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Chromosome structure in mammals is thought to regulate transcription by modulating three-dimensional interactions between enhancers and promoters, notably through CTCF-mediated loops and topologically associating domains (TADs) 1 – 4 . However, how chromosome interactions are actually translated into transcriptional outputs remains unclear. Here, to address this question, we use an assay to position an enhancer at large numbers of densely spaced chromosomal locations relative to a fixed promoter, and measure promoter output and interactions within a genomic region with minimal regulatory and structural complexity. A quantitative analysis of hundreds of cell lines reveals that the transcriptional effect of an enhancer depends on its contact probabilities with the promoter through a nonlinear relationship. Mathematical modelling suggests that nonlinearity might arise from transient enhancer–promoter interactions being translated into slower promoter bursting dynamics in individual cells, therefore uncoupling the temporal dynamics of interactions from those of transcription. This uncovers a potential mechanism of how distal enhancers act from large genomic distances, and of how topologically associating domain boundaries block distal enhancers. Finally, we show that enhancer strength also determines absolute transcription levels as well as the sensitivity of a promoter to CTCF-mediated transcriptional insulation. Our measurements establish general principles for the context-dependent role of chromosome structure in long-range transcriptional regulation. The transcriptional effect of an enhancer depends on its contact probabilities with the promoter through a nonlinear relationship, and enhancer strength determines absolute transcription levels as well as the sensitivity of a promoter to CTCF-mediated transcriptional insulation.

DNA G-quadruplexes (G4s) are guanine-rich sequences that fold into four-stranded structures. Recent progress in the detection and mapping of genomic G4 structures has provided new insights into their functions in regulating transcription and genome stability, and has revealed their potential relevance for cancer therapy. Single-stranded guanine-rich DNA sequences can fold into four-stranded DNA structures called G-quadruplexes (G4s) that arise from the self-stacking of two or more guanine quartets. There has been considerable recent progress in the detection and mapping of G4 structures in the human genome and in biologically relevant contexts. These advancements, many of which align with predictions made previously in computational studies, provide important new insights into the functions of G4 structures in, for example, the regulation of transcription and genome stability, and uncover their potential relevance for cancer therapy.

The transcription factor and oncoprotein MYC is a potent driver of many human cancers and can regulate numerous biological activities that contribute to tumorigenesis. How a single transcription factor can regulate such a diverse set of biological programmes is central to the understanding of MYC function in cancer. In this Perspective, we highlight how multiple proteins that interact with MYC enable MYC to regulate several central control points of gene transcription. These include promoter binding, epigenetic modifications, initiation, elongation and post-transcriptional processes. Evidence shows that a combination of multiple protein interactions enables MYC to function as a potent oncoprotein, working together in a ‘coalition model’, as presented here. Moreover, as MYC depends on its protein interactome for function, we discuss recent research that emphasizes an unprecedented opportunity to target protein interactors to directly impede MYC oncogenesis.This Perspective highlights the importance of protein–protein interactions for the oncogenic functions of MYC and discusses how the MYC protein interactome might be exploited therapeutically.

Key Points Transposable elements (TEs) are increasingly recognized as a potent source of regulatory sequences in eukaryotic genomes The selfish replication cycle of TEs drove the evolution of finely tuned regulatory activities that favoured their propagation and has predisposed them to be co-opted for the regulation of host genes There is a growing number of examples of TE-derived sequences that have been co-opted to regulate important biological processes in organismal development and physiology Dysfunction of TE-derived regulatory sequences is also emerging as a potential driver of diseases including cancer and autoimmunity Functional genomics and genome-editing technologies herald an exciting era for understanding the biological effect of TEs Transposable elements (TEs) are widely known for their deleterious consequences of selfish propagation and mutagenesis. However, as described in this Review, TEs also provide hosts with rich, beneficial gene-regulatory machinery in the form of regulatory DNA elements and TE-derived gene products. The authors highlight the diverse regulatory contributions of TEs to organismal physiology and pathology, provide a framework for responsibly assigning functional roles to TEs and offer visions for the future. Transposable elements (TEs) are a prolific source of tightly regulated, biochemically active non-coding elements, such as transcription factor-binding sites and non-coding RNAs. Many recent studies reinvigorate the idea that these elements are pervasively co-opted for the regulation of host genes. We argue that the inherent genetic properties of TEs and the conflicting relationships with their hosts facilitate their recruitment for regulatory functions in diverse genomes. We review recent findings supporting the long-standing hypothesis that the waves of TE invasions endured by organisms for eons have catalysed the evolution of gene-regulatory networks. We also discuss the challenges of dissecting and interpreting the phenotypic effect of regulatory activities encoded by TEs in health and disease.

