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G-tract RNA removes Polycomb repressive complex 2 from genes

An Author Correction to this article was published on 06 November 2019

This article has been updated

Abstract

Polycomb repressive complex 2 (PRC2) maintains repression of cell-type-specific genes but also associates with genes ectopically in cancer. While it is currently unknown how PRC2 is removed from genes, such knowledge would be useful for the targeted reversal of deleterious PRC2 recruitment events. Here, we show that G-tract RNA specifically removes PRC2 from genes in human and mouse cells. PRC2 preferentially binds G tracts within nascent precursor mRNA (pre-mRNA), especially within predicted G-quadruplex structures. G-quadruplex RNA evicts the PRC2 catalytic core from the substrate nucleosome. In cells, PRC2 transfers from chromatin to pre-mRNA upon gene activation, and chromatin-associated G-tract RNA removes PRC2, leading to H3K27me3 depletion from genes. Targeting G-tract RNA to the tumor suppressor gene CDKN2A in malignant rhabdoid tumor cells reactivates the gene and induces senescence. These data support a model in which pre-mRNA evicts PRC2 during gene activation and provides the means to selectively remove PRC2 from specific genes.

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Fig. 1: PRC2 binds G tracts with the potential to form G4 structures in nascent RNA.
Fig. 2: G4 structures within longer RNAs block PRC2 binding to nucleosomes.
Fig. 3: G4 RNA inhibits interaction of the PRC2 catalytic core with the substrate core nucleosome particle.
Fig. 4: Chromatin-associated G-tract RNA removes PRC2 from specific genes in cells.
Fig. 5: PRC2 transfers from chromatin to nascent pre-mRNA during gene activation.
Fig. 6: G-tract RNA reverses PRC2 recruitment triggered by oncogenic HRasV12.
Fig. 7: G-tract RNA tethering activates CDNK2A and induces cell senescence.

Data availability

Input iCLIP sequencing data have been deposited in the Gene Expression Omnibus (GEO) with accession code GSE120696. Previously published iCLIP sequencing data and RNA-seq data are available in GEO under accession code GSE66829. The positions of predicted G-quadruplex RNA structures and the positions of PRC2 cross-link sites around first 5′ splice sites are provided in Supplementary Table 1. Supplementary Data Set 2 contains t statistics, confidence intervals, effect sizes and degrees of freedom for all significance tests. Raw quantitative PCR data and all other data are available upon reasonable request. Requests for data and materials should be addressed to R.G.J.

Change history

  • 06 November 2019

    An amendment to this paper has been published and can be accessed via a link at the top of the paper.

References

  1. 1.

    Skalska, L., Beltran-Nebot, M., Ule, J. & Jenner, R. G. Regulatory feedback from nascent RNA to chromatin and transcription. Nat. Rev. Mol. Cell Biol. 18, 331–337 (2017).

    CAS  PubMed  Google Scholar 

  2. 2.

    Holoch, D. & Margueron, R. Mechanisms regulating PRC2 recruitment and enzymatic activity. Trends Biochem. Sci. 42, 531–542 (2017).

    CAS  PubMed  Google Scholar 

  3. 3.

    Schuettengruber, B., Bourbon, H. M., Di Croce, L. & Cavalli, G. Genome regulation by polycomb and trithorax: 70 years and counting. Cell 171, 34–57 (2017).

    CAS  PubMed  Google Scholar 

  4. 4.

    Comet, I., Riising, E. M., Leblanc, B. & Helin, K. Maintaining cell identity: PRC2-mediated regulation of transcription and cancer. Nat. Rev. Cancer 16, 803–810 (2016).

    CAS  PubMed  Google Scholar 

  5. 5.

    Qi, W. et al. Selective inhibition of Ezh2 by a small molecule inhibitor blocks tumor cells proliferation. Proc. Natl Acad. Sci. USA 109, 21360–21365 (2012).

    CAS  PubMed  Google Scholar 

  6. 6.

    Knutson, S. K. et al. Durable tumor regression in genetically altered malignant rhabdoid tumors by inhibition of methyltransferase EZH2. Proc. Natl Acad. Sci. USA 110, 7922–7927 (2013).

