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RNA exploits an exposed regulatory site to inhibit the enzymatic activity of PRC2

Nature Structural & Molecular Biology volume 26, pages 237–247 (2019)Cite this article

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Abstract

Polycomb repressive complex 2 (PRC2) is a histone methyltransferase that maintains cell identity during development in multicellular organisms by marking repressed genes and chromatin domains. In addition to four core subunits, PRC2 comprises multiple accessory subunits that vary in their composition during cellular differentiation and define two major holo-PRC2 complexes: PRC2.1 and PRC2.2. PRC2 binds to RNA, which inhibits its enzymatic activity, but the mechanism of RNA-mediated inhibition of holo-PRC2 is poorly understood. Here we present in vivo and in vitro protein-RNA interaction maps and identify an RNA-binding patch within the allosteric regulatory site of human and mouse PRC2, adjacent to the methyltransferase center. RNA-mediated inhibition of holo-PRC2 is relieved by allosteric activation of PRC2 by H3K27me3 and JARID2-K116me3 peptides. Both holo-PRC2.1 and holo-PRC2.2 bind RNA, providing a unified model to explain how RNA and allosteric stimuli antagonistically regulate the enzymatic activity of PRC2.

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Fig. 1: Targeted RBR-ID of PRC2.
Fig. 2: RNA binds to and inhibits both PRC2.1 and PRC2.2.
Fig. 3: Mapping of protein-RNA interactions within PRC2–AEBP2 in vitro.
Fig. 4: Stimulatory peptides relieve the RNA-mediated inhibition of PRC2.
Fig. 5: DNA-independent RNA-mediated inhibition of PRC2.
Fig. 6: The RNA-binding site in the regulatory center of PRC2 is exposed within both PRC2.1 and PRC2.2.
Fig. 7: A model for RNA-mediated inhibition of PRC2.

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Code availability

R scripts used for XL–MS and RBDmap data analysis downstream of pLink and Andromeda, respectively, can be downloaded from GitHub: https://github.com/egmg726/crisscrosslinker.

Data availability

LC–MS raw data for targeted RBR-ID experiments have been deposited at the Chorus project (https://chorusproject.org) with ID 1560. Mass spectrometry data for RBDmap and BS3 XL–MS experiments were deposited at FigShare with DOIs https://doi.org/10.26180/5c3d9751c64ae and https://doi.org/10.26180/5c3d8dd45651b, respectively. Source data for Figs. 2–5 and Supplementary Fig. 2–5 are available within Supplementary Data Set 5–8, respectively.

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Acknowledgements

We would like to thank the Monash Biomedical Proteomics Facility for providing instrumentation and technical support. Q.Z. holds an Australian Research Council (ARC) Discovery Early Career Researcher Award (no. DE180100219). N.J.M. is the Isabella and Marcus Foundation Charlee Ferrar Scholar and is also supported through an Australian Government Research Training Program (RTP) Scholarship. R.W.-T. was supported by NIH training grant no. T32GM008216. E.H.G. holds a Biomedicine Discovery Scholarship and is an EMBL-Australia PhD student. B.M.O. is supported through an Australian Government RTP Scholarship and also by the Monash Graduate Excellence Scholarship. R.B. acknowledges support from the NIH (grant no. R01GM127408) and the March of Dimes Foundation (grant no. 1-FY-15–344). C.D. is an EMBL-Australia Group Leader and acknowledges support from the ARC (grant no. DP190103407) and the NHMRC (grant no. APP1162921).

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Author notes

  1. These authors contributed equally: Qi Zhang, Nicholas McKenzie, Robert Warneford-Thomson.

Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Faculty of Medicine, Nursing and Health Sciences, Monash University, Clayton, Victoria, Australia

    Qi Zhang, Nicholas J. McKenzie, Emma H. Gail, Sarena F. Flanigan, Brady M. Owen, Vitalina Levina, Ralf B. Schittenhelm & Chen Davidovich