The spatial organization of the genome into compartments and topologically associated domains can have an important role in the regulation of gene expression. But could gene expression conversely regulate genome organization? Here, we review recent studies that assessed the requirement of transcription and/or the transcription machinery for the establishment or maintenance of genome topology. The results reveal different requirements at different genomic scales. Transcription is generally not required for higher-level genome compartmentalization, has only moderate effects on domain organization and is not sufficient to create new domain boundaries. However, on a finer scale, transcripts or transcription does seem to have a role in the formation of subcompartments and subdomains and in stabilizing enhancer–promoter interactions. Recent evidence suggests a dynamic, reciprocal interplay between fine-scale genome organization and transcription, in which each is able to modulate or reinforce the activity of the other.Genome organization can regulate gene expression, but can gene expression regulate genome organization? Recent studies reveal that, although not required for higher-level genome organization, transcription has a role in the formation and stabilization of genomic subdomains and enhancer–promoter interactions.

Histones serve to both package and organize DNA within the nucleus. In addition to histone post-translational modification and chromatin remodelling complexes, histone variants contribute to the complexity of epigenetic regulation of the genome. Histone variants are characterized by a distinct protein sequence and a selection of designated chaperone systems and chromatin remodelling complexes that regulate their localization in the genome. In addition, histone variants can be enriched with specific post-translational modifications, which in turn can provide a scaffold for recruitment of variant-specific interacting proteins to chromatin. Thus, through these properties, histone variants have the capacity to endow specific regions of chromatin with unique character and function in a regulated manner. In this Review, we provide an overview of recent advances in our understanding of the contribution of histone variants to chromatin function in mammalian systems. First, we discuss new molecular insights into chaperone-mediated histone variant deposition. Next, we discuss mechanisms by which histone variants influence chromatin properties such as nucleosome stability and the local chromatin environment both through histone variant sequence-specific effects and through their role in recruiting different chromatin-associated complexes. Finally, we focus on histone variant function in the context of both embryonic development and human disease, specifically developmental syndromes and cancer.Histone variants differ from canonical histones in their genomic localization, regulation and function. Incorporation of histone variants endows specific genomic regions with unique features to fine-tune gene expression, contributing to animal development and disease pathogenesis.

Various cis -regulatory functions of genomic loci that produce long non-coding RNAs are revealed, including instances where their promoters have enhancer-like activity and the lncRNA transcripts themselves are not required for activity. The search for lncRNA function Since the discovery of pervasive transcription of long non-coding RNAs (lncRNAs) in mammalian genomes, there has been pressure to determine their functions. Here, Eric Lander and colleagues use a CRISPR/Cas9 deletion approach to uncover various cis -regulatory functions of lncRNAs, including instances in which their promoters have enhancer-like activity and the lncRNA transcripts themselves are often not required for activity. Such effects on neighbouring genes are also seen for protein-coding loci. Mammalian genomes are pervasively transcribed 1 , 2 to produce thousands of long non-coding RNAs (lncRNAs) 3 , 4 . A few of these lncRNAs have been shown to recruit regulatory complexes through RNA–protein interactions to influence the expression of nearby genes 5 , 6 , 7 , and it has been suggested that many other lncRNAs can also act as local regulators 8 , 9 . Such local functions could explain the observation that lncRNA expression is often correlated with the expression of nearby genes 2 , 10 , 11 . However, these correlations have been challenging to dissect 12 and could alternatively result from processes that are not mediated by the lncRNA transcripts themselves. For example, some gene promoters have been proposed to have dual functions as enhancers 13 , 14 , 15 , 16 , and the process of transcription itself may contribute to gene regulation by recruiting activating factors or remodelling nucleosomes 10 , 17 , 18 . Here we use genetic manipulation in mouse cell lines to dissect 12 genomic loci that produce lncRNAs and find that 5 of these loci influence the expression of a neighbouring gene in cis . Notably, none of these effects requires the specific lncRNA transcripts themselves and instead involves general processes associated with their production, including enhancer-like activity of gene promoters, the process of transcription, and the splicing of the transcript. Furthermore, such effects are not limited to lncRNA loci: we find that four out of six protein-coding loci also influence the expression of a neighbour. These results demonstrate that cross-talk among neighbouring genes is a prevalent phenomenon that can involve multiple mechanisms and cis- regulatory signals, including a role for RNA splice sites. These mechanisms may explain the function and evolution of some genomic loci that produce lncRNAs and broadly contribute to the regulation of both coding and non-coding genes.

