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Transcriptional enhancers: from properties to genome-wide predictions

Key Points

  • The development of all organisms relies on differential gene expression, which is controlled by genomic regions called enhancers or cis-regulatory modules (CRMs). Recent studies highlight the importance of enhancers in evolution and disease; however, our understanding of their properties and functions remains incomplete.

  • Enhancers contain short DNA sequences, which are binding sites for transcription factors. In turn, transcription factors recruit cofactors, which modify the nearby chromatin and lead to transcriptional activation.

  • The location of putative enhancers can be predicted genome wide by assessing either the binding of transcription factors and cofactors or post-translational histone modifications by chromatin immunoprecipitation followed by deep sequencing (ChIP–seq). 'Open' chromatin with accessible DNA can be detected by DNase I hypersensitive site sequencing (DNase-seq), micrococcal nuclease sequencing (MNase-seq), formaldehyde-assisted isolation of regulatory elements followed by deep sequencing (FAIRE–seq) or assay for transposase-accessible chromatin using sequencing (ATAC-seq).

  • Distal enhancers can activate target gene expression by looping to promoters. Such spatial contacts can be detected by chromosome conformation capture (3C) assays and its variants circular chromosome conformation capture (4C), chromosome conformation capture carbon copy (5C) and Hi-C methods or by chromatin interaction analysis with paired-end tag sequencing (ChIA–PET, which is a combination of ChIP and various 3C-based methods).

  • The genome-wide prediction of enhancers based on characteristic chromatin features is powerful, but such results have to be interpreted with caution because none of the known features is perfectly predictive.

  • Enhancer activities of candidate sequences can be measured directly in a developmental context using image-based readouts or enhancer-FACS-seq. High-throughput parallel enhancer assays use either ectopic reporters to test thousands of candidates (which are based on DNA barcodes) or genome-wide screens (such as self-transcribing active regulatory region sequencing (STARR-seq)).

  • Our understanding of enhancer biology will be further accelerated by advances in genome editing methods (such as transcription activator-like effector nucleases (TALENs) and the clustered regularly interspaced short palindromic repeat (CRISPR)–Cas9 system), as well as by the development or improvements of methods to assess gene expression, chromatin state and structure in entire genomes and from increasingly few cells (such as thousands of reporters integrated in parallel (TRIP), single-cell RNA sequencing or ChIP–seq, and high-resolution Hi-C).

Abstract

Cellular development, morphology and function are governed by precise patterns of gene expression. These are established by the coordinated action of genomic regulatory elements known as enhancers or cis-regulatory modules. More than 30 years after the initial discovery of enhancers, many of their properties have been elucidated; however, despite major efforts, we only have an incomplete picture of enhancers in animal genomes. In this Review, we discuss how properties of enhancer sequences and chromatin are used to predict enhancers in genome-wide studies. We also cover recently developed high-throughput methods that allow the direct testing and identification of enhancers on the basis of their activity. Finally, we discuss recent technological advances and current challenges in the field of regulatory genomics.

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Figure 1: Enhancers and their features.
Figure 2: Chromatin accessibility and histone marks at regulatory elements.
Figure 3: Genomic methods for predicting enhancers through the detection of transcription factor binding, 'open' chromatin, chromatin marks, or long-range contacts.
Figure 4: Novel approaches to study and manipulate endogenous regulatory activities.

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Acknowledgements

The authors thank O. Bell (Institute of Molecular Biotechnology (IMBA), Vienna, Austria) and members of A.S's group (Research Institute of Molecular Pathology (IMP), Vienna, Austria) for discussions. We apologize to all colleagues whose work could not be discussed or referenced owing to space limitations. D.S. is supported by a European Research Council (ERC) Starting grant (no. 242922) awarded to A.S., and A.S.'s group by the Austrian Science Fund (FWF): F4303-B09. Basic research at the IMP is supported by Boehringer Ingelheim GmbH.

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Glossary

Transcription factor binding motif

(Also known as transcription factor sequence motif, transcription factor motif and transcription factor binding site motif.) A degenerate short (6–10-bp) DNA sequence pattern that summarizes the DNA sequence binding preference of a transcription factor. These motifs are usually represented either as consensus sequences in IUPAC code or by position weight matrices.

Transcription factor motif matches

(Also known as transcription factor motif instances, transcription factor motif occurrences and transcription factor binding sequences). Specific genomic sequences or positions that match transcription factor binding motifs and thus constitute potential transcription factor binding sites. These are also sometimes called transcription factor binding sites, although we prefer to reserve this term for experimentally determined ones.

Position weight matrices

(Also known as position-specific weight matrices or position-specific scoring matrices). Matrices that provide the frequencies at which individual nucleotides are found at the positions of the transcription factor binding motif.

Transcription factor binding sites

Genomic locations of transcription factor binding, typically in vivo. These sites can be determined experimentally (for example, using chromatin immunoprecipitation (ChIP)). ChIP experiments typically reveal that these binding sites and transcription factor motif matches often, but not always, coincide.

Insulator

A chromatin element that acts as a barrier against the influence of positive signals (from enhancers) or negative signals (from silencers and heterochromatin).

Silencers

DNA sequences that cause reduced expression of their target gene (or genes).

Global run-on sequencing

(GRO-seq). A genome-wide method that maps the position and amount of transcriptionally engaged RNA polymerase II.

Chromatin interaction analysis with paired-end tag sequencing

(ChIA–PET). A high-throughput method based on a combination of chromatin immunoprecipitation (ChIP) and chromatin proximity ligation assays to predict long-range chromatin interactions that are mediated by either RNA polymerase II or transcription factors.

Barcodes

Short and typically artificially designed DNA sequences that are used to uniquely identify DNA constructs (for example, those expressing short hairpin RNAs or reporter genes) or cell lines (for example, yeast knockouts). The uniqueness of the barcodes allows screening or testing in parallel using pooling.

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Shlyueva, D., Stampfel, G. & Stark, A. Transcriptional enhancers: from properties to genome-wide predictions. Nat Rev Genet 15, 272–286 (2014). https://doi.org/10.1038/nrg3682

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