Blood glucose levels are tightly controlled by the coordinated action of at least four cell types constituting pancreatic islets. Changes in the proportion and/or function of these cells are ...associated with genetic and molecular pathophysiology of monogenic, type 1, and type 2 (T2D) diabetes. Cellular heterogeneity impedes precise understanding of the molecular components of each islet cell type that govern islet (dys)function, particularly the less abundant delta and gamma/pancreatic polypeptide (PP) cells. Here, we report single-cell transcriptomes for 638 cells from nondiabetic (ND) and T2D human islet samples. Analyses of ND single-cell transcriptomes identified distinct alpha, beta, delta, and PP/gamma cell-type signatures. Genes linked to rare and common forms of islet dysfunction and diabetes were expressed in the delta and PP/gamma cell types. Moreover, this study revealed that delta cells specifically express receptors that receive and coordinate systemic cues from the leptin, ghrelin, and dopamine signaling pathways implicating them as integrators of central and peripheral metabolic signals into the pancreatic islet. Finally, single-cell transcriptome profiling revealed genes differentially regulated between T2D and ND alpha, beta, and delta cells that were undetectable in paired whole islet analyses. This study thus identifies fundamental cell-type-specific features of pancreatic islet (dys)function and provides a critical resource for comprehensive understanding of islet biology and diabetes pathogenesis.
b-Cell heterogeneity is a phenomenon that has been recognized and studied for decades from different perspectives: function (1), telomere length (2), cellular age (3), chromatin structure (4), ...methylation status (5), cyclical or compensatory changes to endoplasmic reticulum (ER) stress and unfolded protein response (6,7), pathophysiologic states like senescence (8), and pliability, such as states of transdifferentiation and dedifferentiation (9). Many studies of b-cell subpopulations have focused on what characterizes their functional profile based on insulin secretion (10,11). Determinants of heterogeneity in b-cell function have been examined, such as glucose sensitivity (12) and the biosynthetic pathways (13). All of these characteristics have been used to describe b-cell subpopulations that vary throughout stages of development, age, and pathology, but defining common mechanisms that determine this heterogeneity has remained difficult.
Pancreatic islet dysfunction and beta cell failure are hallmarks of type 2 diabetes mellitus (T2DM) pathogenesis. In this review, we discuss how genome-wide association studies (GWASs) and recent ...developments in islet (epi)genome and transcriptome profiling (particularly single cell analyses) are providing novel insights into the genetic, environmental, and cellular contributions to islet (dys)function and T2DM pathogenesis. Moving forward, study designs that interrogate and model genetic variation e.g., allelic profiling and (epi)genome editing will be critical to dissect the molecular genetics of T2DM pathogenesis, to build next-generation cellular and animal models, and to develop precision medicine approaches to detect, treat, and prevent islet (dys)function and T2DM.
Regulation of the Oocyte-to-Zygote Transition Stitzel, Michael L.; Seydoux, Geraldine
Science (American Association for the Advancement of Science),
04/2007, Volume:
316, Issue:
5823
Journal Article
Peer reviewed
Oocytes, the female germ cells, contain all the messenger RNAs necessary to start a new life but typically wait until fertilization to begin development. The transition from oocyte to fertilized egg ...(zygote) involves many changes, including protein synthesis, protein and RNA degradation, and organelle remodeling. These changes occur concurrently with the meiotic divisions that produce the haploid maternal genome. Accumulating evidence indicates that the cell-cycle regulators that control the meiotic divisions also regulate the many changes that accompany the oocyte-to-zygote transition. We suggest that the meiotic machinery functions as an internal pacemaker that propels oocytes toward embryogenesis.
Pancreatic islet (dys)function and failure is central to both type 2 diabetes (T2D) genetic risk and pathogenesis. Islet cellular heterogeneity has impeded precise understanding of the specific ...gene(s) and pathway(s) in each cell type that are altered by and/or may contribute to pathophysiology in the prediabetic (PD) and type 2 diabetic (T2D) states. To identify cellular and molecular features of islet dysfunction in PD and T2D states, we completed single cell transcriptome profiling of 137k human islet cells from 11 ND, 6 PD, and 11 T2D donors (⋃5k/individual).
