Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Promiscuous RNA binding by Polycomb repressive complex 2

Nature Structural & Molecular Biology volume 20, pages 1250–1257 (2013)Cite this article

Subjects

Abstract

Polycomb repressive complex 2 (PRC2) is a histone methyltransferase required for epigenetic silencing during development and cancer. Long noncoding RNAs (lncRNAs) recruit PRC2 to chromatin, but the general role of RNA in maintaining repressed chromatin is unknown. Here we measure the binding constants of human PRC2 to various RNAs and find comparable affinity for human lncRNAs targeted by PRC2 as for irrelevant transcripts from ciliates and bacteria. PRC2 binding is size dependent, with lower affinity for shorter RNAs. In vivo, PRC2 predominantly occupies repressed genes; PRC2 is also associated with active genes, but most of those are not regulated by PRC2. These findings support a model in which PRC2's promiscuous binding to RNA transcripts allows it to scan for target genes that have escaped repression, thus leading to maintenance of the repressed state. Such RNAs may also provide a decoy for PRC2.

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

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

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

Figure 1: PRC2 binds the 5′ domain of HOTAIR RNA with submicromolar affinity, in the presence or absence of AEBP2.
Figure 2: Promiscuous binding of RNA by PRC2 in vitro.
Figure 3: Binding of PRC2 to RNA is size dependent.
Figure 4: Interaction between PRC2 and RNA shows little salt dependence.
Figure 5: Widespread binding of RNA by PRC2 in vivo.
Figure 6: PRC2 associates with active genes, in addition to its predominant association with repressed chromatin.
Figure 7: Knockdown of SUZ12 in HEK293T/17 cells, combined with ChIP-seq and RNA-seq, confirms widespread association of EZH2 with H3K4me2/3 and Pol II S5, but expression of the vast majority of these genes does not respond to SUZ12 knockdown.
Figure 8: The junk-mail model for repressed chromatin maintenance by PRC2 using promiscuous RNA binding.

Similar content being viewed by others

Accession codes

Primary accessions

Gene Expression Omnibus

Referenced accessions

NCBI Reference Sequence

Swiss-Prot

References

  1. Margueron, R. & Reinberg, D. The Polycomb complex PRC2 and its mark in life. Nature 469, 343–349 (2011).

    Article  CAS  Google Scholar 

  2. Cao, R. & Zhang, Y. SUZ12 is required for both the histone methyltransferase activity and the silencing function of the EED-EZH2 complex. Mol. Cell 15, 57–67 (2004).

    Article  CAS  Google Scholar 

  3. Margueron, R. et al. Role of the polycomb protein EED in the propagation of repressive histone marks. Nature 461, 762–767 (2009).

    Article  CAS  Google Scholar 

  4. Xu, C. et al. Binding of different histone marks differentially regulates the activity and specificity of polycomb repressive complex 2 (PRC2). Proc. Natl. Acad. Sci. USA 107, 19266–19271 (2010).

    Article  CAS  Google Scholar 

  5. Yuan, W. et al. Dense chromatin activates Polycomb repressive complex 2 to regulate H3 lysine 27 methylation. Science 337, 971–975 (2012).

    Article  CAS  Google Scholar 

  6. Schmitges, F.W. et al. Histone methylation by PRC2 is inhibited by active chromatin marks. Mol. Cell 42, 330–341 (2011).

    Article  CAS  Google Scholar 

  7. Yuan, W. et al. H3K36 methylation antagonizes PRC2-mediated H3K27 methylation. J. Biol. Chem. 286, 7983–7989 (2011).

    Article  CAS  Google Scholar 

  8. Boyer, L.A. et al. Polycomb complexes repress developmental regulators in murine embryonic stem cells. Nature 441, 349–353 (2006).

    Article  CAS  Google Scholar 

  9. Lee, T.I. et al. Control of developmental regulators by Polycomb in human embryonic stem cells. Cell 125, 301–313 (2006).

    Article  CAS  Google Scholar 

  10. Ram, O. et al. Combinatorial patterning of chromatin regulators uncovered by genome-wide location analysis in human cells. Cell 147, 1628–1639 (2011).

