Enhancer Logic and Mechanics in Development and Disease

doi:10.1016/j.tcb.2018.04.003

Trends in Cell Biology

Volume 28, Issue 8, August 2018, Pages 608-630

https://doi.org/10.1016/j.tcb.2018.04.003 Get rights and content

Highlights

Metazoan development requires the orchestration of hundreds of thousands of enhancers to establish precise spatiotemporal gene expression patterns.

Enhancers commonly exist in a ‘suboptimal’ state with respect to their transcription factor binding affinities, and this evolutionary ‘suboptimization’ of both the sequence and binding motif arrangement is key to encoding enhancer tissue-specificity.

Accumulating evidence suggests that enhancers regulate gene transcription by stimulating release of promoter-paused RNA polymerase II into productive elongation.

Bidirectional transcription of enhancer DNA is now appreciated to be a general characteristic of active enhancers, and recent reports document numerous examples of how promoters can function as enhancers to stimulate long-range gene activation. Thus, the distinction between enhancers and promoters is becoming less apparent.

Clusters of cis-regulatory elements appear to be highly interconnected in the nucleus, and these complex regulatory ‘hubs’ are organized into topological domains along the linear chromosome.

Enhancers are distally located genomic cis-regulatory elements that integrate spatiotemporal cues to coordinate gene expression in a tissue-specific manner during metazoan development. Enhancer function depends on a combination of bound transcription factors and cofactors that regulate local chromatin structure, as well as on the topological interactions that are necessary for their activity. Numerous genome-wide studies concur that the vast majority of disease-associated variations occur within non-coding genomic sequences, in other words the ‘cis-regulome’, and this underscores their relevance for human health. Advances in DNA sequencing and genome-editing technologies have dramatically expanded our ability to identify enhancers and investigate their properties in vivo, revealing an extraordinary level of interconnectivity underlying cis-regulatory networks. We discuss here these recently developed methodologies, as well as emerging trends and remaining questions in the field of enhancer biology, and how perturbation of enhancer activities/functions results in enhanceropathies.

Section snippets

Capturing Promoter Accomplices: Enhanced Interrogation of Gene Regulation

Transcriptional enhancers were described nearly 40 years ago when a tandem 72 bp segment of SV40 viral DNA was shown to dramatically boost the expression of a gene, irrespective of its orientation or distance from the gene 1, 2. These properties of augmenting gene expression in a tissue-specific manner became the defining features of enhancers, and these are now appreciated to be crucial for orchestrating proper transcriptional regulation throughout metazoan development. Less than 2% of the

Transcription Factor Gradients in Setting up Embryonic Patterning

Experiments in embryos of Drosophila melanogaster revealed how multiple enhancers can work together to achieve complex expression patterns of a single gene. This concept is exquisitely demonstrated by the archetypical ‘pair-rule’ gene, even-skipped (eve), which is expressed along the length of the embryo in a series of seven alternating stripes (Figure 1A) [3]. While the eve promoter alone is not sufficient to produce this segmental pattern, the surrounding 12 kb region encompassing the eve

Functional Assays

Initial attempts to locate enhancer elements by trial-and-error relied on functional assays to test the ability of a DNA sequence to boost transcription of a reporter gene from a basally active promoter. These types of ‘enhancer bashing’ experiments are low-throughput and not comprehensive because candidate sequences must be manually selected and individually tested. When a sequence is found to have enhancer activity, assigning it to a target gene can be problematic because enhancers are

Chromosome Conformation Capture in Identifying Enhancer–Promoter Interactions

For many of the techniques described above, assigning an enhancer to its target gene can be difficult or unreliable, especially in larger mammalian genomes. Chromosome conformation capture (3C) technologies use DNA proximity ligation to ‘capture’ intrachromosomal interactions, such as enhancer-looping, and map physical chromatin contacts within the nucleus [32]. Briefly, cells are formaldehyde crosslinked, restriction enzyme digested, diluted to promote intramolecular ligation, and amplified by

Regulatory Logic

Enhancers are short, modular, stretches of DNA containing several TF binding sites that impart function through the recruitment of those factors. Enhancers from unrelated genes may respond differently to the presence of a particular factor depending on the number of binding sites available and the affinity of those sites for that TF. For instance, during Drosophila dorsoventral (D/V) patterning, transcription of sog and twist is triggered by a ventral gradient of the transcriptional activator,

Enhancer-Regulated Transcriptional Elongation Control via Pause-Release

Despite decades of research on enhancer function, the precise mechanisms through which enhancers stimulate transcription from their target promoter remain elusive. One can imagine a model in which an enhancer and its target promoter are separately bound by various TFs, and looping of the enhancer to the promoter increases the local concentration of activating factors that are required for Pol II recruitment. While this model is not unreasonable, it conflicts with accumulating evidence that Pol

Dynamic Looping in Enhancer Promoter Communication and Transcriptional Control

As described earlier, the human β-globin LCR directs the transcription of multiple globin genes arranged along the chromosome in the order of their developmental expression. This stepwise activation process was later shown to coincide with loop formation between the LCR and the globin gene expressed at that particular developmental stage [57]. Expression of β-globin in human adult erythrocytes depends on several TFs, including the hematopoietic-specific factors GATA1 and TAL1, as well as the

Life Without H3K4me1 on Enhancers

From flies to humans, H3K4me1 is a highly conserved, but perplexing, feature of enhancer chromatin because its presence does not seem to correlate with enhancer transcriptional status 73, 74. This modification is catalyzed by Trr in Drosophila, and by its mammalian homologs, MLL3/4 (Kmt2c/d), as part of the COMPASS family of H3K4-methylases 75, 76, and work from our laboratory has established that this branch of the COMPASS family is instrumental in facilitating enhancer–promoter communication.

