Project description:Performed ChIP-seq for H3K27ac and H3K4me1 in purified (FACS sorted) islet alpha and beta cell populations. These ChIP-seq experiments can be used to identify cell-type-specific enhancers.
Project description:The SLC30A8 gene encodes the zinc transporter ZnT-8, which provides zinc for insulin-hexamer formation. Genome-wide association studies have shown that a polymorphic variant in SLC30A8 is associated with altered susceptibility to Type 2 diabetes and we recently reported that glucose-stimulated insulin secretion is decreased in islets isolated from Slc30a8-knockout mice. The present study examines the molecular basis for the islet-specific expression of Slc30a8. VISTA analyses identified two conserved regions in Slc30a8 introns 2 and 3, designated enhancers A and B respectively. Transfection experiments demonstrated that enhancer B confers elevated fusion gene expression in both ?TC-3 cells and ?TC-6 cells. In contrast, enhancer A confers elevated fusion gene expression selectively in ?TC-3 and not ?TC-6 cells. These data suggest that enhancer A is an islet ?-cell-specific enhancer and that the mechanisms controlling Slc30a8 expression in ?- and ?-cells are overlapping, but distinct. Gel retardation and ChIP (chromatin immunoprecipitation) assays revealed that the islet-enriched transcription factor Pdx-1 binds enhancer A in vitro and in situ respectively. Mutation of two Pdx-1-binding sites in enhancer A markedly reduces fusion gene expression suggesting that this factor contributes to Slc30a8 expression in ?-cells, a conclusion consistent with developmental studies showing that restriction of Pdx-1 to pancreatic islet ?-cells correlates with the induction of Slc30a8 gene expression and ZnT-8 protein expression in vivo.
Project description:Genetic variants affecting pancreatic islet enhancers are central to T2D risk, but the gene targets of islet enhancer activity are largely unknown. We generate a high-resolution map of islet chromatin loops using Hi-C assays in three islet samples and use loops to annotate target genes of islet enhancers defined using ATAC-seq and published ChIP-seq data. We identify candidate target genes for thousands of islet enhancers, and find that enhancer looping is correlated with islet-specific gene expression. We fine-map T2D risk variants affecting islet enhancers, and find that candidate target genes of these variants defined using chromatin looping and eQTL mapping are enriched in protein transport and secretion pathways. At IGF2BP2, a fine-mapped T2D variant reduces islet enhancer activity and IGF2BP2 expression, and conditional inactivation of IGF2BP2 in mouse islets impairs glucose-stimulated insulin secretion. Our findings provide a resource for studying islet enhancer function and identifying genes involved in T2D risk.
Project description:The pancreatic islet, a cellular community harboring the insulin-producing beta-cells, is known to undergo age-related alterations. However, only a handful of signals associated with aging have been identified. By comparing beta-cells from younger and older zebrafish, here we show that the aging islets exhibit signs of chronic inflammation. These include recruitment of tnf?-expressing macrophages and the activation of NF-kB signaling in beta-cells. Using a transgenic reporter, we show that NF-kB activity is undetectable in juvenile beta-cells, whereas cells from older fish exhibit heterogeneous NF-kB activity. We link this heterogeneity to differences in gene expression and proliferation. Beta-cells with high NF-kB signaling proliferate significantly less compared to their neighbors with low activity. The NF-kB signalinghi cells also exhibit premature upregulation of socs2, an age-related gene that inhibits beta-cell proliferation. Together, our results show that NF-kB activity marks the asynchronous decline in beta-cell proliferation with advancing age.
Project description: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.
Project description:We combined ChIP-seq of chromatin marks and key islet transcription factor with RNA-seq in human islets to map cis-regulatory networks in this primary tissue. The output of this project provides a reference map to dissect genetic variants that alter the susceptibility for Type 2 diabetes, and assist efforts to generate new beta-cells by transcriptional programming strategies
Project description:BACKGROUND: Transcriptional regulation in multi-cellular organisms is a complex process involving multiple modular regulatory elements for each gene. Building whole-genome models of transcriptional networks requires mapping all relevant enhancers and then linking them to target genes. Previous methods of enhancer identification based either on sequence information or on epigenetic marks have different limitations stemming from incompleteness of each of these datasets taken separately. RESULTS: In this work we present a new approach for discovery of regulatory elements based on the combination of sequence motifs and epigenetic marks measured with ChIP-Seq. Our method uses supervised learning approaches to train a model describing the dependence of enhancer activity on sequence features and histone marks. Our results indicate that using combination of features provides superior results to previous approaches based on either one of the datasets. While histone modifications remain the dominant feature for accurate predictions, the models based on sequence motifs have advantages in their general applicability to different tissues. Additionally, we assess the relevance of different sequence motifs in prediction accuracy showing that even tissue-specific enhancer activity depends on multiple motifs. CONCLUSIONS: Based on our results, we conclude that it is worthwhile to include sequence motif data into computational approaches to active enhancer prediction and also that classifiers trained on a specific set of enhancers can generalize with significant accuracy beyond the training set.
