A DNA methylation atlas of normal human cell types.

DNA methylation is a fundamental epigenetic mark that governs gene expression and chromatin organization, thus providing a window into cellular identity and developmental processes1. Current datasets typically include only a fraction of methylation sites and are often based either on cell lines that underwent massive changes in culture or on tissues containing unspecified mixtures of cells2-5. Here we describe a human methylome atlas, based on deep whole-genome bisulfite sequencing, allowing fragment-level analysis across thousands of unique markers for 39 cell types sorted from 205 healthy tissue samples. Replicates of the same cell type are more than 99.5% identical, demonstrating the robustness of cell identity programmes to environmental perturbation. Unsupervised clustering of the atlas recapitulates key elements of tissue ontogeny and identifies methylation patterns retained since embryonic development. Loci uniquely unmethylated in an individual cell type often reside in transcriptional enhancers and contain DNA binding sites for tissue-specific transcriptional regulators. Uniquely hypermethylated loci are rare and are enriched for CpG islands, Polycomb targets and CTCF binding sites, suggesting a new role in shaping cell-type-specific chromatin looping. The atlas provides an essential resource for study of gene regulation and disease-associated genetic variants, and a wealth of potential tissue-specific biomarkers for use in liquid biopsies.

Senescence of human pancreatic beta cells enhances functional maturation through chromatin reorganization and promotes interferon responsiveness.

Senescent cells can influence the function of tissues in which they reside, and their propensity for disease. A portion of adult human pancreatic beta cells express the senescence marker p16, yet it is unclear whether they are in a senescent state, and how this affects insulin secretion. We analyzed single-cell transcriptome datasets of adult human beta cells, and found that p16-positive cells express senescence gene signatures, as well as elevated levels of beta-cell maturation genes, consistent with enhanced functionality. Senescent human beta-like cells in culture undergo chromatin reorganization that leads to activation of enhancers regulating functional maturation genes and acquisition of glucose-stimulated insulin secretion capacity. Strikingly, Interferon-stimulated genes are elevated in senescent human beta cells, but genes encoding senescence-associated secretory phenotype (SASP) cytokines are not. Senescent beta cells in culture and in human tissue show elevated levels of cytoplasmic DNA, contributing to their increased interferon responsiveness. Human beta-cell senescence thus involves chromatin-driven upregulation of a functional-maturation program, and increased responsiveness of interferon-stimulated genes, changes that could increase both insulin secretion and immune reactivity.

miR-17-92 and miR-106b-25 clusters regulate beta cell mitotic checkpoint and insulin secretion in mice.

AIMS/HYPOTHESIS: Adult beta cells in the pancreas are the sole source of insulin in the body. Beta cell loss or increased demand for insulin impose metabolic challenges because adult beta cells are generally quiescent and infrequently re-enter the cell division cycle. The aim of this study is to test the hypothesis that a family of proto-oncogene microRNAs that includes miR-17-92 and miR-106b-25 clusters regulates beta cell proliferation or function in the adult endocrine pancreas. METHODS: To elucidate the role of miR-17-92 and miR-106b-25 clusters in beta cells, we used a conditional miR-17-92/miR-106b-25 knockout mouse model. We employed metabolic assays in vivo and ex vivo, together with advanced microscopy of pancreatic sections, bioinformatics, mass spectrometry and next generation sequencing, to examine potential targets of miR-17-92/miR-106b-25, by which they might regulate beta cell proliferation and function. RESULTS: We demonstrate that miR-17-92/miR-106b-25 regulate the adult beta cell mitotic checkpoint and that miR-17-92/miR-106b-25 deficiency results in reduction in beta cell mass in vivo. Furthermore, we reveal a critical role for miR-17-92/miR-106b-25 in glucose homeostasis and in controlling insulin secretion. We identify protein kinase A as a new relevant molecular pathway downstream of miR-17-92/miR-106b-25 in control of adult beta cell division and glucose homeostasis. CONCLUSIONS/INTERPRETATION: The study contributes to the understanding of proto-oncogene miRNAs in the normal, untransformed endocrine pancreas and illustrates new genetic means for regulation of beta cell mitosis and function by non-coding RNAs. DATA AVAILABILITY: Sequencing data that support the findings of this study have been deposited in GEO with the accession code GSE126516.

