Table 1.

Studies discussed in this review that have cloned and functionally analyzed eRNAs using molecular, biochemical, and cell-based approaches.

Studies	Potential eRNA Regulatory Mechanisms	Method for eRNA Annotation	Notes
A. eRNAs promote target gene transcription
Melo et al. (2013)	Required for efficient transcriptional enhancement of interacting target genes (detailed mechanisms not described).	Cloned 3 regions around the identified p53 enhancer.	Effects of eRNAs on target gene transcription were assessed using luciferase reporter assays.
Lam et al. (2013)	Required for efficient transcriptional enhancement of interacting target genes (detailed mechanisms not described). The Rev-Erb TF act to repress eRNA transcription.	Cloned a fragment based on 5’-GRO- seq GRO-seq data.	Effects of eRNAs on target gene transcription were assessed using luciferase reporter assays.
B. eRNAs drive enhancer-promoter looping
Li et al. (2013)	Increase the strength of enhancer- promoter looping initiated by ERa binding. Cohesin acts, in part, by stabilizing ERa/eRNA-induced enhancer-promoter looping.	Nascent RNA transcripts derived from GRO-seq data.	Effects of eRNAs on target gene transcription were assessed using luciferase reporter assays. Looping mechanism was tested using an eRNA knockdown approach.
Lai et al. (2013)	Interact with Mediator to regulate its chromatin localization and kinase activity towards histone H3 serine 10.	5’ and 3’ RACE.	eRNAs, called ncRNA-a, were polyadenylated and spliced. Looping mechanism was tested using an eRNA knockdown approach.
Tsai et al. (2018)	Interact with cohesion to promote spatially appropriate cohesin loading in trans to regulate gene expression.	RNA-seq from ribosomal RNA- depleted fraction.	The eRNA tested (DKKeRNA) is polyadenylated and spliced.
Panigrahi et al. (2018)	No function was ascribed to the eRNA.	A portion of GREB1 enhancer transcribed region.	eRNA failed to stimulate transcription in a cell-free assay.
C. eRNAs promote the recruitment of transcription factors and coregulators, and regulate their activities
Sigova et al. (2015)	The YY1 TF binds to both enhancers and their cognate eRNAs across the genome. The eRNA stabilizes YY1 at the enhancers.	Fused 60 nt of the eRNA to a sgRNA and tethered the fusion construct to an a YY1 binding site using CRISPR/ Cas9.	RNA tested was derived from the promoter ofAridla. Reduced enhancer transcription diminished YY1 occupancy, whereas artificial tethering of the eRNA enhanced YY1 occupancy.
Bose et al. (2017)	Stimulate CBP/p300 acetyltransferase activity, resulting in eRNA-dependent changes in histone acetylation mediated by CBP, such as H3K27ac.	Nascent RNA transcripts derived from GRO-seq data.	Effect in cells was tested using a knockdown approach.
Rahnamoun et al. (2018)	eRNAs bind to BRD4 to increase BRD4 binding to acetylated histones, promoting transcriptional coregulatory activity.	Nascent RNA transcript derived from GRO-seq data.	BRD4 selectively associates with eRNAs produced from BRD4-bound enhancers. Bromodomains of BRD2, BRD3, BRDT, BRG1, and BRD7 interact directly with eRNAs.
D. eRNAs facilitate RNA Pol II pause-release to promote transcription elongation
Schaukowitch et al. (2014)	Bind to and promote the release of NELF-E, a negative regulator of RNA Pol II elongation, to facilitate RNA Pol II transition to elongation.	Circularized RACE to determine 5’ and 3’ ends.	eRNAs failed to promote enhancer-promoter looping.