  2. Epigenetics Institute, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA

    Robert Warneford-Thomson, Richard Lauman, Benjamin A. Garcia & Roberto Bonasio

  3. Graduate Group in Biochemistry and Biophysics, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA

    Robert Warneford-Thomson & Richard Lauman

  4. Department of Biochemistry and Biophysics, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA

    Benjamin A. Garcia

  5. Monash Biomedical Proteomics Facility, Monash Biomedicine Discovery Institute, Monash University, Clayton, Victoria, Australia

    Ralf B. Schittenhelm

  6. Department of Cell and Developmental Biology, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA

    Roberto Bonasio

  7. EMBL-Australia and the ARC Centre of Excellence in Advanced Molecular Imaging, Clayton, Victoria, Australia

    Chen Davidovich

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Contributions

Q.Z., N.J.M., R.W.-T., S.F.F. and B.M.O. prepared reagents. Q.Z., N.J.M., R.W.-T., S.F.F., B.M.O., R.L., V.L. and R.B.S. carried out experiments. Q.Z., N.J.M., R.W.-T., E.H.G., S.F.F., B.M.O., R.L., V.L., B.A.G. and R.B.S. analyzed data. Q.Z., N.J.M., R.W.-T., R.B. and C.D. wrote the paper. R.B. and C.D. designed and supervised the project.

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Correspondence to Roberto Bonasio or Chen Davidovich.

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Supplementary Figure 1 Additional information about targeted RBR-ID.

a, Summary table of PRC2 RBR-ID data. PRC2 proteins, their Uniprot database accession IDs, percent sequence coverage, and the number of significantly (P < 0.05) depleted RNA-binding peptides identified by RBR-ID are shown for nuclear proteome data (He, C. et al., Mol Cell. 64, 416–430, 2016) and targeted RBR-ID (this study). Because PALI1 was discovered after our proteome-wide study (Conway, E. et al., Mol Cell. 70, 408–421 e8, 2018), we reanalyzed the dataset separately to include its sequence in the database. b, Western blot from representative PRC2 immunoprecipitation for core subunits EED and EZH2 and accessory subunit JARID2 (uncropped blots are in Supplementary Data Set 4. c, Comparison of the portion of mass spectrometry signal mapping to PRC2 subunits in proteome-wide data (He, C. et al. Mol Cell. 64, 416–430, 2016) compared to a representative experiment of SILAC-based targeted RBR-ID. Data are plotted as a percentage of all detected peptides. d, Volcano plot of peptides in the proteome and targeted RBR-ID data, displaying mean log2-fold changes in ± 4SU samples against log-transformed P value, calculated using two-sided Student’s t-test that for proteome data was paired and for targeted RBR-ID data was either paired or unpaired (see Methods). PRC2 peptides from proteome and targeted RBR-ID are displayed in red and blue respectively. Horizontal dashed line represents P = 0.05. e, RBR-ID score plots (see He, C. et al., Mol Cell. 64, 416–430, 2016) for all PRC2 subunits shown in Fig. 1 and discussed in this study. Protein domain schematic is shown below each linear plot

Supplementary Figure 2 PRC2 complex purification and nucleosome reconstitution.

a,b, Full Coomassie blue-stained SDS–PAGE and the corresponding radiogram as shown in Fig. 2c, d. c, Coomassie blue-stained SDS-PAGE gel shows the purity of PRC2 complexes used for HMTase assays in Fig. 2c, d. d, Gel filtration chromatography (Sephacryl S-400 HR resin) of the PRC2 complexes that were used for HMTase assays in Fig. 2c, d. Only fractions corresponding to assembled PRC2 complexes were collected and used. e, Mononucleosomes used for HMTase assays in Fig. 2c, d were analyzed on a 4% polyacrylamide TBE gel and visualized by SYBR Green I post-staining. f, Mononucleosome homogeneity was assessed using negative stain electron microscopy (representative micrograph at x52,000 magnification). g, A Coomassie blue-stained SDS–PAGE gel shows the purity of PRC2-MTF2 and PRC2-MTF2-EPOP complexes. h, Fluorescence anisotropy was carried out to compare the RNA-binding affinities of PRC2-MTF2 and PRC2-MTF2-EPOP. Error bars represent standard deviation based on three independent experiments that were performed on different days. i, Resulting dissociation constants (Kd) and Hill coefficients are indicated, including the corresponding standard errors. Data for PRC2 was imported from Fig 2b, for a direct comparison. j-k, HMTase assays of the indicated complexes were carried out in the presence or absence of 8.0 μM G4 256 RNA. j, A representative Coomassie blue-stained SDS–PAGE and the corresponding radiograms. k, Quantification of HMTase activities from (j), with error bars representing standard deviation calculated from three independent experiments. P values were determined using unpaired two-tailed Student’s t-test; *, P < 0.05.

