Lessons from eRNAs: understanding transcriptional regulation through the lens of nascent RNAs

Abstract

Nascent transcription assays, such as global run-on sequencing (GRO-seq) and precision run-on sequencing (PRO-seq), have uncovered a myriad of unstable RNAs being actively produced from numerous sites genome-wide. These transcripts provide a more complete and immediate picture of the impact of regulatory events. Transcription factors recruit RNA polymerase II, effectively initiating the process of transcription; repressors inhibit polymerase recruitment. Efficiency of recruitment is dictated by sequence elements in and around the RNA polymerase loading zone. A combination of sequence elements and RNA binding proteins subsequently influence the ultimate stability of the resulting transcript. Some of these transcripts are capable of providing feedback on the process, influencing subsequent transcription. By monitoring RNA polymerase activity, nascent assays provide insights into every step of the regulated process of transcription.

Transcription factor enrichment analysis (TFEA) quantifies the activity of multiple transcription factors from a single experiment

“…However, most sites of initiation occur in regions of bidirectional transcription with two closely, oppositely oriented TSSs 24 , 38 , 39 . Many assays are unable to distinguish between the two TSSs within an RNA polymerase loading zone 25 (also see Methods section “Regions of interest”). Therefore, without loss of generality, we assume each assay provides a set of regions of interest (ROI) where each region corresponds to either a single TSS or the midpoint between bidirectional TSSs.…”

“… a Analysis of lipopolysaccharide (LPS) time-series cap analysis gene expression (CAGE) data 10 , 25 using AME and TFEA. Trajectories of activity profiles show LPS triggers immediate activation of the NF- κ β complex (TF65/RelB/NFKB1; yellow), observable at 15 min (blue arrow).…”

“…The majority of altered RNA polymerase initiation sites are at transcription regulatory regions (e.g., enhancers) -not at genes 24 . Thus, by using all polymerase initiation sites (both at enhancers and genes) rather than just the target gene, the assignment problem is sidestepped 25 . Enhancer RNAs (eRNAs) are highly transient unstable transcripts that are essentially undetectable in RNA-seq, yet effectively serve as markers of TF activity.…”

Protocol Variations in Run-On Transcription Dataset Preparation Produce Detectable Signatures in Sequencing Libraries

“…The second common use of run-on sequencing data is to infer which regulators are driving observed patterns of differential transcription [25,29,5]. Alterations in transcription factor activity can be detected by changes in the locations and levels of sites of bidirectional transcription [5,6], the majority of which reside at enhancers [28]. Therefore we next sought to determine whether the alterations observed in eRNA detection (Fig.…”

Genome-wide binding analysis of 195 DNA Binding Proteins reveals “reservoir” promoters and human specific SVA-repeat family regulation

“…Since reservoirs don't have mature transcriptional products despite many promoter binding events, we next examined if reservoirs have "nascent" transcription detected via PRO-seq (reviewed ). These approaches are so precise they can identify specific DBP binding sites through PRO-seq nascent RNA read out [37,38]. Thus, we hypothesized that reservoir promoters would exhibit nascent transcription owing to so many DNA binding events.…”