Skip to main content
Download PDF

Enhancer RNAs: mechanisms in transcriptional regulation and functions in diseases

Cell Communication and Signaling volume 21, Article number: 191 (2023) Cite this article

Abstract

In recent years, increasingly more non-coding RNAs have been detected with the development of high-throughput sequencing technology, including microRNAs (miRNAs), long non-coding RNAs (lncRNAs), circular RNAs (circRNAs), small nucleolar RNAs (snoRNAs), and piwi-interacting RNA (piRNAs). The discovery of enhancer RNAs (eRNAs) in 2010 has further broadened the range of non-coding RNAs revealed. eRNAs are non-coding RNA molecules produced by the transcription of DNA cis-acting elements, enhancer fragments. Recent studies revealed that the transcription of eRNAs may be a biological marker responding to enhancer activity that can participate in the regulation of coding gene transcription. In this review, we discussed the biological characteristics of eRNAs, their functions in transcriptional regulation, the regulation factors of eRNAs production, and the research progress of eRNAs in different diseases.

Video Abstract

Introduction

Cell ability to respond to specific developmental or environmental stimuli requires strict regulation of gene expression, which is controlled by a variety of genomic regulatory elements, including enhancers, promoters, and silencers. Enhancer is a type of non-coding DNA cis-acting element. The 72-bp sequence, isolated from Simian Virus 40 (SV40) in 1981, is the first enhancer to be cloned, with its function tested [1,2,3]. Later studies found that enhancers accounted for a large proportion of genomes. Notably, the expression of more than 20,000 coding genes in the human genome may be controlled by hundreds of thousands of enhancers [4, 5]. Mutations or epigenetic modifications in the enhancer region may lead to the occurrence and development of many diseases, including malignant tumors [6,7,8,9,10,11,12].

Genomic analysis revealed that many enhancers can be combined by RNA polymerase II (RNA Pol II) and actively transcribed to produce non-coding RNAs, namely eRNAs [13, 14]. A number of studies have shown that eRNAs can play a role in the process of transcriptional regulation [15,16,17,18,19], and the transcription of eRNAs may serves as another sign of enhancer activation [20,21,22,23,24,25]. The discovery of eRNAs opens up a new pathway for enhancer's mechanism of action, and adds another layer of complexity to transcriptional regulation. This review summarized the currently available information on the progress of research on eRNAs and discussed their biological properties and functions.

Biological role of enhancers and super-enhancers

During the individual development of eukaryotes, enhancers can activate or enhance gene transcription by binding to transcription factors, cofactors, and chromatin complexes to act on promoters. The dynamic interaction between an enhancer and the corresponding promoter determines its tissue- or cell-specific action mode [26,27,28]. However, the mechanism of this mode of action has not been elucidated. Activated enhancers usually have several characteristics in common: (1) Enhancers are usually found in the open regions of chromatin [29]; (2) More histone H3 lysine 4 monomethylation (H3K4me1) is detected than histone H3 lysine 4 trimethylation (H3K4me3) [30, 31]; (3) Histone acetylation, such as H3 acetylation at lysine 27 (H3K27ac) [32], and the presence of certain histone variants (such as H2AZ [33, 34]); (4) They can be combined with transcription co-activator, such as Mediator [35], p300/CREB-binding protein (CBP) [36,37,38] and bromodomain-containing protein 4 (BRD4) [39]. Therefore, chromatin immunoprecipitation and sequencing (ChIP-seq) has been used in many studies for the detection of the aforementioned factors or epigenetic modifications to reveal enhancer activities [30, 40, 41]. However, the annotation of epigenomic features has generated a large number of putative enhancers (> 400,000 to ~ 1 million) in humans, which is tenfold higher than that of coding genes [42,43,44]. This indicates that additional criteria are needed to more accurately annotate functional enhancers in genomes.

Super-enhancers are a class of cis-regulatory elements with strong transcriptional activation properties, which were first proposed by scholar Richard A. Young in 2013 [45]. They are large clusters of constituent enhancers and usually appear near most of the key genes that determine cell identity. The span range of super enhancers region is usually magnitude higher than typical enhancers [45]. Super-enhancers have higher enrichment densities of transcriptional activation-related histone modifications (H3K27ac, H3K4me1, etc.), Mediator complex, BRD4, p300/CBP and transcription factors than typical enhancers [45, 46]. Like typical enhancers, super enhancers can also be transcribed to form eRNAs, ChIP-Seq results suggested that super-enhancer regions were enriched with more RNA transcription signals than typical enhancer regions, which drove higher levels of target gene expression [46]. Combining the above characteristics, super enhancers have more powerful regulatory functions than typical enhancers.

Discovery and biological characteristics of eRNAs

Using ChIP-seq, two studies in 2010 found that RNA polymerase II could bind to enhancer regions, and the new non-coding transcripts, eRNAs, were detected through RNA-seq technology [13, 14]. Since then, the existence of eRNAs has been confirmed. Research efforts have been focused on the continuing exploration of their functions in gene transcription regulation [15, 19]. Looking back on the previous decades of research, the existence of eRNAs seemed to be traceable. Before the development of high-throughput sequencing technology, researchers had observed enhancer transcription in β-globin genes [47, 48], human growth hormone genes [49], and major histocompatibility complex class II locus control regions (LCR) [50]. However, no in-depth research was done due to the limited technical resources at that time.

According to cap-analysis gene expression (CAGE) technology, the number of eRNAs in humans is approximately 40,000–65,000, which is extremely large [24, 51]. Most eRNAs are bidirectional transcribed, which called 2d-eRNAs, are relatively short in length (within 0.5–2 kb) [52, 53] (Fig. 1A). Such eRNAs do not undergo a complete RNA maturation process. They have a cap structure at the 5′-end but without polyadenylation (polyA) modification at the 3′-end, which also determines their poor stability [13, 19, 53, 54]. Moreover, eRNAs are easily degraded by exosomal complexes in the nucleus [54,55,56]. Although eRNAs are located predominantly in the nucleus, their abundance is generally low [13, 17, 31]. Certain relatively abundant eRNAs are represented by approximately 0.5–20 copies per cell [57]. In addition, about 10% of eRNAs are unidirectionally transcribed (1d-eRNAs), with an average length of more than 4 kb [24] (Fig. 1B). The structure of this part of eRNAs is comparatively stable, with polyA modification at the 3′-end.

Fig. 1
figure 1

Biological characteristics of eRNAs. A Most eRNAs are bidirectional transcribed, which called 2d-eRNAs and with length within 0.5–2 kb, they are unspliced and nonpolyadenylated. B About 10% of eRNAs are unidirectionally transcribed (1d-eRNAs), which average length is more than 4 kb, and they are spliced and polyadenylated

Full size image

Compared with messenger RNA(mRNAs) and lncRNAs, eRNAs mainly present following characteristics: (a) eRNAs are mainly produced by genomic regions marked by high H3K4me1 and low H3K4me3 histone modifications, whereas the promoter regions of lncRNAs genes usually have high levels of H3K4me3 histone modifications [32]; (b) eRNAs are generally unspliced because their corresponding enhancer sequence does not have a U1 splice site, while both mRNAs and lncRNAs can be spliced [24]; (c) According to research, eRNAs have 90–100-fold lower stability than mRNAs and lncRNAs [13]; (d) The expression of eRNAs is positively correlated with the modification level of the active enhancer marker H3K27ac [31, 58]; (e) The enhancer region producing eRNAs is mainly enriched with the serine-5-phosphorylated RNA Pol II, whereas the promoter region that produces mRNAs binds mainly to serine-2-phosphorylated RNA Pol II [52]. However, studies have found that unidirectional eRNAs (1d-eRNAs) and lncRNAs have similar functions. Currently, it is generally believed that no clear boundary exists between them, that is, many annotated 1d-eRNAs may actually be lncRNAs, and vice versa [59, 60].