Beta cell heterogeneity: an evolving concept.

Beta cells are primarily defined by their ability to produce insulin and secrete it in response to appropriate stimuli. It has been known for some time, however, that beta cells are not functionally identical to each other and that the rates of insulin synthesis and release differ from cell to cell, although the functional significance of this variability remains unclear. Recent studies have used heterogeneous gene expression to isolate and evaluate different subpopulations of beta cells and to demonstrate alterations in these subpopulations in diabetes. In the last few years, novel technologies have emerged that permit the detailed evaluation of the proteome (e.g. time-of-flight mass spectroscopy, [CyTOF]) and transcriptome (e.g. massively parallel RNA sequencing) at the single-cell level, and tools for single beta cell metabolomics and epigenomics are quickly maturing. The first wave of single beta cell proteome and transcriptome studies were published in 2016, giving a glimpse into the power, but also the limitations, of these approaches. Despite this progress, it remains unclear if the observed heterogeneity of beta cells represents stable, distinct beta cell types or, alternatively, highly dynamic beta cell states. Here we provide a concise overview of recent developments in the emerging field of beta cell heterogeneity and the implications for our understanding of beta cell biology and pathology.

ß-Cells are not uniform after all-Novel insights into molecular heterogeneity of insulin-secreting cells.

While the ß-cells of the endocrine pancreas are defined as cells with high levels of insulin production and tight stimulus-secretion coupling, the existence of functional heterogeneity among them has been known for decades. Recent advances in molecular technologies, in particular single-cell profiling on both the protein and messenger RNA level, have uncovered that ß-cells exist in several antigenically and molecularly definable states. Using antibodies to cell surface markers or multidimensional clustering of ß-cells using more than 20 protein markers by mass cytometry, 4 distinct groups of ß-cells could be differentiated. However, whether these states represent permanent cell lineages or are readily interconvertible from one group to another remains to be determined. Nevertheless, future analysis of the pathogenesis of type 1 and type 2 diabetes will certainly benefit from a growing appreciation of ß-cell heterogeneity. Here, we aim to summarize concisely the recent advances in the field and their possible impact on our understanding of ß-cell physiology and pathophysiology.

Phosphoproteomic analysis reveals Smarcb1 dependent EGFR signaling in Malignant Rhabdoid tumor cells.

BACKGROUND: The SWI/SNF ATP dependent chromatin remodeling complex is a multi-subunit complex, conserved in eukaryotic evolution that facilitates nucleosomal re-positioning relative to the DNA sequence. In recent years the SWI/SNF complex has emerged to play a role in cancer development as various sub-units of the complex are found to be mutated in a variety of tumors. One core-subunit of the complex, which has been well established as a tumor suppressor gene is SMARCB1 (SNF5/INI1/BAF47). Mutation and inactivation of SMARCB1 have been identified as the underlying mechanism leading to Malignant Rhabdoid Tumors (MRT) and Atypical Teratoid/Rhabdoid Tumors (AT/RT), two highly aggressive forms of pediatric neoplasms. METHODS: We present a phosphoproteomic study of Smarcb1 dependent changes in signaling networks. The SILAC (Stable Isotopic Labeling of Amino Acids in Cell Culture) protocol was used to quantify in an unbiased manner any changes in the phosphoproteomic profile of Smarcb1 deficient murine rhabdoid tumor cell lines following Smarcb1 stable re-expression and under different serum conditions. RESULTS: This study illustrates broad changes in the regulation of multiple biological networks including cell cycle progression, chromatin remodeling, cytoskeletal regulation and focal adhesion. Specifically, we identify Smarcb1 dependent changes in phosphorylation and expression of the EGF receptor, demonstrate downstream signaling and show that inhibition of EGFR signaling specifically hinders the proliferation of Smarcb1 deficient cells. CONCLUSIONS: These results support recent findings regarding the effectivity of EGFR inhibitors in hindering the proliferation of human MRT cells and demonstrate that activation of EGFR signaling in Rhabdoid tumors is SMARCB1 dependent.

