Transcription imparts architecture, function and logic to enhancer units

Abstract

Distal enhancers play pivotal roles in development and disease yet remain one of the least understood regulatory elements. We used massively parallel reporter assays to perform functional comparisons of two leading enhancer models and find that gene-distal transcription start sites (TSSs) are robust predictors of active enhancers with higher resolution than histone modifications. We show active enhancer units are precisely delineated by active TSSs, validate that these boundaries are sufficient for capturing enhancer function, and confirm that core promoter sequences are necessary for this activity. We assay adjacent enhancers and find that their joint activity is often driven by the stronger unit within the cluster. Finally, we validate these results through functional dissection of a distal enhancer cluster using CRISPR-Cas9 deletions. In summary, definition of high-resolution enhancer boundaries enables deconvolution of complex regulatory loci into modular units.

Massively parallel discovery of human-specific substitutions that alter enhancer activity

“…The composition of the MPRA plasmid might also have effects on the observed MPRA activity. It has been proposed that promoter choice ( 41 ) and the presence of additional sequence motifs influencing transcription initiation and pausing ( 42 ) can affect MPRA activity. Enhancer fragments that are inactive in our assay might be active if their native target promoter was used.…”

Multiple modes of regulation control dynamic transcription patterns during the mitosis-G1 transition

“…PRO-seq data allows for the identification of enhancers based on a bidirectional transcription signature similar to that of promoters (Core, Martins, et al, 2014;Danko, Hyland, et al, 2015;Methods). The production of nascent RNA at enhancers is a proxy for enhancer activity (Henriques, Scruggs, et al, 2018;Mikhaylichenko, Bondarenko, et al, 2018;Tippens, Liang, et al, 2020) and therefore allows us to directly compare gene and enhancer kinetics following mitosis. Overall, we find enhancer reactivation appears to be delayed relative to gene bodies (Figure 5A).…”

Systematic comparison of experimental assays and analytical pipelines for identification of active enhancers genome-wide

“… Abstract Mounting evidence supports the idea that transcriptional patterns serve as more specific identifiers of active enhancers than histone marks 1,2 ; however, the optimal strategy to identify active enhancers both experimentally and computationally has not been determined. In this study, we compared 13 genome-wide RNA sequencing assays in K562 cells and showed that the nuclear run-on followed by cap-selection assay (namely, GRO/PRO-cap) has significant advantages in eRNA detection and active enhancer identification.…”

“…However, studies also revealed that enhancers could themselves produce relatively short-lived divergent transcripts, called enhancer RNAs (eRNAs) 5,6 . More recent studies further showed that distal divergent transcription events are more reliable marks for active enhancers than epigenomic signatures 1,2 . Recently we have proposed 7,8 and later experimentally verified 2 the basic unit of active enhancers that are defined by the transcription start sites (TSSs) of the divergent eRNA transcription, and delimited by the promoter-proximal Pol II pause sites flanking these TSSs.…”

“…More recent studies further showed that distal divergent transcription events are more reliable marks for active enhancers than epigenomic signatures 1,2 . Recently we have proposed 7,8 and later experimentally verified 2 the basic unit of active enhancers that are defined by the transcription start sites (TSSs) of the divergent eRNA transcription, and delimited by the promoter-proximal Pol II pause sites flanking these TSSs. Therefore, to identify active enhancers genome-wide in any given sample, it is critical to detect eRNAs and their TSSs with high sensitivity and specificity.…”