Transcriptional enhancers are crucial regulators of gene expression and animal development and the characterization of their genomic organization, spatiotemporal activities and sequence properties is a key goal in modern biology. Here we characterize the in vivo activity of 7,705 Drosophila melanogaster enhancer candidates covering 13.5% of the non-coding non-repetitive genome throughout embryogenesis. 3,557 (46%) candidates are active, suggesting a high density with 50,000 to 100,000 developmental enhancers genome-wide. The vast majority of enhancers display specific spatial patterns that are highly dynamic during development. Most appear to regulate their neighbouring genes, suggesting that the cis-regulatory genome is organized locally into domains, which are supported by chromosomal domains, insulator binding and genome evolution. However, 12 to 21 per cent of enhancers appear to skip non-expressed neighbours and regulate a more distal gene. Finally, we computationally identify cis-regulatory motifs that are predictive and required for enhancer activity, as we validate experimentally. This work provides global insights into the organization of an animal regulatory genome and the make-up of enhancer sequences and confirms and generalizes principles from previous studies. All enhancer patterns are annotated manually with a controlled vocabulary and all results are available through a web interface (http://enhancers.starklab.org), including the raw images of all microscopy slides for manual inspection at arbitrary zoom levels.

The evolution of body shape is thought to be tightly coupled to changes in regulatory sequences, but specific molecular events associated with major morphological transitions in vertebrates have remained elusive. We identified snake-specific sequence changes within an otherwise highly conserved long-range limb enhancer of Sonic hedgehog (Shh). Transgenic mouse reporter assays revealed that the in vivo activity pattern of the enhancer is conserved across a wide range of vertebrates including fish, but not in snakes. Genomic substitution of the mouse enhancer with its human or fish ortholog results in normal limb development. In contrast, replacement with snake orthologs caused severe limb reduction. Synthetic restoration of a single transcription factor binding site lost in the snake lineage reinstated full in vivo function to the snake enhancer. Our results demonstrate changes in a regulatory sequence associated with a major body plan transition and highlight the role of enhancers in morphological evolution.

HOT (highly occupied target) regions bound by many transcription factors are considered to be one of the most intriguing findings of the recent modENCODE reports, yet their functions have remained unclear. We tested 108 Drosophila melanogaster HOT regions in transgenic embryos with site-specifically integrated transcriptional reporters. In contrast to prior expectations, we found 102 (94%) to be active enhancers during embryogenesis and to display diverse spatial and temporal patterns, reminiscent of expression patterns for important developmental genes. Remarkably, HOT regions strongly activate nearby genes and are required for endogenous gene expression, as we show using bacterial artificial chromosome (BAC) transgenesis. HOT enhancers have a distinct cis-regulatory signature with enriched sequence motifs for the global activators Vielfaltig, also known as Zelda, and Trithoraxlike, also known as GAGA. This signature allows the prediction of HOT versus control regions from the DNA sequence alone. Here, we show that Drosophila HOT regions function as transcriptional enhancers with diverse activity patterns. While a large number of bound TFs is the defining feature of HOT regions, many TFs seem to be bound neutrally without any apparent contribution to enhancer activity. HOT enhancers are characterized by a distinct and predictive cis-regulatory signature, which includes motifs for Vielfaltig/Zelda (ZLD), a recently reported activator of the early Drosophila genome (Liang et al. 2008;Harrison et al. 2011;Nien et al. 2011), and Trithoraxlike/GAGA (GAGA), a TF known to form homomeric and heteromeric complexes (Bardwell and Treisman 1994) and to be required for the generation and maintenance of nucleosome-free regions (Croston et al. 1991;Nakayama et al. 2007). Results and Discussion HOT regions function as early embryonic enhancers with diverse patternsWe tested a representative set of 108 D. melanogaster HOT regions (see the Materials and Methods; Supplemental Tables 1, 2) in transgenic embryos with sitespecifically integrated transcriptional reporters (Fig. 1A). Strikingly, 94% (102) of these HOT regions drove reporter expression in a specific pattern during embryogenesis. In contrast, only 39% of control regions (16 of 41) functioned as enhancers, including 11 of 21 regions chosen to contain TF-binding sites and five of 20 regions chosen to contain no known binding sites (see the Materials and Methods; Supplemental Fig. S1; Supplemental Table 1). This enrichment of HOT over control regions is highest at stages 3-10 (corresponding to 1-5 h after fertilization) (Fig. 1B), at which most of the chromatin immunoprecipitation (ChIP) experiments leading to the definition of the HOT regions had been performed (MacArthur et al. 2009; The modENCODE Consortium 2010).Contrary to expectations that HOT enhancers might constitute a particular class of enhancers (for example, with ubiquitous activity), they display highly diverse spatial activity patterns in all major presumptive tissues of the blastoderm embryo ( Fig. 1C; Sup...