Single cell transcriptomes aggregated into distinct clusters of endocrine (alpha, beta, delta, PP/gamma), exocrine (acinar, stellate, ductal), endothelial, and immune cell types. Targeted analysis of each endocrine cell type identified a proliferative alpha cell sub-population comprising ∼0.15-0.3% of endocrine cells and variable insulin production and endoplasmic reticulum stress beta cell states. We compared the proportion of islet endocrine cell type (sub)populations sampled between T2D, PD, and ND donors and found that T2D samples contained significantly fewer beta cells and more alpha cells than ND and, to a lesser degree, PD samples. Delta and PP/gamma cell proportions were similar between disease states. Importantly, no (sub)populations were ND, PD, or T2D-specific.
We compared aggregate T2D, PD, and ND cell type-specific transcriptomes to identify differentially expressed genes in each cell type. Genes related to exocytic granule assembly/organization, including those induced by T2D risk variants (DGKB) or T2D state (GAP43), were among 99 genes induced in T2D beta cells. 103 repressed genes included those associated with glucose transport and implicated as T2D GWAS effector genes such as SLC2A2, SLC5A1, and STARD10, a gene whose deletion in mice impairs islet insulin secretion. Studies are ongoing to assess the functional role(s) of these and additional genes in islet (dys)function.
Disclosure
N. Lawlor: None. R. Kursawe: None. M.L. Stitzel: None.
Funding
American Diabetes Association/Pathway to Stop Diabetes (1-18-ACE-15 to M.L.S.)
Genome-wide association studies (GWASs) have identified more than 70 loci associated with type 2 diabetes (T2D), but for most, the underlying causal variants, associated genes, and functional ...mechanisms remain unknown. At a T2D- and fasting-proinsulin-associated locus on 11q13.4, we have identified a functional regulatory DNA variant, a candidate target gene, and a plausible underlying molecular mechanism. Fine mapping, conditional analyses, and exome array genotyping in 8,635 individuals from the Metabolic Syndrome in Men study confirmed a single major association signal between fasting proinsulin and noncoding variants (p = 7.4 × 10−50). Measurement of allele-specific mRNA levels in human pancreatic islet samples heterozygous for rs11603334 showed that the T2D-risk and proinsulin-decreasing allele (C) is associated with increased ARAP1 expression (p < 0.02). We evaluated four candidate functional SNPs for allelic effects on transcriptional activity by performing reporter assays in rodent pancreatic beta cell lines. The C allele of rs11603334, located near one of the ARAP1 promoters, exhibited 2-fold higher transcriptional activity than did the T allele (p < 0.0001); three other candidate SNPs showed no allelic differences. Electrophoretic mobility shift assays demonstrated decreased binding of pancreatic beta cell transcriptional regulators PAX6 and PAX4 to the rs11603334 C allele. Collectively, these data suggest that the T2D-risk allele of rs11603334 could abrogate binding of a complex containing PAX6 and PAX4 and thus lead to increased promoter activity and ARAP1 expression in human pancreatic islets. This work suggests that increased ARAP1 expression might contribute to T2D susceptibility at this GWAS locus.
Abstract
Motivation
Single-cell RNA-sequencing (scRNA-seq) has brought the study of the transcriptome to higher resolution and makes it possible for scientists to provide answers with more clarity to ...the question of 'differential expression'. However, most computational methods still stick with the old mentality of viewing differential expression as a simple 'up or down' phenomenon. We advocate that we should fully embrace the features of single cell data, which allows us to observe binary (from Off to On) as well as continuous (the amount of expression) regulations.
Results
We develop a method, termed SC2P, that first identifies the phase of expression a gene is in, by taking into account of both cell- and gene-specific contexts, in a model-based and data-driven fashion. We then identify two forms of transcription regulation: phase transition, and magnitude tuning. We demonstrate that compared with existing methods, SC2P provides substantial improvement in sensitivity without sacrificing the control of false discovery, as well as better robustness. Furthermore, the analysis provides better interpretation of the nature of regulation types in different genes.
