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KCNQ1/Kv7, a low-voltage-gated K+ channel, regulates cardiac rhythm and glucose homeostasis. While KCNQ1 mutations are associated with long-QT syndrome and type2 diabetes, its function in human pancreatic cells remains controversial. We identified a homozygous KCNQ1 mutation (R397W) in an individual with permanent neonatal diabetes melitus (PNDM) without cardiovascular symptoms. To decipher the potential mechanism(s), we introduced the mutation into human embryonic stem cells and generated islet-like organoids (SC-islets) using CRISPR-mediated homology-repair. The mutation did not affect pancreatic differentiation, but affected channel function by increasing spike frequency and Ca2+ flux, leading to insulin hypersecretion. With prolonged culturing, the mutant islets decreased their secretion and gradually deteriorated, modeling a diabetic state, which accelerated by high glucose levels. The molecular basis was the downregulated expression of voltage-activated Ca2+ channels and oxidative phosphorylation. Our study provides a better understanding of the role of KCNQ1 in regulating insulin secretion and β-cell survival in hereditary diabetes pathology.

Insulin is an essential regulator of blood glucose homeostasis that is produced exclusively byβcells within the pancreatic islets of healthy individuals. In those affected by diabetes, immune inflammation, damage, and destruction of isletβcells leads to insulin deficiency and hyperglycemia. Current efforts to understand the mechanisms underlyingβcell damage in diabetes rely onin vitro-cultured cadaveric islets. However, isolation of these islets involves removal of crucial matrix and vasculature that supports islets in the intact pancreas. Unsurprisingly, these islets demonstrate reduced functionality over time in standard culture conditions, thereby limiting their value for understanding native islet biology. Leveraging a novel, vascularized micro-organ (VMO) approach, we have recapitulated elements of the native pancreas by incorporating isolated human islets within a three-dimensional matrix nourished by living, perfusable blood vessels. Importantly, these islets show long-term viability and maintain robust glucose-stimulated insulin responses. Furthermore, vessel-mediated delivery of immune cells to these tissues provides a model to assess islet-immune cell interactions and subsequent islet killing-key steps in type 1 diabetes pathogenesis. Together, these results establish the islet-VMO as a novel,ex vivoplatform for studying human islet biology in both health and disease.

Insulin is an essential regulator of blood glucose homeostasis that is produced exclusively byβcells within the pancreatic islets of healthy individuals. In those affected by diabetes, immune inflammation, damage, and destruction of isletβcells leads to insulin deficiency and hyperglycemia. Current efforts to understand the mechanisms underlyingβcell damage in diabetes rely onin vitro-cultured cadaveric islets. However, isolation of these islets involves removal of crucial matrix and vasculature that supports islets in the intact pancreas. Unsurprisingly, these islets demonstrate reduced functionality over time in standard culture conditions, thereby limiting their value for understanding native islet biology. Leveraging a novel, vascularized micro-organ (VMO) approach, we have recapitulated elements of the native pancreas by incorporating isolated human islets within a three-dimensional matrix nourished by living, perfusable blood vessels. Importantly, these islets show long-term viability and maintain robust glucose-stimulated insulin responses. Furthermore, vessel-mediated delivery of immune cells to these tissues provides a model to assess islet-immune cell interactions and subsequent islet killing-key steps in type 1 diabetes pathogenesis. Together, these results establish the islet-VMO as a novel,ex vivoplatform for studying human islet biology in both health and disease.

Dysfunctional pancreatic islet beta cells are a hallmark of type 2 diabetes (T2D), but a comprehensive understanding of the underlying mechanisms, including gene dysregulation, is lacking. Here we integrate information from measurements of chromatin accessibility, gene expression and function in single beta cells with genetic association data to nominate disease-causal gene regulatory changes in T2D. Using machine learning on chromatin accessibility data from 34 nondiabetic, pre-T2D and T2D donors, we identify two transcriptionally and functionally distinct beta cell subtypes that undergo an abundance shift during T2D progression. Subtype-defining accessible chromatin is enriched for T2D risk variants, suggesting a causal contribution of subtype identity to T2D. Both beta cell subtypes exhibit activation of a stress-response transcriptional program and functional impairment in T2D, which is probably induced by the T2D-associated metabolic environment. Our findings demonstrate the power of multimodal single-cell measurements combined with machine learning for characterizing mechanisms of complex diseases.

Pancreatic islet cells derived from human pluripotent stem cells hold great promise for modeling and treating diabetes. Differences between stem-cell-derived and primary islets remain, but molecular insights to inform improvements are limited. Here, we acquire single-cell transcriptomes and accessible chromatin profiles during in vitro islet differentiation and pancreas from childhood and adult donors for comparison. We delineate major cell types, define their regulomes, and describe spatiotemporal gene regulatory relationships between transcription factors. CDX2 emerged as a regulator of enterochromaffin-like cells, which we show resemble a transient, previously unrecognized, serotonin-producing pre-β cell population in fetal pancreas, arguing against a proposed non-pancreatic origin. Furthermore, we observe insufficient activation of signal-dependent transcriptional programs during in vitro β cell maturation and identify sex hormones as drivers of β cell proliferation in childhood. Altogether, our analysis provides a comprehensive understanding of cell fate acquisition in stem-cell-derived islets and a framework for manipulating cell identities and maturity.

Adaptation of the islet β cell insulin-secretory response to changing insulin demand is critical for blood glucose homeostasis, yet the mechanisms underlying this adaptation are unknown. Here, we have shown that nutrient-stimulated histone acetylation plays a key role in adapting insulin secretion through regulation of genes involved in β cell nutrient sensing and metabolism. Nutrient regulation of the epigenome occurred at sites occupied by the chromatin-modifying enzyme lysine-specific demethylase 1 (Lsd1) in islets. β Cell-specific deletion of Lsd1 led to insulin hypersecretion, aberrant expression of nutrient-response genes, and histone hyperacetylation. Islets from mice adapted to chronically increased insulin demand exhibited shared epigenetic and transcriptional changes. Moreover, we found that genetic variants associated with type 2 diabetes were enriched at LSD1-bound sites in human islets, suggesting that interpretation of nutrient signals is genetically determined and clinically relevant. Overall, these studies revealed that adaptive insulin secretion involves Lsd1-mediated coupling of nutrient state to regulation of the islet epigenome.