Supplementary Figure 3 Direct and unbiased detection of protein–RNA interactions within the PRC2-AEBP2 complex.

a, Evidence of UV cross-linking, analyzed using 18% SDS–PAGE and visualized by Coomassie blue and silver staining. Mw: Molecular weight marker; Pre: input before adding LysC or ArgC protease; Inp: input; FT: flow-through; EL: eluate. Scatterplots (bottom) indicate intensities identified by MS/MS for each of the peptides in the input (x-axis) and eluate (y-axis) in four independent RBDmap experiments (in assorted colors). Although the recovered peptides were obtained in quantities below the detection limit of SDS–PAGE (EL lanes in all gels), they were detected by MS/MS only in the +UV sample, indicating the stringency of the purification process. b, PRC2-AEBP2 mutants were evaluated by 10% SDS–PAGE and gel filtration chromatography (Sephacryl S-400 HR resin). c, Fluorescence anisotropy used to quantify the affinity of the mutants to G4 24 RNA. The resulting dissociation constant (Kd), Hill coefficients and the derived ΔΔG are indicated together with details of the mutated amino acids in EZH2 and EED. Error bars in (c) represent standard deviation based on three independent experiments that were performed on different days. Standard errors are indicated in (d) when applicable. e-g, The impaired RNA-binding activity of the mutants and their position on the surface of PRC2 is represented in a ΔΔG heat map using the PRC2-AEBP2 structure (regulatory and substrate peptides are colored in magenta and black respectively). h, Bar plot represents the relative HMTase activities of PRC2-AEBP2 mutations toward the H3 substrate compared to the wild-type, which is indicated as a dashed gray line. Error bars indicate standard deviations as measured across three independent experiments. P values were determined using unpaired two-tailed Student’s t-test; *, P < 0.05. i,j, Representative Coomassie blue-stained SDS–PAGE and the corresponding radiograms used for the HMTase assays are in Fig. 3g and Supplemental Fig. 3h. k, The affinity of PRC2-AEBP2 to 32P-radiolabeled G4 256 RNA was quantified using EMSA. Data points represent three-fold dilutions of PRC2-AEBP2 starting from 50 nM. l, Quantification was done by fitting the EMSA data to an equilibrium binding curve. Error bars indicate standard deviation based on three independent experiments that were performed on different days. The resulting dissociation constant (Kd), Hill coefficient and standard errors are indicated.

Supplementary Figure 4 Stimulatory peptides relieve RNA-mediated inhibition of PRC2.

a,b, HMTase assays were carried out in the presence of 0.5 μM wild-type (WT) or mutant 1 (mt1) PRC2 or PRC2-AEBP2, 2.0 μM nucleosome substrate, in the presence (+) or absence (-) of 80 μM H3K27me3 peptide and in the presence (+) or absence (-) of 4.0 μM G4 256 RNA. Representative Coomassie blue-stained SDS–PAGE (top) and the corresponding radiograms (middle) are presented, with bar plots (bottom) representing the HMTase activities quantified based on three replicates. The data represented by black bars in panels (c) and (d) were used to generate Fig. 4a. e,f, Representative Coomassie blue-stained SDS–PAGE and the corresponding radiograms used for quantifying the HMTase activities presented in Fig. 4c. f, HMTase assays were carried out in the presence of 0.5 μM PRC2-AEBP2, 2.0 μM nucleosome substrate and G4 24 RNA (e) or G4 256 RNA (f) at concentrations of either 0, 4 or 8 μM and stimulatory peptides, as indicated. g, Fluorescence anisotropy used to quantify the affinity of PRC2-AEBP2 to G4 24 RNA in the presence or absence of 10 μM of the EED inhibitor A395 or the negative control A395N. Error bars represent standard deviation based on three independent experiments that were performed on different days. h, Resulting dissociation constants (Kd), Hill coefficients and the derived ΔΔG values are indicated. Standard errors are indicated. i, The coordinates of A395, as previously identified by X-ray crystallography (PDB: 5K0M, He, Y. et al., Nat Chem Biol. 13, 389–395, 2017), are presented on the high-resolution cryo-EM structure of PRC2 (PDB: 6C23) by superimposing EED from both structures. Orange and red spots represent RNA-linked polypeptides that were identified in 2 or 3 independent RBDmap experiments respectively.

