Multi-site Neurogenin3 Phosphorylation Controls Pancreatic Endocrine Differentiation

Roberta Azzarelli,Christopher Hurley,Magdalena K Sznurkowska,Steffen Rulands,Laura Hardwick,Ivonne Gamper,Fahad Ali,Laura Mccracken,Christopher Hindley,Fiona Mcduff,Sonia Nestorowa,Richard Kemp,Kenneth Jones,Berthold Göttgens,Meritxell Huch,Gerard Evan,Benjamin D Simons,Douglas Winton,Anna Philpott

doi:10.1016/j.devcel.2017.04.004

Abstract

SummaryThe proneural transcription factor Neurogenin3 (Ngn3) plays a critical role in pancreatic endocrine cell differentiation, although regulation of Ngn3 protein is largely unexplored. 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 the developing pancreas. Moreover, un(der)phosphorylated Ngn3 maintains insulin expression in adult β cells in the presence of elevated c-Myc and enhances endocrine specification during ductal reprogramming. Mechanistically, preventing multi-site phosphorylation enhances both Ngn3 stability and DNA binding, promoting the increased expression of target genes that drive differentiation. Therefore, multi-site phosphorylation of Ngn3 controls its ability to promote pancreatic endocrine differentiation and to maintain β cell function in the presence of pro-proliferation cues and could be manipulated to promote and maintain endocrine differentiation in vitro and in vivo.

Highlights

While endocrine cells represent only 1%–5% of the entire pancreas, they play a crucial role in physiological processes including glucose homeostasis; loss or dysfunction of b cells can lead to diabetes
Focusing here on its post-translational modification, we show that Ngn3 is phosphorylated on multiple serine-proline (SP) sites by cyclin-dependent kinases (Cdks)
We investigated the functional consequences of preventing Cdk-dependent Ngn3 phosphorylation during pancreas formation

Summary

Introduction

While endocrine cells represent only 1%–5% of the entire pancreas, they play a crucial role in physiological processes including glucose homeostasis; loss or dysfunction of b cells can lead to diabetes. In vitro differentiation and transplantation of bona-fide functional b cells can regulate blood sugar in diabetic mouse models (Pagliuca et al, 2014; Rezania et al, 2014), while b cell protection or replacement in diabetic patients may improve long-term glycemic control. A fasting and refeeding regime may lead to b cell regeneration, with improvement of blood sugar regulation (Cheng et al, 2017). Ngn is a central component of transcription factor reprogramming cocktails that can trans-differentiate adult exocrine acinar cells into functional b cells (Zhou et al, 2008), while cytokine-induced in vivo reprogramming of acinar cells into b cells requires the transition through a Ngn3-positive stage, indicative of reversion back to a progenitor-like phase that re-instates developmental programs of endocrine differentiation (Baeyens et al, 2014). Ngn is required for adult b cell function (Wang et al, 2009)

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