3 adenoviral particles

The transcription factor Neurogenin3 is a master regulator of endocrine pancreas formation, and its deficiency causes diabetes in humans and mice. In the embryonic pancreas, Neurogenin3 is transiently expressed at high levels for a short period to initiate endocrine differentiation in dispersed progenitors. Here, researchers describe a Neurogenin3 positive autoregulatory loop by which the factor may rapidly induce its own levels. The studies show that Neurogenin3 binds to a conserved upstream fragment of its own gene, inducing the deposition of active chromatin marks and transcriptional activation of Neurog3. Furthermore, the ubiquitously expressed endoderm forkhead factors Foxa1 and Foxa2 can act synergistically to amplify Neurogenin3 autoregulation in vitro. However, only Foxa2 colocalizes with Neurogenin3 in pancreatic progenitors, suggesting that this factor plays a major role in regulating Neurogenin3 expression in vivo. Furthermore, in addition to reducing the autoregulatory effects of Neurog3, the knockdown of Foxa2 by RNA interference attenuates Neurogenin3-dependent activation of the endocrine developmental program in cultured ductal mPAC cells. Thus, these data reveal a potential functional collaboration between the endocrine lineage determinant Neurogenin3 and the ubiquitous endoderm progenitor factor Foxa2 in the implementation of the pancreatic endocrine developmental program.

To further understand the molecular cues behind Neurog3 autoactivation, the researchers assessed whether the autoregulatory circuit was dependent on the cellular context. The researchers found that among the cell lines tested, only mouse ductal mPAC and human ductal PANC-1 cells exhibited positive Neurog3 autoregulation (Figure 1A). Indeed, despite correct expression of the NEUROG3 transgene, the endocrine cell lines αTC1.6 (expressing glucagon) and MIN6 (expressing insulin), as well as NIH3T3 fibroblasts, did not induce the endogenous mouse Neurog3 gene (Figure 1A). Furthermore, Neurog3-positive nuclei were detected by immunocytochemistry using an antibody against mouse Neurog3 in mPAC cells treated with adenovirus encoding human NEUROG3, but not in control cells (Figure 1B), confirming that, in addition to mRNA induction, there was accumulation of endogenous mouse Neurog3 protein after introduction of the NEUROG3 transgene.

Figure 1. A, The indicated cell lines were infected with recombinant adenovirus expressing β-galactosidase (B) or NEUROG3 (N). B, Immunocytochemistry using a specific antibody against mouse Neurog3 was performed 48 h after treatment of mPAC and MIN6 cells with the indicated adenoviruses. (Ejarque M, et al., 2013)