SUMMARY A significant portion of the genome is transcribed as long non-coding 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.

STEM CELLS 2007;25:411-418

Previous methods to systematically characterize sequence-intrinsic activity of promoters have been limited by relatively low throughput and the length of sequences that could be tested. Here we present Survey of Regulatory Elements (SuRE), a method to assay more than 108 DNA fragments, each 0.2–2kb in size, for their ability to drive transcription autonomously. In SuRE, a plasmid library is constructed of random genomic fragments upstream of a 20bp barcode and decoded by paired-end sequencing. This library is then transfected into cells and transcribed barcodes are quantified in the RNA by high throughput sequencing. When applied to the human genome, we achieved a 55-fold genome coverage, allowing us to map autonomous promoter activity genome-wide. By computational modeling we delineated subregions within promoters that are relevant for their activity. For instance, we show that antisense promoter transcription is generally dependent on the sense core promoter sequences, and that most enhancers and several families of repetitive elements act as autonomous transcription initiation sites.

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.

Most of the millions of single-nucleotide polymorphisms (SNPs) in the human genome are non-coding, and many overlap with putative regulatory elements. Genome-wide association studies (GWAS) have linked many of these SNPs to human traits or to gene expression levels, but rarely with sufficient resolution to identify the causal SNPs. Functional screens based on reporter assays have previously been of insufficient throughput to test the vast space of SNPs for possible effects on regulatory element activity. Here, we leveraged the throughput and resolution of the SuRE reporter technology to survey the effect of 5.9 million SNPs, including 57% of the known common SNPs, on enhancer and promoter activity. We identified more than 30,000 SNPs that alter the activity of putative regulatory elements, partially in a cell-type specific manner. Integration of this dataset with GWAS results may help pinpoint SNPs that underlie human traits.

Summary It is largely unclear whether genes that are naturally embedded in lamina-associated domains (LADs) are inactive due to their chromatin environment or whether LADs are merely secondary to the lack of transcription. We show that hundreds of human promoters become active when moved from their native LAD position to a neutral context in the same cells, indicating that LADs form a repressive environment. Another set of promoters inside LADs is able to “escape” repression, although their transcription elongation is attenuated. By inserting reporters into thousands of genomic locations, we demonstrate that escaper promoters are intrinsically less sensitive to LAD repression. This is not simply explained by promoter strength but by the interplay between promoter sequence and local chromatin features that vary strongly across LADs. Enhancers also differ in their sensitivity to LAD chromatin. This work provides a general framework for the systematic understanding of gene regulation by repressive chromatin.

Although it was originally believed that enhancers activate only the nearest promoter, recent global analyses enabled by high-throughput technology suggest that the network of enhancer–promoter interactions is far more complex. The mechanisms that determine the specificity of enhancer–promoter interactions are still poorly understood, but they are thought to include biochemical compatibility, constraints imposed by the three-dimensional architecture of chromosomes, insulator elements, and possibly the effects of local chromatin composition. In this review, we assess the current insights into these determinants, and highlight the functional genomic approaches that will lead the way towards better mechanistic understanding.

Housekeeping genes of animal genomes cluster in the same chromosomal regions. It has long been suggested that this organization contributes to their steady expression across all the tissues of the organism. Here, we show that the activity of Drosophila housekeeping gene promoters depends on the expression of their neighbors. By measuring the expression of ∼85,000 reporters integrated in Kc167 cells, we identified the best predictors of expression as chromosomal contacts with the promoters and terminators of active genes. Surprisingly, the chromatin composition at the insertion site and the contacts with enhancers were less informative. These results are substantiated by the existence of genomic "paradoxical" domains, rich in euchromatic features and enhancers, but where the reporters are expressed at low level, concomitant with a deficit of interactions with promoters and terminators. This indicates that the proper function of housekeeping genes relies not on contacts with long distance enhancers but on spatial clustering. Overall, our results suggest that spatial proximity between genes increases their expression and that the linear architecture of the Drosophila genome contributes to this effect.