Approaches to detect eRNAs

Although RNA-seq technology has a wide range of applications, it has not been employed on a large scale in the detection of eRNAs mainly due to their poor stability. Methods such as global run-on sequencing (GRO-seq) and its derivative, the precision nuclear run-on sequencing (PRO-seq) have been used to detect the presence of eRNAs, that is, to determine the transcription at a certain stage of cell development by detecting the sequence of new transcripts in the cell [19, 57, 61,62,63,64,65]. Since eRNAs have a 5'-cap structure, CAGE, precision run-on of capped RNA and sequencing (PRO-cap), 5′-end-selected global run-on followed by sequencing (5'GRO-seq) can also be used for detection [19, 24, 66]. In the functional study of eRNAs, short hairpin RNA (shRNA), small interfering RNA (siRNA), locked nucleic acid (LNA), antisense oligonucleotides (ASOs) and dCas9-KRAB-sgRNA technologies were initially used to knock down the expression of eRNAs transcripts in the nucleus [19, 57, 67]. However, some scholars questioned whether RNA interference (RNAi) can really obstruct eRNAs expression [68]. Therefore, researchers used a combination of two knockout methods to ensure the knockout efficiency, such as siRNAs + LNAs [19, 57], siRNAs + ASOs [19, 69], ASOs + dCas9-KRAB-sgRNA [70] etc., or designed two or more siRNAs/ shRNAs/ LNAs/ ASOs to avoid the off-target effects [17, 71, 72]. Besides, overexpression plasmids [70] or CRISPR activation (CRISPR/dcas9 SAM) [71] were applied to induce the expression of eRNAs.

Functions of eRNAs

Serving as a sign of enhancer activity

Distal-acting enhancers are key elements in the regulation of time- and cell type-specific gene expression patterns. The genome-wide identification of active enhancers is necessary to understand gene expression and developmental and disease-related processes. Initially, researchers assessed the enhancer activity mainly by analyzing the recruitment of stimuli-induced transcriptional activators, epigenetic modifications of histones, and the transcription levels of nearby genes. Later studies found that enhancer transcription and the production of bidirectional eRNAs were also closely related to the enhancer activity [21, 73]. In addition, regions of the human genome with both enhancer chromatin markers and eRNAs transcriptional activity have a higher verification rate of enhancer activity than those with enhancers annotated only by chromatin modification data [24]. However, whether eRNAs can reflect the enhancer activity independently of other markers has not yet been established. Currently, it is generally believed that they can reflect the enhancer activity more accurately than other markers [22,23,24,25, 74]. Moreover, they may also be reliable predictors of cell type-specific enhancer activity [24]. Therefore, the sensitivity of active enhancer annotation can be improved by combining chromatin modification markers and eRNAs transcription.

Promoting the transcription of enhancer target genes

Traditionally, enhancers can be combined with transcription factors, which in turn interact with mediator complexes or other transcription co-activators to promote the recruitment of RNA Pol II and chromatin-modifying enzymes to the promoter region and form chromatin loop (enhancer-promoter loop) to foster the transcription of enhancer target genes. The discovery of eRNAs reveals the complex connection between enhancers that produce eRNAs and target genes, as well as the importance of eRNAs in promoting the transcription of adjacent target genes [15]. In this respect, Kim et al. found that the expression level of eRNAs was positively correlated with that of the coding genes in close proximity [14]. Furthermore, Melo et al. established that p53, a type of important tumour-suppressor proteins, induced target gene transcription by producing eRNAs from distant enhancer regions [16]. In this study, the researchers first identified several p53 binding enhancer regions (p53BER), which have a high affinity with p53, required them to exert their activities, and produced eRNAs in a p53-dependent manner. These authors later found that the suppression of p53-induced eRNAs led to changes in the p53-dependent transcription of the coding gene near p53BER, and the eRNAs expression enhanced the transcriptional activity of the adjacent coding genes. On the other hand, Lam et al. evidenced that the nuclear receptor Rev-Erb in mouse macrophages inhibited gene expression by suppressing the production of eRNAs at the enhancers [19]. These studies confirmed that eRNAs contributed to the transcription of enhancer target genes, indicating that they are not only by-products of gene transcription, but can be classified as a family of regulatory RNAs that enhance gene transcription. With research advances in this area, eRNAs have been found to exert their transcriptional regulatory effects mainly via the pathways described below.

Promoting enhancer-promoter interaction

Studies have found that eRNAs accelerated the transcription process by fostering enhancer-promoter interaction [57, 67]. For example, Li et al. performed GRO-Seq on cells treated with 17β-estradiol (E2) and observed the correlation between the ER-α binding enhancer, the eRNAs induction, and the upregulated expression of estrogen in the neighboring genes [57]. To investigate whether the eRNAs produced by ER-α binding enhancers affect chromatin structure, they applied a three-dimensional DNA selection and ligation (3D-DSL) program. Interestingly, they found that E2 treatment increased the frequency of enhancer-promoter interactions, while that of targeting eRNAs with siRNA/LNA decreased. In addition, Li et al. also established that eRNAs stabilized the enhancer-promoter loops by attracting cohesin complexes, which are key components for the formation and stabilization of chromatin loop structure [57] (Fig. 2). Consistent with this finding, in another examination, the authors used an anti-Rad21 antibody (a subunit of the cohesin complex) for ChIP-seq. They revealed that a subset of Rad21-binding sites overlapped with the enhancer domain of eRNAs induced by E2, which indicated the presence of an interaction between eRNAs and cohesin complexes. Then, the researchers confirmed their physical combination through RNA immunoprecipitation.

Fig. 2
figure 2

eRNAs can promote enhancer-promoter looping. eRNAs stabilized the enhancer-promoter loops by attracting cohesin complexes, which are key components for the formation and stabilization of chromatin loop structure

Full size image

Increasing chromatin accessibility

Mousavi et al. found that the myogenic transcription factors MyoD and MyoG combined extensively in extragenic regions [17]. Among them, multiple enhancers upstream of the MyoD1 gene could be transcribed to produce eRNAs, including eRNA produced by a core enhancer (CE) (CERNA) and that by distal enhancers (DRR) (DRRRNA). By curbing the expression of eRNAs, the authors found that the reduction in CERNA levels significantly decreased the occupancy rate of RNA Pol II in the proximal region of the MyoD1 gene. On the other hand, the knockdown of DRRRNA diminished the occupancy rate of the proximal RNA Pol II of the MyoG gene, which is the downstream gene of MyoD1. The authors also assessed the influence of eRNAs on molecular events before RNA Pol II assembly, namely chromatin accessibility. They found that the knockdown of DRRRNA significantly reduced the chromatin accessibility of MyoG. After the decrease in CERNA expression, the cells were less susceptible to Deoxyribonuclease I (DNase I) treatment at MyoD1 and MyoG. The results of this study shows that eRNAs from the MyoD1 regulatory region promote chromatin recombination/depolymerization and RNA Pol II assembly at specific sites in the muscle-derived gene regulatory network, thereby affecting gene transcription. These findings also suggest that eRNAs can form an open chromatin environment by recruiting key transcription factors and/or chromatin remodeling complexes (Fig. 3A), thus promoting the binding of RNA Pol II, basic transcription factors, and other pre-priming complexes to DNA (Fig. 3C). The research of Tsai et al. confirmed this conclusion [70]. In addition, Pnueli et al. [67]found that the level of active promoter marker H3K4me3 at the target gene promoter was significantly reduced after the knockdown of eRNAs. The level of active enhancer marker H3K27ac at both the promoter and enhancer decreased, but that of repressive histone H3 Lys 27 trimethylation (H3K27me3) increased. It can be seen that eRNAs may also affect the transcription of target genes by adjusting the level of histone modification in the proximal promoter and enhancer regions so as to change the chromatin state (Fig. 3B).

