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.

The core clock transcription factor BMAL1 drives circadian ß-cell proliferation during compensatory regeneration of the endocrine pancreas.

Circadian clocks in pancreatic islets participate in the regulation of glucose homeostasis. Here we examined the role of these timekeepers in ß-cell regeneration after the massive ablation of ß cells by doxycycline-induced expression of diphtheria toxin A (DTA) in Insulin-rtTA/TET-DTA mice. Since we crossed reporter genes expressing α- and ß-cell-specific fluorescent proteins into these mice, we could follow the fate of α- and ß cells separately. As expected, DTA induction resulted in an acute hyperglycemia, which was accompanied by dramatic changes in gene expression in residual ß cells. In contrast, only temporal alterations of gene expression were observed in α cells. Interestingly, ß cells entered S phase preferentially during the nocturnal activity phase, indicating that the diurnal rhythm also plays a role in the orchestration of ß-cell regeneration. Indeed, in arrhythmic Bmal1-deficient mice, which lack circadian clocks, no compensatory ß-cell proliferation was observed, and the ß-cell ablation led to aggravated hyperglycemia, hyperglucagonemia, and fatal diabetes.

Diabetes risk gene and Wnt effector Tcf7l2/TCF4 controls hepatic response to perinatal and adult metabolic demand.

Most studies on TCF7L2 SNP variants in the pathogenesis of type 2 diabetes (T2D) focus on a role of the encoded transcription factor TCF4 in ß cells. Here, a mouse genetics approach shows that removal of TCF4 from ß cells does not affect their function, whereas manipulating TCF4 levels in the liver has major effects on metabolism. In Tcf7l2(-/-) mice, the immediate postnatal surge in liver metabolism does not occur. Consequently, pups die due to hypoglycemia. By combining chromatin immunoprecipitation with gene expression profiling, we identify a TCF4-controlled metabolic gene program that is acutely activated in the postnatal liver. In concordance, adult liver-specific Tcf7l2 knockout mice show reduced hepatic glucose production during fasting and display improved glucose homeostasis when maintained on high-fat diet. Furthermore, liver-specific TCF4 overexpression increases hepatic glucose production. These observations imply that TCF4 directly activates metabolic genes and that inhibition of Wnt signaling may be beneficial in metabolic disease.

Epigenetic liquid biopsies: a novel putative biomarker in immunology and inflammation.

Immune and inflammatory processes occurring within tissues are often undetectable by blood cell counts, standard circulating biomarkers, or imaging, representing an unmet biomedical need. Here, we outline recent advances indicating that liquid biopsies can broadly inform human immune system dynamics. Nucleosome-size fragments of cell-free DNA (cfDNA) released from dying cells into blood contain rich epigenetic information such as methylation, fragmentation, and histone mark patterns. This information allows to infer the cfDNA cell of origin, as well as pre-cell death gene expression patterns. We propose that the analysis of epigenetic features of immune cell-derived cfDNA can shed light on immune cell turnover dynamics in healthy people, and inform the study and diagnosis of cancer, local inflammation, infectious or autoimmune diseases, as well as responses to vaccination.

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.

The histone deacetylase Sirt6 regulates glucose homeostasis via Hif1alpha.

SIRT6 is a member of a highly conserved family of NAD(+)-dependent deacetylases with various roles in metabolism, stress resistance, and life span. SIRT6-deficient mice develop normally but succumb to a lethal hypoglycemia early in life; however, the mechanism underlying this hypoglycemia remained unclear. Here, we demonstrate that SIRT6 functions as a histone H3K9 deacetylase to control the expression of multiple glycolytic genes. Specifically, SIRT6 appears to function as a corepressor of the transcription factor Hif1alpha, a critical regulator of nutrient stress responses. Consistent with this notion, SIRT6-deficient cells exhibit increased Hif1alpha activity and show increased glucose uptake with upregulation of glycolysis and diminished mitochondrial respiration. Our studies uncover a role for the chromatin factor SIRT6 as a master regulator of glucose homeostasis and may provide the basis for novel therapeutic approaches against metabolic diseases, such as diabetes and obesity.

