Functional roles of enhancer RNAs for oestrogen-dependent transcriptional activation


This article has been updated


The functional importance of gene enhancers in regulated gene expression is well established1,2,3. In addition to widespread transcription of long non-coding RNAs (lncRNAs) in mammalian cells4,5,6, bidirectional ncRNAs are transcribed on enhancers, and are thus referred to as enhancer RNAs (eRNAs)7,8,9. However, it has remained unclear whether these eRNAs are functional or merely a reflection of enhancer activation. Here we report that in human breast cancer cells 17β-oestradiol (E2)-bound oestrogen receptor α (ER-α) causes a global increase in eRNA transcription on enhancers adjacent to E2-upregulated coding genes. These induced eRNAs, as functional transcripts, seem to exert important roles for the observed ligand-dependent induction of target coding genes, increasing the strength of specific enhancer–promoter looping initiated by ER-α binding. Cohesin, present on many ER-α-regulated enhancers even before ligand treatment, apparently contributes to E2-dependent gene activation, at least in part by stabilizing E2/ER-α/eRNA-induced enhancer–promoter looping. Our data indicate that eRNAs are likely to have important functions in many regulated programs of gene transcription.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: E2 induction of eRNA in MCF-7 breast cancer cells.
Figure 2: Importance of eRNA for target gene activation.
Figure 3: Ligand-induced eRNA is functionally important.
Figure 4: Role of eRNA in cohesin-dependent gene activation.

Accession codes


Gene Expression Omnibus

Data deposits

The sequencing data sets are deposited in the Gene Expression Omnibus database under accession GSE45822.

Change history

  • 04 June 2013

    The PDF was corrected to remove two duplicated references from the Methods reference list.


  1. 1

    Newman, J. J. & Young, R. A. Connecting transcriptional control to chromosome structure and human disease. Cold Spring Harb. Symp. Quant. Biol. 75, 227–235 (2010)

    CAS  Article  Google Scholar 

  2. 2

    Bulger, M. & Groudine, M. Functional and mechanistic diversity of distal transcription enhancers. Cell 144, 327–339 (2011)

    CAS  Article  Google Scholar 

  3. 3

    Ong, C. T. & Corces, V. G. Enhancer function: new insights into the regulation of tissue-specific gene expression. Nature Rev. Genet. 12, 283–293 (2011)

    CAS  Article  Google Scholar 

  4. 4

    Guttman, M. & Rinn, J. L. Modular regulatory principles of large non-coding RNAs. Nature 482, 339–346 (2012)

    ADS  CAS  Article  Google Scholar 

  5. 5

    Wang, K. C. & Chang, H. Y. Molecular mechanisms of long noncoding RNAs. Mol. Cell 43, 904–914 (2011)

    CAS  Article  Google Scholar 

  6. 6

    Mercer, T. R., Dinger, M. E. & Mattick, J. S. Long non-coding RNAs: insights into functions. Nature Rev. Genet. 10, 155–159 (2009)

    CAS  Article  Google Scholar 

  7. 7

    Kim, T.-K. et al. Widespread transcription at neuronal activity-regulated enhancers. Nature 465, 182–187 (2010)

    ADS  CAS  Article  Google Scholar 

  8. 8

    Hah, N. et al. A rapid, extensive, and transient transcriptional response to estrogen signaling in breast cancer cells. Cell 145, 622–634 (2011)

    CAS  Article  Google Scholar 

  9. 9

    Wang, D. et al. Reprogramming transcription by distinct classes of enhancers functionally defined by eRNA. Nature 474, 390–394 (2011)

    CAS  Article  Google Scholar 

  10. 10

    Welboren, W. J. et al. ChIP-Seq of ERα and RNA polymerase II defines genes differentially responding to ligands. EMBO J. 28, 1418–1428 (2009)

    CAS  Article  Google Scholar 

  11. 11

    Carroll, J. S. et al. Genome-wide analysis of estrogen receptor binding sites. Nature Genet. 38, 1289–1297 (2006)