Establishing causal links between non-coding variants and human phenotypes is an increasing challenge. Here we introduce a high-throughput mouse reporter assay for assessing the pathogenic potential of human enhancer variants in vivo and examine nearly a thousand variants in an enhancer repeatedly linked to polydactyly. We show that 71% of all rare non-coding variants previously proposed as causal led to reporter gene expression in a pattern consistent with their pathogenic role. Variants observed to alter enhancer activity were further confirmed to cause polydactyly in knock-in mice. We also used combinatorial and single-nucleotide mutagenesis to evaluate the in vivo impact of mutations affecting all positions of the enhancer and identified additional functional substitutions, including potentially pathogenic variants hitherto not observed in humans. Our results uncover the functional consequences of hundreds of mutations in a phenotype-associated enhancer and establish a widely applicable strategy for systematic in vivo evaluation of human enhancer variants.

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 the majority of developmental gene expression in both invertebrates and vertebrates, including mammals. Shadow enhancers might provide an important mechanism for buffering 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 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.

The regulation of gene expression is mediated at the transcriptional level by enhancer regions that are bound by sequence-specific transcription factors (TFs). Recent studies have shown that the in vivo binding sites of single TFs differ between developmental or cellular contexts. How this context-specific binding is encoded in the cis-regulatory DNA sequence has, however, remained unclear. We computationally dissect context-specific TF binding sites in Drosophila, Caenorhabditis elegans, mouse, and human and find distinct combinations of sequence motifs for partner factors, which are predictive and reveal specific motif requirements of individual binding sites. We predict that TF binding in the early Drosophila embryo depends on motifs for the early zygotic TFs Vielfaltig (also known as Zelda) and Tramtrack. We validate experimentally that the activity of Twist-bound enhancers and Twist binding itself depend on Vielfaltig motifs, suggesting that Vielfaltig is more generally important for early transcription. Our finding that the motif content can predict contextspecific binding and that the predictions work across different Drosophila species suggests that characteristic motif combinations are shared between sites, revealing context-specific motif codes (cis-regulatory signatures), which appear to be conserved during evolution. Taken together, this study establishes a novel approach to derive predictive cis-regulatory motif requirements for individual TF binding sites and enhancers. Importantly, the method is generally applicable across different cell types and organisms to elucidate cis-regulatory sequence determinants and the corresponding trans-acting factors from the increasing number of tissue-and cell-type-specific TF binding studies.

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Polycomb Response Elements (PREs) are specific DNA sequences that stably maintain the developmental pattern of gene expression. Drosophila PREs are well characterized, whereas the existence of PREs in mammals remains debated. Accumulating evidence supports a model in which CGIs recruit Polycomb-Group complexes (PcG), however, which subset of CGIs are selected to serve as PREs is unclear. Trithorax (Trx) positively regulates gene expression in Drosophila and co-occupies PREs to antagonize Polycomb-dependent silencing. Here, we demonstrate that Trx-dependent H3K4 dimethylation (H3K4me2) marks Drosophila PREs and maintains the developmental expression pattern of nearby genes. Similarly, the mammalian Trx homology, MLL1, deposits H3K4me2 at CpG dense regions that could serve as PREs. In the absence of MLL1 and H3K4me2, H3K27me3 levels, a mark of Polycomb Repressive Complex 2 (PRC2), increase at these loci. By inhibiting PRC2-dependent H3K27me3 in the absence of MLL1, we can rescue expression of these loci, demonstrating a functional balance between MLL1 and PRC2 activities at these sites. Thus, our study provides rules for identifying cell-type specific functional mammalian PREs within the human genome.