The Integrated Physiology of the Exocrine and Endocrine Compartments in Pancreatic Diseases workshop was a 1.5-day scientific conference at the National Institutes of Health (Bethesda, MD) that engaged clinical and basic science investigators interested in diseases of the pancreas. This report provides a summary of the proceedings from the workshop. The goals of the workshop were to forge connections and identify gaps in knowledge that could guide future research directions. Presentations were segregated into six major theme areas, including 1) pancreas anatomy and physiology, 2) diabetes in the setting of exocrine disease, 3) metabolic influences on the exocrine pancreas, 4) genetic drivers of pancreatic diseases, 5) tools for integrated pancreatic analysis, and 6) implications of exocrine-endocrine cross talk. For each theme, multiple presentations were followed by panel discussions on specific topics relevant to each area of research; these are summarized here. Significantly, the discussions resulted in the identification of research gaps and opportunities for the field to address. In general, it was concluded that as a pancreas research community, we must more thoughtfully integrate our current knowledge of normal physiology as well as the disease mechanisms that underlie endocrine and exocrine disorders so that there is a better understanding of the interplay between these compartments.

Insufficient insulin secretion is a hallmark of type 2 diabetes and has been attributed to beta cell identity loss characterized by decreased expression of several key beta cell genes. The pro-inflammatory factor BMP-2 is upregulated in islets of Langerhans from individuals with diabetes and acts as an inhibitor of beta cell function and proliferation. Exposure to BMP-2 induces expression of Id1-4, Hes-1, and Hey-1 which are transcriptional regulators associated with loss of differentiation. The aim of this study was to investigate the mechanism by which BMP-2 induces beta cell dysfunction and loss of cell maturity. Mouse islets exposed to BMP-2 for 10 days showed impaired glucose-stimulated insulin secretion and beta cell proliferation. BMP-2-induced beta cell dysfunction was associated with decreased expression of cell maturity and proliferation markers specific to the beta cell such as Ins1, Ucn3, and Ki67 and increased expression of Id1-4, Hes-1, and Hey-1. The top 30 most regulated proteins significantly correlated with corresponding mRNA expression. BMP-2-induced gene expression changes were associated with a predominant reduction in acetylation of H3K27 and a decrease in NeuroD1 chromatin binding activity. These results show that BMP-2 induces loss of beta cell maturity and suggest that remodeling of H3K27ac and decreased NeuroD1 DNA binding activity participate in the effect of BMP-2 on beta cell dysfunction.

We combined functional genomics and human genetics to investigate processes that affect type 1 diabetes (T1D) risk by mediating beta cell survival in response to proinflammatory cytokines. We mapped 38,931 cytokine-responsive candidate cis-regulatory elements (cCREs) in beta cells using ATAC-seq and snATAC-seq and linked them to target genes using co-accessibility and HiChIP. Using a genome-wide CRISPR screen in EndoC-βH1 cells, we identified 867 genes affecting cytokine-induced survival, and genes promoting survival and up-regulated in cytokines were enriched at T1D risk loci. Using SNP-SELEX, we identified 2,229 variants in cytokine-responsive cCREs altering transcription factor (TF) binding, and variants altering binding of TFs regulating stress, inflammation, and apoptosis were enriched for T1D risk. At the 16p13 locus, a fine-mapped T1D variant altering TF binding in a cytokine-induced cCRE interacted with SOCS1, which promoted survival in cytokine exposure. Our findings reveal processes and genes acting in beta cells during inflammation that modulate T1D risk.

The transition from lean to obese states involves systemic metabolic remodeling that impacts insulin sensitivity, lipid partitioning, inflammation, and glycemic control. Here, we have taken a pharmacological approach to test the role of a nutrient-regulated chromatin modifier, lysine-specific demethylase (LSD1), in obesity-associated metabolic reprogramming. We show that systemic administration of an LSD1 inhibitor (GSK-LSD1) reduces food intake and body weight, ameliorates nonalcoholic fatty liver disease (NAFLD), and improves insulin sensitivity and glycemic control in mouse models of obesity. GSK-LSD1 has little effect on systemic metabolism of lean mice, suggesting that LSD1 has a context-dependent role in promoting maladaptive changes in obesity. In analysis of insulin target tissues we identified white adipose tissue as the major site of insulin sensitization by GSK-LSD1, where it reduces adipocyte inflammation and lipolysis. We demonstrate that GSK-LSD1 reverses NAFLD in a non-hepatocyte-autonomous manner, suggesting an indirect mechanism potentially via inhibition of adipocyte lipolysis and subsequent effects on lipid partitioning. Pair-feeding experiments further revealed that effects of GSK-LSD1 on hyperglycemia and NAFLD are not a consequence of reduced food intake and weight loss. These findings suggest that targeting LSD1 could be a strategy for treatment of obesity and its associated complications including type 2 diabetes and NAFLD.

AbstractThe "Integrated Physiology of the Exocrine and Endocrine Compartments in Pancreatic Diseases" Workshop was a 1.5-day scientific conference at the National Institutes of Health (Bethesda, MD) that engaged clinical and basic science investigators interested in diseases of the pancreas. This report summarizes the workshop proceedings. The goal of the workshop was to forge connections and identify gaps in knowledge that could guide future research directions. Presentations were segregated into 6 major themes, including (a) Pancreas Anatomy and Physiology; (b) Diabetes in the Setting of Exocrine Disease; (c) Metabolic Influences on the Exocrine Pancreas; (d) Genetic Drivers of Pancreatic Diseases; (e) Tools for Integrated Pancreatic Analysis; and (f) Implications of Exocrine-Endocrine Crosstalk. For each theme, there were multiple presentations followed by panel discussions on specific topics relevant to each area of research; these are summarized herein. Significantly, the discussions resulted in the identification of research gaps and opportunities for the field to address. In general, it was concluded that as a pancreas research community, we must more thoughtfully integrate our current knowledge of the normal physiology as well as the disease mechanisms that underlie endocrine and exocrine disorders so that there is a better understanding of the interplay between these compartments.

Detailed characterization of human pancreatic islets is key to elucidating the pathophysiology of all forms of diabetes, especially type 2 diabetes. However, access to human pancreatic islets is limited. Pancreatic tissue for islet retrieval can be obtained from brain-dead organ donors or from individuals undergoing pancreatectomy, often referred to as living donors. Different protocols for human islet procurement can substantially impact islet function. This variability, coupled with heterogeneity between individuals and islets, results in analytical challenges to separate genuine disease pathology or differences between human donors from experimental noise. There are currently no international guidelines for human donor phenotyping, islet procurement and functional characterization. This lack of standardization means that substantial investments from multiple international efforts towards improved understanding of diabetes pathology cannot be fully leveraged. In this Perspective, we overview the status of the field of human islet research, highlight the challenges and propose actions that could accelerate research progress and increase understanding of type 2 diabetes to slow its pandemic spreading.