Fig. 3
figure 3

eRNAs can increase chromatin accessibility. eRNAs can form an open chromatin environment by recruiting chromatin remodeling complexes (A) or adjusting the level of histone modification in the proximal promoter and enhancer regions (B), thus promoting the binding of RNAPII (C), then promotes the transcription of target genes

Full size image

Facilitating RNA Pol II pause-release to enhance transcription extension

RNA Pol II pause is a genome-wide regulatory mechanism of higher eukaryotes with common genes for responses to environmental stimulus [75, 76]. Negative elongation factor (NELF) and DRB sensitivity-inducing factor (DSIF) synergistically trigger RNA Pol II suspension by directly binding to RNA Pol II and nascent RNA [75, 77]. The pause release and subsequent elongation are mediated by the positive transcription elongation factor b (P-TEFb), which phosphorylates the RNA Pol II C-terminal domain (CTD), the DSIF, and the NELF [75, 78]. Schaukowitch et al. [79] found that the suppression of eRNA resulted in decreased expression of specific target genes, whereas the chromosomal loop between the promoter and enhancer was not affected. However, the decline of the eRNA level hindered the effective release of the NELF complex from the promoter of a specific target gene during the transcription induction process, accompanied by a decrease in the extended form of RNA Pol II and target mRNA. Ultraviolet RNA immunoprecipitation (UV-RIP) and in vitro RNA pull-down experiments have shown that the eRNA expressed during neuron stimulation can directly bind to the RNA recognition motif (RRM) of the negative elongation factor complex member E (NELF-E) subunit. Replacing endogenous NELF-E with RRM deletion mutants in neurons significantly reduced the NELF complex binding level at the promoter and mRNA induction. In short, eRNAs can promote the release of NELF by acting as a bait for nascent transcripts, allowing the suspended RNA Pol II to effectively transit to a productive transcription extension (Fig. 4A). Zhao et al. [69] confirmed that Prostate specific antigen (PSA) eRNA activates P-TEFb and promotes RNA Pol II-Ser2 phosphorylation (Pol II-Ser2p) by binding to the subunit CYCLIN T1 of the P-TEFb complex, thereby enhancing the transcription of the target gene, PSA mRNA (Fig. 4B). It becomes evident that eRNAs can affect the target gene transcription by regulating the process of transcription elongation in various ways.

Fig. 4
figure 4

eRNAs can facilitate RNAPII pause-release to enhance transcription extension. A eRNAs can promote the release of NELF by directly bind to NELF subunit, allowing the RNAPII to effectively transit to a productive transcription extension. B eRNAs can facilitate RNAPII pause-release by binding to P-TEFb complex subunit, activating P-TEFb and promoting RNAPII 2 phosphorylation

Full size image

Regulation of eRNAs production

Synthesis

The production of eRNAs can be regulated by a variety of factors (Fig. 5A). At the initiation of eRNAs transcription, the cap-binding complex (CBC) binds to eRNAs, forming a 7-methylguanosine (m7G) cap at the 5′-end. The transcript elongation of the enhancer is controlled by P-TEFb and BRD4, which are recruited by the acetylated histone tail on the enhancer [78, 80, 81]. The interaction of BRD4 and P-TEFb can release the pausing RNA Pol II, thereby promoting the process of transcript elongation [82]. BRD4 is a member of the bromodomain and extra-terminal domain (BET) family. In the observations of the transcription of eRNAs in the enhancer region centered on the BRD4 peak, Kanno et al. [81] established the small molecule BET inhibitor, JQ1, restrained the elongation of eRNA by facilitating its synthesis, which suggests that BRD4 is involved in the eRNAs transcript elongation process. The termination of eRNA transcription is mediated by the Integrator complex, a multi-subunit complex closely related to the CTD of RNA Pol II that possesses endonuclease activity and necessary for the processing of the 3′-end of the nascent transcript [83]. Lai et al. confirmed that Integrator was recruited to enhancers and super-enhancers in a stimulus-dependent manner [62]. The knockdown of its subunit can reduce the signal-dependent production of eRNAs and eliminate the enhancer-promoter chromatin loop induced by stimulation. Meanwhile, it can also lead to RNA Pol II inability for separation from eRNAs, resulting in the accumulation of RNA Pol II-eRNA complexes, which can decrease the mature eRNAs level. The termination of eRNAs also requires the function of WD repeat-containing protein 82 (WDR82) [84], which is an adaptor protein targeting SET1 H3K4 methyltransferase to chromatin. The knockdown of WDR82 causes defective transcription termination, increasing the abundance and length of eRNAs.

Fig. 5
figure 5

The regulation factors of eRNAs production, degradation and modification. A At the initiation of transcription, the cap-binding complex (CBC) form a 7-methylguanosine (m7G) cap at the 5′-end of eRNAs; P-TEFb and BRD4 control the transcript elongation of eRNAs, which are recruited by the acetylated histone tail on the enhancer; The termination of eRNA transcription is mediated by the Integrator complex and WDR82. B The degradation of eRNAs is regulated by RNA exosome complexes. C Chemical modifications including m5C and m6A of eRNAs can stabilize eRNAs. eRNAs m5C modifications is catalyzed by NSUN7; eRNAs m6A methylation and demethylation are catalyzed by MTC and ALKBH5, respectively. MTC: METTL3/METTL14/WTAP m6A methyltransferase complex

Full size image

Degradation

The degradation of eRNAs is mediated by RNA exosome complexes (Fig. 5B), which consists of nine subunits, six of which form a “loop” structure. The other three subunits containing RNA-binding domains form a “cap” structure. The enzymatic activity of the RNA exosome complex is provided by two additional proteins, Rrp44 (Dis3) and Rrp6 (Exosc10) [85,86,87,88]. Pefanis et al. [55] revealed that the expression of non-coding RNAs such as eRNAs increased significantly after the Dis3 and Exosc10 genes were knocked out. During the production of eRNAs, harmful DNA/RNA hybrids, specifically R-loops, may be formed, which may affect the stability of the genome. Among the knockout cells in the Dis3 and Exosc10 genes, Pefanis et al. observed a significant increase in the R-loop structure in the region where eRNAs was transcribed, which suggested that the RNA exosome complex may degrade the harmful secondary structure during the formation of eRNAs to maintain genome stability. Furthermore, another study [24] has detected a 7.4-fold increase in eRNAs peaks after depletion of the exosome complex co-factor MTR4, further confirming that the degradation of eRNAs is mediated by the RNA exosome complex.

