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 s