Francis Lynn

Aims:Insufficient insulin release by β-cells is the primary etiology in type 2 diabetes and coincides with impaired expression of genes essential for β-cell function, but drivers of gene expression dysregulation are not well resolved. Alterations to the genome-wide enrichment and organization of chromatin post-translational modifications may promote gene expression dysregulation. Here, I investigate the role of H3K4me3 in mature β-cells and how its organization in chromatin is linked to the unique β-cell gene transcriptome in health and diabetes. I further test how its enrichment is altered by external challenges in the form of type 2 diabetes-like stresses or perturbation of one carbon metabolism.Methods:To study the functional importance of H3K4me3 in mature β-cells, we depleted H3K4me3 in β-cells of mature mice using an inducible Dpy30 deletion model under control of the Pdx1 or Ins1 promoter and performed a panel of metabolic, transcriptomic, and epigenetic tests. We compared H3K4me3 enrichment patterns with gene expression changes that occur in islets in a mouse model of type 2 diabetes and in human type 2 diabetes. We then examined the metabolic and transcriptomic consequences of folic acid restriction in mouse islets.Results:H3K4me3 contributes to gene expression in mature β-cells. H3K4me3 contributes to H3K27ac levels and, in the absence of H3K4me3, promoter-associated H3K4me1 is partially sufficient to maintain expression. H3K4me3 peak breadth is correlated with gene expression dysregulation in type 2 diabetes in mice and humans. Using a genetic mouse model to impair the methyltransferase activity of trithorax group complexes, we find that reduction of H3K4me3 reduces insulin production and glucose-responsiveness and increases transcriptional entropy. H3K4me3 in mouse β-cells is particularly required for the expression of genes that are dysregulated in a mouse model of type 2 diabetes. While locus-specific alterations are observed, global enrichment of H3K4me3 in islets is robust against external disruption of glucose homeostasis and one-carbon metabolism.Conclusions/interpretation:Overall, this thesis shows that H3K4me3 contributes to expression of genes essential for β-cell identity and function in mature β-cells and implicates dysregulation of H3K4me3 as a factor contributing to β-cell dysfunction in type 2 diabetes by altering gene expression patterns.

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The full abstract for this thesis is available in the body of the thesis, and will be available when the embargo expires.

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Pancreatic β-cells regulate systemic glycemia by releasing the glucose-lowering hormone insulin. Diabetes, a chronic metabolic disease characterized by insulin insufficiency, is linked to β-cell dysfunction with perturbed calcium homeostasis. The activity-induced, calcium-dependent transcription factor, NPAS4, reduced insulin secretion and promoted β-cell health, in part through its target gene, the GTPase-activating protein RGS2. Because our mechanistic understanding of this process remains incomplete, studying the normal physiology of calcium-dependent β-cell function may uncover new avenues for the treatment or prevention of diabetes. The overall goal of my thesis was to establish whether activity-induced NPAS4 and RGS2 expression could optimize β-cell function. Initially, I uncovered a role for CaMKII, calcineurin, and PKB in membrane depolarization-induced Npas4 mRNA and protein expression in MIN6 cells and mouse islets. Calcineurin inhibition and concurrent loss of NPAS4 showed cytotoxic increases in cleaved caspase 3 expression, which was reversed by adenovirally reinstating NPAS4 in MIN6 cells. Co-immunoprecipitation studies in MIN6 cells then uncovered competition between NPAS4 and a related transcription factor, HIF1α, for the shared heterodimerization partner, ARNT. Accordingly, HIF1α target gene expression was lower in human and mouse islets overexpressing Npas4, and higher in β-cell-specific Npas4 knockout mouse islets (N4KO). Because excessive HIF1α signalling compromises β-cell function by switching energy production from oxidative phosphorylation to anaerobic glycolysis, I examined whether N4KO mice developed functional defects. Indeed, N4KO islets showed lower oxygen consumption rate, and HFD-fed N4KO mice developed mild glucose intolerance. To understand how NPAS4 may counteract these defects, I identified shared DNA binding sites of NPAS4 and ARNT in MIN6 cells using ChIP-Seq. Among the shared sites, I observed NPAS4 and ARNT binding near Rgs2, corroborating an earlier study. I then demonstrated that RGS2 is a negative regulator of glucose-stimulated insulin secretion (GSIS), because Rgs2-overexpressing MIN6 cells and mouse islets showed reduced GSIS, due to lower calcium influx and oxygen consumption, whereas Rgs2 knockout cells exhibited increased GSIS. In sum, I demonstrated that NPAS4 and its target gene, RGS2, are important regulators of β-cell function. This suggests that these two factors could be promising therapeutic targets to promote β-cell health and optimize insulin secretion in diabetes.