Modification

Chemical modifications occur on eRNAs can stabilize eRNAs (Fig. 5C), which mainly include 5-methylcytosine (m5C) and N6-methyladenosine (m6A). Aguilo et al. [89] demonstrated that the NOP2/Sun RNA methytransferase 7 (NSUN7) was a key methyltransferase of eRNAs m5C modification. NSUN7 depletion caused an abrupt decrease of eRNAs expression levels and a loss of m5C modification on eRNAs. Lee et al. [90] have detected pervasive m6A signals on eRNAs by using methylation inscribed nascent transcripts sequencing (MINT-Seq). They identified the m6A motif of eRNAs as "GGACT". MINT-Seq results revealed the presence of m6A peaks was on the entire eRNA transcript, but with the highest abundance in the middle of the transcript. In addition, m6A-marked eRNAs (m6A-eRNAs) were longer and possessed higher transcript abundance than non-m6A-marked eRNAs. RNA half-life experiments further confirmed that m6A-eRNAs were more stable. Mechanistically, m6A-eRNAs could recruit the m6A reader protein YTHDC1 to active enhancer regions to promote enhancer transcription and facilitate the formation of transcriptional activator condensates, which in turn promote target genes activation. Interestingly, Xu et al. [91] showed that METTL3/METTL14/WTAP m6A methyltransferase complex (MTC) and m6A demethylase ALKBH5 located in the enhancer region could regulate eRNAs m6A methylation and demethylation, respectively.

eRNAs in diseases

Studies have gradually revealed the regulatory role of eRNAs in a variety of diseases, such as tumors, inflammation, neurological diseases and muscular diseases. We have summarized the eRNAs with confirmed regulatory role in Table 1.

Table 1 eRNAs with confirmed functions in diseases
Full size table

eRNAs in cancer

Enhancers play specific roles in human cells, and their mutations or misregulation can lead to the occurrence of malignant tumors. Therefore, it would be crucially important to understand whether the eRNAs produced by them affect the tumorigenesis process. In a previous study on this topic, Zhang et al. [71] pooled data of large-scale clinical samples and eRNAs expression in tumor cell lines from databases, such as the Cancer Genome Atlas (TCGA), the Cancer Cell Line Encyclopedia (CCLE), Encyclopedia of DNA Elements (ENCODE), Function Annotation of The Mammalian Genome (FANTOM), Roadmap Epigenomics, and 4D Nucleome projects, and found the cancer/lineage-specific expression characteristics of eRNAs. The study identified a total number of 9108 detectable eRNAs in human cancers, which were then divided into three groups: 652 commonly expressed in or above 10 cancer types (ubiquitous eRNAs), and 3124 medium-specific eRNAs expressed in 2–9 cancer types (intermediately specific eRNAs), 5332 eRNAs were expressed in only one cancer type (cancer-type-specific). The ubiquitous eRNAs showed a higher expression level than another two groups.

Meanwhile, a number of studies have confirmed the regulatory roles of eRNAs in tumor progression at the molecular biological level. For example, the HPSE eRNA produced by heparanase can promote cancer progression by driving the chromatin loop and regulating the hnRNPU/p300/EGR1/HPSE axis [72]; Kallikrein 3e (KLK3e), an androgen-induced eRNA, can promote the spatial interaction between the androgen receptor (AR)-dependent gene KLK3 enhancer and KLK2 promoter, and enhance long-distance KLK2 transcription activation [92]. Meanwhile, the study found that KLK3e silencing inhibited the proliferation of prostate cancer cells. Another investigation revealed that eRNAs induced by tumor protein p53 (TP53) are associated with p53-dependent cell-cycle arrest in the breast cancer cell line Michigan Cancer Foundation-7 (MCF-7) [16]. PSA eRNA expression is significantly increased in castration-resistant prostate cancer (CRPC) cells, patient-derived xenografts (PDX), and patient tissues [69]. These findings suggest that eRNAs may be critically involved in tumorigenesis and may thus serve as important biological markers for the diagnosis and treatment of malignant tumors.

eRNAs in inflammation

eRNAs also play a role in the progression of inflammation. For instance, Nicholas et al. [68] found that bacterial lipopolysaccharide (LPS) could stimulate human monocytes to produce 76 differentially expressed eRNAs, among which the IL1β-eRNA is associated with the mRNA production of inflammatory mediators IL1β, CXCL8, and IL6. What’s more, the knockdown of IL1β-eRNA decreased the IL1β mRNA expression and protein release induced by LPS, which suggested that eRNAs were important regulators of human innate immune response. Additionally, another study [95] showed that after stimulation of mouse bone marrow-derived macrophages by synthetic glucocorticoids (such as dexamethasone), eRNAs could be generated at the GR binding site (GBS), termed GR eRNAs. The expression of these GR eRNAs showed glucocorticoid-responsive tissues specific features. They also found that after dexamethasone stimulated macrophages, 81 GR eRNAs were up-regulated, and 108 GR eRNAs were down-regulated. The expression of GR eRNAs and its adjacent genes were also significantly correlated, down-regulated genes adjacent to GBSs with eRNA transcription are mostly enriched in biological processes related to inflammatory response processes such as ‘leukocyte migration’.

eRNAs in neurological diseases

Yao et al. [96] discovered a set of eRNAs specifically expressed in human brain tissues, and constructed a co-expression interaction network of eRNAs-target genes in the human fetal brain and multiple adult brain regions. They also showed that the active enhancer region with the ability to transcribe brain-specific eRNAs was rich in genetic variants associated with the autism spectrum disorder (ASD). Meanwhile, Hauberg et al. [94] also compared 118 differentially transcribed eRNAs in schizophrenia (SCZ) patient samples with control samples and identified schizophrenia-associated gene/ eRNA co-expression modules from the former group. Another study [97] demonstrated that compared with the sham control group, 77 eRNAs were significantly induced in the stroke group, of which 55 eRNAs were exclusively induced in the stroke group. They randomly selected two eRNAs which were significantly induced in the stroke group for knockdown experiment in vivo. Results showed that infarct volumes in the eRNAs knockdown group were larger than the control group, suggesting that eRNAs were associated with the post-stroke neuroprotective response.

eRNAs in myogenesis

As we mentioned above, Mousavi et al. [17]. revealed that CERNA could active MyoD expression in cis by promoting chromatin remodeling and increasing RNA polymerase II occupancy and DNA accessibility, which was a crucial transcription factor in myogenesis. Tsai et al. [70] demonstrated that DRReRNA which was transcribed from the distal enhancer of MyoD (located on mouse chromosome 7) did not regulate the expression of MyoD, but localized to Myogenin locus (located on mouse chromosome 1) and regulated the expression of Myogenin in trans. Furthermore, they found that DRReRNA could bind to SMC3, which was one member of Cohesin complex, and recruit Cohesin at Myogenin in differentiated C2C12 cells, thus promoting myotube differentiation.

Perspectives

It is only more than a decade since the discovery of eRNAs. Although increasing research on eRNAs has been performed in recent years and many of their biological functions have been revealed and confirmed, there are still issues that need to be addressed. For example, most of the functions of eRNAs reported in those literature cannot exist independently of enhancers. Thus, it is critical to elucidate whether they can serve as functional genes similarly to other non-coding RNAs, because most eRNAs have poor stability. In addition, it is worthwhile to establish and develop better tools for the manipulation of eRNAs expression, which would be crucial to the success of functional eRNAs research. In addition, although Zhang et al. has suggested the widespread presence of eRNAs in tumor cells through database integration, the specific mechanisms by which eRNAs function in malignant tumors remain to be explored. Furthermore, the lineage-specific expression signatures of eRNAs may guide the molecular typing and diagnosis of tumors. Their transcriptional regulatory role in tumor genes also suggests the potential to be employed as a target for tumor diagnosis or therapy. In conclusion, many unknowns remain regarding the biological functions and specific mechanisms of eRNAs activities, which require further continuous exploration.

Availability of data and materials

Not applicable.