    CAS  PubMed  Google Scholar 

  7. 7.

    Caganova, M. et al. Germinal center dysregulation by histone methyltransferase EZH2 promotes lymphomagenesis. J. Clin. Invest. 123, 5009–5022 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. 8.

    Beguelin, W. et al. EZH2 is required for germinal center formation and somatic EZH2 mutations promote lymphoid transformation. Cancer Cell 23, 677–692 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. 9.

    McCabe, M. T. et al. EZH2 inhibition as a therapeutic strategy for lymphoma with EZH2-activating mutations. Nature 492, 108–112 (2012).

    CAS  PubMed  Google Scholar 

  10. 10.

    Mohammad, F. et al. EZH2 is a potential therapeutic target for H3K27M-mutant pediatric gliomas. Nat. Med. 23, 483–492 (2017).

    CAS  PubMed  Google Scholar 

  11. 11.

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

    CAS  PubMed  Google Scholar 

  12. 12.

    Bracken, A. P., Dietrich, N., Pasini, D., Hansen, K. H. & Helin, K. Genome-wide mapping of Polycomb target genes unravels their roles in cell fate transitions. Genes Dev. 20, 1123–1136 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. 13.

    Mikkelsen, T. S. et al. Genome-wide maps of chromatin state in pluripotent and lineage-committed cells. Nature 448, 553–560 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. 14.

    Mohn, F. et al. Lineage-specific polycomb targets and de novo DNA methylation define restriction and potential of neuronal progenitors. Mol. Cell 30, 755–766 (2008).

    CAS  PubMed  Google Scholar 

  15. 15.

    Agger, K. et al. The H3K27me3 demethylase JMJD3 contributes to the activation of the INK4A-ARF locus in response to oncogene- and stress-induced senescence. Genes Dev. 23, 1171–1176 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. 16.

    Barradas, M. et al. Histone demethylase JMJD3 contributes to epigenetic control of INK4a/ARF by oncogenic RAS. Genes Dev. 23, 1177–1182 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. 17.

    Berg, T. et al. A transgenic mouse model demonstrating the oncogenic role of mutations in the polycomb-group gene EZH2 in lymphomagenesis. Blood 123, 3914–3924 (2014).

    CAS  PubMed  Google Scholar 

  18. 18.

    Hosogane, M., Funayama, R., Nishida, Y., Nagashima, T. & Nakayama, K. Ras-induced changes in H3K27me3 occur after those in transcriptional activity. PLoS Genet. 9, e1003698 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. 19.

    Kaneda, A. et al. Activation of Bmp2-Smad1 signal and its regulation by coordinated alteration of H3K27 trimethylation in Ras-induced senescence. PLoS Genet. 7, e1002359 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. 20.

    Kondo, Y. et al. Gene silencing in cancer by histone H3 lysine 27 trimethylation independent of promoter DNA methylation. Nat. Genet. 40, 741–750 (2008).

    CAS  PubMed  Google Scholar 

  21. 21.

    Souroullas, G. P. et al. An oncogenic Ezh2 mutation induces tumors through global redistribution of histone 3 lysine 27 trimethylation. Nat. Med. 22, 632–640 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. 22.

    Kia, S. K., Gorski, M. M., Giannakopoulos, S. & Verrijzer, C. P. SWI/SNF mediates polycomb eviction and epigenetic reprogramming of the INK4b-ARF-INK4a locus. Mol. Cell Biol. 28, 3457–3464 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. 23.

    de Vries, N. A. et al. Prolonged Ezh2 depletion in glioblastoma causes a robust switch in cell fate resulting in tumor progression. Cell Reports 10, 383–397 (2015).

    PubMed  Google Scholar 

  24. 24.

    Jermann, P., Hoerner, L., Burger, L. & Schubeler, D. Short sequences can efficiently recruit histone H3 lysine 27 trimethylation in the absence of enhancer activity and DNA methylation. Proc. Natl Acad. Sci. USA 111, E3415–E3421 (2014).

    CAS  PubMed  Google Scholar 

  25. 25.

