Noncoding RNAs (ncRNAs) direct a remarkable number of diverse functions in development and disease through their regulation of transcription, RNA processing and translation. Leading the charge in the RNA revolution is a class of ncRNAs that are synthesized at active enhancers, called enhancer RNAs (eRNAs). Here, we review recent insights into the biogenesis of eRNAs and the mechanisms underlying their multifaceted functions and consider how these findings could inform future investigations into enhancer transcription and eRNA function.
Subscribe to Journal
Get full journal access for 1 year
only $17.42 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Plank, J. L. & Dean, A. Enhancer function: mechanistic and genome-wide insights come together. Mol. Cell 55, 5–14 (2014).
Long, H. K., Prescott, S. L. & Wysocka, J. Ever-changing landscapes: transcriptional enhancers in development and evolution. Cell 167, 1170–1187 (2016).
Rahnamoun, H. et al. Mutant p53 shapes the enhancer landscape of cancer cells in response to chronic immune signaling. Nat. Commun. 8, 754 (2017).
Andersson, R. et al. An atlas of active enhancers across human cell types and tissues. Nature 507, 455–461 (2014).
Franco, H. L., Nagari, A. & Kraus, W. L. TNFα signaling exposes latent estrogen receptor binding sites to alter the breast cancer cell transcriptome. Mol. Cell 58, 21–34 (2015).
Lai, F., Gardini, A., Zhang, A. & Shiekhattar, R. Integrator mediates the biogenesis of enhancer RNAs. Nature 525, 399–403 (2015).
Lam, M. T. et al. Rev-Erbs repress macrophage gene expression by inhibiting enhancer-directed transcription. Nature 498, 511–515 (2013).
ENCODE Project Consortium. An integrated encyclopedia of DNA elements in the human genome. Nature 489, 57–74 (2012).
van Arensbergen, J. et al. High-throughput identification of human SNPs affecting regulatory element activity. Nat. Genet. 51, 1160–1169 (2019).
Schoenfelder, S. & Fraser, P. Long-range enhancer-promoter contacts in gene expression control. Nat. Rev. Genet. 20, 437–455 (2019).
Villar, D. et al. Enhancer evolution across 20 mammalian species. Cell 160, 554–566 (2015).
Arnold, C. D. et al. Quantitative genome-wide enhancer activity maps for five Drosophila species show functional enhancer conservation and turnover during cis-regulatory evolution. Nat. Genet. 46, 685–692 (2014).
Schmidt, D. et al. Five-vertebrate ChIP-seq reveals the evolutionary dynamics of transcription factor binding. Science 328, 1036–1040 (2010).
Blow, M. J. et al. ChIP-seq identification of weakly conserved heart enhancers. Nat. Genet. 42, 806–810 (2010).
May, D. et al. Large-scale discovery of enhancers from human heart tissue. Nat. Genet. 44, 89–93 (2011).
Yang, S. et al. Functionally conserved enhancers with divergent sequences in distant vertebrates. BMC Genomics 16, 882 (2015).
Chatterjee, S., Bourque, G. & Lufkin, T. Conserved and non-conserved enhancers direct tissue specific transcription in ancient germ layer specific developmental control genes. BMC Dev. Biol. 11, 63 (2011).
Hare, E. E., Peterson, B. K., Iyer, V. N., Meier, R. & Eisen, M. B. Sepsid even-skipped enhancers are functionally conserved in Drosophila despite lack of sequence conservation. PLoS Genet. 4, e1000106 (2008).
Beagrie, R. A. & Pombo, A. Gene activation by metazoan enhancers: diverse mechanisms stimulate distinct steps of transcription. Bioessays 38, 881–893 (2016).
Furlong, E. E. M. & Levine, M. Developmental enhancers and chromosome topology. Science 361, 1341–1345 (2018).
Catarino, R. R. & Stark, A. Assessing sufficiency and necessity of enhancer activities for gene expression and the mechanisms of transcription activation. Genes Dev. 32, 202–223 (2018).
Krivega, I. & Dean, A. Enhancer and promoter interactions—long distance calls. Curr. Opin. Genet. Dev. 22, 79–85 (2012).
Deng, W. et al. Controlling long-range genomic interactions at a native locus by targeted tethering of a looping factor. Cell 149, 1233–1244 (2012).
Bartman, C. R., Hsu, S. C., Hsiung, C. C.-S., Raj, A. & Blobel, G. A. Enhancer regulation of transcriptional bursting parameters revealed by forced chromatin looping. Mol. Cell 62, 237–247 (2016).
