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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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.
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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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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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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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DOI: https://doi.org/10.1038/s41594-019-0293-z
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