Enhancer RNAs (eRNAs) represent a class of relatively longnon-coding RNA molecules (50–2000 nucleotides) transcribed from the DNA sequence ofenhancer regions. They were first detected in 2010 through the use of genome-wide techniques such asRNA-seq andChIP-seq.^[1][2] 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.^[3] The expression of a given eRNA correlates with the activity of its corresponding enhancer in target genes.^[4] Increasing evidence suggests that eRNAs actively play a role intranscriptional regulation incis and intrans, and while their mechanisms of action remain unclear, a few models have been proposed.^[3]

Enhancers as sites of extragenictranscription were initially discovered in genome-wide studies that identified enhancers as common regions ofRNA polymerase II (RNA pol II) binding andnon-coding RNA transcription.^[1][2] The level of RNA pol II–enhancer interaction and RNA transcript formation were found to be highly variable among these initial studies. Using explicitchromatin signature peaks, a significant proportion (~70%) ofextragenic RNA Pol II transcription start sites were found to overlap enhancer sites inmurinemacrophages.^[5] Out of 12,000neuronal enhancers in themousegenome, almost 25% of the sites were found to bind RNA Pol II and generatetranscripts.^[6] In parallel studies, 4,588 high confidenceextragenic RNA Pol II binding sites were identified in murine macrophages stimulated with the inflammatory mediaterlipopolysaccharide to induce transcription.^[2] These eRNAs, unlike messenger RNAs (mRNAs), lacked modification bypolyadenylation, 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 apoly A tail.^[7] Furthermore, eRNA levels were correlated withmRNA levels of nearbygenes, suggesting the potential regulatory and functional role of these non-coding enhancerRNAmolecules.^[1]

eRNAs are transcribed fromDNA sequencesupstream anddownstream ofextragenicenhancer regions.^[8] Previously, several model enhancers have demonstrated the capability to directly recruitRNA Pol II and generaltranscription factors and form the pre-initiation complex (PIC) prior to thetranscription start site at thepromoter ofgenes. In certaincell types, activated enhancers have demonstrated the ability to both recruit RNA Pol II and also provide a template for activetranscription of their localsequences.^[2][1]

Depending on the directionality of transcription, enhancer regions generate two different types of non-codingtranscripts, 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 thenucleus.^[9] In general, eRNAs are very unstable and actively degraded by the nuclearexosome. 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 givencell type.^[5]

In most cases, unidirectionaltranscription ofenhancer regions generates long (>4kb) and polyadenylated eRNAs. Enhancers that generate polyA+ eRNAs have a lowerH3K4me1/me3 ratio in theirchromatin signature than 2D-eRNAs.^[7] 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 hostgene's structure except for the alternative firstexon, display poor coding potential.^[10] As a result, polyA+ 1D-eRNAs may represent a mixed group of true enhancer-templated RNAs and multiexonic RNAs.

Bidirectionaltranscription atenhancer sites generates comparatively shorter (0.5-2kb) and non-polyadenylated eRNAs. Enhancers that generate polyA- eRNAs have achromatin 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.^[11]

Arner et al.^[12] identified 65,423transcribed enhancers (producing eRNA) among 33 different cell types under different conditions and different timings of stimulation. The transcription of enhancers generally preceded transcription oftranscription factors which, in turn, generally precededmessenger RNA (mRNA) transcription of genes.

Carullo et al.^[13] 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.^[13] used these cultured neurons to examine the timing of specific enhancer eRNAs compared to themRNAs 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 theimmediate 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 IEGsFOSb andNR4A1, indicating that for many IEGs, eRNA induction precedes mRNA induction in response to neuronal activation.

While some enhancers can activate their targetpromoters at their target genes without transcribing eRNA, most active enhancers do transcribe eRNA during activation of their target promoters.^[14]

The functions for eRNA described below have been reported in diverse biological systems, often demonstrated with a small number of specific enhancer-target gene pairs. It is not clear to what extent the functions of eRNA described here can be generalized to most eRNAs.

The chromosome loops shown in the figure, bringing an enhancer to the promoter of its target gene, may be directed and formed by the eRNA transcribed from the enhancer after the enhancer is activated.

A transcribed enhancer RNA (eRNA) interacting with the complex ofMediator proteins (see Figure), especially Mediator subunit 12 (MED12), appears to be essential in forming the chromosome loop that brings the enhancer into close association with the promoter of the target gene of the enhancer in the case of five genes studied by Lai et al.^[15][16][17] Hou and Kraus,^[18] describe two other studies reporting similar results. Arnold et al.^[19] review another 5 instances where eRNA is active in forming the enhancer-promoter loop.

One well-studied eRNA is the eRNA of the enhancer that interacts with the promoter of the prostate specific antigen (PSA) gene.^[20] The PSA eRNA is strongly up-regulated by theandrogen receptor. High PSA eRNA then has a domino effect. PSA eRNA binds to and activates the positive transcription elongation factorP-TEFb protein complex which can then phosphorylateRNA polymerase II (RNAP II), initiating its activity in producingmRNA. P-TEFb can also phosphorylate thenegative elongation factor NELF (which pauses RNAP II within 60 nucleotides after mRNA initiation begins). Phosphorylated NELF is released from RNAP II, then allowing RNAP II to have productive mRNA progression (see Figure). Up-regulated PSA eRNA thereby increases expression of 586 androgen receptor-responsive genes. Knockdown of PSA eRNA or deleting a set of nucleotides from PSA eRNA causes decreased presence of phosphorylated (active) RNAP II at these genes causing their reduced transcription.