    Lynch, M. D. et al. An interspecies analysis reveals a key role for unmethylated CpG dinucleotides in vertebrate Polycomb complex recruitment. EMBO J. 31, 317–329 (2011).

    PubMed  PubMed Central  Google Scholar 

  26. 26.

    Mendenhall, E. M. et al. GC-rich sequence elements recruit PRC2 in mammalian ES cells. PLoS Genet. 6, e1001244 (2010).

    PubMed  PubMed Central  Google Scholar 

  27. 27.

    Li, H. et al. Polycomb-like proteins link the PRC2 complex to CpG islands. Nature 549, 287–291 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. 28.

    Perino, M. et al. MTF2 recruits polycomb repressive complex 2 by helical-shape-selective DNA binding. Nat. Genet. 50, 1002–1010 (2018).

    CAS  PubMed  Google Scholar 

  29. 29.

    Cooper, S. et al. Jarid2 binds mono-ubiquitylated H2A lysine 119 to mediate crosstalk between Polycomb complexes PRC1 and PRC2. Nat. Commun. 7, 13661 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. 30.

    Kalb, R. et al. Histone H2A monoubiquitination promotes histone H3 methylation in Polycomb repression. Nat. Struct. Mol. Biol. 21, 569–571 (2014).

    CAS  PubMed  Google Scholar 

  31. 31.

    Beltran, M. et al. The interaction of PRC2 with RNA or chromatin is mutually antagonistic. Genome Res. 26, 896–907 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. 32.

    Kaneko, S., Son, J., Shen, S. S., Reinberg, D. & Bonasio, R. PRC2 binds active promoters and contacts nascent RNAs in embryonic stem cells. Nat. Struct. Mol. Biol. 20, 1258–1264 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. 33.

    Kaneko, S., Son, J., Bonasio, R., Shen, S. S. & Reinberg, D. Nascent RNA interaction keeps PRC2 activity poised and in check. Genes Dev. 28, 1983–1988 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. 34.

    Long, Y. et al. Conserved RNA-binding specificity of polycomb repressive complex 2 is achieved by dispersed amino acid patches in EZH2. eLife 6, e31558 (2017).

    PubMed  PubMed Central  Google Scholar 

  35. 35.

    Wang, X. et al. Targeting of polycomb repressive complex 2 to RNA by short repeats of consecutive guanines. Mol. Cell 65, 1056–1067.e5 (2017).

    CAS  PubMed  Google Scholar 

  36. 36.

    Wang, X. et al. Molecular analysis of PRC2 recruitment to DNA in chromatin and its inhibition by RNA. Nat. Struct. Mol. Biol. 24, 1028–1038 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. 37.

    Cifuentes-Rojas, C., Hernandez, A. J., Sarma, K. & Lee, J. T. Regulatory interactions between RNA and polycomb repressive complex 2. Mol. Cell 55, 171–185 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. 38.

    Herzog, V. A. et al. A strand-specific switch in noncoding transcription switches the function of a Polycomb/Trithorax response element. Nat. Genet. 46, 973–981 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  39. 39.

    Zhang, Q. et al. RNA exploits an exposed regulatory site to inhibit the enzymatic activity of PRC2. Nat. Struct. Mol. Biol. 26, 237–247 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. 40.

    Riising, E. M. et al. Gene silencing triggers polycomb repressive complex 2 recruitment to CpG islands genome wide. Mol. Cell 55, 347–360 (2014).

    CAS  PubMed  Google Scholar 

  41. 41.

    Hosogane, M., Funayama, R., Shirota, M. & Nakayama, K. Lack of transcription triggers H3K27me3 accumulation in the gene body. Cell Rep. 16, 696–706 (2016).

    CAS  PubMed  Google Scholar 

  42. 42.

    Rogelj, B. et al. Widespread binding of FUS along nascent RNA regulates alternative splicing in the brain. Sci. Rep. 2, 603 (2012).

    PubMed  PubMed Central  Google Scholar 

  43. 43.

    Konig, J. et al. iCLIP reveals the function of hnRNP particles in splicing at individual nucleotide resolution. Nat. Struct. Mol. Biol. 17, 909–915 (2010).

    PubMed  PubMed Central  Google Scholar 

  44. 44.