Supplementary Figure 5 DNA-independent RNA-mediated inhibition of PRC2.

a, The location of a substrate peptide (JARID2-K116, in black; PDB 6C23) and an oncogenic inhibitory peptide (H3K27M, in brown; PDB 5HYN) within PRC2 (in gray; PDB 6C23) with respect to RNA-linked polypeptides that were identified using RBR-ID (score of > 5; in pink), RBDmap (identified in 2 or 3 independent experiments; represented in orange and red respectively) or in both assays (in yellow). b, The affinity of PRC2-AEBP2 to G4 24 RNA was quantified using fluorescence anisotropy in the presence or absence of a substrate peptide (10 μM JARID2-K116 or 100 μM H3 histone peptide) or an oncogenic peptide (100 μM H3K27M); see panel c for a color code. c, Resulting dissociation constants (Kd), Hill coefficients, the derived ΔΔG values and the number of independent replicates (n) are indicated. Peptide sequences are indicated, with the substrate lysines in red; highlighted in gray, are amino acids that were previously traced in the catalytic center, using high resolution cryo-EM (JARID2-K116; PDB 6C23) or x-ray crystallography (H3K27M; PDB 5HYN). d,e, HMTase assays were carried out in the presence of 0.5 μM PRC2-AEBP2 or PRC2-AEBP2-JARID2, 4.0 μM H3 histone substrate and in the presence or absence of G4 256 RNA or G4 24 RNA at concentrations as indicated under the bar plot. The bar plot represents the relative activity with respect to the no-RNA sample, as recorded by densitometry after SDS–PAGE. See Supplementary Data Set 4 for the uncropped images of the gels and radiograms. f, Uncropped images of the gels shown in Fig. 5a.

Supplementary Figure 6 One face of PRC2 clusters binding sites of multiple regulatory factors.

a, Histogram of distances that were measured between cross-linked lysine pairs within the PRC2 core subunits. BS3 XL-MS data was generated using the three PRC2 complexes as indicated in the color key (see Fig. 6a–c for cross-linking sites) and distances were measured between lysine pairs within the high-resolution cryo-EM structure of the PRC2-AEBP2-JARID2 complex (PDB: 6C23). b, Randomized distances histogram was generated after randomly selecting N lysine pairs and measuring the distances between them over the same structure as in (a), where N is the number of observed cross-linked lysine-pairs in each of the datasets used in (a). c, Front (center) and, rear (left) views, and 20 ° rotation with respect to the front view (right), of the PRC2-AEBP2-JARID2 structure presented in Fig. 6d, using the same color code as in Fig. 6. d, The structure as shown in (c), represented in assorted colors according to the four PRC2 core subunits. AEBP2 and JARID2, as well as the regulatory and substrate peptides, are colored according to the same color key as in Fig. 6d. e, RNA-linked peptides that were identified using targeted RBR-ID (RBR-ID score > 5) were mapped to the highresolution structure of PRC2 (PDB: 6C23 and 5WAI). f, RNA-linked peptides that were identified using RBDmap in 2 or 3 replicates are presented on the same structure and views as in (e), for a direct comparison

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Zhang, Q., McKenzie, N.J., Warneford-Thomson, R. et al. RNA exploits an exposed regulatory site to inhibit the enzymatic activity of PRC2. Nat Struct Mol Biol 26, 237–247 (2019). https://doi.org/10.1038/s41594-019-0197-y

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  • DOI: https://doi.org/10.1038/s41594-019-0197-y

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