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PRC2 binds active promoters and contacts nascent RNAs in embryonic stem cells

Nature Structural & Molecular Biology volume 20, pages 1258–1264 (2013)Cite this article

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Abstract

EZH2 is the catalytic subunit of PRC2, a central epigenetic repressor essential for development processes in vivo and for the differentiation of embryonic stem cells (ESCs) in vitro. The biochemical function of PRC2 in depositing repressive H3K27me3 marks is well understood, but how it is regulated and directed to specific genes before and during differentiation remains unknown. Here, we report that PRC2 binds at low levels to a majority of promoters in mouse ESCs, including many that are active and devoid of H3K27me3. Using in vivo RNA-protein cross-linking, we show that EZH2 directly binds the 5′ region of nascent RNAs transcribed from a subset of these promoters and that these binding events correlate with decreased H3K27me3. Our findings suggest a molecular mechanism by which PRC2 senses the transcriptional state of the cell and translates it into epigenetic information.

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Figure 1: EZH2 binds to RNA in mouse ESCs.
Figure 2: Genome-wide analysis of EZH2 CLIP in mouse ESCs.
Figure 3: EZH2-bound nascent RNAs originate from PRC2+ and H3K27me3− promoters.
Figure 4: Decreased levels of H3K27me3 at ezRNA+ genes.
Figure 5: PRC2 senses transcriptional activity at ESC promoters.

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Acknowledgements

We thank W.W. Tee, L. Vales and P. Voigt for their critical assessment of the manuscript, T. Cech (University of Colorado Boulder) for discussions and the Genome Technology Center at New York University for help with sequencing. This work was supported by grants from the US National Institutes of Health (GM-64844 and R37-37120 to D.R.) and the Howard Hughes Medical Institute (to D.R.). R.B. was supported by a Helen Hay Whitney Foundation postdoctoral fellowship and by the Helen L. and Martin S. Kimmel Center for Stem Cell Biology postdoctoral fellow award.

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  1. Roberto Bonasio

    Present address: Present address: Department of Cell and Developmental Biology, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania, USA.,

Authors and Affiliations

  1. Howard Hughes Medical Institute, New York University School of Medicine, New York, New York, USA

    Syuzo Kaneko, Jinsook Son, Steven S Shen, Danny Reinberg & Roberto Bonasio

  2. Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, New York, New York, USA

    Syuzo Kaneko, Jinsook Son, Steven S Shen, Danny Reinberg & Roberto Bonasio

  3. Center for Health Informatics and Bioinformatics, New York University School of Medicine, New York, New York, USA

    Steven S Shen

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S.K., J.S., S.S.S., D.R. and R.B. designed and performed experiments, analyzed data and wrote the manuscript.

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Correspondence to Danny Reinberg or Roberto Bonasio.

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Integrated supplementary information

Supplementary Figure 1 Generation and validation of mouse ESCs expressing epitope-tagged EZH2.

(a) Schematic diagram indicating the domain structure of EZH2 and the identity and position of the epitope tags in the N3-EZH2 construct. The FLAG tag and Tobacco Etch Virus protease (TEV) recognition site are also indicated although they were not utilized in the experiments described herein. The domain structure of EZH2 refers to NP_031997.2 and is adapted from ref. 24. (b) Western blots from N3-EZH2 ESCs with or without doxycycline (dox) induction. Immunoblots were performed with the indicated antibodies. The position of the epitope-tagged (N3-EZH2) and endogenous (EZH2) protein is indicated in the EZH2 immunoblot. Notice that most endogenous EZH2 is knocked down by an inducible shRNA. (c) View of a large region of mouse chromosome 14, showing profiles for two independent ChIP-seq performed with antibodies against endogenous EZH2, before induction of the N3–EHZ2 transgene, and one ChIP-seq performed with an HA antibody after induction of N3–EZH2. (d) Genome wide correlation profile of endogenous EZH2 ChIP-seq (x axis) and transgenic N3–EZH2 ChIP-seq (y axis) as computed using DiffBind53 over all EZH2 ERs (28,365) identified by MACS52.

