Table 1.

Timeline of enhancer and eRNA discovery and functional characterization

Year	Brief description of discovery	Significance
1958, Pardee, Jacob and Monod [72]	Using genetics models of E. coli, the PaJaMa studies proposed a general mechanism for transcriptional regulation via a repressor system to control protein production of beta-galactosidase. This led to the finding of lac operator, or ‘lac operon’ regulatory element. [73]	This landmark study laid the groundwork for the concept of regulatory elements and the heritability of gene regulation.
1959, Harris H. [13]	Using pulse chase of RNA with labelled nucleotide, a large fraction of nascent RNA was shown to retain in the nucleus. In addition, half-lives of nuclear RNA is significantly shorter compared to cytoplasmic RNA.	Harris deduced that most nuclear RNAs are likely non protein-coding. In a recent Nature Correspondence, Harris connected his 1959 finding with the recent studies of ncRNA [Harris, 2013]. Given the high number of transcribing enhancer units, the comparable frequency of transcription initiation at enhancers, and short eRNA half-lives, this 1959 study is consistent with the observation of pervasive transcription at enhancer elements.
1981, Banerji, J., et al. [1]	Banerji et al showed that a 72 repeat sequence motif from SV40 early gene is sufficient to increase expression of ectopic beta-globin gene by 200 fold. This DNA element is functional over long distance in an orientation independent fashion relative to the beta-globin gene.	Discovery of cis-acting enhancer elements.
1990, Collis et al. [15]	Using nuclear run off assays, Collis et al identified transcription at the LCR of the beta globin region, both in the endogenous locus in MEL cells, as well as a mini-gene construct containing the LCR and beta-globing gene.	One of the first studies demonstrating transcription from enhancer regions.
1992, Tuan et al. [16]	Using RNA protection assays in transfected recombinant plasmids, the authors identified that a non-coding RNA is transcribed from the HS2 enhancer. Only the active enhancer can direct synthesis of enhancer-derived RNA; the same group later showed that disruption of intact HS2-initiated long RNA leads to loss of HS2 enhancer activity and target gene silencing [Ling, 2004].	This study suggested that enhancer ncRNA is only generated from active enhancer and is needed for proper enhancer function.
1997, Ashe et al. [17]	Using nuclear run-on assays and in situ hybridization analysis, the authors found a novel intergenic transcription from the human β-globin locus in K562 but not in HeLa cells. Exogenous expression of the β-globin gene in HeLa cells was sufficient to induce the expression of intergenic ncRNAs from the otherwise silent endogenous chromatin.	Intergenic ncRNA transcript displayed cell line specificity. A ‘trans’ effect is implicated given exogenous plasmid-driven transcription of the globin genes can induce the intergenic ncRNA transcription at the endogenous locus. This mechanism, however, is not well understood.
2000, Gribnau et al. [75]	RNA FISH and DNase I sensitivity assays led to the finding that transcription activity of the human β-globin locus correlates to its sensitivity to DNase I.	Intergenic ncRNA transcription could play a role in maintaining open and active chromatin.
2006, Feng et al. [45]	The ultraconserved region between DLX5/6 was identified to transcribe into Evf-2 ncRNA. This Evf-2 then interacts with DLX2 in vivo to activate the transcription of DLX5 and DLX6 genes.	As ultraconserved regions are frequently enhancers (Pennacchio LA, 2006 Nature), this study implicated that enhancer-derived RNAs can be functionally regulatory.
‘Genomic’ era
2010, Kim et al. [10], 2010 De Santa et al. [26] 2011, Koch et al [Koch, 2011]	Enhancer transcription and transcripts are genome-wide phenomena. Enhancer-templated non-coding RNAs (eRNAs) were usually non-polyadenylated and of lower abundance compared to coding genes. Expression level of eRNAs positively correlated to expression of nearby protein coding genes.	Pervasive genome wide enhancer transcription. The cognate promoter near the enhancer may be required for proper eRNA transcription.
2011, Wang et al. [70] 2011, Hah et al. [22] 2011, Melgar et al. [76] 2013, Hah et al. [35]	Using GRO-seq data, eRNA transcription was found to be induced by stimuli and widely distributed in the genome; their transcription correlated well with the activity of active enhancers.	eRNA transcription serves as a marker of active enhancers. Pharmacological inhibition of eRNA transcription does not inhibit enhancer-promoter looping with 3C.
2010, Orom et al 2013, Lai et al.	From transcripts annotated in GENCODE, the authors identified a cohort of long non-coding RNAs (lncRNAs) that exhibit enhancer-like properties. Unlike eRNAs, this group of ncRNAs, termed enhancer-like lncRNAs or later as ncRNA-activating (ncRNA-a) are spliced, polyadenlyated transcripts expressed from promoter-like regions (i.e. high H3K4me3, low H3K4me1). They activate gene(s) in their vicinity.	The first description of lncRNAs having enhancer-like properties. Later the same group illustrated that ncRNA-a mediated gene regulation via modulation of Mediator complex and chromatin looping [Lai, 2013]. Recent studies of other lncRNA demonstrate novel mechanisms of gene activation, including physical interaction of lncRNA HOTTIP with methyltransferase to drive H3K4 trimethylation and gene expression in the HoxA cluster [KC Wang, 2011]
2013, Melo et al. [38] 2013, Lam et al. [27] 2013, Li et al. [40] 2013, Mousavi et al. [30]	Using siRNA, antisense oligonucleotides and reporter assays, with characterization using qRT-PCR, GRO-seq, and 3D-DSLthese studies found that eRNA transcripts have functional roles in regulating the transcription of neighboring coding genes.	The eRNA transcript per se plays a functional role in regulating gene transcription. A stimulus-induced eRNA transcript is needed for proper enhancer- promoter looping formation and gene activation.
2013, Kaikkonen et al. [31]	Using genomic tools, the authors found that pharmacological inhibition of eRNA transcriptional elongation impaired mono- and dimethylation of histone H3K4 on signal-induced de novo enhancers.	RNA PolII elongation has a role independent of the eRNA transcript in modulating chromatin structure and deposition of enhancer histone marks.