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Multi-site Neurogenin3 Phosphorylation Controls Pancreatic Endocrine Differentiation.

ABSTRACT: The 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.

SUBMITTER: Azzarelli R

PROVIDER: S-EPMC5425251 | biostudies-literature |

REPOSITORIES: biostudies-literature

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

Project description: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.

2017-06-09 | GSE96707 | GEO

Neurogenin3 phosphorylation controls reprogramming efficiency of pancreatic ductal organoids into endocrine cells.

Project description:β-cell replacement has been proposed as an effective treatment for some forms of diabetes, and in vitro methods for β-cell generation are being extensively explored. A potential source of β-cells comes from fate conversion of exocrine pancreatic cells into the endocrine lineage, by overexpression of three regulators of pancreatic endocrine formation and β-cell identity, Ngn3, Pdx1 and MafA. Pancreatic ductal organoid cultures have recently been developed that can be expanded indefinitely, while maintaining the potential to differentiate into the endocrine lineage. Here, using mouse pancreatic ductal organoids, we see that co-expression of Ngn3, Pdx1 and MafA are required and sufficient to generate cells that express insulin and resemble β-cells transcriptome-wide. Efficiency of β-like cell generation can be significantly enhanced by preventing phosphorylation of Ngn3 protein and further augmented by conditions promoting differentiation. Taken together, our new findings underline the potential of ductal organoid cultures as a source material for generation of β-like cells and demonstrate that post-translational regulation of reprogramming factors can be exploited to enhance β-cell generation.

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Role of Neurogenin3 phosphorylation in the reprogramming of pancreatic organoids into endocrine cells

Project description:During pancreatic development, Neurogenin3 (Ngn3) promotes the differentiation of endocrine cells, including insulin-producing β-cells. Our previous work has shown that Ngn3 phosphorylation on multiple serine-proline (SP) sites increases protein stability and endocrine differentiation (Azzarelli et al., DevCell 2017). Here, we aim to exploit our recent data to investigate whether manipulation of Ngn3 phosphostatus might improve in vitro generation of β-cells for replacement therapies in diabetes. A potential source of β-cells comes from fate conversion of exocrine pancreatic cells into the endocrine lineage, by overexpression of 3 transcription factors, Ngn3, Pdx1 and MafA. Using this approach we show that pancreatic ductal cells grown in vitro as 3D organoids can be reprogrammed to express insulin. The efficiency can be strongly enhanced by substituting wild type Ngn3 with a the un(der)phosphorylated and more active Ngn3 form (6S-A Ngn3). Here we perform transcriptomic analysis of a low number of pancreatic organoid cells expressing wild type or phosphomutant Ngn3, alone or in various combination Pdx1 and/or MafA. The analysis revealed endocrine differentiation and in particular β-like cell gene expression in the conditions where Ngn3 was in combination with both Pdx1 and MafA.

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Project description:The superfamily of basic-Helix-Loop-Helix (bHLH) transcription factors influence cell fate in all three embryonic germ layers, and the tissue-specific class II factors have received prominent attention for their potent ability to direct differentiation during development and in cellular reprogramming. The activity of many class II bHLH proteins driving differentiation, and the inhibitory class VI bHLH factor Hes1, is controlled by phosphorylation on multiple sites by Cyclin-dependent kinases (Cdks). As class II proteins are generally thought to be active through hetero-dimerisation with the ubiquitously expressed class I E proteins, regulation of class I transcription factors such as E47 may influence the activity of multiple tissue-specific bHLH proteins. Using differentiation of nerve and muscle in Xenopus frog embryos as a model system, we set out to explore whether with the ubiquitously expressed class I E protein E47 that hetero-dimerises with Class II bHLHs to control their activity, is also regulated by multi-site phosphorylation. We demonstrate that E47 can be readily phosphorylated by Cdks on multiple sites in vitro, while ectopically-expressed E47 exists in multiple phosphorylated forms in Xenopus embryos. Preventing multi-site phosphorylation using a phospho-mutant version of E47 enhances the neurogenic and myogenic activity of three different class II bHLH reprogramming factors, and also when E47 acts in hetero-dimerisation with endogenous proteins. Mechanistically, unlike phospho-regulation of class II bHLH factors, we find that preventing phosphorylation of E47 increases the amount of chromatin-bound E47 protein but without affecting its overall protein stability. Thus, multi-site phosphorylation is a conserved regulatory mechanism across the bHLH superfamily that can be manipulated to enhance cellular differentiation.

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Differentiation of opioid drug effects by hierarchical multi-site phosphorylation.

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Neurogenin3 inhibits proliferation in endocrine progenitors by inducing Cdkn1a.

Project description:During organogenesis, the final size of mature cell populations depends on their rates of differentiation and expansion. Because transient expression of Neurogenin3 (Neurog3) in progenitor cells in the developing pancreas initiates their differentiation to mature islet cells, we examined the role of Neurog3 in cell cycle control during this process. We found that mitotically active pancreatic progenitor cells in mouse embryos exited the cell cycle after the initiation of Neurog3 expression. Transcriptome analysis demonstrated that the Neurog3-expressing cells dramatically up-regulated the mRNA encoding cyclin-dependent kinase inhibitor 1a (Cdkn1a). In Neurog3 null mice, the islet progenitor cells failed to activate Cdkn1a expression and continued to proliferate, showing that their exit from the cell cycle requires Neurog3. Furthermore, induced transgenic expression of Neurog3 in mouse ?-cells in vivo markedly decreased their proliferation, increased Cdkn1a levels, and eventually caused profound hyperglycemia. In contrast, in Cdkn1a null mice, proliferation was incompletely suppressed in the Neurog3-expressing cells. These studies reveal a crucial role for Neurog3 in regulating the cell cycle during the differentiation of islet cells and demonstrate that the subsequent down-regulation of Neurog3 allows the mature islet cell population to expand.

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Role of Neurogenin3 phosphorylation in the reprogramming of pancreatic organoids into endocrine cells

Project description:Role of Neurogenin3 phosphorylation in the reprogramming of pancreatic organoids into endocrine cells

| PRJNA472788 | ENA

Gene Signatures of NEUROGENIN3+ Endocrine Progenitor Cells in the Human Pancreas.

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