Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Identification and analysis of pancreatic islet specific enhancers

ABSTRACT: We anticipated that the identification of cis-regulatory regions active in pancreatic islets would help increase our understanding of islet biology and the pathology of diabetes. Towards this end we used histone H3 lysine 4 monomethylation-based nucleosome predictions genome-wide, in conjunction with binding data for PDX1, FOXA2, MAFA, and NEUROD1, to identify 3,654 putative enhancers that are H3K4me1-enriched uniquely in islets as compared to 14 other tissue or cell-types. We show that these islet-specific enhancers are associated with genes with significantly higher islet specificity than genes associated with non-specific enhancers. Further, islet-specific enhancers were not enriched for typical active or repressive histone methylations in embryonic stem cells and liver, suggesting they are formed by de novo histone methylation during pancreas development. We also identify a subset of enhancers bivalently marked by both H3K4me1 and H3K27me3 in adult pancreatic islets. Further, we show that islet-specific enhancers triple- or quadruple- bound by PDX1, MAFA, NEUROD1 and/or FOXA2 are associated with genes with particularly high islet-specificity, and that these loci are enriched in regions with functional activity in islet cell types. Finally, we demonstrate that cytokines reduce H3K4me1 enrichment levels at selected triple- or quadruple-bound islet-specific enhancers, suggesting that epigenetic changes may contribute to cytokine-induced b-cell dysfunction. In conjunction with data from Hoffman et al Genome Research 2010, an analysis of histone modifications and transcription factor binding sites to identify enhancer regions

ORGANISM(S): Mus musculus

SUBMITTER: Patrick Plettner

PROVIDER: E-GEOD-30298 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Objective: Transcriptional complex activity drives the development and function of pancreatic islet cells to allow for proper glucose regulation. Our prior work highlighted that the LIM-homeodomain transcription factor (TF), Islet-1 (Isl1), and its interacting co-regulator, Ldb1, are vital effectors of developing and adult β-cells. We further found that a member of the Single Stranded DNA-Binding Protein (SSBP) co-regulator family, SSBP3, interacts with Isl1 and Ldb1 in β-cells and primary islets (mouse and human) to impact β-cell target genes MafA and Glp1R. Members of the SSBP family stabilize TF complexes by binding directly to Ldb1 and protecting the complex from ubiquitin-mediated turnover. In this study, we hypothesized that SSBP3 would have critical roles in pancreatic islet cell development and function in vivo, similar to the Isl1::Ldb1 complex. Methods: We first generated a novel SSBP3 LoxP allele mouse line, where Cre-mediated recombination imparts a predicted early protein termination. We bred this mouse to constitutive Cre lines (Pdx1- and Pax6-driven) to recombine of SSBP3 in the developing pancreas and islet (SSBP3DeltaPanc and SSBP3DeltaIslet), respectively. We assessed glucose tolerance and used immunofluorescence to detect changes in islet cell abundance and markers of β-cell identity and function. Using an inducible Cre system we also deleted SSBP3 in the adult β-cell, a model termed SSBP3Deltaβ-cell. We measured glucose tolerance as well as glucose-stimulated insulin secretion (GSIS), both in vivo and in isolated islets in vitro. Using islets from control and SSBP3Deltaβ-cell we conducted RNA-Seq and compared our results to published datasets for similar β-cell specific Ldb1 and Isl1 knockouts to identify commonly regulated target genes. Results: SSBP3DeltaPanc and SSBP3DeltaIslet neonates present with hyperglycemia. SSBP3DeltaIslet mice are glucose intolerant by P21 and exhibit a reduction of β-cell maturity markers MafA, Pdx1, and UCN3. We observe disruptions in islet cell architecture with an increase in glucagon+ α-cells and ghrelin+ ε-cells at P10. Inducible loss of β-cell SSBP3 in SSBP3Deltaβ-cell causes hyperglycemia, glucose intolerance, and reduced GSIS. Transcriptomic analysis of 14-week SSBP3Deltaβ-cell islets revealed a decrease in β-cell function gene expression (Ins, MafA, Ucn3), increased stress and dedifferentiation markers (Neurogenin-3, Aldh1a3, Gastrin), and shared differentially expressed genes between SSBP3, Ldb1, and Isl1 in adult β-cells. Conclusions: SSBP3 drives proper islet development and identity, where its loss causes altered islet-cell abundance and glucose regulatory function. β-cell SSBP3 is required for GSIS and glucose homeostasis, at least partially through shared regulation of Ldb1 and Isl1 target genes.

Project description:FOXA1 is a pioneer factor that is important in hormone dependent cancer cells to stabilise nuclear receptors, such as estrogen receptor (ER) to chromatin. FOXA1 binds to enhancers regions that are enriched in H3K4mono- and dimethylation (H3K4me1, H3K4me2) histone marks and evidence suggests that these marks are requisite events for FOXA1 to associate with enhancers to initate subsequent gene expression events. However, exogenous expression of FOXA1 has been shown to induce H3K4me1 and H3K4me2 signal at enhancer elements and the order of events and the functional importance of these events is not clear. We performed a FOXA1 Rapid Immunoprecipitation Mass Spectrometry of Endogenous Proteins (RIME) screen in ERα-positive MCF-7 breast cancer cells in order to identify FOXA1 interacting partners and we found histone-lysine N-methyltransferase (MLL3) as the top FOXA1 interacting protein. MLL3 is typically thought to induce H3K4me3 at promoter regions, but recent findings suggest it may contribute to H3K4me1 deposition, in line with our observation that MLL3 associates with an enhancer specific protein. We performed MLL3 ChIP-seq in breast cancer cells and unexpectedly found that MLL3 binds mostly at non-promoter regions enhancers, in contrast to the prevailing hypothesis. MLL3 was shown to occupy regions marked by FOXA1 occupancy and as expected, H3K4me1 and H3K4me2. MLL3 binding was dependent on FOXA1, indicating that FOXA1 recruits MLL3 to chromatin. Motif analysis and subsequent genomic mapping revealed a role for Grainy head like protein-2 (GRHL2) which was shown to co-occupy regions of the chromatin with MLL3. Regions occupied by all three factors, namely FOXA1, MLL3 and GRHL2, were most enriched in H3K4me1. MLL3 silencing decreased H3K4me1 at enhancer elements, but had no appreciable impact on H3K4me3 at enhancer elements. We identify a complex relationship between FOXA1, MLL3 and H3K4me1 at enhancers in breast cancer and propose a mechanism whereby the pioneer factor FOXA1 can interact with a chromatin modifier MLL3, recruiting it to chromatin to facilitate the deposition of H3K4me1 histone marks, subsequently demarcating active enhancer elements.

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Identification and analysis of pancreatic islet specific enhancers

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