Comprehensive Genomic Discovery of Non-Coding Transcriptional Enhancers in the African Malaria Vector Anopheles coluzzii

Almost all regulation of gene expression in eukaryotic genomes is mediated by the action of distant non-coding transcriptional enhancers upon proximal gene promoters. Enhancer locations cannot be accurately predicted bioinformatically because of the absence of a defined sequence code, and thus functional assays are required for their direct detection. Here we used a massively parallel reporter assay, Self-Transcribing Active Regulatory Region sequencing (STARR-seq), to generate the first comprehensive genome-wide map of enhancers in Anopheles coluzzii, a major African malaria vector in the Gambiae species complex. The screen was carried out by transfecting reporter libraries created from the genomic DNA of 60 wild A. coluzzii from Burkina Faso into A. coluzzii 4a3A cells, in order to functionally query enhancer activity of the natural population within the homologous cellular context. We report a catalog of 3,288 active genomic enhancers that were significant across three biological replicates, 74% of them located in intergenic and intronic regions. The STARR-seq enhancer screen is chromatin-free and thus detects inherent activity of a comprehensive catalog of enhancers that may be restricted in vivo to specific cell types or developmental stages. Testing of a validation panel of enhancer candidates using manual luciferase assays confirmed enhancer function in 26 of 28 (93%) of the candidates over a wide dynamic range of activity from two to at least 16-fold activity above baseline. The enhancers occupy only 0.7% of the genome, and display distinct composition features. The enhancer compartment is significantly enriched for 15 transcription factor binding site signatures, and displays divergence for specific dinucleotide repeats, as compared to matched non-enhancer genomic controls. The genome-wide catalog of A. coluzzii enhancers is publicly available in a simple searchable graphic format. This enhancer catalogue will be valuable in linking genetic and phenotypic variation, in identifying regulatory elements that could be employed in vector manipulation, and in better targeting of chromosome editing to minimize extraneous regulation influences on the introduced sequences.Importance: Understanding the role of the non-coding regulatory genome in complex disease phenotypes is essential, but even in well-characterized model organisms, identification of regulatory regions within the vast non-coding genome remains a challenge. We used a large-scale assay to generate a genome wide map of transcriptional enhancers. Such a catalogue for the important malaria vector, Anopheles coluzzii, will be an important research tool as the role of non-coding regulatory variation in differential susceptibility to malaria infection is explored and as a public resource for research on this important insect vector of disease.

Quantitative-enhancer-FACS-seq (QeFS) reveals epistatic interactions among motifs within transcriptional enhancers in developing Drosophila tissue

Understanding the contributions of transcription factor DNA binding sites to transcriptional enhancers is a significant challenge. We developed Quantitative enhancer-FACS-Seq for highly parallel quantification of enhancer activities from a genomically integrated reporter in Drosophila melanogaster embryos. We investigate the contributions of the DNA binding motifs of four poorly characterized TFs to the activities of twelve embryonic mesodermal enhancers. We measure quantitative changes in enhancer activity and discover a range of epistatic interactions among the motifs, both synergistic and alleviating. We find that understanding the regulatory consequences of TF binding motifs requires that they be investigated in combination across enhancer contexts.

Prevalent uses of exons as regulatory sequences are possible and inevitable

ABSTRACTIt has long been known that exons can encode transcriptional enhancers. However, the prevalence of such dual-use exons and related questions remain elusive. Our recently predicted highly accurate, large sets of cis-regulatory module candidates (CRMCs) and non-CRMCs in the human genome provide us an opportunity to address these questions. We find that exonic transcription factor binding sites(eTFBSs) occupy at least a third of the total exon lengths, suggesting exonic enhancers(eEHs) are more prevalent than originally thought. Moreover, active eTFBSs significantly overlap experimentally determined active eEHs, and enhance the transcription of nearby genes. Furthermore, both A/T and C/G in eTFBSs are more likely under evolutionary selection than those in non-CRMC exons, indicating the eTFBSs might be in dual-use. Interestingly, eTFBSs in codons tend to encode loops rather than more critical helices and strands in protein structures, while eTFBSs in untranslated regions (UTRs) tend to avoid positions where known UTR-related functions were located. Intriguingly, active eTFBSs are found to be in close physical proximity to distal promoters and involved in the activation of target genes. The close physical proximity between exons and promoters in topologically associating domains might render less critical exons to opt for parts of enhancers when non-exonic sequences are unavailable due to space constraints. It appears that nature avoids the dilemma of evolving a sequence for two unrelated functions by using less-critical, physically available exons for eTFBSs. Therefore, the prevalent dual-use of exons is not only possible but also inevitable.

Transcriptional enhancers and their communication with gene promoters

Transcriptional enhancers play a key role in the initiation and maintenance of gene expression programmes, particularly in metazoa. How these elements control their target genes in the right place and time is one of the most pertinent questions in functional genomics, with wide implications for most areas of biology. Here, we synthesise classic and recent evidence on the regulatory logic of enhancers, including the principles of enhancer organisation, factors that facilitate and delimit enhancer–promoter communication, and the joint effects of multiple enhancers. We show how modern approaches building on classic insights have begun to unravel the complexity of enhancer–promoter relationships, paving the way towards a quantitative understanding of gene control.