    Article  CAS  Google Scholar 

  11. Bernstein, B.E. et al. A bivalent chromatin structure marks key developmental genes in embryonic stem cells. Cell 125, 315–326 (2006).

    Article  CAS  Google Scholar 

  12. Azuara, V. et al. Chromatin signatures of pluripotent cell lines. Nat. Cell Biol. 8, 532–538 (2006).

    Article  CAS  Google Scholar 

  13. Roh, T.Y., Cuddapah, S., Cui, K. & Zhao, K. The genomic landscape of histone modifications in human T cells. Proc. Natl. Acad. Sci. USA 103, 15782–15787 (2006).

    Article  CAS  Google Scholar 

  14. Mousavi, K., Zare, H., Wang, A.H. & Sartorelli, V. Polycomb protein Ezh1 promotes RNA polymerase II elongation. Mol. Cell 45, 255–262 (2012).

    Article  CAS  Google Scholar 

  15. Herz, H.M. et al. Polycomb repressive complex 2-dependent and -independent functions of Jarid2 in transcriptional regulation in Drosophila. Mol. Cell. Biol. 32, 1683–1693 (2012).

    Article  CAS  Google Scholar 

  16. Brookes, E. et al. Polycomb associates genome-wide with a specific RNA polymerase II variant, and regulates metabolic genes in ESCs. Cell Stem Cell 10, 157–170 (2012).

    Article  CAS  Google Scholar 

  17. Ballaré, C. et al. Phf19 links methylated Lys36 of histone H3 to regulation of Polycomb activity. Nat. Struct. Mol. Biol. 19, 1257–1265 (2012).

    Article  Google Scholar 

  18. Musselman, C.A. et al. Molecular basis for H3K36me3 recognition by the Tudor domain of PHF1. Nat. Struct. Mol. Biol. 19, 1266–1272 (2012).

    Article  CAS  Google Scholar 

  19. Brien, G.L. et al. Polycomb PHF19 binds H3K36me3 and recruits PRC2 and demethylase NO66 to embryonic stem cell genes during differentiation. Nat. Struct. Mol. Biol. 19, 1273–1281 (2012).

    Article  CAS  Google Scholar 

  20. Schwartz, Y.B. & Pirrotta, V. Polycomb silencing mechanisms and the management of genomic programmes. Nat. Rev. Genet. 8, 9–22 (2007).

    Article  CAS  Google Scholar 

  21. Cuddapah, S. et al. A novel human polycomb binding site acts as a functional polycomb response element in Drosophila. PLoS ONE 7, e36365 (2012).

    Article  CAS  Google Scholar 

  22. Sing, A. et al. A vertebrate Polycomb response element governs segmentation of the posterior hindbrain. Cell 138, 885–897 (2009).

    Article  CAS  Google Scholar 

  23. Woo, C.J., Kharchenko, P.V., Daheron, L., Park, P.J. & Kingston, R.E. A region of the human HOXD cluster that confers polycomb-group responsiveness. Cell 140, 99–110 (2010).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  25. Rinn, J.L. et al. Functional demarcation of active and silent chromatin domains in human s loci by noncoding RNAs. Cell 129, 1311–1323 (2007).

    Article  CAS  Google Scholar 

  26. 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 

  27. Zhao, J., Sun, B.K., Erwin, J.A., Song, J.J. & Lee, J.T. Polycomb proteins targeted by a short repeat RNA to the mouse X chromosome. Science 322, 750–756 (2008).

    Article  CAS  Google Scholar 

  28. Kanhere, A. et al. Short RNAs are transcribed from repressed polycomb target genes and interact with polycomb repressive complex-2. Mol. Cell 38, 675–688 (2010).

    Article  CAS  Google Scholar 

  29. Zhao, J. et al. Genome-wide identification of polycomb-associated RNAs by RIP-seq. Mol. Cell 40, 939–953 (2010).

    Article  CAS  Google Scholar 

  30. Khalil, A.M. et al. Many human large intergenic noncoding RNAs associate with chromatin-modifying complexes and affect gene expression. Proc. Natl. Acad. Sci. USA 106, 11667–11672 (2009).