Protein biomarkers and alternatively methylated cell-free DNA detect early stage pancreatic cancer.

OBJECTIVE: Pancreatic ductal adenocarcinoma (PDAC) is commonly diagnosed at an advanced stage. Liquid biopsy approaches may facilitate detection of early stage PDAC when curative treatments can be employed. DESIGN: To assess circulating marker discrimination in training, testing and validation patient cohorts (total n=426 patients), plasma markers were measured among PDAC cases and patients with chronic pancreatitis, colorectal cancer (CRC), and healthy controls. Using CA19-9 as an anchor marker, measurements were made of two protein markers (TIMP1, LRG1) and cell-free DNA (cfDNA) pancreas-specific methylation at 9 loci encompassing 61 CpG sites. RESULTS: Comparative methylome analysis identified nine loci that were differentially methylated in exocrine pancreas DNA. In the training set (n=124 patients), cfDNA methylation markers distinguished PDAC from healthy and CRC controls. In the testing set of 86 early stage PDAC and 86 matched healthy controls, CA19-9 had an area under the receiver operating characteristic curve (AUC) of 0.88 (95% CI 0.83 to 0.94), which was increased by adding TIMP1 (AUC 0.92; 95% CI 0.88 to 0.96; p=0.06), LRG1 (AUC 0.92; 95% CI 0.88 to 0.96; p=0.02) or exocrine pancreas-specific cfDNA methylation markers at nine loci (AUC 0.92; 95% CI 0.88 to 0.96; p=0.02). In the validation set of 40 early stage PDAC and 40 matched healthy controls, a combined panel including CA19-9, TIMP1 and a 9-loci cfDNA methylation panel had greater discrimination (AUC 0.86, 95% CI 0.77 to 0.95) than CA19-9 alone (AUC 0.82; 95% CI 0.72 to 0.92). CONCLUSION: A combined panel of circulating markers including proteins and methylated cfDNA increased discrimination compared with CA19-9 alone for early stage PDAC.

A universal cell-free DNA approach for response prediction to preoperative chemoradiation in rectal cancer.

The standard treatment approach for stage II/III rectal cancer is neoadjuvant chemoradiation therapy (nCRT) followed by surgery. In recent years, new treatment approaches have led to higher rates of complete tumor eradication combined with organ-preservation strategies. However, better tools are still needed to personalize therapy for the individual patient. In this prospective observational study, we analyzed colon-derived cell-free (cf)DNA (c-cfDNA) using a tissue-specific DNA methylation signature, and its association with therapy outcomes. Analyzing plasma samples (n = 303) collected during nCRT from 37 patients with locally advanced rectal cancer (LARC), we identified colon-specific methylation markers that discriminated healthy individuals from patients with untreated LARC (area under the curve, 0.81; 95% confidence interval, 0.70-0.92; P < .0001). Baseline c-cfDNA predicted tumor response, with increased levels linked to larger residual cancer. c-cfDNA measured after the first week of therapy identified patients with maximal response and complete cancer eradication, who had significantly lower c-cfDNA compared with those who had residual disease (8.6 vs 57.7 average copies/ml, respectively; P = .013). Increased c-cfDNA after 1 week of therapy was also associated with disease recurrence. Methylation-based liquid biopsy can predict nCRT outcomes and facilitate patient selection for escalation and de-escalation strategies.

Beta-cell death and dysfunction drives hyperglycaemia in organ donors.

