Functional roles of enhancer RNAs for oestrogen-dependent transcriptional activation (original) (raw)

Nature volume 498, pages 516–520 (2013)Cite this article

Subjects

This article has been updated

Abstract

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.

This is a preview of subscription content, access via your institution

Access options

Subscribe to this journal

Receive 51 print issues and online access

$199.00 per year

only $3.90 per issue

Buy this article

Prices may be subject to local taxes which are calculated during checkout

Additional access options:

Similar content being viewed by others

Accession codes

Accessions

Gene Expression Omnibus

Data deposits

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

Change history

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

References

  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)
    Article CAS Google Scholar
  2. Bulger, M. & Groudine, M. Functional and mechanistic diversity of distal transcription enhancers. Cell 144, 327–339 (2011)
    Article CAS Google Scholar
  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)
    Article CAS Google Scholar
  4. Guttman, M. & Rinn, J. L. Modular regulatory principles of large non-coding RNAs. Nature 482, 339–346 (2012)
    Article ADS CAS Google Scholar
  5. Wang, K. C. & Chang, H. Y. Molecular mechanisms of long noncoding RNAs. Mol. Cell 43, 904–914 (2011)
    Article CAS Google Scholar
  6. Mercer, T. R., Dinger, M. E. & Mattick, J. S. Long non-coding RNAs: insights into functions. Nature Rev. Genet. 10, 155–159 (2009)
    Article CAS Google Scholar
  7. Kim, T.-K. et al. Widespread transcription at neuronal activity-regulated enhancers. Nature 465, 182–187 (2010)
    Article ADS CAS Google Scholar
  8. Hah, N. et al. A rapid, extensive, and transient transcriptional response to estrogen signaling in breast cancer cells. Cell 145, 622–634 (2011)
    Article CAS Google Scholar
  9. Wang, D. et al. Reprogramming transcription by distinct classes of enhancers functionally defined by eRNA. Nature 474, 390–394 (2011)
    Article CAS Google Scholar
  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)
    Article CAS Google Scholar
  11. Carroll, J. S. et al. Genome-wide analysis of estrogen receptor binding sites. Nature Genet. 38, 1289–1297 (2006)
    Article CAS Google Scholar
  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)
    Article ADS CAS Google Scholar
  13. Heintzman, N. D. & Ren, B. Finding distal regulatory elements in the human genome. Curr. Opin. Genet. Dev. 19, 541–549 (2009)
    Article CAS Google Scholar
  14. Heintzman, N. D. et al. Histone modifications at human enhancers reflect global cell-type-specific gene expression. Nature 459, 108–112 (2009)
    Article ADS CAS Google Scholar
  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)
    Article ADS CAS Google Scholar
  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)
    Article CAS Google Scholar
  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. Wang, K. C. et al. A long noncoding RNA maintains active chromatin to coordinate homeotic gene expression. Nature 472, 120–124 (2011)
    Article ADS CAS Google Scholar
  19. Melo, C. A. et al. eRNAs are required for p53-dependent enhancer activity and gene transcription. Mol. Cell 49, 524–535 (2013)
    Article CAS Google Scholar
  20. Lai, F. et al. Activating RNAs associate with Mediator to enhance chromatin architecture and transcription. Nature 494, 497–501 (2013)
    Article ADS CAS Google Scholar
  21. Fullwood, M. J. et al. An oestrogen-receptor-α-bound human chromatin interactome. Nature 462, 58–64 (2009)
    Article ADS CAS Google Scholar
  22. Harismendy, O. et al. 9p21 DNA variants associated with coronary artery disease impair interferon-γ signalling response. Nature 470, 264–268 (2011)
    Article ADS CAS Google Scholar
  23. Sanyal, A., Lajoie, B. R., Jain, G. & Dekker, J. The long-range interaction landscape of gene promoters. Nature 489, 109–113 (2012)
    Article ADS CAS Google Scholar
  24. Lieberman-Aiden, E. et al. Comprehensive mapping of long-range interactions reveals folding principles of the human genome. Science 326, 289–293 (2009)
    Article ADS CAS Google Scholar
  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)
    Article CAS Google Scholar
  26. Hadjur, S. et al. Cohesins form chromosomal _cis_-interactions at the developmentally regulated IFNG locus. Nature 460, 410–413 (2009)
    Article ADS CAS Google Scholar
  27. Kagey, M. H. et al. Mediator and cohesin connect gene expression and chromatin architecture. Nature 467, 430–435 (2010)
    Article ADS CAS Google Scholar
  28. Schmidt, D. et al. A CTCF-independent role for cohesin in tissue-specific transcription. Genome Res. 20, 578–588 (2010)
    Article CAS Google Scholar
  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)
    Article CAS Google Scholar
  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)
    Article ADS CAS Google Scholar
  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)
    Article CAS Google Scholar
  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)
    Article CAS Google Scholar
  33. Ingolia, N. T. et al. Genome-wide analysis in vivo of translation with nucleotide resolution using ribosome profiling. Science 324, 218–223 (2009)
    Article ADS CAS Google Scholar
  34. White, A. K. et al. High-throughput microfluidic single-cell RT-qPCR. Proc. Natl Acad. Sci. USA 108, 13999–14004 (2011)
    Article ADS CAS Google Scholar
  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)
    Article CAS Google Scholar
  36. Tsai, M. C. et al. Long non-coding RNA as modular scaffold of histone modification complexes. Science 329, 689–693 (2010)
    Article ADS CAS Google Scholar
  37. Rueden, C. T. et al. Visualization approaches for multidimensional biological image data. Biotechniques 43, 31–36 (2007)
    Article Google Scholar
  38. Lajoie, B. R. et al. My5C: web tools for chromosome conformation capture studies. Nature Methods 6, 690–691 (2009)
    Article CAS Google Scholar
  39. Servant, N. et al. HiTC: exploration of high-throughput ‘C’ experiments. Bioinformatics 28, 2843–2844 (2012)
    Article CAS Google Scholar
  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