    Bedrat, A., Lacroix, L. & Mergny, J. L. Re-evaluation of G-quadruplex propensity with G4Hunter. Nucleic Acids Res. 44, 1746–1759 (2016).

    PubMed  PubMed Central  Google Scholar 

  45. 45.

    Eddy, J. & Maizels, N. Conserved elements with potential to form polymorphic G-quadruplex structures in the first intron of human genes. Nucleic Acids Res. 36, 1321–1333 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  46. 46.

    Engelbrecht, J., Knudsen, S. & Brunak, S. G+C-rich tract in 5′ end of human introns. J. Mol. Biol. 227, 108–113 (1992).

    CAS  PubMed  Google Scholar 

  47. 47.

    Takahama, K. et al. Regulation of telomere length by G-quadruplex telomere DNA- and TERRA-binding protein TLS/FUS. Chem. Biol. 20, 341–350 (2013).

    CAS  PubMed  Google Scholar 

  48. 48.

    Kwok, C. K., Marsico, G., Sahakyan, A. B., Chambers, V. S. & Balasubramanian, S. rG4-seq reveals widespread formation of G-quadruplex structures in the human transcriptome. Nat. Methods 13, 841–844 (2016).

    CAS  PubMed  Google Scholar 

  49. 49.

    Justin, N. et al. Structural basis of oncogenic histone H3K27M inhibition of human polycomb repressive complex 2. Nat. Commun. 7, 11316 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  50. 50.

    Brown, Z. Z. et al. Strategy for ‘detoxification’ of a cancer-derived histone mutant based on mapping its interaction with the methyltransferase PRC2. J. Am. Chem. Soc. 136, 13498–13501 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  51. 51.

    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).

    CAS  PubMed  PubMed Central  Google Scholar 

  52. 52.

    Sigova, A. A. et al. Transcription factor trapping by RNA in gene regulatory elements. Science 350, 978–981 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  53. 53.

    Zalatan, J. G. et al. Engineering complex synthetic transcriptional programs with CRISPR RNA scaffolds. Cell 160, 339–350 (2015).

    CAS  PubMed  Google Scholar 

  54. 54.

    Davidovich, C., Zheng, L., Goodrich, K. J. & Cech, T. R. Promiscuous RNA binding by Polycomb repressive complex 2. Nat. Struct. Mol. Biol. 20, 1250–1257 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  55. 55.

    McCullough, A. J. & Berget, S. M. G triplets located throughout a class of small vertebrate introns enforce intron borders and regulate splice site selection. Mol. Cell Biol. 17, 4562–4571 (1997).

    CAS  PubMed  PubMed Central  Google Scholar 

  56. 56.

    Sirand-Pugnet, P., Durosay, P., Brody, E. & Marie, J. An intronic (A/U)GGG repeat enhances the splicing of an alternative intron of the chicken beta-tropomyosin pre-mRNA. Nucleic Acids Res. 23, 3501–3507 (1995).

    CAS  PubMed  PubMed Central  Google Scholar 

  57. 57.

    Poepsel, S., Kasinath, V. & Nogales, E. Cryo-EM structures of PRC2 simultaneously engaged with two functionally distinct nucleosomes. Nat. Struct. Mol. Biol. 25, 154–162 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  58. 58.

    Chen, S., Jiao, L., Shubbar, M., Yang, X. & Liu, X. Unique structural platforms of Suz12 dictate distinct classes of PRC2 for chromatin binding. Mol. Cell 69, 840–852 e5 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  59. 59.

    Kaneko, S. et al. Interactions between JARID2 and noncoding RNAs regulate PRC2 recruitment to chromatin. Mol. Cell 53, 290–300 (2014).

    CAS  PubMed  Google Scholar 

  60. 60.

    Di Ruscio, A. et al. DNMT1-interacting RNAs block gene-specific DNA methylation. Nature 503, 371–376 (2013).

    PubMed  PubMed Central  Google Scholar 

  61. 61.

    Sayou, C. et al. RNA binding by histone methyltransferases Set1 and Set2. Mol. Cell Biol. 37, e00165-17 (2017).

    PubMed  PubMed Central  Google Scholar 

  62. 62.