Thenegative elongation factor NELF protein can also be released from its interaction with RNAP II by direct interaction with some eRNAs. Schaukowitch et al.^[21] showed that the eRNAs of twoimmediate early genes (IEGs) directly interacted with the NELF protein to release NELF from the RNAP II paused at the promoters of these two genes, allowing these two genes to then be expressed.

In addition, eRNAs appear to interact with as many as 30 other proteins.^[19][17][18]

The notions that not allenhancers are transcribed at the same time and that eRNAtranscription correlates with enhancer-specific activity support the idea that individual eRNAs carry distinct and relevant biological functions.^[3] However, there is still noconsensus on the functional significance of eRNAs. Furthermore, eRNAs can easily be degraded throughexosomes andnonsense-mediated decay, which limits their potential as important transcriptional regulators.^[22] To date, four main models of eRNA function have been proposed,^[3] each supported by different lines ofexperimentalevidence.

Since multiple studies have shown thatRNA Pol II can be found at a very large number ofextragenic regions, it is possible that eRNAs simply represent the product of random "leaky"transcription and carry no functional significance.^[5] The non-specific activity of RNA Pol II would therefore allowextragenic transcriptional noise at sites wherechromatin is already in an open and transcriptionally competent state. This would explain even tissue-specific eRNA expression^[23] as open sites are tissue-specific as well.

RNA Pol II-mediatedgenetranscription induces a local opening ofchromatin state through the recruitment ofhistone acetyltransferases and otherhistone modifiers that promoteeuchromatin formation. It was proposed that the presence of theseenzymes could also induce an opening ofchromatin atenhancer regions, which are usually present at distant locations but can be recruited to targetgenes through looping ofDNA.^[24] In this model, eRNAs are therefore expressed in response toRNA Pol II transcription and therefore carry nobiological function.

While the two previous models implied that eRNAs were not functionally relevant, this mechanism states that eRNAs are functionalmolecules that exhibitcis activity. In this model, eRNAs can locally recruitregulatoryproteins at their own site of synthesis. Supporting this hypothesis, transcripts originating fromenhancers upstream of theCyclin D1 gene are thought to serve as adaptors for the recruitment ofhistone acetyltransferases. It was found that depletion of these eRNAs led to Cyclin D1 transcriptional silencing.^[9]

The last model involvestranscriptional regulation by eRNAs at distantchromosomal locations. Through the differential recruitment ofproteincomplexes, eRNAs can affect the transcriptional competency of specificloci. Evf-2 represents a good example of suchtrans regulatory eRNA as it can induce the expression of Dlx2, which in turn can increase the activity of the Dlx5 and Dlx6enhancers.^[25]Trans-acting eRNAs might also be working incis, and vice versa.

The detection of eRNAs is fairly recent (2010) and has been made possible through the use of genome-wide investigation techniques such asRNA sequencing (RNA-seq) and chromatin immunoprecipitation-sequencing (ChIP-seq).^[1] RNA-seq permits the direct identification of eRNAs by matching the detected transcript to the correspondingenhancer sequence throughbioinformatic analyses.^[26][4] ChIP-seq represents a less direct way to assess enhancertranscription but can also provide crucial information as specificchromatin marks are associated with active enhancers.^[27] Although some data remain controversial, the consensus in the literature is that the best combination of histone post-translational modifications at active enhancers is made ofH2AZ,H3K27ac, and a high ratio of H3K4me1 overH3K4me3.^[27][28][29] ChIP experiments can also be conducted withantibodies that recognizeRNA Pol II, which can be found at sites of activetranscription.^[5] The experimental detection of eRNAs is complicated by their lowendogenous stability conferred byexosome degradation andnonsense-mediated decay.^[22] A comparative study showed that assays enriching forcapped and nascent RNAs (with strategies like nucleirun-on and size selection) could capture more eRNAs compared to canonical RNA-seq.^[30] These assays include Global/Precision Run-on with cap-selection (GRO/PRO-cap), capped-small RNA-seq (csRNA-seq), Native Elongating Transcript-Cap Analysis of Gene Expression (NET-CAGE), and Precision Run-On sequencing (PRO-seq).^[31] Nonetheless, the fact that eRNAs tend to be expressed from active enhancers might make their detection a useful tool to distinguish between active and inactive enhancers.

Evidence that eRNAs cause downstream effects on theefficiency of enhancer activation and gene transcription suggests its functional capabilities and potential importance.^[4] Thetranscription factorp53 has been demonstrated to bindenhancer regions and generate eRNAs in a p53-dependent manner.^[32] Incancer, p53 plays a central role intumor suppression asmutations of thegene are shown to appear in 50% oftumors.^[33] These p53-bound enhancer regions (p53BERs) are shown to interact with multiple local and distal gene targets involved incellproliferation and survival. Furthermore, eRNAs generated by the activation of p53BERs are shown to be required for efficienttranscription of the p53 target genes, indicating the likely important regulatory role of eRNAs in tumor suppression and cancer. Generally, mutations in eRNA have been shown to demonstrate similar phenotypic behavior in oncogenesis as compared to protein-coding RNA.^[34]

Variations in enhancers have been implicated in humandisease but atherapeutic approach to manipulate enhancer activity is currently not available. With the emergence of eRNAs as important components in enhancer activity, powerful therapeutic tools such asRNAi may provide promising routes to target disruption of gene expression.

• Vista Enhancer Database
• Mouse ENCODE ProjectArchived 2012-12-13 at theWayback Machine
• ENCODE Project at UCSC
• PEDB

• RNA
• Gene expression
• Protein biosynthesis
• Molecular genetics
• Spliceosome
• RNA splicing
• Non-coding RNA

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