Dana Avrahami

Aging-Dependent Demethylation of Regulatory Elements Correlates with Chromatin State and Improved β Cell Function

Aging is driven by changes of the epigenetic state that are only partially understood. We performed a comprehensive epigenomic analysis of the pancreatic β cell, key player in glucose homeostasis, in adolescent and very old mice. We observe a global methylation drift resulting in an overall more leveled methylome in old β cells. Importantly, we discover targeted changes in the methylation status of β cell proliferation and function genes that go against the global methylation drift, are specific to β cells, and correlate with repression of the proliferation program and activation of metabolic regulators. These targeted alterations are associated with specific chromatin marks and transcription factor occupancy in young β cells. Strikingly, we find β cell function improved in aged mice, as predicted by the changes in methylome and transcriptome. Thus, aging of terminally differentiated cells in mammals is not always coupled to functional decline.

Single-Cell Transcriptomics of the Human Endocrine Pancreas.

Human pancreatic islets consist of multiple endocrine cell types. To facilitate the detection of rare cellular states and uncover population heterogeneity, we performed single-cell RNA sequencing (RNA-seq) on islets from multiple deceased organ donors, including children, healthy adults, and individuals with type 1 or type 2 diabetes. We developed a robust computational biology framework for cell type annotation. Using this framework, we show that α- and β-cells from children exhibit less well-defined gene signatures than those in adults. Remarkably, α- and β-cells from donors with type 2 diabetes have expression profiles with features seen in children, indicating a partial dedifferentiation process. We also examined a naturally proliferating α-cell from a healthy adult, for which pathway analysis indicated activation of the cell cycle and repression of checkpoint control pathways. Importantly, this replicating α-cell exhibited activated Sonic hedgehog signaling, a pathway not previously known to contribute to human α-cell proliferation. Our study highlights the power of single-cell RNA-seq and provides a stepping stone for future explorations of cellular heterogeneity in pancreatic endocrine cells.

Universal Minicircle Sequence Binding Protein, a CCHC-type Zinc Finger Protein That Binds the Universal Minicircle Sequence of Trypanosomatids PURIFICATION AND CHARACTERIZATION

Replication of kinetoplast DNA minicircles of trypanosomatids initiates at a conserved 12-nucleotide sequence, termed the universal minicircle sequence (UMS, 5′-GGGGTTGGTGTA-3′). A single-stranded nucleic acid binding protein that binds specifically to this origin-associated sequence was purified to apparent homogeneity from Crithidia fasciculata cell extracts. This UMS-binding protein (UMSBP) is a dimer of 27.4 kDa with a 13.7-kDa protomer. UMSBP binds single-stranded DNA as well as single-stranded RNA but not double-stranded or four-stranded DNA structures. Stoichiometry analysis indicates the binding of UMSBP as a protein dimer to the UMS site. The five CCHC-type zinc finger motifs of UMSBP, predicted from its cDNA sequence, are similar to the CCHC motifs found in retroviral Gag polyproteins. The remarkable conservation of this motif in a family of proteins found in eukaryotic organisms from yeast and protozoa to mammals is discussed.

Targeting the cell cycle inhibitor p57Kip2 promotes adult human β cell replication.

Children with focal hyperinsulinism of infancy display a dramatic, non-neoplastic clonal expansion of β cells that have undergone mitotic recombination, resulting in paternal disomy of part of chromosome 11. This disomic region contains imprinted genes, including the gene encoding the cell cycle inhibitor p57Kip2 (CDKN1C), which is silenced as a consequence of the recombination event. We hypothesized that targeting p57Kip2 could stimulate adult human β cell replication. Indeed, when we suppressed CDKN1C expression in human islets obtained from deceased adult organ donors and transplanted them into hyperglycemic, immunodeficient mice, β cell replication increased more than 3-fold. The newly replicated cells retained properties of mature β cells, including the expression of β cell markers such as insulin, PDX1, and NKX6.1. Importantly, these newly replicated cells demonstrated normal glucose-induced calcium influx, further indicating β cell functionality. These findings provide a molecular explanation for the massive β cell replication that occurs in children with focal hyperinsulinism. These data also provided evidence that β cells from older humans, in which baseline replication is negligible, can be coaxed to re-enter and complete the cell cycle while maintaining mature β cell properties. Thus, controlled manipulation of this pathway holds promise for the expansion of β cells in patients with type 2 diabetes.