    Article  CAS  Google Scholar 

  31. Guttman, M. et al. lincRNAs act in the circuitry controlling pluripotency and differentiation. Nature 477, 295–300 (2011).

    Article  CAS  Google Scholar 

  32. Maenner, S. et al. 2-D structure of the A region of Xist RNA and its implication for PRC2 association. PLoS Biol. 8, e1000276 (2010).

    Article  Google Scholar 

  33. Duszczyk, M.M., Wutz, A., Rybin, V. & Sattler, M. The Xist RNA A-repeat comprises a novel AUCG tetraloop fold and a platform for multimerization. RNA 17, 1973–1982 (2011).

    Article  CAS  Google Scholar 

  34. Kaneko, S. et al. Phosphorylation of the PRC2 component Ezh2 is cell cycle-regulated and up-regulates its binding to ncRNA. Genes Dev. 24, 2615–2620 (2010).

    Article  CAS  Google Scholar 

  35. Kowalczykowski, S.C. et al. Cooperative and noncooperative binding of protein ligands to nucleic acid lattices: experimental approaches to the determination of thermodynamic parameters. Biochemistry 25, 1226–1240 (1986).

    Article  CAS  Google Scholar 

  36. Epstein, I.R. Kinetics of nucleic acid-large ligand interactions: exact Monte Carlo treatment and limiting cases of reversible binding. Biopolymers 18, 2037–2050 (1979).

    Article  CAS  Google Scholar 

  37. Broderick, J.A., Salomon, W.E., Ryder, S.P., Aronin, N. & Zamore, P.D. Argonaute protein identity and pairing geometry determine cooperativity in mammalian RNA silencing. RNA 17, 1858–1869 (2011).

    Article  CAS  Google Scholar 

  38. Record, M.T. Jr., Lohman, M.L. & De Haseth, P. Ion effects on ligand-nucleic acid interactions. J. Mol. Biol. 107, 145–158 (1976).

    Article  CAS  Google Scholar 

  39. Makino, D.L., Baumgartner, M. & Conti, E. Crystal structure of an RNA-bound 11-subunit eukaryotic exosome complex. Nature 495, 70–75 (2013).

    Article  CAS  Google Scholar 

  40. Mortazavi, A., Williams, B.A., McCue, K., Schaeffer, L. & Wold, B. Mapping and quantifying mammalian transcriptomes by RNA-Seq. Nat. Methods 5, 621–628 (2008).

    Article  CAS  Google Scholar 

  41. Marks, H. et al. The transcriptional and epigenomic foundations of ground state pluripotency. Cell 149, 590–604 (2012).

    Article  CAS  Google Scholar 

  42. Shaw, G., Morse, S., Ararat, M. & Graham, F.L. Preferential transformation of human neuronal cells by human adenoviruses and the origin of HEK 293 cells. FASEB J. 16, 869–871 (2002).

    Article  CAS  Google Scholar 

  43. Schwartz, J.C. et al. FUS binds the CTD of RNA polymerase II and regulates its phosphorylation at Ser2. Genes Dev. 26, 2690–2695 (2012).

    Article  CAS  Google Scholar 

  44. Ku, M. et al. Genomewide analysis of PRC1 and PRC2 occupancy identifies two classes of bivalent domains. PLoS Genet. 4, e1000242 (2008).

    Article  Google Scholar 

  45. Hansen, K.H. et al. A model for transmission of the H3K27me3 epigenetic mark. Nat. Cell Biol. 10, 1291–1300 (2008).

    Article  CAS  Google Scholar 

  46. Sun, S. et al. Jpx RNA Activates Xist by evicting CTCF. Cell 153, 1537–1551 (2013).

    Article  CAS  Google Scholar 

  47. Mazzone, J. & Pickett, J. The household diary study: mail use & attitudes in FY 2010. (US Postal Service 2011).

  48. Khersonsky, O. & Tawfik, D.S. Enzyme promiscuity: a mechanistic and evolutionary perspective. Annu. Rev. Biochem. 79, 471–505 (2010).

