The liver and pancreas share a common origin and coexpress several transcription factors. To gain insight into the transcriptional networks regulating the function of these tissues, we globally identify binding sites for FOXA2 in adult mouse islets and liver, PDX1 in islets, and HNF4A in liver. Because most eukaryotic transcription factors bind thousands of loci, many of which are thought to be inactive, methods that can discriminate functionally active binding events are essential for the interpretation of genome-wide transcription factor binding data. To develop such a method, we also generated genome-wide H3K4me1 and H3K4me3 localization data in these tissues. By analyzing our binding and histone methylation data in combination with comprehensive gene expression data, we show that H3K4me1 enrichment profiles discriminate transcription factor occupied loci into three classes: those that are functionally active, those that are poised for activation, and those that reflect pioneer-like transcription factor activity. Furthermore, we demonstrate that the regulated presence of H3K4me1-marked nucleosomes at transcription factor occupied promoters and enhancers controls their activity, implicating both tissue-specific transcription factor binding and nucleosome remodeling complex recruitment in determining tissue-specific gene expression. Finally, we apply these approaches to generate novel insights into how FOXA2, PDX1, and HNF4A cooperate to drive islet-and liver-specific gene expression.

SummaryTo date, it remains largely unclear to what extent chromatin machinery contributes to the susceptibility and progression of complex diseases. Here, we combine deep epigenome mapping with single-cell transcriptomics to mine for evidence of chromatin dysregulation in type 2 diabetes. We find two chromatin-state signatures that track β cell dysfunction in mice and humans: ectopic activation of bivalent Polycomb-silenced domains and loss of expression at an epigenomically unique class of lineage-defining genes. β cell-specific Polycomb (Eed/PRC2) loss of function in mice triggers diabetes-mimicking transcriptional signatures and highly penetrant, hyperglycemia-independent dedifferentiation, indicating that PRC2 dysregulation contributes to disease. The work provides novel resources for exploring β cell transcriptional regulation and identifies PRC2 as necessary for long-term maintenance of β cell identity. Importantly, the data suggest a two-hit (chromatin and hyperglycemia) model for loss of β cell identity in diabetes.

We analyzed 8.55 million LongSAGE tags generated from 72 libraries. Each LongSAGE library was prepared from a different mouse tissue. Analysis of the data revealed extensive overlap with existing gene data sets and evidence for the existence of Ϸ24,000 previously undescribed genomic loci. The visual cortex, pancreas, mammary gland, preimplantation embryo, and placenta contain the largest number of differentially expressed transcripts, 25% of which are previously undescribed loci.alternative transcripts ͉ development ͉ serial analysis of gene expression

SUMMARYChromatin is the physical template that stabilizes and specifies transcriptional programs. To date, it remains largely unclear to what extent chromatin machinery contributes to the susceptibility and progression of complex diseases. Here, we combined deep epigenome mapping with single cell transcriptomics to mine for evidence of chromatin dysregulation in type-2 diabetes. We identify two chromatin-state signatures that track the trajectory of β-cell dysfunction in mice and humans: ectopic activation of bivalent Polycomb-domains and a loss of expression at a subclass of highly active domains containing key lineage-defining genes. β-cell specific deletion of Polycomb (Eed/PRC2) triggers parallel transcriptional signatures. Intriguingly, these β-cell Eed-knockouts also exhibit highly penetrant hyperglycemia-independent dedifferentiation indicating that Polycomb dysregulation sensitizes the β-cell for dedifferentiation. These findings provide novel resources for exploring transcriptional and epigenetic control of β-cell (dys)function. They identify PRC2 as necessary for long-term maintenance of β-cell identity. The data suggest a two-hit model for loss of β-cell identity in diabetes and highlight epigenetic therapeutic potential to block dedifferentiation.

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.

Groucho (Gro)/TLE transcriptional corepressors are involved in a variety of developmental mechanisms, including neuronal differentiation. They contain a conserved C-terminal WD40 repeat domain that mediates interactions with several DNA-binding proteins. In particular, Gro/TLE1 interacts with forkhead transcription factor brain factor 1 (BF-1; also termed FoxG1). BF-1 is an essential regulator of neuronal differentiation during cerebral cortex development and represses transcription together with Gro/TLE1. Gro/TLE-related gene product 6 (Grg6) shares with Gro/TLEs a conserved WD40 repeat domain but is more distantly related at its N-terminal half. We demonstrate that Grg6 is expressed in cortical neural progenitor cells and interacts with BF-1. In contrast to Gro/TLE1, however, Grg6 does not promote, but rather suppresses, BF-1-mediated transcriptional repression. Consistent with these observations, Grg6 interferes with the binding of Gro/TLE1 to BF-1 and does not repress transcription when targeted to DNA. Moreover, coexpression of Grg6 and BF-1 in cortical progenitor cells leads to a decrease in the number of proliferating cells and increased neuronal differentiation. Conversely, Grg6 knockdown by RNA interference causes decreased neurogenesis. These results identify a new role for Grg6 in cortical neuron development and establish a functional link between Grg6 and BF-1.

Aims/hypothesis Adult pancreatic islets contain multiple cell types that produce and secrete well characterised hormones, including insulin, glucagon and somatostatin. Although it is increasingly apparent that islets release and respond to more secreted factors than previously thought, systematic analyses are lacking. We therefore sought to identify potential autocrine and/or paracrine islet growth factor loops, and to characterise the function of the netrin family of islet-secreted factors and their receptors, which have been previously unreported in adult islets. Methods Gene expression databases, islet-specific tag sequencing libraries and microarray datasets of FACS purified beta cells were used to compile a list of secreted factors and receptors present in mouse or human islets. Netrins and their receptors were further assessed using RT-PCR, Western blot analysis and immunofluorescence staining. The roles of netrin-1 and netrin-4 in beta cell function, apoptosis and proliferation were also examined. Results We identified 233 secreted factors and 234 secreted factor receptors in islets. The presence of netrins and their receptors was further confirmed. Downregulation of caspase-3 activation was observed when MIN6 cells were exposed to exogenous netrin-1 and netrin-4 under hyperglycaemic conditions. Reduction in caspase-3 cleavage was linked to the decrease in dependence receptors, neogenin and unc-5 homologue A, as well as the activation of Akt and extracellular signal-regulated protein kinase (ERK) signalling. Conclusions/interpretation Our results highlight the large number of potential islet growth factors and point to a context-dependent pro-survival role for netrins in adult beta cells. Since diabetes results from a deficiency in functional beta cell mass, these studies are important steps towards developing novel therapies to improve beta cell survival.

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