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Diabetes is caused by a loss or dysfunction of insulin-producing pancreatic beta-cells. A potential treatment for diabetes is to replace these cells through transplantation. As there is a shortage of donor tissue, efforts to generate an unlimited source of functional insulin-producing beta-cells from human embryonic stem cells (hESCs) are ongoing. During pancreas development, proliferating pancreatic progenitors activate Neurog3, exit the cell cycle, and differentiate. The overarching goal of this thesis was to understand the role of the cell cycle in regulating Neurog3 expression and endocrine cell fate. First, the length of each cell cycle phase of pancreatic progenitors was measured using cumulative EdU labelling, determining an increase in G1 length in Pdx1+ progenitors from 4.5±0.4 to 7.2±0.8 hours between embryonic day (E)11.5 and E13.5. Next, two mouse models were used to show that cell cycle lengthening within pancreatic progenitors stimulates endocrine differentiation. Kras heterozygous loss-of-function mice have increased endocrine cell genesis that was correlated with an increase in progenitor cell cycle length. Ectopic expression of the cyclin-dependent kinase inhibitor Cdkn1b in Sox9+ progenitor cells resulted in a 2.7-fold increase in the number of Neurog3+ cells. As Cdkn1b is an inhibitor of G1-S cyclin-dependent kinases (Cdks), the effect of directly inhibiting Cdk2, Cdk4 and Cdk6 on endocrine differentiation was investigated. Treating embryonic pancreata, ex vivo, for 24 hours with Cdk inhibitors resulted in a 3-fold increase in the number of Neurog3+ cells. To investigate the consequences of CDK inhibition on human endocrine differentiation, a NEUROG3-2A-eGFP (N5-5) knock-in reporter CyT49 hESC line was generated using CRISPR-Cas9. CDK inhibition increased the number of GFP+ endocrine progenitor cells 1.7-fold. These findings suggest that G1 lengthening is required for normal mouse and human organogenesis and that cyclin-dependent kinases act directly to reduce Neurog3 protein. In the final chapter, single-cell transcriptomics was used to profile the gene expression and cell populations present during mouse and human endocrine development. In conclusion, these studies show that progenitor cell-cycle G1 lengthening, through its actions on stabilization of Neurog3, is an essential determinant of normal endocrine cell genesis.

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Pancreatic beta-cells (β-cells) are essential for the maintenance of blood glucose homeostasis, as the primary insulin-secreting cells of the body. During embryogenesis, β-cells differentiate from pancreatic progenitor cells, and following birth, these cells re-enter the cell-cycle and proliferate to maintain a sufficient adult population of β-cells. Transcription factors (TFs) such as neurogenin3 (Neurog3) are essential for endocrine cell specification within the pancreas, while other TFs are required in adult β-cells to maintain their function. Despite the identification of many TFs throughout β-cell development, how TFs regulate the transition between cell states, and how these TFs engage the RNA Polymerase II holoenzyme to regulate transcription is unknown. To address these questions, this thesis examines the role of Sry-related HMG-box 4 (SOX4) and Mediator 15 (MED15) in β-cell development and the adult β-cell state.Work in this thesis has established that in mice, SOX4 is expressed in pancreatic progenitor cells and cooperates with NEUROG3 to activate Neurog3 expression. This demonstrated a requirement for SOX4 in endocrine progenitor cell specification. SOX4 continued to be expressed in endocrine specified cells, and was essential for Neurod1 and Pax4 induction, TFs required for β-cell specification. High-fat diet (HFD)-fed mice with inducible SOX4 deletion in β-cells also exhibited glucose intolerance, due to decreased β-cell mass and replication rate. Loss of Sox4 led to the upregulation of the cell-cycle inhibitor Cdkn1a, a gene that prevents G1-S cell-cycle transition. Additionally, Med15 deletion in pancreatic progenitors demonstrated reduced NEUROG3 expression, and reduced endocrine cell numbers. MED15 deletion following endocrine specification also led to reduced β-cell numbers. Finally, Ins1-Cre facilitated deletion of MED15 in β-cells revealed that its function varied depending on cell-state, with compromised β-cell function if expression is lost during β-cell maturation. These data are the first to determine when SOX4 is required for pancreatic endocrine specification in mice and which targets are directly regulated by SOX4. In addition, the first known in-vivo role for MED15 in mammals is identified, demonstrating that it is an indispensable factor for β-cell differentiation and function. Collectively, these findings contribute to the understanding of how TFs regulate β-cell states.

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Type 2 diabetes is characterized by hyperglycemia associated with reduced insulin secretion from pancreatic beta cells and impaired insulin sensitivity at peripheral target tissues. There is a growing body of evidence that supports the importance of bHLH-PAS domain transcription factors in promoting beta cell function. With the recent identification of neuronal PAS domain protein 4 (NPAS4) within the central nervous system, studies were undertaken to determine whether NPAS4 is expressed in beta cells, how its expression is regulated in response to changing environmental signals and uncover the functional significance of NPAS4 in the maintenance of glucose homeostasis. Together, experiments within this thesis demonstrate that NPAS4 is expressed within the pancreatic beta cell and is rapidly upregulated in response to membrane depolarization and calcium influx. Further, this induction was impaired in a mouse model of beta cell dysfunction and within islets from individuals with T2D. Overexpression studies performed in vitro identified NPAS4 as a novel negative regulator of insulin expression and GLP-1 potentiated insulin secretion. Furthermore, NPAS4 protected beta cells from maladaptive cellular pathways that promote cell dysfunction and death; including endoplasmic reticulum stress and activation of HIF1α. Finally, the characterization of three different Npas4 mouse knockout models suggests that continued NPAS4 expression in the beta cell is required to maintain differentiation status and cellular function. An independent role for NPAS4 in the maintenance of glucose homeostasis was also discovered in other Pdx1-Cre expressing cells, likely within the hypothalamus. Together, the data suggest beta cells induce NPAS4 expression during periods of cellular activity and acts as a protective factor to protect cells in order to promote the maintenance of euglycemia.

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