Cell type-specific gene expression relies on transcription factors (TFs) binding DNA sequence motifs embedded in chromatin. Understanding how motifs are accessed in chromatin is crucial to comprehend differential transcriptional responses and the phenotypic impact of sequence variation. Chromatin obstacles to TF binding range from DNA methylation to restriction of DNA access by nucleosomes depending on their position, composition and modification. In vivo and in vitro approaches now enable the study of TF binding in chromatin at unprecedented resolution. Emerging insights suggest that TFs vary in their ability to navigate chromatin states. However, it remains challenging to link binding and transcriptional outcomes to molecular characteristics of TFs or the local chromatin substrate. Here, we discuss our current understanding of how TFs access DNA in chromatin and novel techniques and directions towards a better understanding of this critical step in genome regulation.In this Review, Isbel et al. describe our current understanding of how transcription factors navigate features of chromatin — particularly DNA methylation and nucleosomes — and how this contributes to specificity of genomic binding and, ultimately, transcriptional regulation.

Shadow enhancers are seemingly redundant transcriptional cis-regulatory elements that regulate the same gene and drive overlapping expression patterns. Recent studies have shown that shadow enhancers are remarkably abundant and control most developmental gene expression in both invertebrates and vertebrates, including mammals. Shadow enhancers might provide an important mechanism for buffering gene expression against mutations in non-coding regulatory regions of genes implicated in human disease. Technological advances in genome editing and live imaging have shed light on how shadow enhancers establish precise gene expression patterns and confer phenotypic robustness. Shadow enhancers can interact in complex ways and may also help to drive the formation of transcriptional hubs within the nucleus. Despite their apparent redundancy, the prevalence and evolutionary conservation of shadow enhancers underscore their key role in emerging metazoan gene regulatory networks.Shadow enhancers regulate a common target gene and drive expression patterns that overlap spatiotemporally. The authors review recent insights into the prevalence and role of shadow enhancers in metazoans, as well as their mechanisms of action to fine-tune gene expression. They also discuss the evolution of shadow enhancers and their implication in disease.

Key Points Enhancers are genetic elements that have major roles in determining cell type-specific gene expression patterns and responses to internal and external signals. Mammalian genomes contain millions of enhancers, but only a subset of them are selected and activated in each cell type in the body. Enhancer selection involves collaborative interactions between 'pioneer' or lineage-determining transcription factors. Such factors are able to prime enhancers in a cell type-specific manner by binding to closely spaced recognition motifs. Signal-dependent transcription factors, such as nuclear receptors and nuclear factor-κB (NF-κB), primarily bind to regions of the genome that are primed in a cell type-specific manner by lineage-determining transcription factors. This enables such broadly expressed, signal-dependent transcription factors to regulate gene expression in a cell type-specific manner. A proportion of the genome contains a very high density of marks of active enhancers. Such enhancer-rich genomic regions, which are called super-enhancers, are different in each cell type and regulate genes required for establishing cell identity and function. An understanding of the mechanisms underlying the cell type-specific selection and function of enhancers will improve our understanding of the effects of natural genetic variation on complex phenotypes and diseases. Many gene expression patterns are dictated by enhancers. Mammalian genomes contain millions of potential enhancers, but only a small subset of them is active in any cell type. Emerging data uncover how cell type-specific enhancer function is established, including the involvement of higher-order genomic organization in the process. The human body contains several hundred cell types, all of which share the same genome. In metazoans, much of the regulatory code that drives cell type-specific gene expression is located in distal elements called enhancers. Although mammalian genomes contain millions of potential enhancers, only a small subset of them is active in a given cell type. Cell type-specific enhancer selection involves the binding of lineage-determining transcription factors that prime enhancers. Signal-dependent transcription factors bind to primed enhancers, which enables these broadly expressed factors to regulate gene expression in a cell type-specific manner. The expression of genes that specify cell type identity and function is associated with densely spaced clusters of active enhancers known as super-enhancers. The functions of enhancers and super-enhancers are influenced by, and affect, higher-order genomic organization.