BACKGROUND: Donor hyperglycaemia following brain death has been attributed to reversible insulin resistance. However, our islet and pancreas transplant data suggest that other mechanisms may be predominant. We aimed to determine the relationships between donor insulin use and markers of beta-cell death and beta-cell function in pancreas donors after brain death. METHODS: In pancreas donors after brain death, we compared clinical and biochemical data in 'insulin-treated' and 'not insulin-treated donors' (IT vs. not-IT). We measured plasma glucose, C-peptide and levels of circulating unmethylated insulin gene promoter cell-free DNA (INS-cfDNA) and microRNA-375 (miR-375), as measures of beta-cell death. Relationships between markers of beta-cell death and islet isolation outcomes and post-transplant function were also evaluated. RESULTS: Of 92 pancreas donors, 40 (43%) required insulin. Glycaemic control and beta-cell function were significantly poorer in IT donors versus not-IT donors [median (IQR) peak glucose: 8 (7-11) vs. 6 (6-8) mmol/L, p = .016; C-peptide: 3280 (3159-3386) vs. 3195 (2868-3386) pmol/L, p = .046]. IT donors had significantly higher levels of INS-cfDNA [35 (18-52) vs. 30 (8-51) copies/ml, p = .035] and miR-375 [1.050 (0.19-1.95) vs. 0.73 (0.32-1.10) copies/nl, p = .05]. Circulating donor miR-375 was highly predictive of recipient islet graft failure at 3 months [adjusted receiver operator curve (SE) = 0.813 (0.149)]. CONCLUSIONS: In pancreas donors, hyperglycaemia requiring IT is strongly associated with beta-cell death. This provides an explanation for the relationship of donor IT with post-transplant beta-cell dysfunction in transplant recipients.

Facultative endocrine progenitor cells in the adult pancreas.

Using a unique injury model of the pancreas in mouse, Xu et al. (2008) now reveal the involvement of neurogenin3, a marker for embryonic-type endocrine progenitor cells, in the formation of new insulin-producing beta cells. These neurogenin3-positive facultative endocrine progenitor cells in the adult pancreas may be of potential value for treating diabetes.

Senolytic elimination of Cox2-expressing senescent cells inhibits the growth of premalignant pancreatic lesions.

OBJECTIVE: Cellular senescence limits tumourigenesis by blocking the proliferation of premalignant cells. Additionally, however, senescent cells can exert paracrine effects influencing tumour growth. Senescent cells are present in premalignant pancreatic intraepithelial neoplasia (PanIN) lesions, yet their effects on the disease are poorly characterised. It is currently unknown whether senolytic drugs, aimed at eliminating senescent cells from lesions, could be beneficial in blocking tumour development. DESIGN: To uncover the functions of senescent cells and their potential contribution to early pancreatic tumourigenesis, we isolated and characterised senescent cells from PanINs formed in a Kras-driven mouse model, and tested the consequences of their targeted elimination through senolytic treatment. RESULTS: We found that senescent PanIN cells exert a tumour-promoting effect through expression of a proinflammatory signature that includes high Cox2 levels. Senolytic treatment with the Bcl2-family inhibitor ABT-737 eliminated Cox2-expressing senescent cells, and an intermittent short-duration treatment course dramatically reduced PanIN development and progression to pancreatic ductal adenocarcinoma. CONCLUSIONS: These findings reveal that senescent PanIN cells support tumour growth and progression, and provide a first indication that elimination of senescent cells may be effective as preventive therapy for the progression of precancerous lesions.

Universal lung epithelium DNA methylation markers for detection of lung damage in liquid biopsies.

BACKGROUND: Circulating biomarkers for lung damage are lacking. Lung epithelium-specific DNA methylation patterns can potentially report the presence of lung-derived cell-free DNA (cfDNA) in blood, as an indication of lung cell death. METHODS: We sorted human lung alveolar and bronchial epithelial cells from surgical specimens, and obtained their methylomes using whole-genome bisulfite sequencing. We developed a PCR sequencing assay determining the methylation status of 17 loci with lung-specific methylation patterns, and used it to assess lung-derived cfDNA in the plasma of healthy volunteers and patients with lung disease. RESULTS: Loci that are uniquely unmethylated in alveolar or bronchial epithelial cells are enriched for enhancers controlling lung-specific genes. Methylation markers extracted from these methylomes revealed that normal lung cell turnover probably releases cfDNA into the air spaces, rather than to blood. People with advanced lung cancer show a massive elevation of lung cfDNA concentration in blood. Among individuals undergoing bronchoscopy, lung-derived cfDNA is observed in the plasma of those later diagnosed with lung cancer, and to a lesser extent in those diagnosed with other lung diseases. Lung cfDNA is also elevated in patients with acute exacerbation of COPD compared with patients with stable disease, and is associated with future exacerbation and mortality in these patients. CONCLUSIONS: Universal cfDNA methylation markers of normal lung epithelium allow for mutation-independent, sensitive and specific detection of lung-derived cfDNA, reporting on ongoing lung injury. Such markers can find broad utility in the study of normal and pathologic human lung dynamics.