    Article  CAS  Google Scholar 

  49. Murphy, F.L., Wang, Y.H., Griffith, J.D. & Cech, T.R. Coaxially stacked RNA helices in the catalytic center of the Tetrahymena ribozyme. Science 265, 1709–1712 (1994).

    Article  CAS  Google Scholar 

  50. Langmead, B., Trapnell, C., Pop, M. & Salzberg, S.L. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 10, R25 (2009).

    Article  Google Scholar 

  51. Quinlan, A.R. & Hall, I.M. BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics 26, 841–842 (2010).

    Article  CAS  Google Scholar 

  52. Li, H. et al. The Sequence Alignment/Map format and SAMtools. Bioinformatics 25, 2078–2079 (2009).

    Article  Google Scholar 

  53. Robinson, J.T. et al. Integrative genomics viewer. Nat. Biotechnol. 29, 24–26 (2011).

    Article  CAS  Google Scholar 

  54. Zhang, Y. et al. Model-based analysis of ChIP-Seq (MACS). Genome Biol. 9, R137 (2008).

    Article  Google Scholar 

  55. Benjamini, Y. & Hochberg, Y. Controlling the false discovery rate: a practical and powerful approach to multiple testing. J. R. Stat. Soc. Ser. B 57, 289–300 (1995).

    Google Scholar 

  56. Schwartz, J.C. et al. FUS binds the CTD of RNA polymerase II and regulates its phosphorylation at Ser2. Genes Dev. 26, 2690–2695 (2012).

    Article  CAS  Google Scholar 

  57. Reimand, J., Kull, M., Peterson, H., Hansen, J. & Vilo, J. g:Profiler: a web-based toolset for functional profiling of gene lists from large-scale experiments. Nucleic Acids Res. 35, W193–W200 (2007).

    Article  Google Scholar 

Download references

Acknowledgements

We thank R. Dowell (University of Colorado Boulder (CU Boulder)) for computational resources, J. Huntley (BioFrontiers Next-Gen Sequencing Facility, CU Boulder) for discussion and assistance with sequencing, R. Kingston (Harvard Medical School) for kindly providing plasmids with PRC2-subunit genes and J.T. Lee (Harvard Medical School) and D. Reinberg (New York University) for discussions. T.R.C. is supported as an investigator of the Howard Hughes Medical Institute. C.D. is supported by the Fulbright Postdoctoral Fellowship and the Machiah Foundation Program. L.Z. is supported by the University of Colorado Medical Scientist Training Program, US National Institutes of Health training grant T32 GM008497 (MSTP).

Author information

Authors and Affiliations

  1. Department of Chemistry & Biochemistry, Howard Hughes Medical Institute, University of Colorado Boulder, Boulder, Colorado, USA

    Chen Davidovich, Karen J Goodrich & Thomas R Cech

  2. BioFrontiers Institute, University of Colorado Boulder, Boulder, Colorado, USA

    Chen Davidovich, Karen J Goodrich & Thomas R Cech

  3. Medical Scientist Training Program, University of Colorado School of Medicine, Aurora, Colorado, USA

    Leon Zheng

Authors
  1. Chen Davidovich
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Leon Zheng
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Karen J Goodrich
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Thomas R Cech
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

C.D. and T.R.C. designed the biochemical experiments, which were carried out by C.D., L.Z. and K.J.G. C.D. carried out the tissue-culture experiments, ChIP-seq, RNA-seq and bioinformatics analysis. C.D. and T.R.C. wrote the manuscript.

Corresponding author

Correspondence to Thomas R Cech.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 In vitro histone methyltransferase (HMTase) assay.

Assays carried out under identical conditions, except for the exclusion of PRC2, H3.1 or both as highlighted. The higher radioactive signal following TCA precipitation, appearing only in the presence of both PRC2 and H3.1 histone, confirmed HMTase activity of PRC2. Some low signal was observed in the absence of histone substrate, possibly because of loaded SAM that was co-precipitated with PRC2 or a weak auto HMTase activity of PRC2, as previously observed4. Error bars represents standard deviations that were generated based on 3 independent samples.

