Multi-site Neurogenin3 phosphorylation controls pancreatic endocrine differentiation: transcriptomic analysis of pancreatic organoids overexpressing wild type and phosphomutant Neurogenin3. Mus musculus

The proneural transcription factor Neurogenin3 (Ngn3) plays a critical role in pancreatic endocrine cell differentiation. While previous studies have focused on Ngn3 gene function, little is known about the regulation of Ngn3 protein. Here we demonstrate that Ngn3 protein undergoes cyclin-dependent kinase (cdk)-mediated phosphorylation on multiple serine-proline sites. Replacing wild-type protein with a phosphomutant form of Ngn3 increases α-cell generation, the earliest endocrine cell type to be formed in developing pancreas. Mechanistically, preventing multi-site phosphorylation enhances Ngn3 stability and DNA binding, and promotes the expression of target genes that drive differentiation. In addition to perturbing embryonic endocrine differentiation, un(der)phosphorylated Ngn3 contributes to maintaining adult β-cell differentiation in the presence of pro-proliferation cue. Here we perform transcriptomic analysis of a low number of pancreatic organoid cells expressing wild type and phosphomutant Ngn3. Analysis of the endocrine differentiation programme activated by Ngn3 in this context revealed the cell fate plasticity of the system and demonstrated that preventing Ngn3 phosphorylation enhances endocrine gene expression. Overall design: RNA-sequencing of pancreatic organoid infected with GFP-WT Ngn3 and GFP-6S-A Ngn3 (phosphomutant version of Ngn3) at 2days and 8days after induction. Control: GFP only. Samples at 2days: 3 biological replicates and 6 technical replicates. Samples at 8days: 3 biological replicates and 1-2-3 technical replicates depending on the sample