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Spatiotemporal gene expression programmes are orchestrated by transcriptional enhancers, which are key regulatory DNA elements that engage in physical contacts with their target-gene promoters, often bridging considerable genomic distances. Recent progress in genomics, genome editing and microscopy methodologies have enabled the genome-wide mapping of enhancer–promoter contacts and their functional dissection. In this Review, we discuss novel concepts on how enhancer–promoter interactions are established and maintained, how the 3D architecture of mammalian genomes both facilitates and constrains enhancer–promoter contacts, and the role they play in gene expression control during normal development and disease.For appropriate control of gene expression, enhancers must communicate with the right target genes at the right time, typically over large genomic distances. In this Review, Schoenfelder and Fraser discuss our latest understanding of long-range enhancer–promoter crosstalk, including target-gene specificity, interaction dynamics, protein and RNA architects of interactions, roles of 3D genome organization and the pathological consequences of regulatory rewiring.

The proper activities of enhancers and gene promoters are essential for coordinated transcription within a cell. Although diverse methodologies have been developed to identify enhancers and promoters, most have tacitly assumed that these elements are distinct. However, studies have unexpectedly shown that regulatory elements may have both enhancer and promoter functions. Here we review these results, focusing on the factors that determine the promoter and/or enhancer activity of regulatory elements. We discuss emerging models that define regulatory elements by accessible DNA and their non-mutually-exclusive abilities to drive transcription initiation (promoter activity) and/or to enhance transcription at other such regions (enhancer activity).Activating transcriptional regulatory elements have traditionally been categorized into promoters, which define transcription start sites, and enhancers, which act distally to stimulate transcription. In this Review, Andersson and Sandelin discuss the latest findings from methodologies for profiling and testing transcriptional regulatory elements at scale. They explain how the data support an updated, nuanced model that accounts for the numerous overlapping molecular properties of promoters and enhancers.

Structural and quantitative chromosomal rearrangements, collectively referred to as structural variation (SV), contribute to a large extent to the genetic diversity of the human genome and thus are of high relevance for cancer genetics, rare diseases and evolutionary genetics. Recent studies have shown that SVs can not only affect gene dosage but also modulate basic mechanisms of gene regulation. SVs can alter the copy number of regulatory elements or modify the 3D genome by disrupting higher-order chromatin organization such as topologically associating domains. As a result of these position effects, SVs can influence the expression of genes distant from the SV breakpoints, thereby causing disease. The impact of SVs on the 3D genome and on gene expression regulation has to be considered when interpreting the pathogenic potential of these variant types.

Long non-coding RNAs (lncRNAs) are largely heterogeneous and functionally uncharacterized. Here, using FANTOM5 cap analysis of gene expression (CAGE) data, we integrate multiple transcript collections to generate a comprehensive atlas of 27,919 human lncRNA genes with high-confidence 5′ ends and expression profiles across 1,829 samples from the major human primary cell types and tissues. Genomic and epigenomic classification of these lncRNAs reveals that most intergenic lncRNAs originate from enhancers rather than from promoters. Incorporating genetic and expression data, we show that lncRNAs overlapping trait-associated single nucleotide polymorphisms are specifically expressed in cell types relevant to the traits, implicating these lncRNAs in multiple diseases. We further demonstrate that lncRNAs overlapping expression quantitative trait loci (eQTL)-associated single nucleotide polymorphisms of messenger RNAs are co-expressed with the corresponding messenger RNAs, suggesting their potential roles in transcriptional regulation. Combining these findings with conservation data, we identify 19,175 potentially functional lncRNAs in the human genome. A catalogue of human long non-coding RNA genes and their expression profiles across samples from major human primary cell types, tissues and cell lines. A catalogue of long non-coding RNAs Alistair Forrest, Piero Carninci and colleagues of the FANTOM Consortium provide a catalogue of human long non-coding RNA (lncRNA) genes and their expression profiles across samples from human primary cell types, tissues and cell lines. They used combined analyses of multiple data sets to identify 27,919 lncRNA genes with high-confidence 5′ ends, as well as a subset of 19,175 potentially functional lncRNA loci. The lncRNA catalogue and annotations are available through an open web resource.