ObjectiveThe contribution of beta-cell dysfunction to type 2 diabetes (T2D) is not restricted to insulinopenia in the late stages of the disease. Elevated fasting insulinemia in normoglycemic humans is a major factor predicting the onset of insulin resistance and T2D, demonstrating an early alteration of beta-cell function in T2D. Moreover, an early and chronic increase in fasting insulinemia contributes to insulin resistance in high-fat diet (HFD)-fed mice. However, whether there are genetic factors that promote beta-cell-initiated insulin resistance remains undefined. Human variants of the mitochondrial transporter ABCB10, which regulates redox by increasing bilirubin synthesis, have been associated with an elevated risk of T2D. The effects of T2D ABCB10 variants on ABCB10 expression and the actions of ABCB10 in beta-cells are unknown.MethodsThe expression of beta-cell ABCB10 was analyzed in published transcriptome datasets from human beta-cells carrying the T2D-risk ABCB10 variant. Insulin sensitivity, beta-cell proliferation, and secretory function were measured in beta-cell-specific ABCB10 KO mice (Ins1Cre-Abcb10flox/flox). The short-term role of beta-cell ABCB10 activity on glucose-stimulated insulin secretion (GSIS) was determined in isolated islets.ResultsCarrying the T2Drisk allele G of ABCB10 rs348330 variant was associated with increased ABCB10 expression in human beta-cells. Constitutive deletion of Abcb10 in beta-cells protected mice from hyperinsulinemia and insulin resistance by limiting HFD-induced beta-cell expansion. An early limitation in GSIS and H2O2-mediated signaling caused by elevated ABCB10 activity can initiate an over-compensatory expansion of beta-cell mass in response to HFD. Accordingly, increasing ABCB10 expression was sufficient to limit GSIS capacity. In health, ABCB10 protein was decreased during islet maturation, with maturation restricting beta-cell proliferation and elevating GSIS. Finally, ex-vivo and short-term deletion of ABCB10 in islets isolated from HFD-fed mice increased H2O2 and GSIS, which was reversed by bilirubin treatments.ConclusionsBeta-cell ABCB10 is required for HFD to induce insulin resistance in mice by amplifying beta-cell mass expansion to maladaptive levels that cause fasting hyperinsulinemia.

BackgroundThe pancreatic β cell, as the sole source of the vital hormone insulin, has been under intensive study for more than a century. Given the potential of newly created insulin-producing cells as a treatment or even cure of type 1 diabetes (T1D) and possibly in severe cases of type 2 diabetes (T2D), multiple academic and commercial laboratories are working to derive surrogate glucose-responsive, insulin-producing cells.Scope of reviewThe recent development of advanced phenotyping technologies, including molecular, epigenomic, histological, or functional, have greatly improved our understanding of the critical properties of human β cells. Using this information, here we summarize the salient features of normal, fully functional adult human β cells, and propose minimal criteria for what should rightfully be termed 'β cells' as opposed to insulin-producing but not fully-functional surrogates that we propose should be referred to as 'β-like' cells or insulin-producing cells.Major conclusionsClear criteria can be established to differentiate fully functional, mature β cells from 'β-like' surrogates. In addition, we outline important knowledge gaps that must be addressed to enable a greater understanding of the β cell.

Genes involved in distinct diabetes types suggest shared disease mechanisms. Here we show that One Cut Homeobox 1 (ONECUT1) mutations cause monogenic recessive syndromic diabetes in two unrelated patients, characterized by intrauterine growth retardation, pancreas hypoplasia and gallbladder agenesis/hypoplasia, and early-onset diabetes in heterozygous relatives. Heterozygous carriers of rare coding variants of ONECUT1 define a distinctive subgroup of diabetic patients with early-onset, nonautoimmune diabetes, who respond well to diabetes treatment. In addition, common regulatory ONECUT1 variants are associated with multifactorial type 2 diabetes. Directed differentiation of human pluripotent stem cells revealed that loss of ONECUT1 impairs pancreatic progenitor formation and a subsequent endocrine program. Loss of ONECUT1 altered transcription factor binding and enhancer activity and NKX2.2/NKX6.1 expression in pancreatic progenitor cells. Collectively, we demonstrate that ONECUT1 controls a transcriptional and epigenetic machinery regulating endocrine development, involved in a spectrum of diabetes, encompassing monogenic (recessive and dominant) as well as multifactorial inheritance. Our findings highlight the broad contribution of ONECUT1 in diabetes pathogenesis, marking an important step toward precision diabetes medicine.

Pancreatic β-cells secrete insulin commensurate to circulating nutrient levels to maintain normoglycemia. The ability of β-cells to couple insulin secretion to nutrient stimuli is acquired during a postnatal maturation process. In mature β-cells the insulin secretory response adapts to changes in nutrient state. Both β-cell maturation and functional adaptation rely on the interplay between extracellular cues and cell type-specific transcriptional programs. Here we review emerging evidence that developmental and homeostatic regulation of β-cell function involves collaboration between lineage-determining and signal-dependent transcription factors (LDTFs and SDTFs, respectively). A deeper understanding of β-cell SDTFs and their cognate signals would delineate mechanisms of β-cell maturation and functional adaptation, which has direct implications for diabetes therapies and for generating mature β-cells from stem cells.

Genetic risk variants that have been identified in genome-wide association studies of complex diseases are primarily non-coding1. Translating these risk variants into mechanistic insights requires detailed maps of gene regulation in disease-relevant cell types2. Here we combined two approaches: a genome-wide association study of type 1 diabetes (T1D) using 520,580 samples, and the identification of candidate cis-regulatory elements (cCREs) in pancreas and peripheral blood mononuclear cells using single-nucleus assay for transposase-accessible chromatin with sequencing (snATAC-seq) of 131,554 nuclei. Risk variants for T1D were enriched in cCREs that were active in T cells and other cell types, including acinar and ductal cells of the exocrine pancreas. Risk variants at multiple T1D signals overlapped with exocrine-specific cCREs that were linked to genes with exocrine-specific expression. At the CFTR locus, the T1D risk variant rs7795896 mapped to a ductal-specific cCRE that regulated CFTR; the risk allele reduced transcription factor binding, enhancer activity and CFTR expression in ductal cells. These findings support a role for the exocrine pancreas in the pathogenesis of T1D and highlight the power of large-scale genome-wide association studies and single-cell epigenomics for understanding the cellular origins of complex disease.

