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.
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References
Margueron, R. & Reinberg, D. The Polycomb complex PRC2 and its mark in life. Nature 469, 343–349 (2011).
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).
Margueron, R. et al. Role of the polycomb protein EED in the propagation of repressive histone marks. Nature 461, 762–767 (2009).
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).
Yuan, W. et al. Dense chromatin activates Polycomb repressive complex 2 to regulate H3 lysine 27 methylation. Science 337, 971–975 (2012).
Schmitges, F.W. et al. Histone methylation by PRC2 is inhibited by active chromatin marks. Mol. Cell 42, 330–341 (2011).
Yuan, W. et al. H3K36 methylation antagonizes PRC2-mediated H3K27 methylation. J. Biol. Chem. 286, 7983–7989 (2011).
Boyer, L.A. et al. Polycomb complexes repress developmental regulators in murine embryonic stem cells. Nature 441, 349–353 (2006).
Lee, T.I. et al. Control of developmental regulators by Polycomb in human embryonic stem cells. Cell 125, 301–313 (2006).
Ram, O. et al. Combinatorial patterning of chromatin regulators uncovered by genome-wide location analysis in human cells. Cell 147, 1628–1639 (2011).
Bernstein, B.E. et al. A bivalent chromatin structure marks key developmental genes in embryonic stem cells. Cell 125, 315–326 (2006).
Azuara, V. et al. Chromatin signatures of pluripotent cell lines. Nat. Cell Biol. 8, 532–538 (2006).
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).
Mousavi, K., Zare, H., Wang, A.H. & Sartorelli, V. Polycomb protein Ezh1 promotes RNA polymerase II elongation. Mol. Cell 45, 255–262 (2012).
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).
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).
Ballaré, C. et al. Phf19 links methylated Lys36 of histone H3 to regulation of Polycomb activity. Nat. Struct. Mol. Biol. 19, 1257–1265 (2012).
Musselman, C.A. et al. Molecular basis for H3K36me3 recognition by the Tudor domain of PHF1. Nat. Struct. Mol. Biol. 19, 1266–1272 (2012).
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).
Schwartz, Y.B. & Pirrotta, V. Polycomb silencing mechanisms and the management of genomic programmes. Nat. Rev. Genet. 8, 9–22 (2007).
Cuddapah, S. et al. A novel human polycomb binding site acts as a functional polycomb response element in Drosophila. PLoS ONE 7, e36365 (2012).
Sing, A. et al. A vertebrate Polycomb response element governs segmentation of the posterior hindbrain. Cell 138, 885–897 (2009).
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).
Tsai, M.C. et al. Long noncoding RNA as modular scaffold of histone modification complexes. Science 329, 689–693 (2010).
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).
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).
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).
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).
Zhao, J. et al. Genome-wide identification of polycomb-associated RNAs by RIP-seq. Mol. Cell 40, 939–953 (2010).
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).
Guttman, M. et al. lincRNAs act in the circuitry controlling pluripotency and differentiation. Nature 477, 295–300 (2011).
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).
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).
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).
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).
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).
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).
Record, M.T. Jr., Lohman, M.L. & De Haseth, P. Ion effects on ligand-nucleic acid interactions. J. Mol. Biol. 107, 145–158 (1976).
Makino, D.L., Baumgartner, M. & Conti, E. Crystal structure of an RNA-bound 11-subunit eukaryotic exosome complex. Nature 495, 70–75 (2013).
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).
Marks, H. et al. The transcriptional and epigenomic foundations of ground state pluripotency. Cell 149, 590–604 (2012).
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).
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).
Ku, M. et al. Genomewide analysis of PRC1 and PRC2 occupancy identifies two classes of bivalent domains. PLoS Genet. 4, e1000242 (2008).
Hansen, K.H. et al. A model for transmission of the H3K27me3 epigenetic mark. Nat. Cell Biol. 10, 1291–1300 (2008).
Sun, S. et al. Jpx RNA Activates Xist by evicting CTCF. Cell 153, 1537–1551 (2013).
Mazzone, J. & Pickett, J. The household diary study: mail use & attitudes in FY 2010. (US Postal Service 2011).
Khersonsky, O. & Tawfik, D.S. Enzyme promiscuity: a mechanistic and evolutionary perspective. Annu. Rev. Biochem. 79, 471–505 (2010).
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).
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).
Quinlan, A.R. & Hall, I.M. BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics 26, 841–842 (2010).
Li, H. et al. The Sequence Alignment/Map format and SAMtools. Bioinformatics 25, 2078–2079 (2009).
Robinson, J.T. et al. Integrative genomics viewer. Nat. Biotechnol. 29, 24–26 (2011).
Zhang, Y. et al. Model-based analysis of ChIP-Seq (MACS). Genome Biol. 9, R137 (2008).
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).
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).
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).
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).
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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.
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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)
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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
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DOI: https://doi.org/10.1038/nsmb.2679
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