Blood vessels restrain pancreas branching, differentiation and growth.

How organ size and form are controlled during development is a major question in biology. Blood vessels have been shown to be essential for early development of the liver and pancreas, and are fundamental to normal and pathological tissue growth. Here, we report that, surprisingly, non-nutritional signals from blood vessels act to restrain pancreas growth. Elimination of endothelial cells increases the size of embryonic pancreatic buds. Conversely, VEGF-induced hypervascularization decreases pancreas size. The growth phenotype results from vascular restriction of pancreatic tip cell formation, lateral branching and differentiation of the pancreatic epithelium into endocrine and acinar cells. The effects are seen both in vivo and ex vivo, indicating a perfusion-independent mechanism. Thus, the vasculature controls pancreas morphogenesis and growth by reducing branching and differentiation of primitive epithelial cells.

Disrupted RNA editing in beta cells mimics early-stage type 1 diabetes.

A major hypothesis for the etiology of type 1 diabetes (T1D) postulates initiation by viral infection, leading to double-stranded RNA (dsRNA)-mediated interferon response and inflammation; however, a causal virus has not been identified. Here, we use a mouse model, corroborated with human islet data, to demonstrate that endogenous dsRNA in beta cells can lead to a diabetogenic immune response, thus identifying a virus-independent mechanism for T1D initiation. We found that disruption of the RNA editing enzyme adenosine deaminases acting on RNA (ADAR) in beta cells triggers a massive interferon response, islet inflammation, and beta cell failure and destruction, with features bearing striking similarity to early-stage human T1D. Glycolysis via calcium enhances the interferon response, suggesting an actionable vicious cycle of inflammation and increased beta cell workload.

DNA Methylation-Based Assessment of Cell Composition in Human Pancreas and Islets.

Assessment of pancreas cell type composition is crucial to the understanding of the genesis of diabetes. Current approaches use immunodetection of protein markers, for example, insulin as a marker of ß-cells. A major limitation of these methods is that protein content varies in physiological and pathological conditions, complicating the extrapolation to actual cell number. Here, we demonstrate the use of cell type-specific DNA methylation markers for determining the fraction of specific cell types in human islet and pancreas specimens. We identified genomic loci that are uniquely demethylated in specific pancreatic cell types and applied targeted PCR to assess the methylation status of these loci in tissue samples, enabling inference of cell type composition. In islet preparations, normalization of insulin secretion to ß-cell DNA revealed similar ß-cell function in pre-type 1 diabetes (T1D), T1D, and type 2 diabetes (T2D), which was significantly lower than in donors without diabetes. In histological pancreas specimens from recent-onset T1D, this assay showed ß-cell fraction within the normal range, suggesting a significant contribution of ß-cell dysfunction. In T2D pancreata, we observed increased α-cell fraction and normal ß-cell fraction. Methylation-based analysis provides an accurate molecular alternative to immune detection of cell types in the human pancreas, with utility in the interpretation of insulin secretion assays and the assessment of pancreas cell composition in health and disease.

Megakaryocyte- and erythroblast-specific cell-free DNA patterns in plasma and platelets reflect thrombopoiesis and erythropoiesis levels.

