Enhancer RNAs (eRNAs) represent a class of relatively long non-coding RNA molecules (50–2000 nucleotides) transcribed from the DNA sequence of enhancer regions. They were first detected in 2010 through the use of genome-wide techniques such as RNA-seq and ChIP-seq. eRNAs can be subdivided into two main classes: 1D eRNAs and 2D eRNAs, which differ primarily in terms of their size, polyadenylation state, and transcriptional directionality. The expression of a given eRNA correlates with the activity of its corresponding enhancer in target genes. Increasing evidence suggests that eRNAs actively play a role in transcriptional regulation in cis and in trans, and while their mechanisms of action remain unclear, a few models have been proposed.

Enhancers as sites of extragenic transcription were initially discovered in genome-wide studies that identified enhancers as common regions of RNA polymerase II (RNA pol II) binding and non-coding RNA transcription. The level of RNA pol II–enhancer interaction and RNA transcript formation were found to be highly variable among these initial studies. Using explicit chromatin signature peaks, a significant proportion (~70%) of extragenic RNA Pol II transcription start sites were found to overlap enhancer sites in murine macrophages. Out of 12,000 neuronal enhancers in the mouse genome, almost 25% of the sites were found to bind RNA Pol II and generate transcripts. In parallel studies, 4,588 high confidence extragenic RNA Pol II binding sites were identified in murine macrophages stimulated with the inflammatory mediater lipopolysaccharide to induce transcription. These eRNAs, unlike messenger RNAs (mRNAs), lacked modification by polyadenylation, were generally short and non-coding, and were bidirectionally transcribed. Later studies revealed the transcription of another type of eRNAs, generated through unidirectional transcription, that were longer and contained a poly A tail. Furthermore, eRNA levels were correlated with mRNA levels of nearby genes, suggesting the potential regulatory and functional role of these non-coding enhancer RNA molecules.

eRNAs are transcribed from DNA sequences upstream and downstream of extragenic enhancer regions. Previously, several model enhancers have demonstrated the capability to directly recruit RNA Pol II and general transcription factors and form the pre-initiation complex (PIC) prior to the transcription start site at the promoter of genes. In certain cell types, activated enhancers have demonstrated the ability to both recruit RNA Pol II and also provide a template for active transcription of their local sequences.

Depending on the directionality of transcription, enhancer regions generate two different types of non-coding transcripts, 1D-eRNAs and 2D-eRNAs. The nature of the pre-initiation complex and specific transcription factors recruited to the enhancer may control the type of eRNAs generated. After transcription, the majority of eRNAs remain in the nucleus. In general, eRNAs are very unstable and actively degraded by the nuclear exosome. Not all enhancers are transcribed, with non-transcribed enhancers greatly outnumbering the transcribed ones in the order of magnitude of dozens of thousands in every given cell type.

In most cases, unidirectional transcription of enhancer regions generates long (>4kb) and polyadenylated eRNAs. Enhancers that generate polyA+ eRNAs have a lower H3K4me1/me3 ratio in their chromatin signature than 2D-eRNAs. PolyA+ eRNAs are distinct from long multiexonic poly transcripts (meRNAs) that are generated by transcription initiation at intragenic enhancers. These long non-coding RNAs, which accurately reflect the host gene's structure except for the alternative first exon, display poor coding potential. As a result, polyA+ 1D-eRNAs may represent a mixed group of true enhancer-templated RNAs and multiexonic RNAs.

Bidirectional transcription at enhancer sites generates comparatively shorter (0.5-2kb) and non-polyadenylated eRNAs. Enhancers that generate polyA- eRNAs have a chromatin signature with a higher H3K4me1/me3 ratio than 1D-eRNAs. In general, enhancer transcription and production of bidirectional eRNAs demonstrate a strong correlation of enhancer activity on gene transcription.

Arner et al. identified 65,423 transcribed enhancers (producing eRNA) among 33 different cell types under different conditions and different timings of stimulation. The transcription of enhancers generally preceded transcription of transcription factors which, in turn, generally preceded messenger RNA (mRNA) transcription of genes.

Carullo et al. examined one particular cell type, neurons (from primary neuron cultures). They exhibited 28,492 putative enhancers generating eRNAs. These eRNAs were often transcribed from both strands of the enhancer DNA in opposite directions. Carullo et al. used these cultured neurons to examine the timing of specific enhancer eRNAs compared to the mRNAs of their target genes. The cultured neurons were activated and RNA was isolated from those neurons at 0, 3.75, 5, 7.5, 15, 30, and 60 minutes after activation. In these experimental conditions, they found that 2 of the 5 enhancers of the immediate early gene (IEG) FOS, that is FOS enhancer 1 and FOS enhancer 3, became activated and initiated transcription of their eRNAs (eRNA1 and eRNA3). FOS eRNA1 and eRNA3 were significantly up-regulated within 7.5 minutes, whereas FOS mRNA was only upregulated 15 minutes after stimulation. Similar patterns occurred at IEGs FOSb and NR4A1, indicating that for many IEGs, eRNA induction precedes mRNA induction in response to neuronal activation.