Supplementary Figure 2 PAR-CLIP for EZH2 in mouse ESCs.

(a) Correlation plot for CLIP tag density on RefSeq gene bodies obtained in 4 separate biological replicates. Because of limited coverage, the replicates were paired 2 by 2. A similar correlation was observed when the order of pairing was inverted. (b) Same as a, but the gene bodies of the 250 genes containing the most RCSs were divided in 10 windows, to analyze the degree of spatial consistency between replicates. (c) Additional examples of EZH2 CLIP tags mapping to the 5' of nascent transcripts originating from protein-coding genes. A track for EZH2 ChIP-seq is also shown and the scale is indicated to the right in reads per 10 million mapped (RP10M). RCSs called by PARalyzer are shown as red bars. (d) Density profile of CLIP tags over the 250 unique RefSeq transcripts with the highest CLIP tag density. Transcripts were normalized for length and divided in 100 bins. The position of the transcription start site (TSS) and termination site (TTS) is shown. The profile includes the 10 kb upstream and downstream, each divided in 50 200 nts bins. The profile for RNA-seq reads (gray dashed line) is shown for comparison.

Supplementary Figure 3 5' bias of EZH2 binding as a function of bioinformatic cutoffs.

The smoothed density profile for RCSs (a) and CLIP tags (b) is shown as in Fig. 2c and Supplementary Fig. 2d over an increasingly restricted set of RefSeq genes (numbers are indicated in the plots) obtained by varying the cutoff of number of RCSs (a) or CLIP tags (b) required for inclusion.

Supplementary Figure 4 ezRNAs originate from PRC2+ and H3K27me−, nonbivalent promoters.

(a) Normalized and sorted EZH2 occupancy heatmap (bottom) for all unique RefSeq transcripts in E14 cells grown in serum. The distribution of ezRNA+ genes relative to the heatmap is shown by black lines (middle) and a smoothed density plot (top). The control is a random permutation of the same distribution (dashed gray line). Data was obtained from GSM59011531. (b) As in a, but for H3K27me3. Data was obtained from GSM59013231. (c, d) Venn diagram showing lack of overlap between ezRNA+ genes and bivalent genes, as defined by Mikkelsen et al.32 (c) or Marks et al.31 (d). (e) Normalized heatmaps for chromatin occupancy of EZH2 and SUZ12 in E14 mouse ESCs on all unique RefSeq TSSs. Both heatmaps were sorted by EZH2 occupancy (indicated by the arrow) and show the average of two biological replicates.

Supplementary Figure 5 Relationship between H3K27me3 levels and ezRNA production.

(a) Correlation analysis between H3K27me3 and EZH2 densities at single ezRNA+ genes (dark gray points, black regression line) or a control set (light gray points, dashed gray regression line) equalized by PRC2 levels as in Fig. 4a (left panel) or RPKMs as in Fig. 4b (right panel). Densities are plotted as log2(RP10M) in each 10 kb window spanning the TSS. (b) Density profiles for H3K27me3 in E14 (left panels) or TNGA (right panels) mouse ESCs grown in serum (top) or 2i (bottom) conditions. The profiles were calculated on the promoters of 1,108 ezRNA+ transcripts (black line) or an equally sized control set of promoters (gray dashed line) matched individually for EZH2 occupancy in E14 cells.

Supplementary Figure 6 Whole blot images for all experiments.

(a) Relative to Fig. 1a. (b) Relative to Fig. 1b. (c) Relative to Fig. 1d. (d) Relative to Fig. 2f.

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Kaneko, S., Son, J., Shen, S. et al. PRC2 binds active promoters and contacts nascent RNAs in embryonic stem cells. Nat Struct Mol Biol 20, 1258–1264 (2013). https://doi.org/10.1038/nsmb.2700

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