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

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.

Resolving systematic errors in widely used enhancer activity assays in human cells.

The identification of transcriptional enhancers in the human genome is a prime goal in biology. Enhancers are typically predicted via chromatin marks, yet their function is primarily assessed with plasmid-based reporter assays. Here, we show that such assays are rendered unreliable by two previously reported phenomena relating to plasmid transfection into human cells: (i) the bacterial plasmid origin of replication (ORI) functions as a conflicting core promoter and (ii) a type I interferon (IFN-I) response is activated. These cause confounding false positives and negatives in luciferase assays and STARR-seq screens. We overcome both problems by employing the ORI as core promoter and by inhibiting two IFN-I-inducing kinases, enabling genome-wide STARR-seq screens in human cells. In HeLa-S3 cells, we uncover strong enhancers, IFN-I-induced enhancers, and enhancers endogenously silenced at the chromatin level. Our findings apply to all episomal enhancer activity assays in mammalian cells and are key to the characterization of human enhancers.

Promoting transcription over long distances.

Promoters and enhancers have long been regarded as distinct elements, a notion that has been challenged more recently. Two new studies now identify promoters that function as long-range enhancers in vivo to regulate the transcription of distal genes.