Towards a comprehensive catalogue of validated and target-linked human enhancers

Abstract

The human gene catalogue is essentially complete, but we lack an equivalently vetted inventory of bona fide human enhancers. Hundreds of thousands of candidate enhancers have been nominated via biochemical annotations; however, only a handful of these have been validated and confidently linked to their target genes. Here we review emerging technologies for discovering, characterizing and validating human enhancers at scale. We furthermore propose a new framework for operationally defining enhancers that accommodates the heterogeneous and complementary results that are emerging from reporter assays, biochemical measurements and CRISPR screens.

Figure 1.. Approaches for identifying, validating and characterizing enhancers.

a ∣ Biochemical annotations of candidate enhancers. a schematic depiction of an enhancer and target gene marked with the biochemical annotations used to nominate candidate enhancers and other features of non-coding DNA. Although the enhancer has been depicted in 3D proximity to its target promoter, we note that the mechanistic importance of such enhancer–promoter proximity is far from settled. We refer the reader to the section “Emerging approaches for biochemical annotation: 3D conformation mapping” for a discussion of open questions of enhancer–promoter communication and the importance of chromatin looping. b ∣ Episomal reporter assay: a candidate enhancer and reporter gene located in cis on an episomal vector. The candidate enhancer may increase expression of the reporter gene by recruiting transcriptional machinery. The degree of enhancer-mediated activation is measured by the abundance of reporter transcripts or the quantity of the reporter-encoded protein. c ∣ Massively parallel reporter assays (MPRAs): many candidate enhancers can be interrogated simultaneously in a reporter assay if a barcode is encoded in the reporter transcript. The relative abundance of barcodes can be used to estimate the relative activities of the candidate enhancers to which they are linked. We show here just one of many formats of MPRAs that have been developed. 3C, chromosome conformation capture; 4C, chromosome conformation capture-on-chip; ATAC-seq, assay for transposase-accessible chromatin using sequencing; ChIP-seq, chromatin immunoprecipitation followed-by sequencing; DNase-seq, DNaseI hypersensitivity sequencing; MNase-seq, micrococcal nuclease digestion combined with sequencing; PRO-seq, precision run-on sequencing; POL, RNA polymerase; TF, transcription factor. Part a is adapted from REF.

Figure 2.. CRISPR-based approaches for perturbing enhancers.

The CRISPR system has been repurposed with four main perturbation methods that can disrupt enhancer activity a ∣ Single-cut small sequence insertion or deletion (indel). An active CRISPR nuclease such as Cas9 is directed to make a single-cut that by inaccurate repair will usually create a small indel <10 bp. This indel can sometimes disrupt an enhancer’s function if, for example, it overlaps a key transcription factor (TF) binding site. b ∣ Dual-cut long sequence deletions. To guarantee that a perturbation disrupts the enhancer’s functional sequence, the entire enhancer can be deleted by directing two flanking cuts to either side. In some cells, due to inaccurate repair, deletions may occur between the two cuts. However, this is inefficient and will only be one of several possible repair outcomes, which must be accounted for in experimental design. c ∣ CRISPRi-based epigenetic repression. The nuclease domain of the CRISPR enzyme is rendered inactive (‘dead’, such as dCas9) but is tethered to a repressive domain (e.g. KRAB) which is known to disrupt enhancer activity and expression. d ∣ CRISPRa-based epigenetic activation. A dead-CRISPR enzyme is tethered to an activating domain (e.g. a fusion of VP64, p65, and rtTA) that can potentially induce activation of a target gene when targeted to a primed enhancer. POL, RNA polymerase.

Figure 3.. CRISPR-based screens of enhancer–gene links.

In all such screens, guide RNA (gRNA)-based perturbations are designed to candidate enhancers and delivered to mammalian cells as a pool. a ∣ In most screens, cells are separated by expression of a single or few genes, and perturbations are tested for enrichment in high or low expression bins. b ∣ In ‘whole-transcriptome’ screens, single-cell RNA sequencing (RNA-seq) is used to evaluate the expression of any gene against each perturbation. c ∣ The future of such screens would benefit from higher standards (and better methods) to validate screen results (e.g. by deletion of individual elements), investigating why all such screens have had a low ‘hit rate’ thus far, and comparison of results with massively parallel reporter assay (MPRA) readouts of activity.

Figure 4.

A tiered framework to describe the level of support for the enhancer candidacy of a non-coding sequence. We propose ‘validated and target-linked’ support as the degree of evidence that we should be aiming for in cataloguing non-coding sequences as bona fide human enhancers. If the evidence falls short of that, as it currently does for nearly all candidate enhancers, we propose strong, moderate, and weak tiers to describe candidate enhancers with less or conflicting evidence. The vast majority of candidate human enhancers are presently only weakly supported.

Figure 5:. The blind men and the elephant of human enhancer biology.

Much like blind men inspecting an elephant, operational definitions of enhancers are merely a means to characterize the underlying biological phenomenon, but fall short of the phenomenon itself. As we work to develop a catalogue of bona fide biological enhancers, an updated operational definition that accommodates the heterogeneous and complementary results that are emerging from reporter assays, biochemical measurements, and CRISPR screens is likely to be necessary. In our view, the catalogue can and should aim for knowledge of the cell-type specificity of each element, strong and multifaceted support for each element's role as a bona fide enhancer, and knowledge of each element’s target gene(s).