ARNO through Its Coiled-coil Domain Regulates Endocytosis at the Apical Surface of Polarized Epithelial Cells

ARNO is a guanine-nucleotide exchange protein for the ARF family of GTPases. Here we show that in polarized epithelial cells, ARNO is localized exclusively to the apical plasma membrane, where it regulates endocytosis. Expression of ARNO stimulates apical endocytosis of the polymeric immunoglobulin receptor, and coexpression of ARF6 with ARNO leads to a synergistic stimulation of apical endocytosis. Expression of a dominant negative ARF6 mutant, ARF6-T27N, antagonizes this stimulatory effect. Deletion of the N-terminal coiled-coil (CC) domain of ARNO causes the mutant ARNO to localize to both the apical and basolateral plasma membranes. Expression of the CC domain alone abolishes ARNO-induced apical endocytosis as well as co-localization of IgA-receptor complexes with ARNO and clathrin. These results suggest that the CC domain contributes to the specificity of apical localization of ARNO through association with components of the apical plasma membrane. We conclude that ARNO acts together with ARF6 to regulate apical endocytosis.

PAX6 maintains β cell identity by repressing genes of alternative islet cell types

Type 2 diabetes is thought to involve a compromised &bgr; cell differentiation state, but the mechanisms underlying this dysfunction remain unclear. Here, we report a key role for the TF PAX6 in the maintenance of adult &bgr; cell identity and function. PAX6 was downregulated in &bgr; cells of diabetic db/db mice and in WT mice treated with an insulin receptor antagonist, revealing metabolic control of expression. Deletion of Pax6 in &bgr; cells of adult mice led to lethal hyperglycemia and ketosis that were attributed to loss of &bgr; cell function and expansion of &agr; cells. Lineage-tracing, transcriptome, and chromatin analyses showed that PAX6 is a direct activator of &bgr; cell genes, thus maintaining mature &bgr; cell function and identity. In parallel, we found that PAX6 binds promoters and enhancers to repress alternative islet cell genes including ghrelin, glucagon, and somatostatin. Chromatin analysis and shRNA-mediated gene suppression experiments indicated a similar function of PAX6 in human &bgr; cells. We conclude that reduced expression of PAX6 in metabolically stressed &bgr; cells may contribute to &bgr; cell failure and &agr; cell dysfunction in diabetes.

Epigenetic regulation of pancreas development and function

Multiple signaling systems and transcription factor cascades control pancreas development and endocrine cell fate determination. Epigenetic processes contribute to the control of this transcriptional hierarchy, involving both histone modifications and DNA methylation. Here, we summarize recent advances in the field that demonstrate the importance of epigenetic regulation in pancreas development, β-cell proliferation, and cell fate choice. These breakthroughs were made using the phenotypic analysis of mice with mutations in genes that encode histone modifying enzymes and related proteins; by application of activators or inhibitors of the enzymes that acetylate or methylate histones to fetal pancreatic explants in culture; and by genomic approaches that determined the patterns of histone modifications and chromatin state genome-wide.

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.

Effects of ageing and senescence on pancreatic β-cell function

Ageing is generally associated with deterioration of organ function and regenerative potential. In the case of pancreatic β‐cells, an age‐related decline in proliferative potential is well documented, and was proposed to contribute to the increased prevalence of type 2 diabetes in the elderly. The effects of ageing on β‐cell function, namely glucose‐stimulated insulin secretion (GSIS), have not been studied as extensively. Recent work revealed that, surprisingly, β‐cells of mature mice and humans secrete more insulin than young β‐cells in response to high glucose concentrations, potentially serving to counteract age‐related peripheral insulin resistance. This functional change appears to be orchestrated by p16Ink4A‐driven cellular senescence and downstream remodelling of chromatin structure and DNA methylation, enhancing the expression of genes controlling β‐cell function. We propose that activation of the cellular senescence program drives life‐long functional maturation of β‐cells, due to β‐cell hypertrophy, enhanced glucose uptake and more efficient mitochondrial metabolism, in parallel to locking these cells in a non‐replicative state. We speculate that the beneficial aspects of this process can be harnessed to enhance GSIS. Other age‐related mechanisms, which are currently poorly understood, act to increase basal insulin secretion levels also in low glucose conditions. This leads to an overall reduction in the amplitude of insulin secretion between low and high glucose at old age, which may contribute to a deterioration in metabolic control.

β-Cells are not uniform after all-Novel insights into molecular heterogeneity of insulin-secreting cells: AVRAHAMI et al.

While the β‐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 β‐cells exist in several antigenically and molecularly definable states. Using antibodies to cell surface markers or multidimensional clustering of β‐cells using more than 20 protein markers by mass cytometry, 4 distinct groups of β‐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 β‐cell heterogeneity. Here, we aim to summarize concisely the recent advances in the field and their possible impact on our understanding of β‐cell physiology and pathophysiology.