Deregulation of Transcriptional Enhancers in Cancer

Epigenetic regulations can shape a cell’s identity by reversible modifications of the chromatin that ultimately control gene expression in response to internal and external cues. In this review, we first discuss the concept of cell plasticity in cancer, a process that is directly controlled by epigenetic mechanisms, with a particular focus on transcriptional enhancers as the cornerstone of epigenetic regulation. In the second part, we discuss mechanisms of enhancer deregulation in adult stem cells and epithelial-to-mesenchymal transition (EMT), as two paradigms of cell plasticity that are dependent on epigenetic regulation and serve as major sources of tumour heterogeneity. Finally, we review how genetic variations at enhancers and their epigenetic modifiers contribute to tumourigenesis, and we highlight examples of cancer drugs that target epigenetic modifications at enhancers.

Enhancing gonadotrope gene expression through regulatory lncRNAs

The world of long non-coding RNAs (lncRNAs) has opened up massive new prospects in understanding the regulation of gene expression. Not only are there seemingly almost infinite numbers of lncRNAs in the mammalian cell, but they have highly diverse mechanisms of action. In the nucleus, some are chromatin-associated, transcribed from transcriptional enhancers (eRNAs) and/or direct changes in the epigenetic landscape with profound effects on gene expression. The pituitary gonadotrope is responsible for activation of reproduction through production and secretion of appropriate levels of the gonadotropic hormones. As such, it exemplifies a cell whose function is defined through changes in developmental and temporal patterns of gene expression, including those that are hormonally-induced. Roles for diverse distal regulatory elements and eRNAs in gonadotrope biology have only just begun to emerge. Here, we will present an overview of the different kinds of lncRNAs that alter gene expression, and what is known about their roles in regulating some of the key gonadotrope genes. We will also review various screens that have detected differentially expressed pituitary lncRNAs associated with changes in reproductive state, and those whose expression is found to play a role in gonadotrope-derived non-functioning pituitary adenomas. We hope to shed light on this exciting new field, emphasize the open questions, and encourage research to illuminate the roles of lncRNAs in various endocrine systems.

EVI1 activates tumor-promoting transcriptional enhancers in pancreatic cancer

Cancer cells utilize epigenetic alterations to acquire autonomous capabilities for tumor maintenance. Here, we show that pancreatic ductal adenocarcinoma (PDA) cells utilize super-enhancers (SEs) to activate the transcription factor EVI1 (ecotropic viral integration site 1) gene, resulting in activation of an EVI1-dependent transcription program conferring PDA tumorigenesis. Our data indicate that SE is the vital cis-acting element to maintain aberrant EVI1 transcription in PDA cells. Consistent with disease progression and inferior survival outcomes of PDA patients, we further show that EVI1 upregulation is a major cause of aggressive tumor phenotypes. Specifically, EVI1 promotes anchorage-independent growth and motility in vitro and enhances tumor propagation in vivo. Mechanistically, EVI1-dependent activation of tumor-promoting gene expression programs through the stepwise configuration of the active enhancer chromatin attributes to these phenotypes. In sum, our findings support the premise that EVI1 is a crucial driver of oncogenic transcription programs in PDA cells. Further, we emphasize the instructive role of epigenetic aberrancy in establishing PDA tumorigenesis.

Widespread reorganisation of pluripotent factor binding and gene regulatory interactions between human pluripotent states

The transition from naive to primed pluripotency is accompanied by an extensive reorganisation of transcriptional and epigenetic programmes. However, the role of transcriptional enhancers and three-dimensional chromatin organisation in coordinating these developmental programmes remains incompletely understood. Here, we generate a high-resolution atlas of gene regulatory interactions, chromatin profiles and transcription factor occupancy in naive and primed human pluripotent stem cells, and develop a network-graph approach to examine the atlas at multiple spatial scales. We uncover highly connected promoter hubs that change substantially in interaction frequency and in transcriptional co-regulation between pluripotent states. Small hubs frequently merge to form larger networks in primed cells, often linked by newly-formed Polycomb-associated interactions. We identify widespread state-specific differences in enhancer activity and interactivity that correspond with an extensive reconfiguration of OCT4, SOX2 and NANOG binding and target gene expression. These findings provide multilayered insights into the chromatin-based gene regulatory control of human pluripotent states.

Topography of transcriptionally active chromatin in glioblastoma

Molecular profiling of the most aggressive brain tumor glioblastoma (GBM) on the basis of gene expression, DNA methylation, and genomic variations advances both cancer research and clinical diagnosis. The enhancer architectures and regulatory circuitries governing tumor-intrinsic transcriptional diversity and subtype identity are still elusive. Here, by mapping H3K27ac deposition, we analyze the active regulatory landscapes across 95 GBM biopsies, 12 normal brain tissues, and 38 cell line counterparts. Analyses of differentially regulated enhancers and super-enhancers uncovered previously unrecognized layers of intertumor heterogeneity. Integrative analysis of variant enhancer loci and transcriptome identified topographies of transcriptional enhancers and core regulatory circuitries in four molecular subtypes of primary tumors: AC1-mesenchymal, AC1-classical, AC2-proneural, and AC3-proneural. Moreover, this study reveals core oncogenic dependency on super-enhancer–driven transcriptional factors, long noncoding RNAs, and druggable targets in GBM. Through profiling of transcriptional enhancers, we provide clinically relevant insights into molecular classification, pathogenesis, and therapeutic intervention of GBM.