Supplementary Figure 2 Sequence source for HOTAIR 1–300 RNA and HOTAIR 400 RNA.

The sequence of HOTAIR 1–300 (in red) originated from a previously generated cDNA clone57 and is in accord with the HOTAIR RNA examined in previous studies24,34. We found this RNA construct prone to aggregation under various conditions, evidenced by the presence of radiolabeled RNA in wells and across lanes following non-denaturing gel electrophoresis, in the absence or presence of protein (Fig 1b). RNA aggregation was eliminated by adding approximately 50 nucleotides upstream and downstream of HOTAIR 1–300, forming HOTAIR 400 RNA (see Supplementary Methods for complete details of DNA templates used for in vitro RNA transcription). Importantly, all bases that were added (in light blue) were not arbitrarily tailored but appear within cellular HOTAIR transcripts (in blue).

Supplementary Figure 3 PRC2-RNA direct binding assay after prolonged incubation.

PRC2-RNA direct binding assay after prolonged incubation. To verify that dissociation constants were derived under equilibrium conditions, incubation time was increased eightfold, from 30 min to 240 min. The fraction of bound RNA was not increased following this prolonged incubation (panel a). An increase of 43% in equilibrium dissociation constant (panel b) is most likely due to loss of similar fraction of RNA binding activity by PRC2 due to the prolonged incubation period (4 hours at 30 °C) prior to the EMSA experiment.

Supplementary Figure 4 Direct binding assay of PRC2 with various RNAs.

(a) EMSA of different RNAs after incubation in the presence or absence of PRC2 complex confirms similar affinity of PRC2 to the following: a 400 base RNA from the 5' domain of HOTAIR RNA (HOTAIR 400 for sense RNA and as HOTAIR 400 for antisense RNA), an approximately 500 base RNA representing the entire A-region from the RepA gene including all tandem repeats (A-region for sense and asA-region for antisense strand), the 397 base mouse telomerase RNA (mouse TR), a 157 base RNA representing the wild type (wt) P4-P6 region within the Tetrahymena group I intron, and a mutant P4-P6 RNA that cannot form tertiary structure48. (b) Direct binding EMSA of the antisense strand of HOTAIR 1–300 (as HOTAIR 1–300, binding curve presented in Fig 1c).

Supplementary Figure 5 Secondary structure prediction of MBP 1–300.

Secondary structure prediction of MBP 1–300 performed by mFold using default settings. Presented are the top three hits, those with the lowest ΔGs. These predictions failed to identify the two-hairpin motif (in dashed frame) that was previously suggested to be enriched in short ncRNAs associated with PRC228.

Supplementary Figure 6 Gene Ontology (GO) analysis for genes that were upregulated after SUZ12 knockdown.

GO analysis yielded a notable network of significant GO terms for developmental processes, with specification at neuron development. GO term IDs indicated in brackets, p-values indicated under each GO term name.

Supplementary Figure 7 Original western blots used to generate Figure 7a.

Full lanes shown as captured, without further cropping.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–7 and Supplementary Tables 6 and 7 (PDF 1298 kb)

Supplementary Table 1

Genes observed with Ezh2-FE >3 (XLSX 40 kb)

Supplementary Table 2

GO analysis for genes with high Ezh2-FE and coverage (XLSX 13 kb)

Supplementary Table 3

Data for Figure 5b (XLSX 26 kb)

Supplementary Table 4

PRC2-regulated and EZH2-associated genes (XLSX 274 kb)

Supplementary Table 5

GO analysis for genes upregulated by SUZ12 KD (XLSX 16 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Davidovich, C., Zheng, L., Goodrich, K. et al. Promiscuous RNA binding by Polycomb repressive complex 2. Nat Struct Mol Biol 20, 1250–1257 (2013). https://doi.org/10.1038/nsmb.2679

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb.2679

This article is cited by

Search

Advanced search

Quick links

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