Gene expression patterns in metazoans range from widespread expression in multiple cell types, such as expression of genes required for the maintenance of basic cellular functions, to complex spatiotemporal expression of genes with pleiotropic functions. Examples of transcription factors (TFs) involved in such complex regulation are PAX6, which is crucial for development of the eye and also of sensory organs and specific neural and epidermal tissues; Sonic Hedgehog (SHH), which is involved in development of many systems, as diverse as limb and brain; and TBX5, which is involved in heart and forelimb development.

The precise and complex spatiotemporal expression of genes often requires the deployment of additional cis-regulatory elements, physically displaced from the promoter. These promoter-distal cis-regulatory elements bind TFs that are cell-lineage-specific and those that are expressed in the presence of external signals as hormones, for example, at specific time points such as differentiation or proliferation. By integrating different cues, these elements coordinate complex patterns of gene expression in different tissues and time points (Figure 1a).

Enhancers are a class of cis-regulatory elements that promote gene expression and often are essential for eliciting the complex expression patterns of developmental genes. These elements typically span a few hundred base pairs (bp) and are composed of clusters of transcription factor binding sites (6- to 20-bp motifs) to which combinations of trans-activating and repressive factors bind in sequence-specific manner. They can be located in intergenic regions, introns and exons, tens to hundreds of kilobases from their target genes ( 1 , reviewed in 2 ).

Although these elements have been studied for decades through careful dissection of individual examples 3 , the advent of genome-wide chromatin immunoprecipitation (ChIP), an experimental technique that locates DNA sites where specific proteins are bound (reviewed in 4 ) enabled the largely unbiased identification of tens of thousands of putative elements in a single experiment and the discovery of global patterns that are shedding light on how enhancers act (reviewed in 5 ). Studies using this technique have confirmed and expanded our appreciation of the importance of cis-regulatory elements during development and in adult function, changing the way we view gene regulation in metazoans.

Here we review recent findings, obtained mainly from genome-wide studies, of how enhancers are activated, the role of enhancer features in mammalian development, and the involvement of this class of cis-regulatory elements in disease. Although earlier discoveries have attributed enhancer variation to several human diseases (reviewed in 2 6 ), these studies have been largely limited to rare Mendelian disorders, which commonly involve single gene disruptions and follow simple patterns of inheritance. We discuss the previously unappreciated role of promoter-distal cis-regulatory variation in common disease susceptibility from genome-wide association studies (GWASs) and discuss how a variety of genome annotations can additionally be exploited to expedite discovery of causal variants.

Enhancers are recognized by the cellular machinery through a combination of chromatin modifications and sequence-specific binding of TFs. Given that DNA is compacted into chromatin, enhancers must be localized to sites accessible to proteins, that is, in euchromatin regions with exposed DNA. However, enhancers are not always accessible and may require appropriate stimuli to become 'open'. For example, chromatin containing distal enhancers that have become active has been shown to undergo dynamic nucleosome repositioning following T-cell activation 7 , androgen receptor treatment 8 and erythrocyte differentiation 9 . These stimuli and other cellular processes cause nucleosome repositioning, which involves chromatin remodeling complexes such as BAF (reviewed in 10 ). The specificity of these complexes to particular enhancers seems to be mediated by 'pioneer' factors, FOXA1 being the best characterized example (reviewed in 11 ). These proteins bind to nucleosomal DNA, recruiting chromatin remodelers that facilitate chromatin opening and the subsequent binding of TFs 11 .

The binding of chromatin remodelers might also involve chemical groups present in nucleosomes 12 13 . Histones, the proteins that constitute nucleosomes, can be dynamically modified (for example, acetylated, methylated or phosphorylated) at different residues (reviewed in 12 ). The role of histone modifications in enhancer function is still unclear. One possibility is that the cell machinery recognizes a code of DNA elements based on combinations of histone modifications 13 . Given that there is a wide assortment of histone modifications, discovering those few that are sufficient to distinguish DNA elements and enhancer states is important. Indeed, recent studies using ChIP have uncovered genome-wide patterns that allow certain DNA elements to be distinguished (reviewed in 5 ). For example, whereas trimethylation of lysine 4 of histone 3 (H3K4me3) is predominantly present in active promoters, distal enhancers are associated with monomethylation (H3K4me1) 14 , which is largely tissue-specific 15 16 .