SOX9 (Sex-determining region Y Box 9) is a well-characterized transcription factor that is a marker for progenitor cells in various tissues. In the liver, cells delineated by SOX9 are responsible for regenerating liver parenchyma when cell proliferation is impaired following chronic injury. However, whether these SOX9+ cells play a role in liver carcinogenesis has not been fully understood, although high SOX9 expression has been linked to poor survival outcome in liver cancer patients. To address this question, we developed a liver cancer mouse model (PtenloxP/loxP; Sox9-CreERT+; R26RYFP) where tumor suppressor Pten (phosphatase and tensin homolog deleted on chromosome ten) is deleted in SOX9+ cells following tamoxifen injection. In this paper, we employ lineage-tracing to demonstrate the tumorigenicity potential of the Pten-, SOX9+ cells. We show that these cells are capable of giving rise to mixed-lineage tumors that manifest features of both hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (CCA). Our results suggest that PTEN loss induces the transformation of SOX9+ cells. We further show that to activate these transformed SOX9+ cells, the presence of liver injury is crucial. Liver injury, induced by hepatotoxin 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) or high-fat diet (HFD), substantially increases tumor incidence and accelerates liver carcinogenesis from SOX9+ cells in Pten null mice but not in control mice. We further examine the mechanisms underlying tumor formation in this model to show that concurrent with the induction of niche signal (i.e., Wnt signaling), liver injury significantly stimulates the expansion of tumor-initiating cells (TICs). Together, these data show that (1) SOX9+ cells have the potential to become TICs following the primary transformation (i.e. Pten deletion) and that (2) liver injury is necessary for promoting the activation and proliferation of transformed SOX9+ cells, resulting in the genesis of mixed-lineage liver tumors.

Single-nucleus assay for transposase-accessible chromatin using sequencing (snATAC-seq) creates new opportunities to dissect cell type-specific mechanisms of complex diseases. Since pancreatic islets are central to type 2 diabetes (T2D), we profiled 15,298 islet cells by using combinatorial barcoding snATAC-seq and identified 12 clusters, including multiple alpha, beta and delta cell states. We cataloged 228,873 accessible chromatin sites and identified transcription factors underlying lineage- and state-specific regulation. We observed state-specific enrichment of fasting glucose and T2D genome-wide association studies for beta cells and enrichment for other endocrine cell types. At T2D signals localized to islet-accessible chromatin, we prioritized variants with predicted regulatory function and co-accessibility with target genes. A causal T2D variant rs231361 at the KCNQ1 locus had predicted effects on a beta cell enhancer co-accessible with INS and genome editing in embryonic stem cell-derived beta cells affected INS levels. Together our findings demonstrate the power of single-cell epigenomics for interpreting complex disease genetics.

Many sequence variants have been linked to complex human traits and diseases1, but deciphering their biological functions remains challenging, as most of them reside in noncoding DNA. Here we have systematically assessed the binding of 270 human transcription factors to 95,886 noncoding variants in the human genome using an ultra-high-throughput multiplex protein-DNA binding assay, termed single-nucleotide polymorphism evaluation by systematic evolution of ligands by exponential enrichment (SNP-SELEX). The resulting 828 million measurements of transcription factor-DNA interactions enable estimation of the relative affinity of these transcription factors to each variant in vitro and evaluation of the current methods to predict the effects of noncoding variants on transcription factor binding. We show that the position weight matrices of most transcription factors lack sufficient predictive power, whereas the support vector machine combined with the gapped k-mer representation show much improved performance, when assessed on results from independent SNP-SELEX experiments involving a new set of 61,020 sequence variants. We report highly predictive models for 94 human transcription factors and demonstrate their utility in genome-wide association studies and understanding of the molecular pathways involved in diverse human traits and diseases.

Genetic variants associated with type 2 diabetes (T2D) risk affect gene regulation in metabolically relevant tissues, such as pancreatic islets. Here, we investigated contributions of regulatory programs active during pancreatic development to T2D risk. Generation of chromatin maps from developmental precursors throughout pancreatic differentiation of human embryonic stem cells (hESCs) identifies enrichment of T2D variants in pancreatic progenitor-specific stretch enhancers that are not active in islets. Genes associated with progenitor-specific stretch enhancers are predicted to regulate developmental processes, most notably tissue morphogenesis. Through gene editing in hESCs, we demonstrate that progenitor-specific enhancers harboring T2D-associated variants regulate cell polarity genes LAMA1 and CRB2. Knockdown of lama1 or crb2 in zebrafish embryos causes a defect in pancreas morphogenesis and impairs islet cell development. Together, our findings reveal that a subset of T2D risk variants specifically affects pancreatic developmental programs, suggesting that dysregulation of developmental processes can predispose to T2D.

Cell type specification during pancreatic development is tightly controlled by a transcriptional and epigenetic network. The precise role of most transcription factors, however, has been only described in mice. To convey such concepts to human pancreatic development, alternative model systems such as pancreatic in vitro differentiation of human pluripotent stem cells can be employed. Here, we analyzed stage-specific RNA-, ChIP-, and ATAC-sequencing data to dissect transcriptional and regulatory mechanisms during pancreatic development. Transcriptome and open chromatin maps of pancreatic differentiation from human pluripotent stem cells provide a stage-specific pattern of known pancreatic transcription factors and indicate ONECUT1 as a crucial fate regulator in pancreas progenitors. Moreover, our data suggest that ONECUT1 is also involved in preparing pancreatic progenitors for later endocrine specification. The dissection of the transcriptional and regulatory circuitry revealed an important role for ONECUT1 within such network and will serve as resource to study human development and disease.