    Nozawa, R. S. et al. SAF-A regulates interphase chromosome structure through oligomerization with chromatin-associated RNAs. Cell 169, 1214–1227.e18 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  63. 63.

    Dominguez, A. A., Lim, W. A. & Qi, L. S. Beyond editing: repurposing CRISPR-Cas9 for precision genome regulation and interrogation. Nat. Rev. Mol. Cell Biol. 17, 5–15 (2016).

    CAS  PubMed  Google Scholar 

  64. 64.

    Kearns, N. A. et al. Cas9 effector-mediated regulation of transcription and differentiation in human pluripotent stem cells. Development 141, 219–223 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  65. 65.

    Labun, K., Montague, T. G., Gagnon, J. A., Thyme, S. B. & Valen, E. CHOPCHOP v2: a web tool for the next generation of CRISPR genome engineering. Nucleic Acids Res. 44, W272–W276 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  66. 66.

    Huppertz, I. et al. iCLIP: protein-RNA interactions at nucleotide resolution. Methods 65, 274–287 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  67. 67.

    Bouwman, R. D. et al. Human immunodeficiency virus Tat associates with a specific set of cellular RNAs. Retrovirology 11, 53 (2014).

    PubMed  PubMed Central  Google Scholar 

  68. 68.

    Kwok, C. K. & Balasubramanian, S. Targeted detection of G-quadruplexes in cellular RNAs. Angew. Chem. Int Ed. Engl. 54, 6751–6754 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  69. 69.

    Bartke, T. et al. Nucleosome-interacting proteins regulated by DNA and histone methylation. Cell 143, 470–484 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  70. 70.

    Saravanan, M. et al. Interactions between the nucleosome histone core and Arp8 in the INO80 chromatin remodeling complex. Proc. Natl Acad. Sci. USA 109, 20883–20888 (2012).

    CAS  PubMed  Google Scholar 

  71. 71.

    Dyer, P. N. et al. Reconstitution of nucleosome core particles from recombinant histones and DNA. Methods Enzym. 375, 23–44 (2004).

    CAS  Google Scholar 

  72. 72.

    Trapnell, C. et al. Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation. Nat. Biotechnol. 28, 511–515 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  73. 73.

    Kim, D. et al. TopHat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions. Genome Biol. 14, R36 (2013).

    PubMed  PubMed Central  Google Scholar 

  74. 74.

    Katz, Y., Wang, E. T., Airoldi, E. M. & Burge, C. B. Analysis and design of RNA sequencing experiments for identifying isoform regulation. Nat. Methods 7, 1009–1015 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  75. 75.

    Hon, G. C. et al. 5mC oxidation by Tet2 modulates enhancer activity and timing of transcriptome reprogramming during differentiation. Mol. Cell 56, 286–297 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank the UCL Cancer Institute Genomics Core Facility and Bill Lyons Informatics Centre, both supported by the Cancer Research UK–UCL Centre (award C416/A18088). We thank A. Bracken (Trinity College Dublin), N. Brockdorff (University of Oxford), A. Fisher (London Institute for Medical Sciences) and B. Vanhaesebroeck (UCL) for cell lines. We also thank I. Ruiz de los Mozos and J. Ule for assistance with iCount and feedback on the manuscript and to M. Vila de Mucha for assistance with flow cytometry. The research was funded by grants from the European Research Council (ERC, 311704), Worldwide Cancer Research (13-0256) and Bloodwise (18008) to R.G.J., CoNaCyT (411064) to M.T., ERC (309952) and the Helmholtz Society to T.B., and Cancer Research UK (FC001078), Medical Research Council (FC001078) and Wellcome Trust (FC001078) grants to the Francis Crick Institute (funding N.J., S.K., S.J.G. and J.R.W.).

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Contributions

M.B. co-designed and performed all experiments, except where noted below. M.T. performed the nucleosome IPs with different linker DNA lengths. N.J., assisted by S.K., measured competition between G4 RNA and the substrate core nucleosome particle for the PRC2 catalytic core in experiments co-designed by J.R.W. G.K. performed bioinformatics analysis, assisted by J.A. and R.G.J. K.B.W. helped with qRT−PCR experiments. B.M.F. and A.T. produced nucleosomes. J.H., T.B., S.J.G., J.R.W. and R.G.J. supervised the research. R.G.J. co-designed experiments and wrote the paper with help from all authors.