Sawado, T., Halow, J., Bender, M. A. & Groudine, M. The β-globin locus control region (LCR) functions primarily by enhancing the transition from transcription initiation to elongation. Genes Dev. 17, 1009–1018 (2003).
Song, S.-H. et al. Multiple functions of Ldb1 required for β-globin activation during erythroid differentiation. Blood 116, 2356–2364 (2010).
Li, W. et al. Functional roles of enhancer RNAs for oestrogen-dependent transcriptional activation. Nature 498, 516–520 (2013).
Rahnamoun, H. et al. RNAs interact with BRD4 to promote enhanced chromatin engagement and transcription activation. Nat. Struct. Mol. Biol. 25, 687–697 (2018).
Tsai, P.-F. et al. A muscle-specific enhancer RNA mediates cohesin recruitment and regulates transcription in trans. Mol. Cell 71, 129–141.e8 (2018).
Schaukowitch, K. et al. Enhancer RNA facilitates NELF release from immediate early genes. Mol. Cell 56, 29–42 (2014).
Lai, F. et al. Activating RNAs associate with Mediator to enhance chromatin architecture and transcription. Nature 494, 497–501 (2013).
Ivaldi, M. S. et al. Fetal γ-globin genes are regulated by the BGLT3 long noncoding RNA locus. Blood 132, 1963–1973 (2018).
De Santa, F. et al. A large fraction of extragenic RNA Pol II transcription sites overlap enhancers. PLoS Biol. 8, e1000384 (2010).
Kim, T.-K. et al. Widespread transcription at neuronal activity-regulated enhancers. Nature 465, 182–187 (2010).
Franco, H. L. et al. Enhancer transcription reveals subtype-specific gene expression programs controlling breast cancer pathogenesis. Genome Res. 28, 159–170 (2018).
Hah, N., Murakami, S., Nagari, A., Danko, C. G. & Kraus, W. L. Enhancer transcripts mark active estrogen receptor binding sites. Genome Res. 23, 1210–1223 (2013).
Core, L. J. et al. Analysis of nascent RNA identifies a unified architecture of initiation regions at mammalian promoters and enhancers. Nat. Genet. 46, 1311–1320 (2014).
Hah, N. et al. Inflammation-sensitive super enhancers form domains of coordinately regulated enhancer RNAs. Proc. Natl Acad. Sci. USA 112, E297–E302 (2015).
Hah, N. et al. A rapid, extensive, and transient transcriptional response to estrogen signaling in breast cancer cells. Cell 145, 622–634 (2011).
Kwak, H., Fuda, N. J., Core, L. J. & Lis, J. T. Precise maps of RNA polymerase reveal how promoters direct initiation and pausing. Science 339, 950–953 (2013).
Mahat, D. B. et al. Base-pair-resolution genome-wide mapping of active RNA polymerases using precision nuclear run-on (PRO-seq). Nat. Protoc. 11, 1455–1476 (2016).
Carninci, P. et al. Genome-wide analysis of mammalian promoter architecture and evolution. Nat. Genet. 38, 626–635 (2006).
Shiraki, T. et al. Cap analysis gene expression for high-throughput analysis of transcriptional starting point and identification of promoter usage. Proc. Natl Acad. Sci. USA 100, 15776–15781 (2003).
Arner, E. et al. Transcribed enhancers lead waves of coordinated transcription in transitioning mammalian cells. Science 347, 1010–1014 (2015).
Meers, M. P. et al. Transcription start site profiling uncovers divergent transcription and enhancer-associated RNAs in Drosophila melanogaster. BMC Genomics 19, 157 (2018).
Henriques, T. et al. Widespread transcriptional pausing and elongation control at enhancers. Genes Dev. 32, 26–41 (2018).
Chen, R. A.-J. et al. The landscape of RNA polymerase II transcription initiation in C. elegans reveals promoter and enhancer architectures. Genome Res. 23, 1339–1347 (2013).
Chepelev, I., Wei, G., Wangsa, D., Tang, Q. & Zhao, K. Characterization of genome-wide enhancer-promoter interactions reveals co-expression of interacting genes and modes of higher order chromatin organization. Cell Res. 22, 490–503 (2012).
Heintzman, N. D. et al. Histone modifications at human enhancers reflect global cell-type-specific gene expression. Nature 459, 108–112 (2009).