Availability and implementation
SC2P is implemented as an open source R package publicly available at https://github.com/haowulab/SC2P.
Supplementary information
Supplementary data are available at Bioinformatics online.
Cis-Regulatory elements (cis-REs) include promoters, enhancers, and insulators that regulate gene expression programs via binding of transcription factors. ATAC-seq technology effectively identifies ...active cis-REs in a given cell type (including from single cells) by mapping accessible chromatin at base-pair resolution. However, these maps are not immediately useful for inferring specific functions of cis-REs. For this purpose, we developed a deep learning framework (CoRE-ATAC) with novel data encoders that integrate DNA sequence (reference or personal genotypes) with ATAC-seq cut sites and read pileups. CoRE-ATAC was trained on 4 cell types (n = 6 samples/replicates) and accurately predicted known cis-RE functions from 7 cell types (n = 40 samples) that were not used in model training (mean average precision = 0.80, mean F1 score = 0.70). CoRE-ATAC enhancer predictions from 19 human islet samples coincided with genetically modulated gain/loss of enhancer activity, which was confirmed by massively parallel reporter assays (MPRAs). Finally, CoRE-ATAC effectively inferred cis-RE function from aggregate single nucleus ATAC-seq (snATAC) data from human blood-derived immune cells that overlapped with known functional annotations in sorted immune cells, which established the efficacy of these models to study cis-RE functions of rare cells without the need for cell sorting. ATAC-seq maps from primary human cells reveal individual- and cell-specific variation in cis-RE activity. CoRE-ATAC increases the functional resolution of these maps, a critical step for studying regulatory disruptions behind diseases.
Molecular mechanisms remain unknown for most type 2 diabetes genome-wide association study identified loci. Variants associated with type 2 diabetes and fasting glucose levels reside in introns of
, ...a gene that encodes adenylate cyclase 5. Adenylate cyclase 5 catalyzes the production of cyclic AMP, which is a second messenger molecule involved in cell signaling and pancreatic β-cell insulin secretion. We demonstrated that type 2 diabetes risk alleles are associated with decreased
expression in human islets and examined candidate variants for regulatory function. rs11708067 overlaps a predicted enhancer region in pancreatic islets. The type 2 diabetes risk rs11708067-A allele showed fewer H3K27ac ChIP-seq reads in human islets, lower transcriptional activity in reporter assays in rodent β-cells (rat 832/13 and mouse MIN6), and increased nuclear protein binding compared with the rs11708067-G allele. Homozygous deletion of the orthologous enhancer region in 832/13 cells resulted in a 64% reduction in expression level of
, but not adjacent gene
, and a 39% reduction in insulin secretion. Together, these data suggest that rs11708067-A risk allele contributes to type 2 diabetes by disrupting an islet enhancer, which results in reduced
expression and impaired insulin secretion.
We used an unbiased genome-wide approach to identify exonic variants segregating with diabetes in a multigenerational Finnish family. At least eight members of this family presented with diabetes ...with age of diagnosis ranging from 18 to 51 years and a pattern suggesting autosomal dominant inheritance. We sequenced the exomes of four affected members of this family and performed follow-up genotyping of additional affected and unaffected family members. We uncovered a novel nonsynonymous variant (p.Trp314Arg) in the Wolfram syndrome 1 (WFS1) gene that segregates completely with the diabetic phenotype. Multipoint parametric linkage analysis with 13 members of this family identified a single linkage signal with maximum logarithm of odds score 3.01 at 4p16.2-p16.1, corresponding to a region harboring the WFS1 locus. Functional studies demonstrate a role for this variant in endoplasmic reticulum stress, which is consistent with the β-cell failure phenotype seen in mutation carriers. This represents the first compelling report of a mutation in WFS1 associated with dominantly inherited nonsyndromic adult-onset diabetes.
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