Abbreviations

miRNAs:

MicroRNAs

lncRNAs:

Long non-coding RNAs

circRNAs:

Circular RNAs

snoRNAs:

Small nucleolar RNAs

piRNAs:

Piwi-interacting RNA

eRNAs:

Enhancer RNAs

SV40:

Simian virus 40

RNA Pol II:

RNA polymerase II

H3K4me1:

Histone H3 lysine 4 monomethylation

H3K4me3:

Histone H3 lysine 4 trimethylation

H3K27ac:

Histone H3 acetylation at lysine 27

CBP:

CREB-binding protein

BRD4:

Bromodomain-containing protein 4

ChIP-seq:

Chromatin immunoprecipitation and sequencing

LCR:

Locus control regions

CAGE:

Cap-analysis gene expression

polyA:

Polyadenylation

mRNAs:

Messenger RNA

GRO-seq:

Global run-on sequencing

PRO-seq:

Precision nuclear run-on sequencing

PRO-cap:

Precision run-on of capped RNA and sequencing

5'GRO-seq:

5′-End-selected global run-on followed by sequencing

shRNA:

Short hairpin RNA

siRNA:

Small interfering RNA

LNA:

Locked nucleic acid

ASOs:

Antisense oligonucleotides

RNAi:

RNA interference

p53BER:

P53 binding enhancer regions

3D-DSL:

Three-dimensional DNA selection and ligation

DNase I:

Deoxyribonuclease I

H3K27me3:

Histone H3 Lys 27 trimethylation

NELF:

Negative elongation factor

DSIF:

DRB sensitivity-inducing factor

P-TEFb:

Positive transcription elongation factor b

CTD:

C-terminal domain

UV-RIP:

Ultraviolet RNA immunoprecipitation

RRM:

RNA recognition motif

NELF-E:

Negative elongation factor complex member E

PSA:

Prostate specific antigen

Pol II-Ser2p:

RNA Pol II-Ser2 phosphorylation

CBC:

Cap-binding complex

m7G:

7-Methylguanosine

BET:

Bromodomain and extra-terminal domain

WDR82:

WD repeat-containing protein 82

m5C:

5-Methylcytosine

m6A:

N6-methyladenosine

NSUN7:

NOP2/Sun RNA methytransferase 7

MINT-Seq:

Methylation inscribed nascent transcripts sequencing

MTC:

METTL3/METTL14/WTAP m6A methyltransferase complex

TCGA:

The Cancer Genome Atlas

CCLE:

The Cancer Cell Line Encyclopedia

ENCODE:

Encyclopedia of DNA Element

FANTOM:

Function Annotation of The Mammalian Genome

KLK3e:

Kallikrein 3e

AR:

Androgen receptor

MCF-7:

Michigan Cancer Foundation-7

CRPC:

Castration-resistant prostate cancer

PDX:

Patient-derived xenografts

LPS:

Lipopolysaccharide

GBS:

GR binding site

ASD:

Autism spectrum disorder

SCZ:

Schizophrenia

References

  1. Benoist C, Chambon P. In vivo sequence requirements of the SV40 early promotor region. Nature. 1981;290(5804):304–10.

    CAS  PubMed  Google Scholar 

  2. Moreau P, Hen R, Wasylyk B, Everett R, Gaub MP, Chambon P. The SV40 72 base repair repeat has a striking effect on gene expression both in SV40 and other chimeric recombinants. Nucleic Acids Res. 1981;9(22):6047–68.

    CAS  PubMed  PubMed Central  Google Scholar 

  3. Banerji J, Rusconi S, Schaffner W. Expression of a beta-globin gene is enhanced by remote SV40 DNA sequences. Cell. 1981;27(2 Pt 1):299–308.

    CAS  PubMed  Google Scholar 

  4. Consortium EP. An integrated encyclopedia of DNA elements in the human genome. Nature. 2012;489(7414):57–74.

    Google Scholar 

  5. Cusanovich DA, Hill AJ, Aghamirzaie D, Daza RM, Pliner HA, Berletch JB, et al. A single-cell atlas of in vivo mammalian chromatin accessibility. Cell. 2018;174(5):1309-24 e18.

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Chen H, Li C, Peng X, Zhou Z, Weinstein JN, Cancer Genome Atlas Research N, et al. A pan-cancer analysis of enhancer expression in nearly 9000 patient samples. Cell. 2018;173(2):386-99 e12.

    CAS  PubMed  PubMed Central  Google Scholar 

  7. Hazan I, Monin J, Bouwman BAM, Crosetto N, Aqeilan RI. Activation of oncogenic super-enhancers is coupled with DNA repair by RAD51. Cell Rep. 2019;29(3):560-72 e4.

    CAS  PubMed  PubMed Central  Google Scholar 

  8. Chai P, Yu J, Jia R, Wen X, Ding T, Zhang X, et al. Generation of onco-enhancer enhances chromosomal remodeling and accelerates tumorigenesis. Nucleic Acids Res. 2020;48(21):12135–50.

    CAS  PubMed  PubMed Central  Google Scholar 

  9. Bell E, Curry EW, Megchelenbrink W, Jouneau L, Brochard V, Tomaz RA, et al. Dynamic CpG methylation delineates subregions within super-enhancers selectively decommissioned at the exit from naive pluripotency. Nat Commun. 2020;11(1):1112.

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Wilson MR, Reske JJ, Holladay J, Neupane S, Ngo J, Cuthrell N, et al. ARID1A mutations promote P300-dependent endometrial invasion through super-enhancer hyperacetylation. Cell Rep. 2020;33(6): 108366.

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Bell RE, Golan T, Sheinboim D, Malcov H, Amar D, Salamon A, et al. Enhancer methylation dynamics contribute to cancer plasticity and patient mortality. Genome Res. 2016;26(5):601–11.

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Song Y, van den Berg PR, Markoulaki S, Soldner F, Dall’Agnese A, Henninger JE, et al. Dynamic enhancer DNA methylation as basis for transcriptional and cellular heterogeneity of ESCs. Mol Cell. 2019;75(5):905-20 e6.

    CAS  PubMed  PubMed Central  Google Scholar 

  13. De Santa F, Barozzi I, Mietton F, Ghisletti S, Polletti S, Tusi BK, et al. A large fraction of extragenic RNA pol II transcription sites overlap enhancers. PLoS Biol. 2010;8(5): e1000384.

    PubMed  PubMed Central  Google Scholar 

  14. Kim TK, Hemberg M, Gray JM, Costa AM, Bear DM, Wu J, et al. Widespread transcription at neuronal activity-regulated enhancers. Nature. 2010;465(7295):182–7.

    CAS  PubMed  PubMed Central  Google Scholar 

  15. Onoguchi M, Hirabayashi Y, Koseki H, Gotoh Y. A noncoding RNA regulates the neurogenin1 gene locus during mouse neocortical development. Proc Natl Acad Sci U S A. 2012;109(42):16939–44.

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Melo CA, Drost J, Wijchers PJ, van de Werken H, de Wit E, Oude Vrielink JA, et al. eRNAs are required for p53-dependent enhancer activity and gene transcription. Mol Cell. 2013;49(3):524–35.

    CAS  PubMed  Google Scholar 

  17. Mousavi K, Zare H, Dell’orso S, Grontved L, Gutierrez-Cruz G, Derfoul A, et al. eRNAs promote transcription by establishing chromatin accessibility at defined genomic loci. Mol Cell. 2013;51(5):606–17.

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Lam MT, Li W, Rosenfeld MG, Glass CK. Enhancer RNAs and regulated transcriptional programs. Trends Biochem Sci. 2014;39(4):170–82.

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Lam MT, Cho H, Lesch HP, Gosselin D, Heinz S, Tanaka-Oishi Y, et al. Rev-Erbs repress macrophage gene expression by inhibiting enhancer-directed transcription. Nature. 2013;498(7455):511–5.

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Melgar MF, Collins FS, Sethupathy P. Discovery of active enhancers through bidirectional expression of short transcripts. Genome Biol. 2011;12(11):R113.