The activating receptor NKp46 is essential for the development of type 1 diabetes.

The mechanism of action of natural killer (NK) cells in type 1 diabetes is still unknown. Here we show that the activating receptor NKp46 recognizes mouse and human ligands on pancreatic beta cells. NK cells appeared in the pancreas when insulitis progressed to type 1 diabetes, and NKp46 engagement by beta cells led to degranulation of NK cells. NKp46-deficient mice had less development of type 1 diabetes induced by injection of a low dose of streptozotocin. Injection of soluble NKp46 proteins into nonobese diabetic mice during the early phase of insulitis and the prediabetic stage prevented the development of type 1 diabetes. Our findings demonstrate that NKp46 is essential for the development of type 1 diabetes and highlight potential new therapeutic modalities for this disease.

Biphasic dynamics of beta cell mass in a mouse model of congenital hyperinsulinism: implications for type 2 diabetes.

AIMS/HYPOTHESIS: Acute hyperglycaemia stimulates pancreatic beta cell proliferation in the mouse whereas chronic hyperglycaemia appears to be toxic. We hypothesise that this toxic effect is mediated by increased beta cell workload, unrelated to hyperglycaemia per se. METHODS: To test this hypothesis, we developed a novel mouse model of cell-autonomous increased beta cell glycolytic flux caused by a conditional heterozygous beta cell-specific mutation that activates glucokinase (GCK), mimicking key aspects of the rare human genetic disease GCK-congenital hyperinsulinism. RESULTS: In the mutant mice, we observed random and fasting hypoglycaemia (random 4.5-5.4 mmol/l and fasting 3.6 mmol/l) that persisted for 15 months. GCK activation led to increased beta cell proliferation as measured by Ki67 expression (2.7% vs 1.5%, mutant and wild-type (WT), respectively, p < 0.01) that resulted in a 62% increase in beta cell mass in young mice. However, by 8 months of age, mutant mice developed impaired glucose tolerance, which was associated with decreased absolute beta cell mass from 2.9 mg at 1.5 months to 1.8 mg at 8 months of age, with preservation of individual beta cell function. Impaired glucose tolerance was further exacerbated by a high-fat/high-sucrose diet (AUC 1796 vs 966 mmol/l × min, mutant and WT, respectively, p < 0.05). Activation of GCK was associated with an increased DNA damage response and an elevated expression of Chop, suggesting metabolic stress as a contributor to beta cell death. CONCLUSIONS/INTERPRETATION: We propose that increased workload-driven biphasic beta cell dynamics contribute to decreased beta cell function observed in long-standing congenital hyperinsulinism and type 2 diabetes.

Towards systematic nomenclature for cell-free DNA.

Cell-free DNA (cfDNA) has become widely recognized as a promising candidate biomarker for minimally invasive characterization of various genomic disorders and other clinical scenarios. However, among the obstacles that currently challenge the general progression of the research field, there remains an unmet need for unambiguous universal cfDNA nomenclature. To address this shortcoming, we classify in this report the different types of cfDNA molecules that occur in the human body based on its origin, genetic traits, and locality. We proceed by assigning existing terms to each of these cfDNA subtypes, while proposing new terms and abbreviations where clarity is lacking and more precise stratification would be beneficial. We then suggest the proper usage of these terms within different contexts and scenarios, focusing mainly on the nomenclature as it relates to the domains of oncology, prenatal testing, and post-transplant surgery surveillance. We hope that these recommendations will serve as useful considerations towards the establishment of universal cfDNA nomenclature in the future. In addition, it is conceivable that many of these recommendations can be transposed to cell-free RNA nomenclature by simply exchanging "DNA" with "RNA" in each acronym/abbreviation. Similarly, when describing DNA and RNA collectively, the suffix can be replaced with "NAs" to indicate nucleic acids.