Creyghton, M. P. et al. Histone H3K27ac separates active from poised enhancers and predicts developmental state. Proc. Natl Acad. Sci. USA 107, 21931–21936 (2010).
Rada-Iglesias, A. et al. A unique chromatin signature uncovers early developmental enhancers in humans. Nature 470, 279–283 (2011).
Wang, D. et al. Reprogramming transcription by distinct classes of enhancers functionally defined by eRNA. Nature 474, 390–394 (2011).
Mikhaylichenko, O. et al. The degree of enhancer or promoter activity is reflected by the levels and directionality of eRNA transcription. Genes Dev. 32, 42–57 (2018).
Pekowska, A. et al. H3K4 tri-methylation provides an epigenetic signature of active enhancers. EMBO J. 30, 4198–4210 (2011).
Koch, F. et al. Transcription initiation platforms and GTF recruitment at tissue-specific enhancers and promoters. Nat. Struct. Mol. Biol. 18, 956–963 (2011).
Natoli, G. & Andrau, J. C. Noncoding transcription at enhancers: general principles and functional models. Annu. Rev. Genet. 46, 1–19 (2012).
Flynn, R. A., Almada, A. E., Zamudio, J. R. & Sharp, P. A. Antisense RNA polymerase II divergent transcripts are P-TEFb dependent and substrates for the RNA exosome. Proc. Natl Acad. Sci. USA 108, 10460–10465 (2011).
Scruggs, B. S. et al. Bidirectional transcription arises from two distinct hubs of transcription factor binding and active chromatin. Mol. Cell 58, 1101–1112 (2015).
Preker, P. et al. PROMoter uPstream Transcripts share characteristics with mRNAs and are produced upstream of all three major types of mammalian promoters. Nucleic Acids Res. 39, 7179–7193 (2011).
Kowalczyk, M. S. et al. Intragenic enhancers act as alternative promoters. Mol. Cell 45, 447–458 (2012).
Andersson, R. & Sandelin, A. Determinants of enhancer and promoter activities of regulatory elements. Nat. Rev. Genet. 21, 71–87 (2020).
Kim, T. K. & Shiekhattar, R. Architectural and functional commonalities between enhancers and promoters. Cell 162, 948–959 (2015).
Hirose, Y. & Manley, J. L. RNA polymerase II and the integration of nuclear events. Genes Dev. 14, 1415–1429 (2000).
Proudfoot, N. J., Furger, A. & Dye, M. J. Integrating mRNA processing with transcription. Cell 108, 501–512 (2002).
Shatkin, A. J. & Manley, J. L. The ends of the affair: capping and polyadenylation. Nat. Struct. Biol. 7, 838–842 (2000).
Hintermair, C. et al. Threonine-4 of mammalian RNA polymerase II CTD is targeted by Polo-like kinase 3 and required for transcriptional elongation. EMBO J. 31, 2784–2797 (2012).
Hsin, J. P. & Manley, J. L. The RNA polymerase II CTD coordinates transcription and RNA processing. Genes Dev. 26, 2119–2137 (2012).
Mayer, A. et al. CTD tyrosine phosphorylation impairs termination factor recruitment to RNA polymerase II. Science 336, 1723–1725 (2012).
Corden, J. L. Tails of RNA polymerase II. Trends Biochem. Sci. 15, 383–387 (1990).
West, M. L. & Corden, J. L. Construction and analysis of yeast RNA polymerase II CTD deletion and substitution mutations. Genetics 140, 1223–1233 (1995).
Eick, D. & Geyer, M. The RNA polymerase II carboxy-terminal domain (CTD) code. Chem. Rev. 113, 8456–8490 (2013).
Bonn, S. et al. Tissue-specific analysis of chromatin state identifies temporal signatures of enhancer activity during embryonic development. Nat. Genet. 44, 148–156 (2012).
Descostes, N. et al. Tyrosine phosphorylation of RNA polymerase II CTD is associated with antisense promoter transcription and active enhancers in mammalian cells. Elife 3, e02105 (2014).
Hsin, J. P., Li, W., Hoque, M., Tian, B. & Manley, J. L. RNAP II CTD tyrosine 1 performs diverse functions in vertebrate cells. Elife 3, e02112 (2014).
Bentley, D. L. Coupling mRNA processing with transcription in time and space. Nat. Rev. Genet. 15, 163–175 (2014).
Shah, N. et al. Tyrosine-1 of RNA polymerase II CTD controls global termination of gene transcription in mammals. Mol. Cell 69, 48–61.e6 (2018).