Key Points Enhancers are distal regulatory genomic elements that determine spatiotemporal and quantitative gene transcription programmes in response to developmental or environmental cues. Global transcriptomic and (epi)genomic advances have found that enhancers are pervasively transcribed into non-coding RNAs (ncRNAs), which are named enhancer RNAs (eRNAs). The expression levels of eRNAs are highly correlated with the activity of the functional enhancers, both in developmental and rapid signal-regulated transcriptional programmes. eRNAs bear several common and unique features compared to other long ncRNAs or coding mRNAs. Heterogeneity of eRNAs exists in terms of both transcriptional processes and RNA properties, which might be linked to their functional diversity. There is evidence supporting a functional role of both the eRNA transcripts per se, and the act of transcription, in the activation of target coding genes. However, for the large majority of eRNAs in the mammalian genome, evidence of their functional roles is still limited. Mechanisms underlying the roles of specific eRNAs include modulating the chromatin accessibility of cognate promoters, stabilizing enhancer–promoter interactions, trapping transcription factors on genomic sites, and regulating the RNA polymerase II pause release at cognate promoters. The act of enhancer transcription may facilitate histone modifications, remodel large areas of chromatin and induce transcriptional interference. Enhancer transcription may take part in generating several important biological phenomena, including transcription-dependent R-loops, genomic mutations and instability at enhancers, and potentially facilitate new gene birth during evolution. Further evidence is needed to fully understand the functions of different categories of enhancers as transcription units and the roles of eRNAs per se , in gene regulation, development and disease. The observation that many, if not all, functional enhancers generate non-coding enhancer RNAs (eRNAs) has raised critical questions regarding the potential biological roles of the enhancer transcription process and, indeed, of eRNAs. This article reviews fundamental insights into the inter-regulation of enhancers and promoters and discusses unresolved questions regarding the functional role of enhancers as transcription units in genome regulation. Networks of regulatory enhancers dictate distinct cell identities and cellular responses to diverse signals by instructing precise spatiotemporal patterns of gene expression. However, 35 years after their discovery, enhancer functions and mechanisms remain incompletely understood. Intriguingly, recent evidence suggests that many, if not all, functional enhancers are themselves transcription units, generating non-coding enhancer RNAs. This observation provides a fundamental insight into the inter-regulation between enhancers and promoters, which can both act as transcription units; it also raises crucial questions regarding the potential biological roles of the enhancer transcription process and non-coding enhancer RNAs. Here, we review research progress in this field and discuss several important, unresolved questions regarding the roles and mechanisms of enhancers in gene regulation.

Precise patterns of gene expression in metazoans are controlled by three classes of regulatory elements: promoters, enhancers and boundary elements. During differentiation and development, these elements form specific interactions in dynamic higher-order chromatin structures. However, the relationship between genome structure and its function in gene regulation is not completely understood. Here we review recent progress in this field and discuss whether genome structure plays an instructive role in regulating gene expression or is a reflection of the activity of the regulatory elements of the genome.In this Review, Oudelaar and Higgs discuss the relationship between genome structure and gene regulation, with a focus on whether genome organization has an instructive role or largely reflects the activity of regulatory elements.

The human gene catalogue is essentially complete, but we lack an equivalently vetted inventory of bona fide human enhancers. Hundreds of thousands of candidate enhancers have been nominated via biochemical annotations; however, only a handful of these have been validated and confidently linked to their target genes. Here we review emerging technologies for discovering, characterizing and validating human enhancers at scale. We furthermore propose a new framework for operationally defining enhancers that accommodates the heterogeneous and complementary results that are emerging from reporter assays, biochemical measurements and CRISPR screens.In this Review, Gasperini, Tome and Shendure discuss the evolving definitions of transcriptional enhancers, as well as diverse modern experimental tools to identify them. The authors describe how these diverse mindsets and methods provide differing but complementary insights into enhancers, each with notable strengths and caveats. They discuss how such views and approaches might be combined in a comprehensive catalogue of functional enhancers.