BackgroundCo-localized combinations of histone modifications ("chromatin states") have been shown to correlate with promoter and enhancer activity. Changes in chromatin states over multiple time points ("chromatin state trajectories") have previously been analyzed at promoter and enhancers separately. With the advent of time series Hi-C data it is now possible to connect promoters and enhancers and to analyze chromatin state trajectories at promoter-enhancer pairs.ResultsWe present TimelessFlex, a framework for investigating chromatin state trajectories at promoters and enhancers and at promoter-enhancer pairs based on Hi-C information. TimelessFlex extends our previous approach Timeless, a Bayesian network for clustering multiple histone modification data sets at promoter and enhancer feature regions. We utilize time series ATAC-seq data measuring open chromatin to define promoters and enhancer candidates. We developed an expectation-maximization algorithm to assign promoters and enhancers to each other based on Hi-C interactions and jointly cluster their feature regions into paired chromatin state trajectories. We find jointly clustered promoter-enhancer pairs showing the same activation patterns on both sides but with a stronger trend at the enhancer side. While the promoter side remains accessible across the time series, the enhancer side becomes dynamically more open towards the gene activation time point. Promoter cluster patterns show strong correlations with gene expression signals, whereas Hi-C signals get only slightly stronger towards activation. The code of the framework is available at https://github.com/henriettemiko/TimelessFlex .ConclusionsTimelessFlex clusters time series histone modifications at promoter-enhancer pairs based on Hi-C and it can identify distinct chromatin states at promoter and enhancer feature regions and their changes over time.

FOXA pioneer transcription factors (TFs) associate with primed enhancers in endodermal organ precursors. Using a human stem cell model of pancreas differentiation, we here discover that only a subset of pancreatic enhancers is FOXA-primed, whereas the majority is unprimed and engages FOXA upon lineage induction. Primed enhancers are enriched for signal-dependent TF motifs and harbor abundant and strong FOXA motifs. Unprimed enhancers harbor fewer, more degenerate FOXA motifs, and FOXA recruitment to unprimed but not primed enhancers requires pancreatic TFs. Strengthening FOXA motifs at an unprimed enhancer near NKX6.1 renders FOXA recruitment pancreatic TF-independent, induces priming, and broadens the NKX6.1 expression domain. We make analogous observations about FOXA binding during hepatic and lung development. Our findings suggest a dual role for FOXA in endodermal organ development: first, FOXA facilitates signal-dependent lineage initiation via enhancer priming, and second, FOXA enforces organ cell type-specific gene expression via indirect recruitment by lineage-specific TFs.

Developmental progression depends on temporally defined changes in gene expression mediated by transient exposure of lineage intermediates to signals in the progenitor niche. To determine whether cell-intrinsic epigenetic mechanisms contribute to signal-induced transcriptional responses, here we manipulate the signalling environment and activity of the histone demethylase LSD1 during differentiation of hESC-gut tube intermediates into pancreatic endocrine cells. We identify a transient requirement for LSD1 in endocrine cell differentiation spanning a short time-window early in pancreas development, a phenotype we reproduced in mice. Examination of enhancer and transcriptome landscapes revealed that LSD1 silences transiently active retinoic acid (RA)-induced enhancers and their target genes. Furthermore, prolonged RA exposure phenocopies LSD1 inhibition, suggesting that LSD1 regulates endocrine cell differentiation by limiting the duration of RA signalling. Our findings identify LSD1-mediated enhancer silencing as a cell-intrinsic epigenetic feedback mechanism by which the duration of the transcriptional response to a developmental signal is limited.

Pancreatic ductal adenocarcinoma (PDAC) is associated with metaplastic changes in the pancreas but the transcriptional program underlying these changes is incompletely understood. The zinc finger transcription factor, PRDM3, is lowly expressed in normal pancreatic acini and its expression increases during tumorigenesis. Although PRDM3 promotes proliferation and migration of PDAC cell lines, the role of PRDM3 during tumor initiation from pancreatic acinar cells in vivo is unclear. In this study, we showed that high levels of PRDM3 expression in human pancreas was associated with pancreatitis, and well-differentiated but not poorly differentiated carcinoma. We examined PRDM3 function in pancreatic acinar cells during tumor formation and pancreatitis by inactivating Prdm3 using a conditional allele (Ptf1aCreER;Prdm3flox/flox mice) in the context of oncogenic Kras expression and supraphysiological cerulein injections, respectively. In Prdm3-deficient mice, KrasG12D-driven preneoplastic lesions were more abundant and progressed to high-grade precancerous lesions more rapidly. This is consistent with our observations that low levels of PRDM3 in human PDAC was correlated significantly with poorer survival in patient. Moreover, loss of Prdm3 in acinar cells elevated exocrine injury, enhanced immune cell activation and infiltration, and greatly increased acinar-to-ductal cell reprogramming upon cerulein-induced pancreatitis. Whole transcriptome analyses of Prdm3 knockout acini revealed that pathways involved in inflammatory response and Hif-1 signaling were significantly upregulated in Prdm3-depleted acinar cells. Taken together, our results suggest that Prdm3 favors the maintenance of acinar cell homeostasis through modulation of their response to inflammation and oncogenic Kras activation, and thus plays a previously unexpected suppressive role during PDAC initiation.

Long noncoding RNAs (lncRNAs) are a heterogenous group of RNAs, which can encode small proteins. The extent to which developmentally regulated lncRNAs are translated and whether the produced microproteins are relevant for human development is unknown. Using a human embryonic stem cell (hESC)-based pancreatic differentiation system, we show that many lncRNAs in direct vicinity of lineage-determining transcription factors (TFs) are dynamically regulated, predominantly cytosolic, and highly translated. We genetically ablated ten such lncRNAs, most of them translated, and found that nine are dispensable for pancreatic endocrine cell development. However, deletion of LINC00261 diminishes insulin+ cells, in a manner independent of the nearby TF FOXA2. One-by-one disruption of each of LINC00261's open reading frames suggests that the RNA, rather than the produced microproteins, is required for endocrine development. Our work highlights extensive translation of lncRNAs during hESC pancreatic differentiation and provides a blueprint for dissection of their coding and noncoding roles.

A comprehensive understanding of mechanisms that underlie the development and function of human cells requires human cell models. For the pancreatic lineage, protocols have been developed to differentiate human pluripotent stem cells (hPSCs) into pancreatic endocrine and exocrine cells through intermediates resembling in vivo development. In recent years, this differentiation system has been employed to decipher mechanisms of pancreatic development, congenital defects of the pancreas, as well as genetic forms of diabetes and exocrine diseases. In this review, we summarize recent insights gained from studies of pancreatic hPSC models. We discuss how genome-scale analyses of the differentiation system have helped elucidate roles of chromatin state, transcription factors, and noncoding RNAs in pancreatic development and how the analysis of cells with disease-relevant mutations has provided insight into the molecular underpinnings of genetically determined diseases of the pancreas.