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Correspondence to Richard G. Jenner.

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Integrated supplementary information

Supplementary Figure 1 Preferential binding of PRC2 to G-tract RNA in conditions favoring G4 formation.

(a) The positional distribution of the top 10 PRC2 8-mers (by z-score) relative to PRC2 (bright red) or input (dark red) RNA crosslink sites, and the distribution of the A8 k-mer relative to PRC2 (bright blue) or input (dark blue) RNA crosslink sites, normalized by the mean frequency from 100 randomisations. G-tract-containing 8-mers peak around the crosslink site (the dip at the crosslink site (dashed line) is due to the known bias for UV-C crosslinking at uracil). The A-rich k-mer is depleted from PRC2 crosslink sites. (b) Native polyacrylamide gel electrophoresis of 32P end-labeled [G4A4]5 or [GA]20 RNA oligonucleotides in folding or pull-down buffer in the presence of 150 mM K+ or Li+. RNA folded into a G4 structure is labeled. (c) Immunoblotting for EZH2 after pull-down of recombinant PRC2 (EZH2, SUZ12, EED, RBBP4/7; 1.5 ng/μl) with 10-fold dilutions of biotinylated G4-forming [G4A4]5 or control [GA]20 RNA oligonucleotides in 150 mM K+ or Li+ buffer. Representative of three independent experiments. (d) Immunoblotting for SUZ12 and H3 after incubation of recombinant PRC2 (1.5 ng/μl) with biotinylated nucleosomes (50 nM) in the presence of 2, 20 or 200 ng/μl G4-forming [G4A4]5 or control [GA]20 RNA in K+ or Li+ buffer. Representative of three independent experiments.

Supplementary Figure 2 PRC2 binds nascent RNA at predicted G4-forming sequences at the 5′ end of the first intron.

(a) Alternative splicing events caused by Suz12 deletion in mouse ESC (blue) and for comparison alternative splicing events occurring during differentiation of ESC to neural progenitor cells. Alternative splicing events were identified with MISO and divided into 5 different types: skipped exons (SE), mutually exclusive exons (MXE), retained introns (RI), alternative 5′ splice sites (A5SS) and alternative 3′ splice sites (A3SS). (b) As Fig. 1c, but displaying the RNA crosslink-density at splice-sites across the genes that either contain (red) or do not contain (blue) a predicted G4 forming sequence -30 to +300 nt around the first 5′ splice site. (c) As Fig. 1c, except that the crosslink density for non-G4 junctions has been normalised by the non-G4 G-nucleotide frequency vs G4 G-nucleotide frequency ratio at each position (PRC2 P=2 × 10−16, FUS P=2 × 10−16). (d) Characterisation of predicted G4-forming sequences at first exon/intron junctions (-30 to 300 nt) that are either crosslinked or not crosslinked to PRC2 in cells (iCLIP FDR < 0.05) in terms of the number of G-tracts, G nucleotides per tract, nucleotides per loop between G-tracts, loop base composition, expression level of the host gene and position of the crosslinked G within G-tracts. This analysis shows that PRC2 does not have a preference for any particular G4 features, except for a lower number of G-tracts per G4 (P=0.02).

Supplementary Figure 3 G4 structures within longer RNAs bind PRC2 and antagonise its interaction with nucleosomes.