Project description:AIMS/HYPOTHESIS:The paucity of information on the epigenetic barriers that are blocking reprogramming protocols, and on what makes a beta cell unique, has hampered efforts to develop novel beta cell sources. Here, we aimed to identify enhancers in pancreatic islets, to understand their developmental ontologies, and to identify enhancers unique to islets to increase our understanding of islet-specific gene expression. METHODS:We combined H3K4me1-based nucleosome predictions with pancreatic and duodenal homeobox 1 (PDX1), neurogenic differentiation 1 (NEUROD1), v-Maf musculoaponeurotic fibrosarcoma oncogene family, protein A (MAFA) and forkhead box A2 (FOXA2) occupancy data to identify enhancers in mouse islets. RESULTS:We identified 22,223 putative enhancer loci in in vivo mouse islets. Our validation experiments suggest that nearly half of these loci are active in regulating islet gene expression, with the remaining regions probably poised for activity. We showed that these loci have at least nine developmental ontologies, and that islet enhancers predominately acquire H3K4me1 during differentiation. We next discriminated 1,799 enhancers unique to islets and showed that these islet-specific enhancers have reduced association with annotated genes, and identified a subset that are instead associated with novel islet-specific long non-coding RNAs (lncRNAs). CONCLUSIONS/INTERPRETATIONS:Our results indicate that genes with islet-specific expression and function tend to have enhancers devoid of histone methylation marks or, less often, that are bivalent or repressed, in embryonic stem cells and liver. Further, we identify a subset of enhancers unique to islets that are associated with novel islet-specific genes and lncRNAs. We anticipate that these data will facilitate the development of novel sources of functional beta cell mass.
Project description:Genome-wide association studies (GWASs) and functional genomics approaches implicate enhancer disruption in islet dysfunction and type 2 diabetes (T2D) risk. We applied genetic fine-mapping and functional (epi)genomic approaches to a T2D- and proinsulin-associated 15q22.2 locus to identify a most likely causal variant, determine its direction of effect, and elucidate plausible target genes. Fine-mapping and conditional analyses of proinsulin levels of 8,635 non-diabetic individuals from the METSIM study support a single association signal represented by a cluster of 16 strongly associated (p < 10-17) variants in high linkage disequilibrium (r2 > 0.8) with the GWAS index SNP rs7172432. These variants reside in an evolutionarily and functionally conserved islet and ? cell stretch or super enhancer; the most strongly associated variant (rs7163757, p = 3 × 10-19) overlaps a conserved islet open chromatin site. DNA sequence containing the rs7163757 risk allele displayed 2-fold higher enhancer activity than the non-risk allele in reporter assays (p < 0.01) and was differentially bound by ? cell nuclear extract proteins. Transcription factor NFAT specifically potentiated risk-allele enhancer activity and altered patterns of nuclear protein binding to the risk allele in vitro, suggesting that it could be a factor mediating risk-allele effects. Finally, the rs7163757 proinsulin-raising and T2D risk allele (C) was associated with increased expression of C2CD4B, and possibly C2CD4A, both of which were induced by inflammatory cytokines, in human islets. Together, these data suggest that rs7163757 contributes to genetic risk of islet dysfunction and T2D by increasing NFAT-mediated islet enhancer activity and modulating C2CD4B, and possibly C2CD4A, expression in (patho)physiologic states.
Project description:RATIONALE:The transcription factor Islet-1 is a marker of cardiovascular progenitors during embryogenesis. The isolation of Islet-1-positive (Islet-1(+)) cells from early postnatal hearts suggested that Islet-1 also marks cardiac progenitors in adult life. OBJECTIVE:We investigated the distribution and identity of Islet-1(+) cells in adult murine heart and evaluated whether their number or distribution change with age or after myocardial infarction. METHODS AND RESULTS:Distribution of Islet-1(+) cells in adult heart was investigated using gene targeted mice with nuclear ?-galactosidase inserted into the Islet-1 locus. nLacZ-positive cells were only present in 3 regions of the adult heart: clusters in the interatrial septum and around the pulmonary veins, scattered within the wall of the great vessels, and a strictly delimited cluster between the right atrium and superior vena cava. Islet-1(+) cells in the first type of clusters coexpressed markers for parasympathetic neurons. Positive cells in the great arteries coexpressed smooth muscle actin and myosin heavy chain, indicating a smooth muscle cell identity. Very few Islet-1(+) cells within the outflow tract expressed the cardiomyocyte marker ?-actinin. Islet-1(+) cells in the right atrium coexpressed the sinoatrial node pacemaker cell marker HCN4. Cell number and localization remained unchanged between 1 to 18 months of age. Consistently Islet-1 mRNA was detected in human sinoatrial node. Islet-1(+) cells could not be detected in the infarct zone 2 to 28 days after myocardial infarction, aside from 10 questionable cells in 1/13 hearts. CONCLUSIONS:Our results identify Islet-1 as a novel marker of the adult sinoatrial node and do not provide evidence for Islet-1(+) cells to serve as cardiac progenitors.