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

ABSTRACT: 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.

ORGANISM(S): Mus musculus

PROVIDER: GSE96707 | GEO | 2017/06/09

SECONDARY ACCESSION(S): PRJNA379454

REPOSITORIES: GEO

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Project description:Aims/hypothesis: Duct cells isolated from adult human pancreas can be reprogrammed to express islet beta cell genes by adenoviral transduction of the developmental transcription factor neurogenin3 (Ngn3). In this study we aimed to fully characterize the extent of this reprogramming and intended to improve it. Methods: The extent of the Ngn3-mediated duct-to-endocrine cell reprogramming was measured employing genome wide mRNA profiling. By modulation of the Delta-Notch signaling or addition of pancreatic endocrine transcription factors Myt1, MafA and Pdx1 we intended to improve the reprogramming. Results: Ngn3 stimulates duct cells to express a focused set of genes that are abundant in islet endocrine cells and/or neural tissues. This neuro-endocrine shift, however, covers a minor fraction (5%) of the estimated genome-wide transcriptome difference between duct and islet endocrine cells. Interestingly, transduction of exogenous Ngn3 activates endogenous Ngn3 suggesting auto-activation of this gene. Furthermore, pancreatic endocrine reprogramming of human duct cells can be moderately enhanced by inhibition of Delta-Notch signaling as well as by co-expressing the transcription factor Myt1, but not MafA and Pdx1. Conclusions/interpretation: The results provide further insight into the plasticity of adult human duct cells and suggest measurable routes to enhance Ngn3-mediated in vitro reprogramming protocols for regenerative beta cell therapy in diabetes. We used Affymetrix HG133A and HG133B to get a comprehensive view on the reprogramming potential in vitro of human pancreatic duct cell cultures (n=3-4) at 3 and 14/20 days after ectopic adenoviral expression of murine neurogenin 3 as compared to GFP-expressing control vectors. The microarray analysis was performed on 3 independent samples that each contained RNA extracted from a pool of 3 independent donor pancreata. The total number of non-selected donor organs is 9. Transcripts were considered as differentially regulated by Ngn3 when 1.5 fold (LCB, unpaired P < 0.05) up- or down-regulated in AdGFP-Ngn3 versus AdGFP controls, at 3 and/or 14 dpi. Transcripts that showed differential expression between day 3 and day 14 in AdGFP-Ngn3 duct cells but not in AdGFP control cells, were also considered Ngn3-regulated

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

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