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. 2013 Nov;20(11):1258-64.
doi: 10.1038/nsmb.2700. Epub 2013 Oct 20.

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

Syuzo Kaneko  1 , Jinsook Son, Steven S Shen, Danny Reinberg, Roberto Bonasio
Affiliations

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

Syuzo Kaneko et al. Nat Struct Mol Biol. 2013 Nov.

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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Figures

Figure 1
Figure 1
EZH2 binds to RNA in mESCs. (a) CLIP blots for HA-tagged EZH2 in control cells and cells induced with doxycycline and before or after irradiation with UVC. The autoradiography is shown at the top and the approximate position of HA-EZH2 is indicated. The corresponding HA immunoblot is shown at the bottom. (b) PAR-CLIP autoradiography (top) and western blot (bottom). Different 4-SU concentrations, UV wavelength, and RNAse treatments were tested. (c) Scheme of the purification strategy used for PAR-CLIP-seq experiments. (d) Autoradiography of 3 biological replicates (rep1–3) utilized for PAR-CLIP-seq library construction. The dashed red boxes indicate the position of the excised bands. (e) Histogram plot for the mutation frequencies in PAR-CLIP-seq reads. The bars represent the average % of unique mapped CLIP tags containing the indicated mutation from the 4 biological replicates + s.d. (f) Genome browser view of EZH2 CLIP tags mapping to the Meg3 lncRNA (top) or Kcnq1ot1 antisense ncRNA (bottom). The 4 biological replicates are plotted separately. RCSs called by PARalyzer are shown as red bars. UCSC gene models are displayed. Rep1–4, biological replicate.
Figure 2
Figure 2
Genome-wide analysis of EZH2 CLIP in mouse ESCs. (a) