Ignasi Moran

Pancreatic islet enhancer clusters enriched in type 2 diabetes risk-associated variants

Type 2 diabetes affects over 300 million people, causing severe complications and premature death, yet the underlying molecular mechanisms are largely unknown. Pancreatic islet dysfunction is central in type 2 diabetes pathogenesis, and understanding islet genome regulation could therefore provide valuable mechanistic insights. We have now mapped and examined the function of human islet cis-regulatory networks. We identify genomic sequences that are targeted by islet transcription factors to drive islet-specific gene activity and show that most such sequences reside in clusters of enhancers that form physical three-dimensional chromatin domains. We find that sequence variants associated with type 2 diabetes and fasting glycemia are enriched in these clustered islet enhancers and identify trait-associated variants that disrupt DNA binding and islet enhancer activity. Our studies illustrate how islet transcription factors interact functionally with the epigenome and provide systematic evidence that the dysregulation of islet enhancers is relevant to the mechanisms underlying type 2 diabetes.

Human β Cell Transcriptome Analysis Uncovers lncRNAs That Are Tissue-Specific, Dynamically Regulated, and Abnormally Expressed in Type 2 Diabetes

A significant portion of the genome is transcribed as long noncoding RNAs (lncRNAs), several of which are known to control gene expression. The repertoire and regulation of lncRNAs in disease-relevant tissues, however, has not been systematically explored. We report a comprehensive strand-specific transcriptome map of human pancreatic islets and β cells, and uncover >1100 intergenic and antisense islet-cell lncRNA genes. We find islet lncRNAs that are dynamically regulated and show that they are an integral component of the β cell differentiation and maturation program. We sequenced the mouse islet transcriptome and identify lncRNA orthologs that are regulated like their human counterparts. Depletion of HI-LNC25, a β cell-specific lncRNA, downregulated GLIS3 mRNA, thus exemplifying a gene regulatory function of islet lncRNAs. Finally, selected islet lncRNAs were dysregulated in type 2 diabetes or mapped to genetic loci underlying diabetes susceptibility. These findings reveal a new class of islet-cell genes relevant to β cell programming and diabetes pathophysiology.

Derepression of Polycomb targets during pancreatic organogenesis allows insulin-producing beta-cells to adopt a neural gene activity program.

The epigenome changes that underlie cellular differentiation in developing organisms are poorly understood. To gain insights into how pancreatic beta-cells are programmed, we profiled key histone methylations and transcripts in embryonic stem cells, multipotent progenitors of the nascent embryonic pancreas, purified beta-cells, and 10 differentiated tissues. We report that despite their endodermal origin, beta-cells show a transcriptional and active chromatin signature that is most similar to ectoderm-derived neural tissues. In contrast, the beta-cell signature of trimethylated H3K27, a mark of Polycomb-mediated repression, clusters with pancreatic progenitors, acinar cells and liver, consistent with the epigenetic transmission of this mark from endoderm progenitors to their differentiated cellular progeny. We also identified two H3K27 methylation events that arise in the beta-cell lineage after the pancreatic progenitor stage. One is a wave of cell-selective de novo H3K27 trimethylation in non-CpG island genes. Another is the loss of bivalent and H3K27me3-repressed chromatin in a core program of neural developmental regulators that enables a convergence of the gene activity state of beta-cells with that of neural cells. These findings reveal a dynamic regulation of Polycomb repression programs that shape the identity of differentiated beta-cells.

The miRNA profile of human pancreatic islets and beta-cells and relationship to type 2 diabetes pathogenesis.