Circulating cell-free DNA (cfDNA) fragments are a biological analyte with extensive utility in diagnostic medicine. Understanding the source of cfDNA and mechanisms of release is crucial for designing and interpreting cfDNA-based liquid biopsy assays. Using cell type-specific methylation markers as well as genome-wide methylation analysis, we determine that megakaryocytes, the precursors of anuclear platelets, are major contributors to cfDNA (~26%), while erythroblasts contribute 1-4% of cfDNA in healthy individuals. Surprisingly, we discover that platelets contain genomic DNA fragments originating in megakaryocytes, contrary to the general understanding that platelets lack genomic DNA. Megakaryocyte-derived cfDNA is increased in pathologies involving increased platelet production (Essential Thrombocythemia, Idiopathic Thrombocytopenic Purpura) and decreased upon reduced platelet production due to chemotherapy-induced bone marrow suppression. Similarly, erythroblast cfDNA is reflective of erythrocyte production and is elevated in patients with thalassemia. Megakaryocyte- and erythroblast-specific DNA methylation patterns can thus serve as biomarkers for pathologies involving increased or decreased thrombopoiesis and erythropoiesis, which can aid in determining the etiology of aberrant levels of erythrocytes and platelets.

Extensive elimination of acinar cells during normal postnatal pancreas growth.

While programmed cell death plays important roles during morphogenetic stages of development, post-differentiation organ growth is considered an efficient process whereby cell proliferation increases cell number. Here we demonstrate that early postnatal growth of the pancreas unexpectedly involves massive acinar cell elimination. Measurements of cell proliferation and death in the human pancreas in comparison to the actual increase in cell number predict daily elimination of 0.7% of cells, offsetting 88% of cell formation over the first year of life. Using mouse models, we show that death is associated with mitosis, through a failure of dividing cells to generate two viable daughters. In p53-deficient mice, acinar cell death and proliferation are reduced, while organ size is normal, suggesting that p53-dependent developmental apoptosis triggers compensatory proliferation. We propose that excess cell turnover during growth of the pancreas, and presumably other organs, facilitates robustness to perturbations and supports maintenance of tissue architecture.

The DNA methylome of human vascular endothelium and its use in liquid biopsies.

BACKGROUND: Vascular endothelial cells (VECs) are an essential component of each tissue, contribute to multiple pathologies, and are targeted by important drugs. Yet, there is a shortage of biomarkers to assess VEC turnover. METHODS: To develop DNA methylation-based liquid biopsies for VECs, we determined the methylome of VECs isolated from freshly dissociated human tissues. FINDINGS: A comparison with a human cell-type methylome atlas yielded thousands of loci that are uniquely unmethylated in VECs. These sites are typically gene enhancers, often residing adjacent to VEC-specific genes. We also identified hundreds of genomic loci that are differentially methylated in organotypic VECs, indicating that VECs feeding specific organs are distinct cell types with a stable epigenetic identity. We established universal and lung-specific VEC markers and evaluated their presence in circulating cell-free DNA (cfDNA). Nearly 2.5% of cfDNA in the plasma of healthy individuals originates from VECs. Sepsis, graft versus host disease, and cardiac catheterization are associated with elevated levels of VEC-derived cfDNA, indicative of vascular damage. Lung-specific VEC cfDNA is selectively elevated in patients with chronic obstructive pulmonary disease (COPD) or lung cancer, revealing tissue-specific vascular turnover. CONCLUSIONS: VEC cfDNA biomarkers inform vascular dynamics in health and disease, potentially contributing to early diagnosis and monitoring of pathologies, and assessment of drug activity. FUNDING: This work was supported by the Beutler Research Program, Helmsley Charitable Trust, JDRF, Grail and the DON Foundation (to Y.D.). Y.D holds the Walter & Greta Stiel Chair in heart studies. B.G., R.S., J.M., D.N., T.K., and Y.D. filed patents on cfDNA analysis.

Glycemic control releases regenerative potential of pancreatic beta cells blocked by severe hyperglycemia.