Native pancreatic islets interact with neighboring cells by establishing three-dimensional (3D) structures, and are surrounded by perfusion at an interstitial flow level. However, flow effects are generally ignored in islet culture models, although cell perfusion is known to improve the cell microenvironment and to mimic in vivo physiology better than static culture systems. Here, we have developed functional islet spheroids using a microfluidic chip that mimics interstitial flow conditions with reduced shear cell damage. Dynamic culture, compared to static culture, enhanced islet health and maintenance of islet endothelial cells, reconstituting the main component of islet extracellular matrix within spheroids. Optimized flow condition allowed localization of secreted soluble factors near spheroids, facilitating diffusion-mediated paracrine interactions within islets, and enabled long-term maintenance of islet morphology and function for a month. The proposed model can aid islet preconditioning before transplantation and has potential applications as an in vitro model for diabetic drug testing.

Pancreatic endocrine cell differentiation is orchestrated by the action of transcription factors that operate in a gene regulatory network to activate endocrine lineage genes and repress lineage-inappropriate genes. MicroRNAs (miRNAs) are important modulators of gene expression, yet their role in endocrine cell differentiation has not been systematically explored. Here we characterize miRNA-regulatory networks active in human endocrine cell differentiation by combining small RNA sequencing, miRNA over-expression, and network modeling approaches. Our analysis identified Let-7g, Let-7a, miR-200a, miR-127, and miR-375 as endocrine-enriched miRNAs that drive endocrine cell differentiation-associated gene expression changes. These miRNAs are predicted to target different transcription factors, which converge on genes involved in cell cycle regulation. When expressed in human embryonic stem cell-derived pancreatic progenitors, these miRNAs induce cell cycle exit and promote endocrine cell differentiation. Our study delineates the role of miRNAs in human endocrine cell differentiation and identifies miRNAs that could facilitate endocrine cell reprogramming.

It is now recognized that cell identity is more fluid, and tissues more plastic, than previously thought. The plasticity of cells is relevant to diverse fields, most notably developmental and stem cell biology, regenerative medicine, and cancer biology. To date, a comprehensive and uniform nomenclature to define distinct cell states and their injury-induced interconversions has been elusive. The first Keystone Symposium devoted exclusively to cellular plasticity in regeneration and tumorigenesis was held on January 2019 in Keystone, Colorado, and featured a workshop on terminology in the cell plasticity field. Definitions for terms such as plasticity, de- and transdifferentiation, reversion, and paligenosis were discussed. Here, we summarize the content and tenor of the symposium and nomenclature-focused workshop with regard to terms in the field. We outline the challenges with current definitions and recommend best practices and approaches to developing an accurate and acceptable nomenclature in the future.

Genetic variants affecting pancreatic islet enhancers are central to T2D risk, but the gene targets of islet enhancer activity are largely unknown. We generate a high-resolution map of islet chromatin loops using Hi-C assays in three islet samples and use loops to annotate target genes of islet enhancers defined using ATAC-seq and published ChIP-seq data. We identify candidate target genes for thousands of islet enhancers, and find that enhancer looping is correlated with islet-specific gene expression. We fine-map T2D risk variants affecting islet enhancers, and find that candidate target genes of these variants defined using chromatin looping and eQTL mapping are enriched in protein transport and secretion pathways. At IGF2BP2, a fine-mapped T2D variant reduces islet enhancer activity and IGF2BP2 expression, and conditional inactivation of IGF2BP2 in mouse islets impairs glucose-stimulated insulin secretion. Our findings provide a resource for studying islet enhancer function and identifying genes involved in T2D risk.

OBJECTIVE: Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive tumour thought to arise from ductal cells via pancreatic intraepithelial neoplasia (PanIN) precursor lesions. Modelling of different genetic events in mice suggests both ductal and acinar cells can give rise to PDAC. However, the impact of cellular context alone on tumour development and phenotype is unknown. DESIGN: We examined the contribution of cellular origin to PDAC development by inducing PDAC-associated mutations, KrasG12D expression and Trp53 loss, specifically in ductal cells (Sox9CreER;KrasLSL-G12D;Trp53flox/flox (Duct:KPcKO )) or acinar cells (Ptf1aCreER;KrasLSL-G12D;Trp53flox/flox (Acinar:KPcKO )) in mice. We then performed a thorough analysis of the resulting histopathological changes. RESULTS: Both mouse models developed PDAC, but Duct:KPcKO mice developed PDAC earlier than Acinar:KPcKO mice. Tumour development was more rapid and associated with high-grade murine PanIN (mPanIN) lesions in Duct:KPcKO mice. In contrast, Acinar:KPcKO mice exhibited widespread metaplasia and low-grade as well as high-grade mPanINs with delayed progression to PDAC. Acinar-cell-derived tumours also had a higher prevalence of mucinous glandular features reminiscent of early mPanIN lesions. CONCLUSION: These findings indicate that ductal cells are primed to form carcinoma in situ that become invasive PDAC in the presence of oncogenic Kras and Trp53 deletion, while acinar cells with the same mutations appear to require a prolonged period of transition or reprogramming to initiate PDAC. Our findings illustrate that PDAC can develop in multiple ways and the cellular context in which mutations are acquired has significant impact on precursor lesion initiation, disease progression and tumour phenotype.

Background & aimsActivating mutation of the KRAS gene is common in some cancers, such as pancreatic cancer, but rare in other cancers. Chronic pancreatitis is a predisposing condition for pancreatic ductal adenocarcinoma (PDAC), but how it synergizes with KRAS mutation is not known.MethodsWe used a mouse model to express an activating mutation of Kras in conjunction with obstruction of the main pancreatic duct to recapitulate a common etiology of human chronic pancreatitis. Because the cell of origin of PDAC is not clear, Kras mutation was introduced into either duct cells or acinar cells.ResultsAlthough KrasG12D expression in both cell types was protective against damage-associated cell death, chronic pancreatitis induced p53, p21, and growth arrest only in acinar-derived cells. Mutant duct cells did not elevate p53 or p21 expression and exhibited increased proliferation driving the appearance of PDAC over time.ConclusionsOne mechanism by which tissues may be susceptible or resistant to KRASG12D-initiated tumorigenesis is whether they undergo a p53-mediated damage response. In summary, we have uncovered a mechanism by which inflammation and intrinsic cellular programming synergize for the development of PDAC.