Recent advances in the understanding of the genetics of type 2 diabetes (T2D) susceptibility have focused attention on the regulation of transcriptional activity within the pancreatic beta-cell. MicroRNAs (miRNAs) represent an important component of regulatory control, and have proven roles in the development of human disease and control of glucose homeostasis. We set out to establish the miRNA profile of human pancreatic islets and of enriched beta-cell populations, and to explore their potential involvement in T2D susceptibility. We used Illumina small RNA sequencing to profile the miRNA fraction in three preparations each of primary human islets and of enriched beta-cells generated by fluorescence-activated cell sorting. In total, 366 miRNAs were found to be expressed (i.e. >100 cumulative reads) in islets and 346 in beta-cells; of the total of 384 unique miRNAs, 328 were shared. A comparison of the islet-cell miRNA profile with those of 15 other human tissues identified 40 miRNAs predominantly expressed (i.e. >50% of all reads seen across the tissues) in islets. Several highly-expressed islet miRNAs, such as miR-375, have established roles in the regulation of islet function, but others (e.g. miR-27b-3p, miR-192-5p) have not previously been described in the context of islet biology. As a first step towards exploring the role of islet-expressed miRNAs and their predicted mRNA targets in T2D pathogenesis, we looked at published T2D association signals across these sites. We found evidence that predicted mRNA targets of islet-expressed miRNAs were globally enriched for signals of T2D association (p-values <0.01, q-values <0.1). At six loci with genome-wide evidence for T2D association (AP3S2, KCNK16, NOTCH2, SCL30A8, VPS26A, and WFS1) predicted mRNA target sites for islet-expressed miRNAs overlapped potentially causal variants. In conclusion, we have described the miRNA profile of human islets and beta-cells and provide evidence linking islet miRNAs to T2D pathogenesis.

TEAD and YAP regulate the enhancer network of human embryonic pancreatic progenitors

The genomic regulatory programmes that underlie human organogenesis are poorly understood. Pancreas development, in particular, has pivotal implications for pancreatic regeneration, cancer and diabetes. We have now characterized the regulatory landscape of embryonic multipotent progenitor cells that give rise to all pancreatic epithelial lineages. Using human embryonic pancreas and embryonic-stem-cell-derived progenitors we identify stage-specific transcripts and associated enhancers, many of which are co-occupied by transcription factors that are essential for pancreas development. We further show that TEAD1, a Hippo signalling effector, is an integral component of the transcription factor combinatorial code of pancreatic progenitor enhancers. TEAD and its coactivator YAP activate key pancreatic signalling mediators and transcription factors, and regulate the expansion of pancreatic progenitors. This work therefore uncovers a central role for TEAD and YAP as signal-responsive regulators of multipotent pancreatic progenitors, and provides a resource for the study of embryonic development of the human pancreas.

Human Pancreatic β Cell lncRNAs Control Cell-Specific Regulatory Networks

Summary Recent studies have uncovered thousands of long non-coding RNAs (lncRNAs) in human pancreatic β cells. β cell lncRNAs are often cell type specific and exhibit dynamic regulation during differentiation or upon changing glucose concentrations. Although these features hint at a role of lncRNAs in β cell gene regulation and diabetes, the function of β cell lncRNAs remains largely unknown. In this study, we investigated the function of β cell-specific lncRNAs and transcription factors using transcript knockdowns and co-expression network analysis. This revealed lncRNAs that function in concert with transcription factors to regulate β cell-specific transcriptional networks. We further demonstrate that the lncRNA PLUTO affects local 3D chromatin structure and transcription of PDX1, encoding a key β cell transcription factor, and that both PLUTO and PDX1 are downregulated in islets from donors with type 2 diabetes or impaired glucose tolerance. These results implicate lncRNAs in the regulation of β cell-specific transcription factor networks.