Nojima, T. et al. Deregulated expression of mammalian lncRNA through loss of SPT6 induces R-loop formation, replication stress, and cellular senescence. Mol. Cell 72, 970–984.e7 (2018).
Wang, A. H. et al. The elongation factor Spt6 maintains ESC pluripotency by controlling super-enhancers and counteracting Polycomb proteins. Mol. Cell 68, 398–413.e6 (2017).
Lin, C., Garruss, A. S., Luo, Z., Guo, F. & Shilatifard, A. The RNA Pol II elongation factor Ell3 marks enhancers in ES cells and primes future gene activation. Cell 152, 144–156 (2013).
Gil, N. & Ulitsky, I. Production of spliced long noncoding RNAs specifies regions with increased enhancer activity. Cell Syst. 7, 537–547.e3 (2018).
Vučićević, D., Corradin, O., Ntini, E., Scacheri, P. C. & Ørom, U. A. Long ncRNA expression associates with tissue-specific enhancers. Cell Cycle 14, 253–260 (2015).
Core, L. J. et al. Defining the status of RNA polymerase at promoters. Cell Rep. 2, 1025–1035 (2012).
Kaikkonen, M. U. et al. Remodeling of the enhancer landscape during macrophage activation is coupled to enhancer transcription. Mol. Cell 51, 310–325 (2013).
Andersson, R. et al. Nuclear stability and transcriptional directionality separate functionally distinct RNA species. Nat. Commun. 5, 5336 (2014).
Pefanis, E. et al. RNA exosome-regulated long non-coding RNA transcription controls super-enhancer activity. Cell 161, 774–789 (2015).
Lubas, M. et al. The human nuclear exosome targeting complex is loaded onto newly synthesized RNA to direct early ribonucleolysis. Cell Rep. 10, 178–192 (2015).
Austenaa, L. M. et al. Transcription of mammalian cis-regulatory elements is restrained by actively enforced early termination. Mol. Cell 60, 460–474 (2015).
Ulitsky, I. & Bartel, D. P. lincRNAs: genomics, evolution, and mechanisms. Cell 154, 26–46 (2013).
Guttman, M. & Rinn, J. L. Modular regulatory principles of large non-coding RNAs. Nature 482, 339–346 (2012).
Cabili, M. N. et al. Integrative annotation of human large intergenic noncoding RNAs reveals global properties and specific subclasses. Genes Dev. 25, 1915–1927 (2011).
Almada, A. E., Wu, X., Kriz, A. J., Burge, C. B. & Sharp, P. A. Promoter directionality is controlled by U1 snRNP and polyadenylation signals. Nature 499, 360–363 (2013).
Paralkar, V. R. et al. Unlinking an lncRNA from its associated cis element. Mol. Cell 62, 104–110 (2016).
Groff, A. F. et al. In vivo characterization of Linc-p21 reveals functional cis-regulatory DNA elements. Cell Rep. 16, 2178–2186 (2016).
Kopp, F. & Mendell, J. T. Functional classification and experimental dissection of long noncoding RNAs. Cell 172, 393–407 (2018).
Alvarez-Dominguez, J. R., Knoll, M., Gromatzky, A. A. & Lodish, H. F. The super-enhancer-derived alncRNA-EC7/Bloodlinc potentiates red blood cell development in trans. Cell Rep. 19, 2503–2514 (2017).
Espinosa, J. M. On the origin of lncRNAs: missing link found. Trends Genet. 33, 660–662 (2017).
Cinghu, S. et al. Intragenic enhancers attenuate host gene expression. Mol. Cell 68, 104–117.e6 (2017).
Shechner, D. M., Hacisuleyman, E., Younger, S. T. & Rinn, J. L. Multiplexable, locus-specific targeting of long RNAs with CRISPR-Display. Nat. Methods 12, 664–670 (2015).
Lee, J. S. & Mendell, J. T. Antisense-mediated transcript knockdown triggers premature transcription termination. Mol. Cell 77, 1044–1054.e3 (2020).
Lai, F., Damle, S. S., Ling, K. K. & Rigo, F. Directed RNase H cleavage of nascent transcripts causes transcription termination. Mol. Cell 77, 1032–1043.e4 (2020).
Yang, Y. et al. Enhancer RNA-driven looping enhances the transcription of the long noncoding RNA DHRS4-AS1, a controller of the DHRS4 gene cluster. Sci. Rep. 6, 20961 (2016).