Diabetogenic ablation of beta cells in mice triggers a regenerative response whereby surviving beta cells proliferate and euglycemia is regained. Here, we identify and characterize heterogeneity in response to beta cell ablation. Efficient beta cell elimination leading to severe hyperglycemia (>28 mmol/L), causes permanent diabetes with failed regeneration despite cell cycle engagement of surviving beta cells. Strikingly, correction of glycemia via insulin, SGLT2 inhibition, or a ketogenic diet for about 3 weeks allows partial regeneration of beta cell mass and recovery from diabetes, demonstrating regenerative potential masked by extreme glucotoxicity. We identify gene expression changes in beta cells exposed to extremely high glucose levels, pointing to metabolic stress and downregulation of key cell cycle genes, suggesting failure of cell cycle completion. These findings reconcile conflicting data on the impact of glucose on beta cell regeneration and identify a glucose threshold converting glycemic load from pro-regenerative to anti-regenerative.

B cell-derived cfDNA after primary BNT162b2 mRNA vaccination anticipates memory B cells and SARS-CoV-2 neutralizing antibodies.

BACKGROUND: Much remains unknown regarding the response of the immune system to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) vaccination. METHODS: We employed circulating cell-free DNA (cfDNA) to assess the turnover of specific immune cell types following administration of the Pfizer/BioNTech vaccine. FINDINGS: The levels of B cell cfDNA after the primary dose correlated with development of neutralizing antibodies and memory B cells after the booster, revealing a link between early B cell turnover-potentially reflecting affinity maturation-and later development of effective humoral response. We also observed co-elevation of B cell, T cell, and monocyte cfDNA after the booster, underscoring the involvement of innate immune cell turnover in the development of humoral and cellular adaptive immunity. Actual cell counts remained largely stable following vaccination, other than a previously demonstrated temporary reduction in neutrophil and lymphocyte counts. CONCLUSIONS: Immune cfDNA dynamics reveal the crucial role of the primary SARS-CoV-2 vaccine in shaping responses of the immune system following the booster vaccine. FUNDING: This work was supported by a generous gift from Shlomo Kramer. Supported by grants from Human Islet Research Network (HIRN UC4DK116274 and UC4DK104216 to R.S. and Y.D.), Ernest and Bonnie Beutler Research Program of Excellence in Genomic Medicine, The Alex U Soyka Pancreatic Cancer Fund, The Israel Science Foundation, the Waldholtz/Pakula family, the Robert M. and Marilyn Sternberg Family Charitable Foundation, the Helmsley Charitable Trust, Grail, and the DON Foundation (to Y.D.). Y.D. holds the Walter and Greta Stiel Chair and Research Grant in Heart Studies. I.F.-F. received a fellowship from the Glassman Hebrew University Diabetes Center.

Early sample tagging and pooling enables simultaneous SARS-CoV-2 detection and variant sequencing.

Most severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) diagnostic tests have relied on RNA extraction followed by reverse transcription quantitative polymerase chain reaction (RT-qPCR) assays. Whereas automation improved logistics and different pooling strategies increased testing capacity, highly multiplexed next-generation sequencing (NGS) diagnostics remain a largely untapped resource. NGS tests have the potential to markedly increase throughput while providing crucial SARS-CoV-2 variant information. Current NGS-based detection and genotyping assays for SARS-CoV-2 are costly, mostly due to parallel sample processing through multiple steps. Here, we have established ApharSeq, in which samples are barcoded in the lysis buffer and pooled before reverse transcription. We validated this assay by applying ApharSeq to more than 500 clinical samples from the Clinical Virology Laboratory at Hadassah hospital in a robotic workflow. The assay was linear across five orders of magnitude, and the limit of detection was Ct 33 (~1000 copies/ml, 95% sensitivity) with >99.5% specificity. ApharSeq provided targeted high-confidence genotype information due to unique molecular identifiers incorporated into this method. Because of early pooling, we were able to estimate a 10- to 100-fold reduction in labor, automated liquid handling, and reagent requirements in high-throughput settings compared to current testing methods. The protocol can be tailored to assay other host or pathogen RNA targets simultaneously. These results suggest that ApharSeq can be a promising tool for current and future mass diagnostic challenges.

Remote immune processes revealed by immune-derived circulating cell-free DNA.

