Effects of overexpressed Atoh8 on the transcriptional profile of mouse ductal cells mPAC in the absence or presence of co-expressed Neurogenin3

The basic helix-loop-helix (bHLH) transcription factors of the Drosophila’s atonal-related superfamily Neurogenin3 (Neurog3) and NeuroD1 promote endocrine differentiation in the gastrointestinal tract. Atonal Homolog 8 (Atoh8/Math6) is a newly identified member of the atonal-related family whose expression is induced by Neurog3 and NeuroD1 in cell culture, indicating a possible role for this gene in the endocrine differentiation program downstream of these two pro-endocrine factors. Intriguingly, available experimental evidence based on a reduced number of genes suggests that Atoh8 may negatively regulate Neurog3-targeting events. In this study, we have analyzed global changes in gene expression profiles upon exogenous expression of Atoh8 alone or in combination with Neurog3 in mouse pancreatic duct (mPAC) cells. These cells activate neuroendocrine-specific gene expression in response to Neurog3 and NeuroD1 and thus serve as an optimal model to evaluate the proendocrine activity of Atoh8. We have compared transcriptional profiles between mPAC cells treated with a recombinant adenovirus expressing Atoh8 (Ad-Atoh8) or a control adenovirus encoding B-galactosidase (Ad-Bgal), and between cells treated with Ad-Neurog3+Ad-Bgal or cells treated with Ad-Neurog3+Ad-Atoh8. The results obtained show that Atoh8 exhibits a very modest transcriptional activity in these cells thus confirming that Atoh8 does not function as a proendocrine gene. Furthermore, our data also confirm the ability of Atoh8 to block Neurog3-dependent transcriptional activation events. However, since repression is only seen for a small subset of Neurog3 gene targets, we discard a general role of Atoh8 as a negative regulator of Neurog3 pro-endocrine activity. mPAC cells (mouse pancreatic duct cells) were divided in 6 groups according to the treatment received: 1) non-treated (control for the infection effect), 2) treated with recombinant adenoviruses coding for β-galactosidase (control for infection with one adenovirus), 3) treated with recombinant adenoviruses coding for Atonal Homolog 8, 4) treated with recombinant adenoviruses coding for Neurogenin 3, 5) treated with recombinant adenoviruses coding for β-galactosidase and Neurogenin 3 (control for the effect of two adenoviruses, being one of them Neurogenin 3), 6) treated with recombinant adenoviruses coding for Neurogenin 3 and Atonal Homolog 8. Experiment was performed three independent times (3 independent biological replicates).