Chu, C., Qu, K., Zhong, F. L., Artandi, S. E. & Chang, H. Y. Genomic maps of long noncoding RNA occupancy reveal principles of RNA-chromatin interactions. Mol. Cell 44, 667–678 (2011).
Femino, A. M., Fay, F. S., Fogarty, K. & Singer, R. H. Visualization of single RNA transcripts in situ. Science 280, 585–590 (1998).
Bose, D. A. et al. RNA binding to CBP stimulates histone acetylation and transcription. Cell 168, 135–149.e22 (2017).
Hsieh, C.-L. et al. Enhancer RNAs participate in androgen receptor-driven looping that selectively enhances gene activation. Proc. Natl Acad. Sci. USA 111, 7319–7324 (2014).
Mousavi, K. et al. eRNAs promote transcription by establishing chromatin accessibility at defined genomic loci. Mol. Cell 51, 606–617 (2013).
Sigova, A. A. et al. Transcription factor trapping by RNA in gene regulatory elements. Science 350, 978–981 (2015).
Zhao, Y. et al. Activation of P-TEFb by androgen receptor-regulated enhancer RNAs in castration-resistant prostate cancer. Cell Rep. 15, 599–610 (2016).
Tan, S. H. et al. The enhancer RNA ARIEL activates the oncogenic transcriptional program in T-cell acute lymphoblastic leukemia. Blood 134, 239–251 (2019).
Aguilera, A. & García-Muse, T. R loops: from transcription byproducts to threats to genome stability. Mol. Cell 46, 115–124 (2012).
Meng, F. L. et al. Convergent transcription at intragenic super-enhancers targets AID-initiated genomic instability. Cell 159, 1538–1548 (2014).
Aguilo, F. et al. Deposition of 5-methylcytosine on enhancer RNAs enables the coactivator function of PGC-1α. Cell Rep. 14, 479–492 (2016).
Maris, C., Domínguez, C. & Allain, F. H. The RNA recognition motif, a plastic RNA-binding platform to regulate post-transcriptional gene expression. FEBS J. 272, 2118–2131 (2005).
Grishin, N. V. KH domain: one motif, two folds. Nucleic Acids Res. 29, 638–643 (2001).
Chang, K. Y. & Ramos, A. The double-stranded RNA-binding motif, a versatile macromolecular docking platform. FEBS J. 272, 2109–2117 (2005).
Nair, S. J. et al. Phase separation of ligand-activated enhancers licenses cooperative chromosomal enhancer assembly. Nat. Struct. Mol. Biol. 26, 193–203 (2019).
Banani, S. F., Lee, H. O., Hyman, A. A. & Rosen, M. K. Biomolecular condensates: organizers of cellular biochemistry. Nat. Rev. Mol. Cell Biol. 18, 285–298 (2017).
Ruthenburg, A. J., Li, H., Patel, D. J. & Allis, C. D. Multivalent engagement of chromatin modifications by linked binding modules. Nat. Rev. Mol. Cell Biol. 8, 983–994 (2007).
Hnisz, D., Shrinivas, K., Young, R. A., Chakraborty, A. K. & Sharp, P. A. A phase separation model for transcriptional control. Cell 169, 13–23 (2017).
Polymenidou, M. The RNA face of phase separation. Science 360, 859–860 (2018).
Boija, A. et al. Transcription factors activate genes through the phase-separation capacity of their activation domains. Cell 175, 1842–1855.e16 (2018).
Sabari, B. R. et al. Coactivator condensation at super-enhancers links phase separation and gene control. Science 361, eaar3958 (2018).
Boeynaems, S. et al. Protein phase separation: a new phase in cell biology. Trends Cell Biol. 28, 420–435 (2018).
We thank Jim Kadonaga for reading the manuscript and providing helpful suggestions. Research in the Lauberth laboratory is supported by a grant from the NIH/National Institute of General Medical Sciences (R35 GM128900) to S.L., and research in the Sartorelli laboratory is supported by the National Institute of Arthritis and Musculoskeletal and Skin Diseases Intramural Research Program of the NIH (grants AR041126 and AR041164).
The authors declare no competing interests.
Peer review information Beth Moorefield was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
About this article
Cite this article
Sartorelli, V., Lauberth, S.M. Enhancer RNAs are an important regulatory layer of the epigenome. Nat Struct Mol Biol 27, 521–528 (2020). https://doi.org/10.1038/s41594-020-0446-0