Pioneer Factors and Enhancer Function

Given the degenerate nature of enhancer TF binding sequences, a given genome might contain millions of DNA sequences with the potential to act as enhancers, but only a relatively small subset of those sequences are functionally active in a given cell type. By providing a physical barrier to TF binding, the vast majority of putative cis-regulatory elements remain inaccessible due to nucleosome occupancy; however, a subset of sequence-specific master TFs called ‘pioneer factors’ possess the

Shadow Enhancers

Exactly as clusters of Ubx binding sites within a single enhancer were necessary to drive proper expression of shavenbaby under temperature stress, some genes require seemingly redundant ‘shadow’ enhancers to ensure robust transcription under suboptimal conditions. This was first demonstrated in Drosophila for enhancers targeted by Dorsal, and this was later shown to be a general feature for precise patterning of all Gap genes during embryogenesis, as well as at Hox loci in mammals 34, 88, 89.

Superenhancers (SEs) or Stretch Enhancers

Enhancer Mutation

In the past several years a plethora of genome-wide association studies (GWAS) have established that the vast majority of disease-associated sequence variation occurs within non-coding regulatory DNA, implicating enhancer mutations in a variety of diseases 97, 98, 99, termed enhanceropathies. For example, partial disruption of the human globin gene LCR caused by a DNA translocation was found to be responsible for a large number of β-thalassemias [19]. Similarly, mutations 1 Mb away from Sonic

Concluding Remarks and Future Perspectives

Tremendous progress has been made over the past decade toward deciphering the cis-regulatory logic underlying gene expression in development and disease, but several questions remain unanswered (see Outstanding Questions). As we discussed, the natural tendency for enhancers to adopt suboptimal binding sequences complicates our attempts to predict their locations based on known TF binding motifs alone. Advances in DNA sequencing have allowed headway toward identifying human enhancers; however,

Acknowledgments

The authors would like to thank Drs Edwin Smith, Andrea Piunti, and Marc Morgan for insightful discussions during the preparation of this manuscript. R.R. is supported by a National Cancer Institute (NCI) Predoctoral to Postdoctoral Fellow Transition Award (F99CA222988). Studies in the laboratory of A.S. are supported by NCI Research Project Grant R01CA214035 and the Outstanding Investigator Award R35CA197569.

Glossary

Cis-regulatory element: non-coding DNA sequences that are bound by trans-acting factors to regulate the transcription of one or more genes that are usually located on the same DNA molecule.
CRISPR/Cas9: genome-editing technology used to create DNA double-strand breaks at a genomic region of interest: can create short insertions/deletions or large deletions through the non-homologous end-joining (NHEJ) pathway. Alternatively, the homology-directed repair (HDR) pathway can be exploited to introduce

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Trends in Cell Biology

Feature ReviewEnhancer Logic and Mechanics in Development and Disease

Highlights

Section snippets

Capturing Promoter Accomplices: Enhanced Interrogation of Gene Regulation

Transcription Factor Gradients in Setting up Embryonic Patterning

Functional Assays

Chromosome Conformation Capture in Identifying Enhancer–Promoter Interactions

Regulatory Logic

Enhancer-Regulated Transcriptional Elongation Control via Pause-Release

Dynamic Looping in Enhancer Promoter Communication and Transcriptional Control

Life Without H3K4me1 on Enhancers

Pioneer Factors and Enhancer Function

Shadow Enhancers

Superenhancers (SEs) or Stretch Enhancers

Enhancer Mutation

Concluding Remarks and Future Perspectives

Acknowledgments

Glossary

Cell

Cell

Cell

Cell

Cell

Cell

Cell Rep.

Cell

Curr. Opin. Genet. Dev.

Curr. Top. Dev. Biol.

Cell

Cell

Cell

Mol. Cell

Cell

Mol. Cell

Cell

Cell

Cell

Cell

Cell

Cell

Mol. Cell

Mol. Cell

Mech. Dev.

Cell Rep.

Mol. Cell

Cell

Cell

Cell

Cell

The SV40 72 base repair repeat has a striking effect on gene expression both in SV40 and other chimeric recombinants

Nucleic Acids Res.

Regulation of a segmentation stripe by overlapping activators and repressors in the Drosophila embryo

Science

The eve stripe 2 enhancer employs multiple modes of transcriptional synergy

Development

A self-organizing system of repressor gradients establishes segmental complexity in Drosophila

Nature

Enhancer biology and enhanceropathies

Nat. Struct. Mol. Biol.

Transcriptional activation by recruitment

Nature

Genes regulating the remote wing margin enhancer in the Drosophila cut locus

Genetics

Drosophila nipped-B protein supports sister chromatid cohesion and opposes the stromalin/Scc3 cohesion factor to facilitate long-range activation of the cut gene

Mol. Cell. Biol.

Mediator and cohesin connect gene expression and chromatin architecture

Nature

Genome-wide quantitative enhancer activity maps identified by STARR-seq

Science

Dissection of thousands of cell type-specific enhancers identifies dinucleotide repeat motifs as general enhancer features

Genome Res.

FIREWACh: high-throughput functional detection of transcriptional regulatory modules in mammalian cells

Nat. Methods

Genome-scale functional characterization of Drosophila developmental enhancers in vivo

Nature

Enhancer loops appear stable during development and are associated with paused polymerase

Nature

Beta-globin gene inactivation by DNA translocation in gamma beta-thalassaemia

Nature

Feature Review
Enhancer Logic and Mechanics in Development and Disease