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Mikhaylichenko O, Bondarenko V, Harnett D, Schor IE, Males M, Viales RR, et al. The degree of enhancer or promoter activity is reflected by the levels and directionality of eRNA transcription. Genes Dev. 2018;32(1):42–57.

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Wang D, Garcia-Bassets I, Benner C, Li W, Su X, Zhou Y, et al. Reprogramming transcription by distinct classes of enhancers functionally defined by eRNA. Nature. 2011;474(7351):390–4.

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Zhu Y, Sun L, Chen Z, Whitaker JW, Wang T, Wang W. Predicting enhancer transcription and activity from chromatin modifications. Nucleic Acids Res. 2013;41(22):10032–43.

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Andersson R, Gebhard C, Miguel-Escalada I, Hoof I, Bornholdt J, Boyd M, et al. An atlas of active enhancers across human cell types and tissues. Nature. 2014;507(7493):455–61.

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Chen H, Liang H. A high-resolution map of human enhancer RNA Loci characterizes super-enhancer activities in cancer. Cancer Cell. 2020;38(5):701-15 e5.

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Heinz S, Romanoski CE, Benner C, Glass CK. The selection and function of cell type-specific enhancers. Nat Rev Mol Cell Biol. 2015;16(3):144–54.

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Zaret KS. Pioneering the chromatin landscape. Nat Genet. 2018;50(2):167–9.

    CAS  PubMed  Google Scholar 

  28. Fernandez Garcia M, Moore CD, Schulz KN, Alberto O, Donague G, Harrison MM, et al. Structural features of transcription factors associating with nucleosome binding. Mol Cell. 2019;75(5):921-32 e6.

    CAS  PubMed  Google Scholar 

  29. Sheffield NC, Thurman RE, Song L, Safi A, Stamatoyannopoulos JA, Lenhard B, et al. Patterns of regulatory activity across diverse human cell types predict tissue identity, transcription factor binding, and long-range interactions. Genome Res. 2013;23(5):777–88.

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Heintzman ND, Stuart RK, Hon G, Fu Y, Ching CW, Hawkins RD, et al. Distinct and predictive chromatin signatures of transcriptional promoters and enhancers in the human genome. Nat Genet. 2007;39(3):311–8.

    CAS  PubMed  Google Scholar 

  31. Djebali S, Davis CA, Merkel A, Dobin A, Lassmann T, Mortazavi A, et al. Landscape of transcription in human cells. Nature. 2012;489(7414):101–8.

    CAS  PubMed  PubMed Central  Google Scholar 

  32. Heintzman ND, Hon GC, Hawkins RD, Kheradpour P, Stark A, Harp LF, et al. Histone modifications at human enhancers reflect global cell-type-specific gene expression. Nature. 2009;459(7243):108–12.

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Brunelle M, NordellMarkovits A, Rodrigue S, Lupien M, Jacques PE, Gevry N. The histone variant H2A. Z is an important regulator of enhancer activity. Nucleic Acids Res. 2015;43(20):9742–56.

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Hu G, Cui K, Northrup D, Liu C, Wang C, Tang Q, et al. H2A. Z facilitates access of active and repressive complexes to chromatin in embryonic stem cell self-renewal and differentiation. Cell Stem Cell. 2013;12(2):180–92.

    CAS  PubMed  Google Scholar 

  35. Kagey MH, Newman JJ, Bilodeau S, Zhan Y, Orlando DA, van Berkum NL, et al. Mediator and cohesin connect gene expression and chromatin architecture. Nature. 2010;467(7314):430–5.

    CAS  PubMed  PubMed Central  Google Scholar 

  36. Ghisletti S, Barozzi I, Mietton F, Polletti S, De Santa F, Venturini E, et al. Identification and characterization of enhancers controlling the inflammatory gene expression program in macrophages. Immunity. 2010;32(3):317–28.

    CAS  PubMed  Google Scholar 

  37. Rada-Iglesias A, Bajpai R, Swigut T, Brugmann SA, Flynn RA, Wysocka J. A unique chromatin signature uncovers early developmental enhancers in humans. Nature. 2011;470(7333):279–83.

    CAS  PubMed  Google Scholar 

  38. Vernimmen D, Lynch MD, De Gobbi M, Garrick D, Sharpe JA, Sloane-Stanley JA, et al. Polycomb eviction as a new distant enhancer function. Genes Dev. 2011;25(15):1583–8.

    CAS  PubMed  PubMed Central  Google Scholar 

  39. Zhang W, Prakash C, Sum C, Gong Y, Li Y, Kwok JJ, et al. Bromodomain-containing protein 4 (BRD4) regulates RNA polymerase II serine 2 phosphorylation in human CD4+ T cells. J Biol Chem. 2012;287(51):43137–55.

    CAS  PubMed  PubMed Central  Google Scholar 

  40. Barski A, Cuddapah S, Cui K, Roh TY, Schones DE, Wang Z, et al. High-resolution profiling of histone methylations in the human genome. Cell. 2007;129(4):823–37.

    CAS  PubMed  Google Scholar 

  41. Visel A, Blow MJ, Li Z, Zhang T, Akiyama JA, Holt A, et al. ChIP-seq accurately predicts tissue-specific activity of enhancers. Nature. 2009;457(7231):854–8.

    CAS  PubMed  PubMed Central  Google Scholar 

  42. Calo E, Wysocka J. Modification of enhancer chromatin: what, how, and why? Mol Cell. 2013;49(5):825–37.

    CAS  PubMed  Google Scholar 

  43. Shlyueva D, Stampfel G, Stark A. Transcriptional enhancers: from properties to genome-wide predictions. Nat Rev Genet. 2014;15(4):272–86.

    CAS  PubMed  Google Scholar 

  44. Rivera CM, Ren B. Mapping human epigenomes. Cell. 2013;155(1):39–55.

    CAS  PubMed  Google Scholar 

  45. Whyte WA, Orlando DA, Hnisz D, Abraham BJ, Lin CY, Kagey MH, et al. Master transcription factors and mediator establish super-enhancers at key cell identity genes. Cell. 2013;153(2):307–19.

    CAS  PubMed  PubMed Central  Google Scholar 

  46. Hnisz D, Abraham BJ, Lee TI, Lau A, Saint-Andre V, Sigova AA, et al. Super-enhancers in the control of cell identity and disease. Cell. 2013;155(4):934–47.

    CAS  PubMed  Google Scholar 

  47. Tuan D, Kong S, Hu K. Transcription of the hypersensitive site HS2 enhancer in erythroid cells. Proc Natl Acad Sci U S A. 1992;89(23):11219–23.

    CAS  PubMed  PubMed Central  Google Scholar 

  48. Ashe HL, Monks J, Wijgerde M, Fraser P, Proudfoot NJ. Intergenic transcription and transinduction of the human beta-globin locus. Genes Dev. 1997;11(19):2494–509.

    CAS  PubMed  PubMed Central  Google Scholar 

  49. Rogan DF, Cousins DJ, Santangelo S, Ioannou PA, Antoniou M, Lee TH, et al. Analysis of intergenic transcription in the human IL-4/IL-13 gene cluster. Proc Natl Acad Sci U S A. 2004;101(8):2446–51.

    CAS  PubMed  PubMed Central  Google Scholar 

  50. Masternak K, Peyraud N, Krawczyk M, Barras E, Reith W. Chromatin remodeling and extragenic transcription at the MHC class II locus control region. Nat Immunol. 2003;4(2):132–7.

    CAS  PubMed  Google Scholar 

  51. Arner E, Daub CO, Vitting-Seerup K, Andersson R, Lilje B, Drablos F, et al. Transcribed enhancers lead waves of coordinated transcription in transitioning mammalian cells. Science. 2015;347(6225):1010–4.