    CAS  Article  Google Scholar 

  12. 12

    Kwon, Y. S. et al. Sensitive ChIP-DSL technology reveals an extensive estrogen receptor α-binding program on human gene promoters. Proc. Natl Acad. Sci. USA 104, 4852–4857 (2007)

    ADS  CAS  Article  Google Scholar 

  13. 13

    Heintzman, N. D. & Ren, B. Finding distal regulatory elements in the human genome. Curr. Opin. Genet. Dev. 19, 541–549 (2009)

    CAS  Article  Google Scholar 

  14. 14

    Heintzman, N. D. et al. Histone modifications at human enhancers reflect global cell-type-specific gene expression. Nature 459, 108–112 (2009)

    ADS  CAS  Article  Google Scholar 

  15. 15

    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)

    ADS  CAS  Article  Google Scholar 

  16. 16

    Ahlenstiel, C. L. et al. Direct evidence of nuclear Argonaute distribution during transcriptional silencing links the actin cytoskeleton to nuclear RNAi machinery in human cells. Nucleic Acids Res. 40, 1579–1595 (2012)

    CAS  Article  Google Scholar 

  17. 17

    Mayer, C., Schmitz, K. M., Li, J., Grummt, I. & Santoro, R. Intergenic transcripts regulate the epigenetic state of rRNA genes. Mol. Cell 5, 351–361 (2006)

    Article  Google Scholar 

  18. 18

    Wang, K. C. et al. A long noncoding RNA maintains active chromatin to coordinate homeotic gene expression. Nature 472, 120–124 (2011)

    ADS  CAS  Article  Google Scholar 

  19. 19

    Melo, C. A. et al. eRNAs are required for p53-dependent enhancer activity and gene transcription. Mol. Cell 49, 524–535 (2013)

    CAS  Article  Google Scholar 

  20. 20

    Lai, F. et al. Activating RNAs associate with Mediator to enhance chromatin architecture and transcription. Nature 494, 497–501 (2013)

    ADS  CAS  Article  Google Scholar 

  21. 21

    Fullwood, M. J. et al. An oestrogen-receptor-α-bound human chromatin interactome. Nature 462, 58–64 (2009)

    ADS  CAS  Article  Google Scholar 

  22. 22

    Harismendy, O. et al. 9p21 DNA variants associated with coronary artery disease impair interferon-γ signalling response. Nature 470, 264–268 (2011)

    ADS  CAS  Article  Google Scholar 

  23. 23

    Sanyal, A., Lajoie, B. R., Jain, G. & Dekker, J. The long-range interaction landscape of gene promoters. Nature 489, 109–113 (2012)

    ADS  CAS  Article  Google Scholar 

  24. 24

    Lieberman-Aiden, E. et al. Comprehensive mapping of long-range interactions reveals folding principles of the human genome. Science 326, 289–293 (2009)

    ADS  CAS  Article  Google Scholar 

  25. 25

    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)

    CAS  Article  Google Scholar 

  26. 26

    Hadjur, S. et al. Cohesins form chromosomal cis-interactions at the developmentally regulated IFNG locus. Nature 460, 410–413 (2009)

    ADS  CAS  Article  Google Scholar 

  27. 27

    Kagey, M. H. et al. Mediator and cohesin connect gene expression and chromatin architecture. Nature 467, 430–435 (2010)

    ADS  CAS  Article  Google Scholar 

  28. 28

    Schmidt, D. et al. A CTCF-independent role for cohesin in tissue-specific transcription. Genome Res. 20, 578–588 (2010)

    CAS  Article  Google Scholar 

  29. 29

    Cai, S. & Kohwi-Shigematsu, T. Intranuclear relocalization of matrix binding sites during T cell activation detected by amplified fluorescence in situ hybridization. Methods 19, 394–402 (1999)

    CAS  Article  Google Scholar 

  30. 30

    Core, L. J., Waterfall, J. J. & Lis, J. T. Nascent RNA sequencing reveals widespread pausing and divergent initiation at human promoters. Science 322, 1845–1848 (2008)