Different approaches have investigated the effects of different extracellular matrices (ECMs) and three-dimensional (3D) culture on islet function, showing encouraging results. Ideally, the proper scaffold should mimic the biochemical composition of the native tissue as it drives numerous signaling pathways involved in tissue homeostasis and functionality. Tissue-derived decellularized biomaterials can preserve the ECM composition of the native tissue making it an ideal scaffold for 3D tissue engineering applications. However, the decellularization process may affect the retention of specific components, and the choice of a proper detergent is fundamental in preserving the native ECM composition. In this study, we evaluated the effect of different decellularization protocols on the mechanical properties and biochemical composition of pancreatic ECM (pECM) hydrogels. Fresh porcine pancreas tissue was harvested, cut into small pieces, rinsed in water, and treated with two different detergents (sodium dodecyl sulfate [SDS] or Triton X-100) for 1 day followed by 3 days in water. Effective decellularization was confirmed by PicoGreen assay, Hoescht, and H&E staining, showing no differences among groups. Use of a protease inhibitor (PI) was also evaluated. Effective decellularization was confirmed by PicoGreen assay and hematoxylin and eosin (H&E) staining, showing no differences among groups. Triton-treated samples were able to form a firm hydrogel under appropriate conditions, while the use of SDS had detrimental effects on the gelation properties of the hydrogels. ECM biochemical composition was characterized both in the fresh porcine pancreas and all decellularized pECM hydrogels by quantitative mass spectrometry analysis. Fibrillar collagen was the major ECM component in all groups, with all generated hydrogels having a higher amount compared with fresh pancreas. This effect was more pronounced in the SDS-treated hydrogels when compared with the Triton groups, showing very little retention of other ECM molecules. Conversely, basement membrane and matricellular proteins were better retained when the tissue was pretreated with a PI and decellularized in Triton X-100, making the hydrogel more similar to the native tissue. In conclusion, we showed that all the protocols evaluated in the study showed effective tissue decellularization, but only when the tissue was pretreated with a PI and decellularized in Triton detergent, the biochemical composition of the hydrogel was closer to the native tissue ECM. Impact Statement The article compares different methodologies for the generation of a pancreas-derived hydrogel for tissue engineering applications. The biochemical characterization of the newly generated hydrogel shows that the material retains all the extracellular molecules of the native tissue and is capable of sustaining functionality of the encapsulated beta-cells.

Pancreatic β cell physiology changes substantially throughout life, yet the mechanisms that drive these changes are poorly understood. Here, we performed comprehensive in vivo quantitative proteomic profiling of pancreatic islets from juvenile and 1-year-old mice. The analysis revealed striking differences in abundance of enzymes controlling glucose metabolism. We show that these changes in protein abundance are associated with higher activities of glucose metabolic enzymes involved in coupling factor generation as well as increased activity of the coupling factor-dependent amplifying pathway of insulin secretion. Nutrient tracing and targeted metabolomics demonstrated accelerated accumulation of glucose-derived metabolites and coupling factors in islets from 1-year-old mice, indicating that age-related changes in glucose metabolism contribute to improved glucose-stimulated insulin secretion with age. Together, our study provides an in-depth characterization of age-related changes in the islet proteome and establishes metabolic rewiring as an important mechanism for age-associated changes in β cell function.

The mechanisms that restrict regeneration and maintain cell identity following injury are poorly characterized in higher vertebrates. Following β-cell loss, 1-2% of the glucagon-producing α-cells spontaneously engage in insulin production in mice. Here we explore the mechanisms inhibiting α-cell plasticity. We show that adaptive α-cell identity changes are constrained by intra-islet insulin- and Smoothened-mediated signalling, among others. The combination of β-cell loss or insulin-signalling inhibition, with Smoothened inactivation in α- or δ-cells, stimulates insulin production in more α-cells. These findings suggest that the removal of constitutive 'brake signals' is crucial to neutralize the refractoriness to adaptive cell-fate changes. It appears that the maintenance of cell identity is an active process mediated by repressive signals, which are released by neighbouring cells and curb an intrinsic trend of differentiated cells to change.

Genetic drivers of cancer can be dysregulated through epigenetic modifications of DNA. Although the critical role of DNA 5-methylcytosine (5mC) in the regulation of transcription is recognized, the functions of other non-canonical DNA modifications remain obscure. Here, we report the identification of novel N6-methyladenine (N6-mA) DNA modifications in human tissues and implicate this epigenetic mark in human disease, specifically the highly malignant brain cancer glioblastoma. Glioblastoma markedly upregulated N6-mA levels, which co-localized with heterochromatic histone modifications, predominantly H3K9me3. N6-mA levels were dynamically regulated by the DNA demethylase ALKBH1, depletion of which led to transcriptional silencing of oncogenic pathways through decreasing chromatin accessibility. Targeting the N6-mA regulator ALKBH1 in patient-derived human glioblastoma models inhibited tumor cell proliferation and extended the survival of tumor-bearing mice, supporting this novel DNA modification as a potential therapeutic target for glioblastoma. Collectively, our results uncover a novel epigenetic node in cancer through the DNA modification N6-mA.

The American Diabetes Association convened a research symposium, "Epigenetics and Epigenomics: Implications for Diabetes and Obesity" on 17-19 November 2017. International experts in genetics, epigenetics, computational biology, and physiology discussed the current state of understanding of the relationships between genetics, epigenetics, and environment in diabetes and examined existing evidence for the role of epigenetic factors in regulating metabolism and the risk of diabetes and its complications. The authors summarize the presentations, which highlight how the complex interactions between genes and environment may in part be mediated through epigenetic changes and how information about nutritional and other environmental stimuli can be transmitted to the next generation. In addition, the authors present expert consensus on knowledge gaps and research recommendations for the field.