Islet cells share promoter hypomethylation independently of expression, but exhibit cell-type-specific methylation in enhancers.

DNA methylation at promoters is an important determinant of gene expression. Earlier studies suggested that the insulin gene promoter is uniquely unmethylated in insulin-expressing pancreatic ß-cells, providing a classic example of this paradigm. Here we show that islet cells expressing insulin, glucagon, or somatostatin share a lack of methylation at the promoters of the insulin and glucagon genes. This is achieved by rapid demethylation of the insulin and glucagon gene promoters during differentiation of Neurogenin3+ embryonic endocrine progenitors, regardless of the specific endocrine cell-type chosen. Similar methylation dynamics were observed in transgenic mice containing a human insulin promoter fragment, pointing to the responsible cis element. Whole-methylome comparison of human α- and ß-cells revealed generality of the findings: genes active in one cell type and silent in the other tend to share demethylated promoters, while methylation differences between α- and ß-cells are concentrated in enhancers. These findings suggest an epigenetic basis for the observed plastic identity of islet cell types, and have implications for ß-cell reprogramming in diabetes and diagnosis of ß-cell death using methylation patterns of circulating DNA.

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.

Principles of DNA methylation and their implications for biology and medicine.

DNA methylation represents an annotation system for marking the genetic text, thus providing instruction as to how and when to read the information and control transcription. Unlike sequence information, which is inherited, methylation patterns are established in a programmed process that continues throughout development, thus setting up stable gene expression profiles. This DNA methylation paradigm is a key player in medicine. Some changes in methylation closely correlate with age providing a marker for biological ageing, and these same sites could also play a part in cancer. The genome continues to undergo programmed variation in methylation after birth in response to environmental inputs, serving as a memory device that could affect ageing and predisposition to various metabolic, autoimmune, and neurological diseases. Taking advantage of tissue-specific differences, methylation can be used to detect cell death and thereby monitor many common diseases with a simple cell-free circulating-DNA blood test.

Identification of tissue-specific cell death using methylation patterns of circulating DNA.

Minimally invasive detection of cell death could prove an invaluable resource in many physiologic and pathologic situations. Cell-free circulating DNA (cfDNA) released from dying cells is emerging as a diagnostic tool for monitoring cancer dynamics and graft failure. However, existing methods rely on differences in DNA sequences in source tissues, so that cell death cannot be identified in tissues with a normal genome. We developed a method of detecting tissue-specific cell death in humans based on tissue-specific methylation patterns in cfDNA. We interrogated tissue-specific methylome databases to identify cell type-specific DNA methylation signatures and developed a method to detect these signatures in mixed DNA samples. We isolated cfDNA from plasma or serum of donors, treated the cfDNA with bisulfite, PCR-amplified the cfDNA, and sequenced it to quantify cfDNA carrying the methylation markers of the cell type of interest. Pancreatic ß-cell DNA was identified in the circulation of patients with recently diagnosed type-1 diabetes and islet-graft recipients; oligodendrocyte DNA was identified in patients with relapsing multiple sclerosis; neuronal/glial DNA was identified in patients after traumatic brain injury or cardiac arrest; and exocrine pancreas DNA was identified in patients with pancreatic cancer or pancreatitis. This proof-of-concept study demonstrates that the tissue origins of cfDNA and thus the rate of death of specific cell types can be determined in humans. The approach can be adapted to identify cfDNA derived from any cell type in the body, offering a minimally invasive window for diagnosing and monitoring a broad spectrum of human pathologies as well as providing a better understanding of normal tissue dynamics.

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.