Blood cell counts often fail to report on immune processes occurring in remote tissues. Here, we use immune cell type-specific methylation patterns in circulating cell-free DNA (cfDNA) for studying human immune cell dynamics. We characterized cfDNA released from specific immune cell types in healthy individuals (N = 242), cross sectionally and longitudinally. Immune cfDNA levels had no individual steady state as opposed to blood cell counts, suggesting that cfDNA concentration reflects adjustment of cell survival to maintain homeostatic cell numbers. We also observed selective elevation of immune-derived cfDNA upon perturbations of immune homeostasis. Following influenza vaccination (N = 92), B-cell-derived cfDNA levels increased prior to elevated B-cell counts and predicted efficacy of antibody production. Patients with eosinophilic esophagitis (N = 21) and B-cell lymphoma (N = 27) showed selective elevation of eosinophil and B-cell cfDNA, respectively, which were undetectable by cell counts in blood. Immune-derived cfDNA provides a novel biomarker for monitoring immune responses to physiological and pathological processes that are not accessible using conventional methods.

ß-Cell DNA Damage Response Promotes Islet Inflammation in Type 1 Diabetes.

Type 1 diabetes (T1D) is an autoimmune disease where pancreatic ß-cells are destroyed by islet-infiltrating T cells. Although a role for ß-cell defects has been suspected, ß-cell abnormalities are difficult to demonstrate. We show a ß-cell DNA damage response (DDR), presented by activation of the 53BP1 protein and accumulation of p53, in biopsy and autopsy material from patients with recently diagnosed T1D as well as a rat model of human T1D. The ß-cell DDR is more frequent in islets infiltrated by CD45+ immune cells, suggesting a link to islet inflammation. The ß-cell toxin streptozotocin (STZ) elicits DDR in islets, both in vivo and ex vivo, and causes elevation of the proinflammatory molecules IL-1ß and Cxcl10. ß-Cell-specific inactivation of the master DNA repair gene ataxia telangiectasia mutated (ATM) in STZ-treated mice decreases the expression of proinflammatory cytokines in islets and attenuates the development of hyperglycemia. Together, these data suggest that ß-cell DDR is an early event in T1D, possibly contributing to autoimmunity.

Postnatal Exocrine Pancreas Growth by Cellular Hypertrophy Correlates with a Shorter Lifespan in Mammals.

Developmental processes in different mammals are thought to share fundamental cellular mechanisms. We report a dramatic increase in cell size during postnatal pancreas development in rodents, accounting for much of the increase in organ size after birth. Hypertrophy of pancreatic acinar cells involves both higher ploidy and increased biosynthesis per genome copy; is maximal adjacent to islets, suggesting endocrine to exocrine communication; and is partly driven by weaning-related processes. In contrast to the situation in rodents, pancreas cell size in humans remains stable postnatally, indicating organ growth by pure hyperplasia. Pancreatic acinar cell volume varies 9-fold among 24 mammalian species analyzed, and shows a striking inverse correlation with organismal lifespan. We hypothesize that cellular hypertrophy is a strategy for rapid postnatal tissue growth, entailing life-long detrimental effects.

The Genetic Program of Pancreatic ß-Cell Replication In Vivo.

The molecular program underlying infrequent replication of pancreatic ß-cells remains largely inaccessible. Using transgenic mice expressing green fluorescent protein in cycling cells, we sorted live, replicating ß-cells and determined their transcriptome. Replicating ß-cells upregulate hundreds of proliferation-related genes, along with many novel putative cell cycle components. Strikingly, genes involved in ß-cell functions, namely, glucose sensing and insulin secretion, were repressed. Further studies using single-molecule RNA in situ hybridization revealed that in fact, replicating ß-cells double the amount of RNA for most genes, but this upregulation excludes genes involved in ß-cell function. These data suggest that the quiescence-proliferation transition involves global amplification of gene expression, except for a subset of tissue-specific genes, which are "left behind" and whose relative mRNA amount decreases. Our work provides a unique resource for the study of replicating ß-cells in vivo.