A Loss-of-Function Splice Acceptor Variant in IGF2 Is Protective for Type 2 Diabetes

Type 2 diabetes (T2D) affects more than 415 million people worldwide, and its costs to the health care system continue to rise. To identify common or rare genetic variation with potential therapeutic implications for T2D, we analyzed and replicated genome-wide protein coding variation in a total of 8,227 individuals with T2D and 12,966 individuals without T2D of Latino descent. We identified a novel genetic variant in the IGF2 gene associated with ∼20% reduced risk for T2D. This variant, which has an allele frequency of 17% in the Mexican population but is rare in Europe, prevents splicing between IGF2 exons 1 and 2. We show in vitro and in human liver and adipose tissue that the variant is associated with a specific, allele-dosage–dependent reduction in the expression of IGF2 isoform 2. In individuals who do not carry the protective allele, expression of IGF2 isoform 2 in adipose is positively correlated with both incidence of T2D and increased plasma glycated hemoglobin in individuals without T2D, providing support that the protective effects are mediated by reductions in IGF2 isoform 2. Broad phenotypic examination of carriers of the protective variant revealed no association with other disease states or impaired reproductive health. These findings suggest that reducing IGF2 isoform 2 expression in relevant tissues has potential as a new therapeutic strategy for T2D, even beyond the Latin American population, with no major adverse effects on health or reproduction.

Human pancreatic islet 3D chromatin architecture provides insights into the genetics of type 2 diabetes

Genetic studies promise to provide insight into the molecular mechanisms underlying type 2 diabetes (T2D). Variants associated with T2D are often located in tissue-specific enhancer regions (enhancer clusters, stretch enhancers or super-enhancers). So far, such domains have been defined through clustering of enhancers in linear genome maps rather than in 3D-space. Furthermore, their target genes are generally unknown. We have now created promoter capture Hi-C maps in human pancreatic islets. This linked diabetes-associated enhancers with their target genes, often located hundreds of kilobases away. It further revealed sets of islet enhancers, super-enhancers and active promoters that form 3D higher-order hubs, some of which show coordinated glucose-dependent activity. Hub genetic variants impact the heritability of insulin secretion, and help identify individuals in whom genetic variation of islet function is important for T2D. Human islet 3D chromatin architecture thus provides a framework for interpretation of T2D GWAS signals.

A Comprehensive Reanalysis Of Publicly Available GWAS Datasets Reveals An X Chromosome Rare Regulatory Variant Associated With High Risk For Type 2 Diabetes.

The reanalysis of publicly available GWAS data represents a powerful and cost-effective opportunity to gain insights into the genetics and pathophysiology of complex diseases. We demonstrate this by gathering and reanalyzing public type 2 diabetes (T2D) GWAS data for 70,127 subjects, using an innovative imputation and association strategy based on multiple reference panels (1000G and UK10K). This approach led us replicate and fine map 50 known T2D loci, and identify seven novel associated regions: five driven by common variants in or near LYPLAL1, NEUROG3, CAMKK2, ABO and GIP genes; one by a low frequency variant near EHMT2; and one driven by a rare variant in chromosome Xq23, associated with a 2.7-fold increased risk for T2D in males, and located within an active enhancer associated with the expression of Angiotensin II Receptor type 2 gene (AGTR2), a known modulator of insulin sensitivity. We further show that the risk T allele reduces binding of a nuclear protein, resulting in increased enhancer activity in muscle cells. Beyond providing novel insights into the genetics and pathophysiology of T2D, these results also underscore the value of reanalyzing publicly available data using novel analytical approaches.

Insights into β-cell biology and type 2 diabetes pathogenesis from studies of the islet transcriptome

Human β-cells play a pivotal role in the pathogenesis of type 2 diabetes (T2D). Consequently, improved understanding of the molecular and cellular processes critical to the normal function of these cells, and the ways in which these processes are disturbed during disease development, is central to efforts to develop novel therapeutic strategies. Detailed exploration of the transcriptomic, proteomic and metabolomic composition of islet cells provides a platform for defining cellular function. The recent advent of next-generation sequencing approaches is enabling ever more complete inventories of the islet transcriptome, and these data are already providing important insights into islet biology. For example, transcriptome data have contributed to: (a) definition of transcript candidacy at T2D-associated loci; (b) identification of novel disease biomarkers; (c) characterisation of novel regulatory mechanisms (such as those involving non-coding RNAs), and (d) discovery of factors of potential relevance to β-cell reprogramming efforts.