H3K4me1 was largely accepted as a general enhancer marker, and several studies have used ChIP of H3K4me1 coupled with high-throughput sequencing to locate tens of thousands of distal enhancers in various cells and tissues (for example, 15 17 18 ). However, it was found that not all H3K4me1 regions correspond to active enhancers 16 17 18 19 . A recent study demonstrated that presence of acetylation of lysine 27 of histone 3 (H3K27ac) was associated with active enhancers identified by H3K4me1 in several cell types, whereas a sub-population comprised seemingly inactive H3K4me1 regions that were devoid of this acetylation and were deemed 'poised' 16 . Histone acetylation is catalyzed by acetyltransferases, such as p300, which are recruited by bound TFs and thought to bind chromatin remodelers 13 . Given its role in enhancer activation, p300 has also been used to locate enhancers 14 15 18 20 , but its presence may not distinguish between active and poised enhancers 16 19 , suggesting that factors other than the presence of acetyltransferases are necessary for enhancer activation.

In addition to the active enhancer mark H3K27ac, a recent study found that H3K4me3 is associated with enhancer activation 21 , contrary to the widely accepted notion that H3K4me3 is mostly a promoter histone modification. Similarly to H3K27ac, distal enhancers marked by H3K4me1 that became active during T-cell differentiation gained H3K4me3, whereas inactive enhancers remained marked solely by H3K4me1.

The outcome of enhancer activation by acetylation, nucleosome repositioning and TF binding is gene transcription. Active enhancers are believed to initiate gene expression through physical interaction with their target promoters. The prevailing model proposes that they directly contact promoters by looping out intervening chromatin (Figure 1b; reviewed in 22 ). This is demonstrated by techniques that allow the determination of physical interactions between segments in the genome, such as chromatin interaction analysis (ChIA) 23 and chromatin conformation capture (3C) and variants 24 . By contacting promoters, enhancers would supply trans-factors and activate transcription.

It has been recently shown that at least a fraction of active enhancers are transcribed by RNA polymerase II (Pol II), resulting in 'enhancer RNA' molecules (eRNA) 25 26 . It is unclear whether eRNAs have a regulatory role per se or whether they are simply a byproduct associated with Pol II recruitment produced when Pol II passes enhancers as it attempts to recruit methyl- and acetyltransferases 25 26 . Alternatively, assuming that enhancers directly interact with promoters, eRNAs could be the result of transcription of the wrong DNA sequence, with no biological function. This idea is consistent with the dependence of eRNAs on their target promoters, the correlation of eRNAs with mRNA levels and the bi-directionality of eRNA transcription 25 26 . Regardless of their function, eRNAs and presence of Pol II are useful in the identification of active enhancers, in addition to H3K27ac.

In summary, enhancers are epigenetically distinguishable from other DNA elements and undergo activation through chemical modification of specific histone residues, typically acetylation, catalyzed by acetyltransferases such as p300. Recruitment of chromatin remodelers that reposition nucleosomes, through binding either to acetyl-lysine groups or to pioneer factors that bind nucleosomal DNA, enables sequence-specific binding of TFs to DNA (Figure 1b).

Most enhancer features were initially described at developmental loci. One important reason for this identification bias is the dynamic nature of development. Indeed, comparisons between distinct developmental stages might reveal novel features not identifiable in a more static differentiated cell lineage. Later studies might also find some of these features in non-developmental enhancers, but it is possible that they are more frequent among developmental ones, given the complexity and variability of developmental processes.

One possible distinction of developmental enhancers is their enrichment in evolutionarily conserved sequences. Because of the functional importance of cis-regulatory elements in general, a significant proportion of enhancer sequences are evolutionarily conserved 27 . However, the conservation of developmental enhancers seems to be even more pronounced. This is alluded to by studies that found a biased association of transcription factors/developmental genes with both higher densities of conserved sequences 28 and the presence of sequences harboring particularly deep conservation 29 30 . More studies are needed to directly compare conservation levels of developmental and other enhancers to fully clarify this issue.

Another feature that might be particular to developmental enhancers is functional redundancy to ensure accurate expression. Shadow enhancers are regulatory elements that drive similar expression patterns to their primary enhancers 31 but together drive more faithful expression, especially under suboptimal conditions 32 . Although shadow enhancers were identified in Drosophila, they may not be exclusive to invertebrates, as redundant enhancers have also been observed in mammals 33 34 . However, it remains to be established whether non-developmental genes also rely on shadow enhancers.

Given the importance of regulatory elements during development, the misregulation of these sequences is likely to carry phenotypic consequences. Similar to protein-coding mutations, variation in enhancer elements has been previously attributed to several Mendelian disorders (reviewed in 2 6 ). However, the functional impact of mutations in cis-regulatory elements can differ significantly from that of protein-coding mutations, even if both are connected to the same gene. Mutations in enhancers are largely limited to cis effects on transcription, whereas those within protein-coding sequences can alter broader aspects of gene regulation, such as mRNA processing and stability, translation initiation and elongation or even protein structure and folding 49 . In addition, as cis-regulatory elements are modular and can act independently to regulate their target genes, disruptions to the regulatory elements are restricted to a spatial and temporal subset of the global function of the gene, and they are therefore predicted to result in a less detrimental effect than coding mutations, with which pleiotropic effects could be more prevalent 50 51 .

