Assessing sufficiency and necessity of enhancer activities for gene expression and the mechanisms of transcription activation

Abstract

Enhancers are important genomic regulatory elements directing cell type-specific transcription. They assume a key role during development and disease, and their identification and functional characterization have long been the focus of scientific interest. The advent of next-generation sequencing and clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9-based genome editing has revolutionized the means by which we study enhancer biology. In this review, we cover recent developments in the prediction of enhancers based on chromatin characteristics and their identification by functional reporter assays and endogenous DNA perturbations. We discuss that the two latter approaches provide different and complementary insights, especially in assessing enhancer sufficiency and necessity for transcription activation. Furthermore, we discuss recent insights into mechanistic aspects of enhancer function, including findings about cofactor requirements and the role of post-translational histone modifications such as monomethylation of histone H3 Lys4 (H3K4me1). Finally, we survey how these approaches advance our understanding of transcription regulation with respect to promoter specificity and transcriptional bursting and provide an outlook covering open questions and promising developments.

Keywords: enhancer prediction; enhancers; gene expression; genetic screens; reporter assays; transcription regulation.

Figure 1.

Overview: enhancers and (core) promoters. (A) Gene (black bars) transcription starts at the TSSs (straight arrow) within core promoter elements (light brown). Enhancers (blue boxes) are cis-regulatory DNA sequences that activate expression of their target genes and are often found in introns or distal intergenic regions both upstream and downstream. (B) Nucleosomes (black circles) bind to DNA and decrease accessibility to other proteins, such as TFs (colored rods). Enhancers contain TF-binding motifs (colored boxes), sequences specifically recognized by TFs and to which TFs bind in competition with nucleosomes. Enhancer-bound TFs recruit transcriptional cofactors (colored polygons) and activate gene expression from a distal gene. Cofactors often have catalytic activity and post-translationally modify TFs, histones, and other proteins in the vicinity of enhancers and promoters (small colored circles indicate such modifications).

Figure 2.

Contributions of cofactors and histone tail modifications to enhancer activity. (A) The methyltransferases Mll3/4/Trr are required for enhancer activity and transcription, but their methyltransferase activity is not (Dorighi et al. 2017; Rickels et al. 2017). (Top) An active enhancer with methyltransferase and H3K4me1-marked flanking histones activates gene expression. (Middle) Mutation of the catalytic domain results in the loss of H3K4me1 but maintains enhancer activity and gene expression. (Bottom) Knockout of the methyltransferase leads to the loss of gene expression. (B) Enzymatic targets of acetyltransferases P300/CBP, which have been reported to acetylate many proteins, including TFs, cofactors, histones, and members of the PIC, including Pol II (for references, see the text). (C) H3K27ac may have an indirect role in preventing PRC2-mediated silencing. (Top) PRC2 (brown) catalyzes H3K27me3, which is blocked by H3K27ac, preventing PRC2-mediated silencing. (Bottom) Mutations of H3K27 to methionine (M; as observed frequently in pediatric gliomas) or arginine (R) prevent both acetylation and methylation. Both mutations induce changes in gene expression that mimic PRC2 loss-of-function H3K27M in a dominant fashion, as indicated by the dashed cross (for references, see the text).

Figure 3.

Ectopic reporter assays measure enhancer activities quantitatively. (A) Ectopic assays remove candidate sequences from their endogenous loci and test them in a heterologous setup using reporter genes (green) such as β-galactosidase (lacZ) or luciferase. Such reporter constructs can be used with nonintegrating (episomal) plasmids or can be integrated into the genome. (B) High-throughput reporter assays vastly increase the number of candidates per experiment by replacing the reporter gene with either a barcode or the enhancer itself. MPRAs uniquely assign each candidate to a barcode and define enhancer activities by quantifying the barcode-containing transcripts. STARR-seq uses each candidate as its own barcode, which simplifies library cloning and increases throughput. Both types of assays typically include ORFs (green) to stabilize the reporter mRNA.

Figure 4.

Clustered regularly interspaced short palindromic repeat (CRISPR)–Cas9-based approaches to assess endogenous enhancer activities. (A) Endogenous enhancer activities are typically assessed by genetic perturbation and assays that detect loss of enhancer function. Transcription of an endogenous target gene is driven by the enhancer (blue) and is lost upon enhancer mutation or deletion by Cas9 and guide RNAs (gRNAs). Deletions can be repaired through homologous recombination to insert exogenous sequences (purple), allowing essentially arbitrary manipulations such as the exchange of enhancers with homologous sequences from other species (e.g., Kvon et al. 2016). (B) Endogenous high-throughput screens rely on cell selection to enrich for gRNAs that perturb enhancer activities. In a typical screen, a pool of gRNAs is transfected into cells, which introduces mutations or deletions in candidate regions. gRNAs that target active enhancers (blue) disrupt target gene expression and can be enriched by selecting for a cellular phenotype (e.g., increased proliferation [left] or reporter gene expression [right]). The gRNAs enriched in the selected cells can identify the enhancers targeted (for references, see the text).

Figure 5.

Ectopic enhancer activity assays and genetic perturbations of endogenous enhancers are complementary, and the outcomes need to be interpreted with care. Each row represents a different scenario in which the candidate (blue) is an active cellular enhancer or not (ground truth; left columns). The right columns indicate the respective outcomes of ectopic enhancer activity assays and genetic perturbations. (Green checkmark) Enhancer activity detected; (red cross) no enhancer activity detected; (yellow checkmark) outcome depends on degree of redundancy. See the text for details.