BACKGROUND:The plasticity of pancreatic acinar cells to undergo acinar to ductal metaplasia (ADM) has been demonstrated to contribute to the regeneration of the pancreas in response to injury. Sox9 is critical for ductal cell fate and important in the formation of ADM, most likely in concert with a complex hierarchy of, as yet, not fully elucidated transcription factors. RESULTS:By using a mouse model of acute pancreatitis and three dimensional organoid culture of primary pancreatic ductal cells, we herein characterize the Ets-transcription factor Etv5 as a pivotal regulator of ductal cell identity and ADM that acts upstream of Sox9 and is essential for Sox9 expression in ADM. Loss of Etv5 is associated with increased severity of acute pancreatitis and impaired ADM formation leading to delayed tissue regeneration and recovery in response to injury. CONCLUSIONS:Our data provide new insights in the regulation of ADM with implications in our understanding of pancreatic homeostasis, pancreatitis and epithelial plasticity. Developmental Dynamics 247:854-866, 2018. © 2018 Wiley Periodicals, Inc.

BACKGROUND & AIMS:Intraductal papillary mucinous neoplasias (IPMNs) are precancerous cystic lesions that can develop into pancreatic ductal adenocarcinomas (PDACs). These large macroscopic lesions are frequently detected during medical imaging, but it is unclear how they form or progress to PDAC. We aimed to identify cells that form IPMNs and mutations that promote IPMN development and progression. METHODS:We generated mice with disruption of Pten specifically in ductal cells (Sox9CreERT2;Ptenflox/flox;R26RYFP or PtenΔDuct/ΔDuct mice) and used PtenΔDuct/+ and Pten+/+ mice as controls. We also generated KrasG12D;PtenΔDuct/ΔDuct and KrasG12D;PtenΔDuct/+ mice. Pancreata were collected when mice were 28 weeks to 14.5 months old and analyzed by histology, immunohistochemistry, and electron microscopy. We performed multiplexed droplet digital polymerase chain reaction to detect spontaneous Kras mutations in PtenΔDuct/ΔDuct mice and study the effects of Ras pathway activation on initiation and progression of IPMNs. We obtained 2 pancreatic sections from a patient with an invasive pancreatobiliary IPMN and analyzed the regions with and without the invasive IPMN (control tissue) by immunohistochemistry. RESULTS:Mice with ductal cell-specific disruption of Pten but not control mice developed sporadic, macroscopic, intraductal papillary lesions with histologic and molecular features of human IPMNs. PtenΔDuct/ΔDuct mice developed IPMNs of several subtypes. In PtenΔDuct/ΔDuct mice, 31.5% of IPMNs became invasive; invasion was associated with spontaneous mutations in Kras. KrasG12D;PtenΔDuct/ΔDuct mice all developed invasive IPMNs within 1 month. In KrasG12D;PtenΔDuct/+ mice, 70% developed IPMN, predominately of the pancreatobiliary subtype, and 63.3% developed PDAC. In all models, IPMNs and PDAC expressed the duct-specific lineage tracing marker yellow fluorescent protein. In immunohistochemical analyses, we found that the invasive human pancreatobiliary IPMN tissue had lower levels of PTEN and increased levels of phosphorylated (activated) ERK compared with healthy pancreatic tissue. CONCLUSIONS:In analyses of mice with ductal cell-specific disruption of Pten, with or without activated Kras, we found evidence for a ductal cell origin of IPMNs. We also showed that PTEN loss and activated Kras have synergistic effects in promoting development of IPMN and progression to PDAC.

Cadherin-mediated cell-cell adhesion plays an important role in organ development and changes in cadherin expression are often linked to morphogenetic and pathogenic events. Cadherins interact with other intracellular components to form adherens junctions (AJs) and provide mechanical attachments between adjacent cells. E-cadherin (Cdh1) represents an integral component of these intercellular junctions. To elucidate the function of E-cadherin in the developing pancreas, we generated and studied pancreas-specific Cdh1-knockout (Cdh1ΔPan/ΔPan) mice. Cdh1ΔPan/ΔPan mice exhibit normal body size at birth, but fail to gain weight and become hypoglycemic soon afterward. We found that E-cadherin is not required for the establishment of apical-basal polarity or pancreatic exocrine cell identity at birth. However, four days after birth, the pancreata of Cdh1ΔPan/ΔPan mutants display progressive deterioration of exocrine architecture and dysregulation of Wnt and YAP signaling. At this time point, the acinar cells of Cdh1ΔPan/ΔPan mutants begin to exhibit ductal phenotypes, suggesting acinar-to-ductal metaplasia (ADM) in the E-cadherin-deficient pancreas. Our findings demonstrate that E-cadherin plays an integral role in the maintenance of exocrine architecture and regulation of homeostatic signaling. The present study provides insights into the involvement of cadherin-mediated cell-cell adhesion in pathogenic conditions such as pancreatitis or pancreatic cancer.

BackgroundBlood glucose levels are tightly controlled by the coordinated actions of hormone-producing endocrine cells that reside in pancreatic islets. Islet cell malfunction underlies diabetes development and progression. Due to the cellular heterogeneity within islets, it has been challenging to uncover how specific islet cells contribute to glucose homeostasis and diabetes pathogenesis. Recent advances in single-cell technologies and computational methods have opened up new avenues to resolve islet heterogeneity and study islet cell states in health and disease.Scope of reviewIn the past year, a multitude of studies have been published that used single-cell approaches to interrogate the transcriptome and proteome of the different islet cell types. Here, we summarize the conclusions of these studies, as well as discuss the technologies used and the challenges faced with computational analysis of single-cell data from islet studies.Major conclusionsBy analyzing single islet cells from rodents and humans at different ages and disease states, the studies reviewed here have provided new insight into endocrine cell function and facilitated a high resolution molecular characterization of poorly understood processes, including regeneration, maturation, and diabetes pathogenesis. Gene expression programs and pathways identified in these studies pave the way for the discovery of new targets and approaches to prevent, monitor, and treat diabetes.

Pancreatic β cell mass for appropriate blood glucose control is established during early postnatal life. β cell proliferative capacity declines postnatally, but the extrinsic cues and intracellular signals that cause this decline remain unknown. To obtain a high-resolution map of β cell transcriptome dynamics after birth, we generated single-cell RNA-seq data of β cells from multiple postnatal time points and ordered cells based on transcriptional similarity using a new analytical tool. This analysis captured signatures of immature, proliferative β cells and established high expression of amino acid metabolic, mitochondrial, and Srf/Jun/Fos transcription factor genes as their hallmark feature. Experimental validation revealed high metabolic activity in immature β cells and a role for reactive oxygen species and Srf/Jun/Fos transcription factors in driving postnatal β cell proliferation and mass expansion. Our work provides the first high-resolution molecular characterization of state changes in postnatal β cells and paves the way for the identification of novel therapeutic targets to stimulate β cell regeneration.