Acknowledgements

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

Author notes

  1. Wenbo Li and Dimple Notani: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Medicine, Howard Hughes Medical Institute, School of Medicine, University of California, San Diego, La Jolla, California 92093, USA,
    Wenbo Li, Dimple Notani, Qi Ma, Bogdan Tanasa, Esperanza Nunez, Aaron Yun Chen, Daria Merkurjev, Jie Zhang, Kenneth Ohgi, Xiaoyuan Song, Soohwan Oh, Hong-Sook Kim & Michael G. Rosenfeld
  2. Graduate Program in Bioinformatics, University of California, San Diego, La Jolla, California 92093, USA,
    Qi Ma & Daria Merkurjev
  3. Graduate Program, Kellogg School of Science and Technology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA,
    Bogdan Tanasa
  4. Graduate Program in Biological Sciences, University of California, San Diego, La Jolla, California 92093, USA,
    Soohwan Oh
  5. Department of Medicine, Cellular and Molecular Medicine, University of California, San Diego, La Jolla, California 92093, USA,
    Christopher K. Glass

Authors

  1. Wenbo Li
    You can also search for this author inPubMed Google Scholar
  2. Dimple Notani
    You can also search for this author inPubMed Google Scholar
  3. Qi Ma
    You can also search for this author inPubMed Google Scholar
  4. Bogdan Tanasa
    You can also search for this author inPubMed Google Scholar
  5. Esperanza Nunez
    You can also search for this author inPubMed Google Scholar
  6. Aaron Yun Chen
    You can also search for this author inPubMed Google Scholar
  7. Daria Merkurjev
    You can also search for this author inPubMed Google Scholar
  8. Jie Zhang
    You can also search for this author inPubMed Google Scholar
  9. Kenneth Ohgi
    You can also search for this author inPubMed Google Scholar
  10. Xiaoyuan Song
    You can also search for this author inPubMed Google Scholar
  11. Soohwan Oh
    You can also search for this author inPubMed Google Scholar
  12. Hong-Sook Kim
    You can also search for this author inPubMed Google Scholar
  13. Christopher K. Glass
    You can also search for this author inPubMed Google Scholar
  14. Michael G. Rosenfeld
    You can also search for this author inPubMed Google Scholar

Contributions

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 toMichael G. Rosenfeld.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

PowerPoint slides

Rights 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). https://doi.org/10.1038/nature12210

Download citation

Editorial Summary

Regulatory role for eRNAs

Bidirectional non-coding RNAs are transcribed from enhancer elements, but it is unclear whether these enhancer-derived RNAs (eRNAs) have a functional role or are merely a reflection of enhancer activity. Two manuscripts in this issue of Nature examine this question in the context of the positive and negative transcriptional functions of different nuclear receptors. Wenbo Li et al. provide evidence for the functional importance of eRNA transcription during the activation of genes by the oestrogen receptor in breast cancer cell lines; and Michael Lam et al. show that the repressive functions of Rev-Erb nuclear receptors in macrophages are linked to their ability to inhibit the transcription of eRNAs. Taken together these studies provide evidence for a role for eRNAs in contributing to enhancer functions.