Identification and analysis of murine pancreatic islet enhancers

Abstract

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.

An evolutionarily conserved DNA architecture determines target specificity of the TWIST family bHLH transcription factors

“…While there is significant but subtle specificity within the E-box sequence itself for binding by some bHLH proteins, most notably at the "NN" nucleotides in the CANNTG sequence and possibly extending to nucleotides just outside of the consensus, these variations are not enough to account for the significant specificity in target gene regulation shown by the vast set of bHLH proteins. Thus, it was remarkable that upon surveying published ChIP-seq studies of bHLH transcription factors, including NEUROD1 (Tennant et al 2013), E2A , and c-MYC (Chen et al 2008), we did not detect the double E-box motif, suggesting that the novel double E-box motif is unique to the TWIST proteins and confers its target specificity. Unlike many known bHLH transcription factors that recognize specific nucleotide sequences surrounding the canonical E-box motif, the TWIST subfamily of bHLH proteins has evolved a unique strategy to recognize the spatial architecture of the double E-box motif to achieve specificity.…”

“…To determine whether recognition of the double E-box motif is unique to the TWIST family of bHLH transcription factors, we analyzed the publicly available ChIP-seq data for NEUROD1 (murine Islet cells) (Tennant et al 2013), E47 (murine B cells) , and c-MYC (murine embryonic stem [ES] cells) (Chen et al 2008). Remarkably, the double E-box motif is not enriched in the ChIP-seq data with these bHLH proteins (Fig.…”

PAX6 maintains β cell identity by repressing genes of alternative islet cell types

“…We overlapped the PAX6-bound sites with previously published whole-genome maps of monomethylated histone H3 at lysine 4 (H3K4me1, associated with enhancers); acetylated histone H3 at lysine 27 (H3K27ac, associated with active enhancers); acetylated histone H3 at lysine 9 (H3K9ac, associated with active promoters); and histone H3 lysine 27 trimethylation (H3K27me3, associated with repression) -all obtained from sorted mouse β cells (52). In addition, we overlapped PAX6 ChIP-seq peaks with whole-genome chromatin-binding maps of MAFA, NKX6.1, PDX1, FOXA2, and NEUROD1 (6,52,53). Genes activated by PAX6 (downregulated in mutant β cells) were enriched for the active enhancer mark H3K27ac, while genes repressed by PAX6 were enriched for the repressive mark H3K27me3, consistent with the idea that PAX6 functions as a direct repressor of these genes.…”

Hippo Signaling Influences HNF4A and FOXA2 Enhancer Switching during Hepatocyte Differentiation

“…For RNA-seq analysis, RNA was prepared using TRIzol (Invitrogen) and sequenced on an Illumina Genome Analyzer as previously described (Griffith et al, 2010). The following NCBI data sets were used in this study: SRA008281, GSM751034, and GSM751035 (Hoffman et al, 2010; Tennant et al, 2013). Unthresholded TF chIP-seq data can be visualized in the UCSC Genome Browser using the following URLs.…”