(a) Top: In vitro transcribed WT PIM1 RNA, control RNA lacking the central 24 nt G4-forming sequence (ΔG4), control RNA in which Gs within the G4-forming sequence are mutated to non-Gs (G-to-H) and control RNA in which Gs within the G4-forming sequence are mutated to non-Gs and an equal number of non-G residues outside of the G4-forming sequence are mutated to Gs to maintain the overall G-content (G-rich). Bottom: as top, except for biotinylated RNAs. (b) Reverse transcriptase stalling assays performed for the sequences described in (a) in either K+ or Li+ buffer. Full-length cDNA, truncated cDNAs produced by RT stalling products, and free RT primer are labeled. (c) Significant PRC2 RNA crosslink sites (FDR < 0.05) at the last exon of murine Pim1. Significant crosslinks are also marked for input RNA and IgG controls and for PRC2 in Suz12−/− cells. Counts of Watson and Crick strand crosslinks per base are shown by positive and negative integers, respectively. Nuclear and total RNA-seq read densities (reads per million) are shown below. The position of the orthologous human PIM1 RNA sequence that was used in pull-down assays in Fig. 2 is indicated. (d) Replicates of Fig. 2a. Immunoblotting for SUZ12 after pull-down of recombinant PRC2 with 10-fold dilutions of biotinylated PIM1 RNA or control PIM1 RNA lacking G4-forming sequence (ΔG4) in KCl or LiCl-containing buffer. (e) Replicates of Fig. 2c. Immunoblotting for SUZ12 and H3 after pull-down of recombinant PRC2 with biotinylated nucleosomes (reconstituted with 185 bp DNA) in the presence of 2, 20 or 200 ng/μl PIM1 or ΔG4 RNA. (f) Immunoblotting for H3K27me1 after incubation of 30 nM PRC2 with 0.8 μM nucleosomes (30 mins, 25oC). PRC2 retains its activity in K+ and Li+ buffers.

Supplementary Figure 4 The PRC2 catalytic core binds G4 RNA and this blocks it interaction with the nucleosome core particle.

(a) Fluorescence anisotropy measuring binding of the PRC2 catalytic core (EZH2, EED, SUZ12 VEFS domain) directly to fluorescein-labeled [G4A4]4 RNA in nucleosome binding buffer (40 mM KCl). Mean and S.E., n=3. (b) As (a), except for the G4-forming 24 nt sequence within PIM1 RNA. (c) Fluorescence intensity measuring binding of the PRC2 catalytic core directly to MDCC-labeled wild type core nucleosome particles (reconstituted with 147 bp DNA) in the presence or absence of competing 500 nM [G4A4]4 RNA (mean and S.E., n=3). (d) Fluorescence anisotropy measuring binding of the PRC2 catalytic core to fluorescein-labeled [G4A4]4 RNA in the presence of unlabeled PIM1 G4 RNA or an unlabeled non-G4-forming part of PIM1 RNA. Mean and S.E., n=3. (e) Immunoblotting for SUZ12, JARID2, HMGN1 and H3 after co-immunoprecipitation of PRC2 from Jarid2GT/GT or matched wild-type E14 ESC with nucleosomes containing HA-tagged histone H2A (reconstituted with either 185 bp or 147 bp DNA) from mock or RNaseA-treated nuclear extract. Representative of 2 independent experiments. (f) Immunoblotting for SUZ12, AEBP2, HMGN1 and H3 after co-immunoprecipitation of PRC2 from Aebp2GT/GT or paired WT ESC with nucleosomes containing HA-tagged histone H2A (reconstituted with either 185 bp or 147 bp DNA) from mock or RNaseA-treated nuclear extract. Representative of 2 independent experiments.

Supplementary Figure 5 Tethering G-tract RNA to chromatin evicts PRC2 and depletes H3K27me3 at specific genes in cells.