    CAS  PubMed  PubMed Central  Google Scholar 

  52. Koch F, Fenouil R, Gut M, Cauchy P, Albert TK, Zacarias-Cabeza J, et al. Transcription initiation platforms and GTF recruitment at tissue-specific enhancers and promoters. Nat Struct Mol Biol. 2011;18(8):956–63.

    CAS  PubMed  Google Scholar 

  53. Natoli G, Andrau JC. Noncoding transcription at enhancers: general principles and functional models. Annu Rev Genet. 2012;46:1–19.

    CAS  PubMed  Google Scholar 

  54. Andersson R, Refsing Andersen P, Valen E, Core LJ, Bornholdt J, Boyd M, et al. Nuclear stability and transcriptional directionality separate functionally distinct RNA species. Nat Commun. 2014;5:5336.

    CAS  PubMed  Google Scholar 

  55. Pefanis E, Wang J, Rothschild G, Lim J, Kazadi D, Sun J, et al. RNA exosome-regulated long non-coding RNA transcription controls super-enhancer activity. Cell. 2015;161(4):774–89.

    CAS  PubMed  PubMed Central  Google Scholar 

  56. Lubas M, Andersen PR, Schein A, Dziembowski A, Kudla G, Jensen TH. The human nuclear exosome targeting complex is loaded onto newly synthesized RNA to direct early ribonucleolysis. Cell Rep. 2015;10(2):178–92.

    CAS  PubMed  Google Scholar 

  57. Li W, Notani D, Ma Q, Tanasa B, Nunez E, Chen AY, et al. Functional roles of enhancer RNAs for oestrogen-dependent transcriptional activation. Nature. 2013;498(7455):516–20.

    CAS  PubMed  PubMed Central  Google Scholar 

  58. Kaikkonen MU, Spann NJ, Heinz S, Romanoski CE, Allison KA, Stender JD, et al. Remodeling of the enhancer landscape during macrophage activation is coupled to enhancer transcription. Mol Cell. 2013;51(3):310–25.

    CAS  PubMed  PubMed Central  Google Scholar 

  59. Vucicevic D, Corradin O, Ntini E, Scacheri PC, Orom UA. Long ncRNA expression associates with tissue-specific enhancers. Cell Cycle. 2015;14(2):253–60.

    CAS  PubMed  PubMed Central  Google Scholar 

  60. Leveille N, Melo CA, Rooijers K, Diaz-Lagares A, Melo SA, Korkmaz G, et al. Genome-wide profiling of p53-regulated enhancer RNAs uncovers a subset of enhancers controlled by a lncRNA. Nat Commun. 2015;6:6520.

    CAS  PubMed  Google Scholar 

  61. Allen MA, Andrysik Z, Dengler VL, Mellert HS, Guarnieri A, Freeman JA, et al. Global analysis of p53-regulated transcription identifies its direct targets and unexpected regulatory mechanisms. Elife. 2014;3: e02200.

    PubMed  PubMed Central  Google Scholar 

  62. Lai F, Gardini A, Zhang A, Shiekhattar R. Integrator mediates the biogenesis of enhancer RNAs. Nature. 2015;525(7569):399–403.

    CAS  PubMed  PubMed Central  Google Scholar 

  63. Hah N, Benner C, Chong LW, Yu RT, Downes M, Evans RM. Inflammation-sensitive super enhancers form domains of coordinately regulated enhancer RNAs. Proc Natl Acad Sci U S A. 2015;112(3):E297-302.

    CAS  PubMed  PubMed Central  Google Scholar 

  64. Hah N, Murakami S, Nagari A, Danko CG, Kraus WL. Enhancer transcripts mark active estrogen receptor binding sites. Genome Res. 2013;23(8):1210–23.

    CAS  PubMed  PubMed Central  Google Scholar 

  65. Mahat DB, Kwak H, Booth GT, Jonkers IH, Danko CG, Patel RK, et al. Base-pair-resolution genome-wide mapping of active RNA polymerases using precision nuclear run-on (PRO-seq). Nat Protoc. 2016;11(8):1455–76.

    PubMed  PubMed Central  Google Scholar 

  66. Core LJ, Martins AL, Danko CG, Waters CT, Siepel A, Lis JT. Analysis of nascent RNA identifies a unified architecture of initiation regions at mammalian promoters and enhancers. Nat Genet. 2014;46(12):1311–20.

    CAS  PubMed  PubMed Central  Google Scholar 

  67. Pnueli L, Rudnizky S, Yosefzon Y, Melamed P. RNA transcribed from a distal enhancer is required for activating the chromatin at the promoter of the gonadotropin alpha-subunit gene. Proc Natl Acad Sci U S A. 2015;112(14):4369–74.

    CAS  PubMed  PubMed Central  Google Scholar 

  68. Ne II, Heward JA, Roux B, Tsitsiou E, Fenwick PS, Lenzi L, et al. Long non-coding RNAs and enhancer RNAs regulate the lipopolysaccharide-induced inflammatory response in human monocytes. Nat Commun. 2014;5:3979.

    Google Scholar 

  69. Zhao Y, Wang L, Ren S, Wang L, Blackburn PR, McNulty MS, et al. Activation of P-TEFb by androgen receptor-regulated enhancer RNAs in castration-resistant prostate cancer. Cell Rep. 2016;15(3):599–610.

    CAS  PubMed  PubMed Central  Google Scholar 

  70. Tsai PF, Dell’Orso S, Rodriguez J, Vivanco KO, Ko KD, Jiang K, et al. A muscle-specific enhancer RNA mediates cohesin recruitment and regulates transcription in trans. Mol Cell. 2018;71(1):129-41 e8.

    CAS  PubMed  PubMed Central  Google Scholar 

  71. Zhang Z, Lee JH, Ruan H, Ye Y, Krakowiak J, Hu Q, et al. Transcriptional landscape and clinical utility of enhancer RNAs for eRNA-targeted therapy in cancer. Nat Commun. 2019;10(1):4562.

    PubMed  PubMed Central  Google Scholar 

  72. Jiao W, Chen Y, Song H, Li D, Mei H, Yang F, et al. HPSE enhancer RNA promotes cancer progression through driving chromatin looping and regulating hnRNPU/p300/EGR1/HPSE axis. Oncogene. 2018;37(20):2728–45.

    CAS  PubMed  Google Scholar 

  73. Carullo NVN, Phillips Iii RA, Simon RC, Soto SAR, Hinds JE, Salisbury AJ, et al. Enhancer RNAs predict enhancer-gene regulatory links and are critical for enhancer function in neuronal systems. Nucleic Acids Res. 2020;48(17):9550–70.

    CAS  PubMed  PubMed Central  Google Scholar 

  74. Banerjee AR, Kim YJ, Kim TH. A novel virus-inducible enhancer of the interferon-beta gene with tightly linked promoter and enhancer activities. Nucleic Acids Res. 2014;42(20):12537–54.

    PubMed  PubMed Central  Google Scholar 

  75. Adelman K, Lis JT. Promoter-proximal pausing of RNA polymerase II: emerging roles in metazoans. Nat Rev Genet. 2012;13(10):720–31.

    CAS  PubMed  PubMed Central  Google Scholar 

  76. Gilchrist DA, Fromm G, dos Santos G, Pham LN, McDaniel IE, Burkholder A, et al. Regulating the regulators: the pervasive effects of Pol II pausing on stimulus-responsive gene networks. Genes Dev. 2012;26(9):933–44.

