Conserved Cis-Acting Range Extender Element Mediates Extreme Long-Range Enhancer Activity in Mammals.

While most mammalian enhancers regulate their cognate promoters over moderate distances of tens of kilobases (kb), some enhancers act over distances in the megabase range. The sequence features enabling such extreme-distance enhancer-promoter interactions remain elusive. Here, we used in vivo enhancer replacement experiments in mice to show that short- and medium-range enhancers cannot initiate gene expression at extreme-distance range. We uncover a novel conserved cis-acting element, Range EXtender (REX), that confers extreme-distance regulatory activity and is located next to a long-range enhancer of Sall1. The REX element itself has no endogenous enhancer activity. However, addition of the REX to other short- and mid-range enhancers substantially increases their genomic interaction range. In the most extreme example observed, addition of the REX increased the range of an enhancer by an order of magnitude, from its native 71kb to 840kb. The REX element contains highly conserved [C/T]AATTA homeodomain motifs. These motifs are enriched around long-range limb enhancers genome-wide, including the ZRS, a benchmark long-range limb enhancer of Shh. Mutating the [C/T]AATTA motifs within the ZRS does not affect its limb-specific enhancer activity at short range, but selectively abolishes its long-range activity, resulting in severe limb reduction in knock-in mice. In summary, we identify a sequence signature globally associated with long-range enhancer-promoter interactions and describe a prototypical REX element that is necessary and sufficient to confer extreme-distance gene activation by remote enhancers.

Increased enhancer-promoter interactions during developmental enhancer activation in mammals.

Remote enhancers are thought to interact with their target promoters via physical proximity, yet the importance of this proximity for enhancer function remains unclear. Here we investigate the three-dimensional (3D) conformation of enhancers during mammalian development by generating high-resolution tissue-resolved contact maps for nearly a thousand enhancers with characterized in vivo activities in ten murine embryonic tissues. Sixty-one percent of developmental enhancers bypass their neighboring genes, which are often marked by promoter CpG methylation. The majority of enhancers display tissue-specific 3D conformations, and both enhancer-promoter and enhancer-enhancer interactions are moderately but consistently increased upon enhancer activation in vivo. Less than 14% of enhancer-promoter interactions form stably across tissues; however, these invariant interactions form in the absence of the enhancer and are likely mediated by adjacent CTCF binding. Our results highlight the general importance of enhancer-promoter physical proximity for developmental gene activation in mammals.

Structural variants drive context-dependent oncogene activation in cancer.

Higher-order chromatin structure is important for the regulation of genes by distal regulatory sequences1,2. Structural variants (SVs) that alter three-dimensional (3D) genome organization can lead to enhancer-promoter rewiring and human disease, particularly in the context of cancer3. However, only a small minority of SVs are associated with altered gene expression4,5, and it remains unclear why certain SVs lead to changes in distal gene expression and others do not. To address these questions, we used a combination of genomic profiling and genome engineering to identify sites of recurrent changes in 3D genome structure in cancer and determine the effects of specific rearrangements on oncogene activation. By analysing Hi-C data from 92 cancer cell lines and patient samples, we identified loci affected by recurrent alterations to 3D genome structure, including oncogenes such as MYC, TERT and CCND1. By using CRISPR-Cas9 genome engineering to generate de novo SVs, we show that oncogene activity can be predicted by using 'activity-by-contact' models that consider partner region chromatin contacts and enhancer activity. However, activity-by-contact models are only predictive of specific subsets of genes in the genome, suggesting that different classes of genes engage in distinct modes of regulation by distal regulatory elements. These results indicate that SVs that alter 3D genome organization are widespread in cancer genomes and begin to illustrate predictive rules for the consequences of SVs on oncogene activation.