Key Points The development of all organisms relies on differential gene expression, which is controlled by genomic regions called enhancers or cis -regulatory modules (CRMs). Recent studies highlight the importance of enhancers in evolution and disease; however, our understanding of their properties and functions remains incomplete. Enhancers contain short DNA sequences, which are binding sites for transcription factors. In turn, transcription factors recruit cofactors, which modify the nearby chromatin and lead to transcriptional activation. The location of putative enhancers can be predicted genome wide by assessing either the binding of transcription factors and cofactors or post-translational histone modifications by chromatin immunoprecipitation followed by deep sequencing (ChIP–seq). 'Open' chromatin with accessible DNA can be detected by DNase I hypersensitive site sequencing (DNase-seq), micrococcal nuclease sequencing (MNase-seq), formaldehyde-assisted isolation of regulatory elements followed by deep sequencing (FAIRE–seq) or assay for transposase-accessible chromatin using sequencing (ATAC-seq). Distal enhancers can activate target gene expression by looping to promoters. Such spatial contacts can be detected by chromosome conformation capture (3C) assays and its variants circular chromosome conformation capture (4C), chromosome conformation capture carbon copy (5C) and Hi-C methods or by chromatin interaction analysis with paired-end tag sequencing (ChIA–PET, which is a combination of ChIP and various 3C-based methods). The genome-wide prediction of enhancers based on characteristic chromatin features is powerful, but such results have to be interpreted with caution because none of the known features is perfectly predictive. Enhancer activities of candidate sequences can be measured directly in a developmental context using image-based readouts or enhancer-FACS-seq. High-throughput parallel enhancer assays use either ectopic reporters to test thousands of candidates (which are based on DNA barcodes) or genome-wide screens (such as self-transcribing active regulatory region sequencing (STARR-seq)). Our understanding of enhancer biology will be further accelerated by advances in genome editing methods (such as transcription activator-like effector nucleases (TALENs) and the clustered regularly interspaced short palindromic repeat (CRISPR)–Cas9 system), as well as by the development or improvements of methods to assess gene expression, chromatin state and structure in entire genomes and from increasingly few cells (such as thousands of reporters integrated in parallel (TRIP), single-cell RNA sequencing or ChIP–seq, and high-resolution Hi-C). Enhancers are DNA elements that are key regulators of gene expression, but their complexities and context dependence makes their identification and characterization challenging. This Review discusses how an improved understanding of the varied properties of enhancers is being used in diverse approaches for the systematic prediction of enhancers genome wide. Cellular development, morphology and function are governed by precise patterns of gene expression. These are established by the coordinated action of genomic regulatory elements known as enhancers or cis -regulatory modules. More than 30 years after the initial discovery of enhancers, many of their properties have been elucidated; however, despite major efforts, we only have an incomplete picture of enhancers in animal genomes. In this Review, we discuss how properties of enhancer sequences and chromatin are used to predict enhancers in genome-wide studies. We also cover recently developed high-throughput methods that allow the direct testing and identification of enhancers on the basis of their activity. Finally, we discuss recent technological advances and current challenges in the field of regulatory genomics.

The prevailing view of metazoan gene regulation is that individual genes are independently regulated by their own dedicated sets of transcriptional enhancers. Past studies have reported long-range gene–gene associations 1 – 3 , but their functional importance in regulating transcription remains unclear. Here we used quantitative single-cell live imaging methods to provide a demonstration of co-dependent transcriptional dynamics of genes separated by large genomic distances in living Drosophila embryos. We find extensive physical and functional associations of distant paralogous genes, including co-regulation by shared enhancers and co-transcriptional initiation over distances of nearly 250 kilobases. Regulatory interconnectivity depends on promoter-proximal tethering elements, and perturbations in these elements uncouple transcription and alter the bursting dynamics of distant genes, suggesting a role of genome topology in the formation and stability of co-transcriptional hubs. Transcriptional coupling is detected throughout the fly genome and encompasses a broad spectrum of conserved developmental processes, suggesting a general strategy for long-range integration of gene activity. In Drosophila , there are extensive physical and functional associations of distant paralogous genes, including co-regulation by shared enhancers and co-transcriptional initiation over distances of nearly 250 kilobases.