    CAS  PubMed  PubMed Central  Google Scholar 

  77. Missra A, Gilmour DS. Interactions between DSIF (DRB sensitivity inducing factor), NELF (negative elongation factor), and the Drosophila RNA polymerase II transcription elongation complex. Proc Natl Acad Sci U S A. 2010;107(25):11301–6.

    CAS  PubMed  PubMed Central  Google Scholar 

  78. Fujinaga K, Irwin D, Huang Y, Taube R, Kurosu T, Peterlin BM. Dynamics of human immunodeficiency virus transcription: P-TEFb phosphorylates RD and dissociates negative effectors from the transactivation response element. Mol Cell Biol. 2004;24(2):787–95.

    CAS  PubMed  PubMed Central  Google Scholar 

  79. Schaukowitch K, Joo JY, Liu X, Watts JK, Martinez C, Kim TK. Enhancer RNA facilitates NELF release from immediate early genes. Mol Cell. 2014;56(1):29–42.

    CAS  PubMed  PubMed Central  Google Scholar 

  80. Sigova AA, Abraham BJ, Ji X, Molinie B, Hannett NM, Guo YE, et al. Transcription factor trapping by RNA in gene regulatory elements. Science. 2015;350(6263):978–81.

    CAS  PubMed  PubMed Central  Google Scholar 

  81. Kanno T, Kanno Y, LeRoy G, Campos E, Sun HW, Brooks SR, et al. BRD4 assists elongation of both coding and enhancer RNAs by interacting with acetylated histones. Nat Struct Mol Biol. 2014;21(12):1047–57.

    CAS  PubMed  PubMed Central  Google Scholar 

  82. Bisgrove DA, Mahmoudi T, Henklein P, Verdin E. Conserved P-TEFb-interacting domain of BRD4 inhibits HIV transcription. Proc Natl Acad Sci U S A. 2007;104(34):13690–5.

    CAS  PubMed  PubMed Central  Google Scholar 

  83. Baillat D, Hakimi MA, Naar AM, Shilatifard A, Cooch N, Shiekhattar R. Integrator, a multiprotein mediator of small nuclear RNA processing, associates with the C-terminal repeat of RNA polymerase II. Cell. 2005;123(2):265–76.

    CAS  PubMed  Google Scholar 

  84. Austenaa LM, Barozzi I, Simonatto M, Masella S, Della Chiara G, Ghisletti S, et al. Transcription of mammalian cis-regulatory elements is restrained by actively enforced early termination. Mol Cell. 2015;60(3):460–74.

    CAS  PubMed  Google Scholar 

  85. Houseley J, LaCava J, Tollervey D. RNA-quality control by the exosome. Nat Rev Mol Cell Biol. 2006;7(7):529–39.

    CAS  PubMed  Google Scholar 

  86. Januszyk K, Lima CD. Structural components and architectures of RNA exosomes. Adv Exp Med Biol. 2011;702:9–28.

    PubMed  Google Scholar 

  87. Liu Q, Greimann JC, Lima CD. Reconstitution, activities, and structure of the eukaryotic RNA exosome. Cell. 2006;127(6):1223–37.

    CAS  PubMed  Google Scholar 

  88. Lorentzen E, Basquin J, Tomecki R, Dziembowski A, Conti E. Structure of the active subunit of the yeast exosome core, Rrp44: diverse modes of substrate recruitment in the RNase II nuclease family. Mol Cell. 2008;29(6):717–28.

    CAS  PubMed  Google Scholar 

  89. Aguilo F, Li S, Balasubramaniyan N, Sancho A, Benko S, Zhang F, et al. Deposition of 5-Methylcytosine on enhancer RNAs enables the coactivator function of PGC-1alpha. Cell Rep. 2016;14(3):479–92.

    CAS  PubMed  PubMed Central  Google Scholar 

  90. Lee JH, Wang R, Xiong F, Krakowiak J, Liao Z, Nguyen PT, et al. Enhancer RNA m6A methylation facilitates transcriptional condensate formation and gene activation. Mol Cell. 2021;81(16):3368-85 e9.

    CAS  PubMed  PubMed Central  Google Scholar 

  91. Xu W, He C, Kaye EG, Li J, Mu M, Nelson GM, et al. Dynamic control of chromatin-associated m(6)A methylation regulates nascent RNA synthesis. Mol Cell. 2022;82(6):1156-68 e7.

    CAS  PubMed  PubMed Central  Google Scholar 

  92. Hsieh CL, Fei T, Chen Y, Li T, Gao Y, Wang X, et al. Enhancer RNAs participate in androgen receptor-driven looping that selectively enhances gene activation. Proc Natl Acad Sci U S A. 2014;111(20):7319–24.

    CAS  PubMed  PubMed Central  Google Scholar 

  93. Stone JK, Vukadin L, Ahn EE. eNEMAL, an enhancer RNA transcribed from a distal MALAT1 enhancer, promotes NEAT1 long isoform expression. PLoS ONE. 2021;16(5): e0251515.

    CAS  PubMed  PubMed Central  Google Scholar 

  94. Hauberg ME, Fullard JF, Zhu L, Cohain AT, Giambartolomei C, Misir R, et al. Differential activity of transcribed enhancers in the prefrontal cortex of 537 cases with schizophrenia and controls. Mol Psychiatry. 2019;24(11):1685–95.

    CAS  PubMed  Google Scholar 

  95. Greulich F, Bielefeld KA, Scheundel R, Mechtidou A, Strickland B, Uhlenhaut NH. Enhancer RNA expression in response to glucocorticoid treatment in murine macrophages. Cells. 2021;11(1):28.

    PubMed  PubMed Central  Google Scholar 

  96. Yao P, Lin P, Gokoolparsadh A, Assareh A, Thang MW, Voineagu I. Coexpression networks identify brain region-specific enhancer RNAs in the human brain. Nat Neurosci. 2015;18(8):1168–74.

    CAS  PubMed  Google Scholar 

  97. Bhattarai S, Akella A, Gandhi A, Dharap A. Modulation of brain pathology by enhancer RNAs in cerebral ischemia. Mol Neurobiol. 2021;58(4):1482–90.

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

None.

Funding

This study was supported by National Natural Scientific Foundation of China (No. 82072854; 82203248); Natural Science Foundation of Guangdong Province [No. 2022A1515012293]; the Project for Key Medicine Discipline Construction of Guangzhou Municipality, CHN [No. 2021–2023-17]; Science and Technology Projects in Guangzhou [No. 202201020093].

Author information

Authors and Affiliations

  1. Department of Obstetrics and Gynecology, Department of Gynecologic Oncology Research Office, Guangzhou Key Laboratory of Targeted Therapy for Gynecologic Oncology, Guangdong Provincial Key Laboratory of Major Obstetric Diseases, The Third Affiliated Hospital of Guangzhou Medical University, No.63 Duobao Road, Liwan District, Guangdong Province, Guangzhou City, 510150, People’s Republic of China

    Qianhui Li, Xin Liu, Jingtao Wen, Xi Chen, Bumin Xie & Yang Zhao

Authors
  1. Qianhui Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Xin Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jingtao Wen
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Xi Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Bumin Xie
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Yang Zhao
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

QHL, XL, JTW, XC, and BMX collected relevant information, QHL wrote the manuscript, YZ designed and revised the manuscript. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Yang Zhao.

Ethics declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Li, Q., Liu, X., Wen, J. et al. Enhancer RNAs: mechanisms in transcriptional regulation and functions in diseases. Cell Commun Signal 21, 191 (2023). https://doi.org/10.1186/s12964-023-01206-0

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1186/s12964-023-01206-0

Keywords

Cell Communication and Signaling

ISSN: 1478-811X