(a) Immunoblotting for HA-dCas9 in 3T3 cells before and after induction with dox and after subsequent washout of dox. The 0, 6 and 12 day time-points were selected to perform ChIP-qPCR. (b) G-tract, G-rich and control A-tract RNA tethering construct design. (c) HA-dCas9, SUZ12, H3K27me3 and total H3 occupancy (as % input, with non-specific IgG control) at Fgf11 (A and B primer pairs, Fig. 4b), Pax7 and Actb before and after induction of dCas9 in cells expressing sgRNA targeted to Fgf11 and appended with either G-tract, G-rich or A-tract RNA. Representative data from a single set of ChIP experiments are shown (mean and S.D. from 3 technical replicates). Data from 3 independent experiments are shown in Fig. 4b. (d) Spliced (mRNA) and unspliced (pre-mRNA) Fgf11, Adcy6 and Sorcs2 RNA, relative to spliced or unspliced Actb RNA, respectively, in cells expressing sgRNAs specific for these genes before and after induction of dCas9 expression (mean and S.D., n=3). (e) H2AK119ub, H3K27ac, and total H3 occupancy (as % input, with non-specific IgG control) at Fgf11 (A and B primer pairs), Pax7 and Actb before and after induction of dCas9. Data from 3 independent experiments are shown in Fig. 4c. (f) Top: position of the 3′ sgRNA target and primer pairs A, B and C within Fgf11. Bottom: Change in HA-dCas9, SUZ12, H3K27me3 and total H3 occupancy occupancy (as % input, with non-specific IgG control) at Fgf11 before and after induction of dCas9 in cells expressing sgRNA targeted to the 3′ end of Fgf11. (g) HA-dCas9, SUZ12, H3K27me3, total H3 occupancy (with IgG control) at Fgf11, Pax7 and Actb before and after dox treatment and after subsequent dox washout (mean and S.D., n=2 dox inductions). Data from 3 independent experiments are shown in Fig. 4e.

Supplementary Figure 6 G-tract RNA tethering mimics dynamic changes in PRC2 gene occupancy.

(a) Immunoblotting for HRasV12 in parental NIH/3T3 cells and in cells stably expressing HRasV12. (b) Change in NIH/3T3 cell morphology upon expression of HRasV12. Scale bars indicate 400 μm. (c) Change in expression of Adcy7, Sorcs2 and Smad6 nascent unspliced RNA (top) and mature spliced mRNA (bottom) upon expression of HRasV12 relative to nascent or mature Actb mRNA, respectively. Nascent RNA: Adcy7 P=0.01, Sorcs2 P=0.005, Smad6 P=0.04. mRNA: Adcy7 P=0.005, Sorcs2 P=0.0003, Smad6 P=0.002). (d) HA-dCas9, SUZ12, H3K27me3 and total H3 occupancy (as % input, with non-specific IgG control) at Adcy7, Sorcs2 and Actb before and after induction of dCas9 in cells expressing sgRNA targeted to Adcy7 and appended with either G-tract or A-tract RNA. Representative data from a single set of ChIP experiments are shown (mean and S.D. from 3 technical replicates). Data from 3 independent experiments are shown in Fig. 5d. (e) As D, except in cells expressing sgRNA to Sorcs2. Data from 3 independent experiments are shown in Fig. 5e. (f) As D, except in cells expressing HRasV12 and sgRNA targeted to Smad6. Data from 3 independent experiments are shown in Fig. 6c. (g) Spliced (mRNA) and unspliced (pre-mRNA) Smad6 RNA, relative to spliced or unspliced Actb RNA, respectively, in HRasV12 cells expressing sgRNAs specific Smad6 before and after induction of dCas9 expression (mean and S.D., n=3).

Supplementary Figure 7 G-tract RNA removes PRC2 from CDKN2A and induces senescence in MRT cells.

(a) Immunoblotting for HA-dCas9 and ACTB in a stable G-401 cell line before and treatment with dox. (b) HA-dCas9, SUZ12, H3K27me3 and total H3 occupancy (as % input, with non-specific IgG control) at CDNK2A (A and B primer pairs), EVX2 and ACTB before and after induction of dCas9 in G-401 cells expressing sgRNA targeted to CDNK2A and appended with either G-tract or A-tract RNA. Representative data from a single set of ChIP experiments are shown (mean and S.D. from 3 technical replicates). Data from 2 independent experiments are shown in Fig. 7a. (c) Cell size (forward scatter) versus beta-galactosidase staining, measured by flow cytometry, in G-401 cells expressing sgRNA targeted to CDKN2A and appended with either G-tract or A-tract RNA after no treatment, doxycycline-mediated induction of HA-dCas9 for 6 days, or treatment with 3.3 μM cisplatin for 24 hrs. Data from unstained cells are shown to the left.

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Beltran, M., Tavares, M., Justin, N. et al. G-tract RNA removes Polycomb repressive complex 2 from genes. Nat Struct Mol Biol 26, 899–909 (2019). https://doi.org/10.1038/s41594-019-0293-z

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