Aside from these constraints, cis-regulatory mutations have the potential to generate a plethora of transcriptional alterations through both loss- and gain-of-function effects, leading to a gradient of phenotypic severities. A clear illustration of this is seen in the dysregulation of SHH expression and limb malformations. SHH expression in a region of limb buds known as the zone of polarizing activity (ZPA) is necessary for limb patterning 2 . This expression pattern is governed by a long-range enhancer element about 1 megabase from SHH, known as the ZPA regulatory sequence (ZRS). Point mutations within this element have been linked to a congenital disease leading to extra digits known as preaxial polydactyly 1 , whereas deletion of the entire ZRS in mice led to a truncation of limbs 52 .

Importantly, these phenotypic hallmarks are not exclusive to enhancer elements but encompass a broader range of regulatory sequences, as is highlighted by a mutation at the α-globin locus in the Melanesian population 53 . The regulatory mutation identified by De Gobbi et al. 53 produced a novel GATA1 binding site, leading to the formation of a promoter-like element within the locus that induced a decrease in expression of downstream α-globin genes, leading to α-thalassemia.

Enhancers are specified and activated through complex mechanisms involving epigenetic modifications and TF binding. By acting as independent gene switches that respond to different cues, enhancers regulate complex expression patterns. Their disruption may cause perturbations in gene expression, leading to disease. Although their role in Mendelian disorders has been previously established, recent GWAS functional analyses have extended their contributions as the underlying cause of several common diseases.

Although a number of studies have greatly expanded our knowledge on the role of enhancers in development, we are only beginning to understand genome-wide aspects of enhancer function. Unraveling developmental programs on a genome scale will require not only mapping enhancers, but also elucidating the TFs that regulate them. Efforts using ChIP to map binding sites of known TFs in a few cell lineages have been carried out, but more studies will be necessary. Current large-scale projects such as ENCODE will greatly augment the field in this respect 90 . Comparisons of these maps across developmental stages will deliver a full account of how transcriptional regulation during development unrolls.

Given that ChIP is limited to known TFs, it will be necessary to couple proteomics techniques with ChIP to identify partnering TFs ab initio in specific contexts 93 . Such studies will expand the number of known pioneer factors and TFs, revealing as-yet unknown regulatory networks. In addition, studies analyzing the phenotypic impact of TF knockouts will be needed to functionally characterize new regulators.

As hinted by the discovery that enhancers can be active or poised, it is likely that enhancers are less mechanistically and functionally homogeneous than we think, and identifying and understanding their different subgroups will be required for a full comprehension of the role of enhancers in gene regulation.

Despite a trend for finding regulatory variation in common disease susceptibilities, additional regulatory mechanisms besides direct sequence variation may be important. As epigenetic states are crucial to cis-regulatory function, the misregulation of histone modifications or DNA methylation at an enhancer element could also lead to disease, even if there is no genetic mutation within the element per se. This could result in alterations of enhancer accessibility that induce fluctuations in gene expression, similar to the effects of direct sequence variants. In this case, indirect genetic mutations in trans-factors or even environmental perturbations could have a role. For example, poor maternal nutrition during gestation in rodents led to epigenetic misregulation at a Hnf4a enhancer element in offspring, generating perturbations in promoter-enhancer communication and lowered Hnf4a transcriptional output in pancreatic islets 94 . These environmentally mediated transgenerational epigenetic changes have also been demonstrated for hippocampal expression of the glucocorticoid receptor gene (Nr3c1), which is involved in stress response 95 . The duration of maternal care affected the degree of DNA methylation, histone acetylation and binding of the nerve growth factor inducible TF NGFI-A at the Nr3c1 promoter in rodent offspring. Indeed, similar epigenetic modifications have been identified and correlated with childhood abuse at the orthologous promoter in humans 96 . Although these studies have suggested a novel level of regulatory control carrying phenotypic consequences, a more systematic and agnostic strategy is necessary to address the prevalence of such mechanisms on a broader, genome-wide scale. Proposed epigenome-wide association studies may provide much needed information on such regulatory alterations 61 .

Although GWASs have been successful in mapping disease-associated regions, the collective genetic effects of these loci contribute only a minority to the heritability of common disease risk, warranting the use of different strategies to identify additional disease variants 97 . Current next-generation sequencing efforts may define rarer, more deleterious cis-regulatory mutations, and analyses of structural variation could further uncover genetic disruptions spanning regulatory elements leading to common disease susceptibility.

A combination of these efforts will produce a clearer picture of the role of cis-regulatory elements in development as well as their contributions to common disease risk.

The authors declare that they have no competing interests.