    ADS  CAS  Article  Google Scholar 

  31. 31

    Abukhdeir, A. M. et al. Physiologic estrogen receptor α signaling in non-tumorigenic human mammary epithelial cells. Breast Cancer Res. Treat. 99, 23–33 (2006)

    CAS  Article  Google Scholar 

  32. 32

    Heinz, S. et al. Simple combinations of lineage-determining transcription factors prime cis-regulatory elements required for macrophage and B cell identities. Mol. Cell 38, 576–589 (2010)

    CAS  Article  Google Scholar 

  33. 33

    Ingolia, N. T. et al. Genome-wide analysis in vivo of translation with nucleotide resolution using ribosome profiling. Science 324, 218–223 (2009)

    ADS  CAS  Article  Google Scholar 

  34. 34

    White, A. K. et al. High-throughput microfluidic single-cell RT-qPCR. Proc. Natl Acad. Sci. USA 108, 13999–14004 (2011)

    ADS  CAS  Article  Google Scholar 

  35. 35

    Zhong, J. F. et al. A microfluidic processor for gene expression profiling of single human embryonic stem cells. Lab Chip 8, 68–74 (2008)

    CAS  Article  Google Scholar 

  36. 36

    Tsai, M. C. et al. Long non-coding RNA as modular scaffold of histone modification complexes. Science 329, 689–693 (2010)

    ADS  CAS  Article  Google Scholar 

  37. 37

    Rueden, C. T. et al. Visualization approaches for multidimensional biological image data. Biotechniques 43, 31–36 (2007)

    Article  Google Scholar 

  38. 38

    Lajoie, B. R. et al. My5C: web tools for chromosome conformation capture studies. Nature Methods 6, 690–691 (2009)

    CAS  Article  Google Scholar 

  39. 39

    Servant, N. et al. HiTC: exploration of high-throughput ‘C’ experiments. Bioinformatics 28, 2843–2844 (2012)

    CAS  Article  Google Scholar 

  40. 40

    Stadhouders, R. et al. Dynamic long-range chromatin interactions control Myb proto-oncogene transcription during erythroid development. EMBO J. 31, 986–999 (2011)

    Article  Google Scholar 

Download references


We thank K. Hutt for help with statistical analyses; M. Ghassemian from the University of California, San Diego (UCSD) Biomolecular/Proteomics Mass Spectrometry Facility for assistance with mass spectrometry; C. Nelson for cell culture assistance; J. Hightower for assistance with figure and manuscript preparation. We thank H. Chang for providing the BoxB, λN–GAL4 constructs. We acknowledge the UCSD Cancer Center Specialized Support Grant P30 CA23100 for confocal microscopy. W.L. and D.N. are supported by Department of Defense (DoD) postdoctoral fellowships, BC110381 and BC103858, respectively. M.G.R. is an investigator with the Howard Hughes Medical Institute. This work was supported by grants DK 039949, DK018477, NS034934, HL065445, CA173903 to C.K.G., and from the DoD.

Author information




M.G.R., W.L., D.N., E.N. and C.K.G. conceived the project. W.L. and D.N. performed most of the experiments reported, with contributions from E.N. and A.Y.C. (FISH). Q.M., B.T. and D.M. performed bioinformatic analyses. Q.M., E.N. and B.T. made equivalent contributions to this study. Additional experiments/methods were contributed by X.S., S.O. and H.-S.K. J.Z. and K.O. assisted in deep-sequencing library preparations and sequencing. W.L., D.N. and M.G.R. wrote the final paper with input from C.K.G.

Corresponding author

Correspondence to Michael G. Rosenfeld.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains Supplementary Figures 1-9 and Supplementary Tables 1-8. (PDF 3919 kb)

PowerPoint slides

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Li, W., Notani, D., Ma, Q. et al. Functional roles of enhancer RNAs for oestrogen-dependent transcriptional activation. Nature 498, 516–520 (2013).

Download citation

Further reading


By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.


Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing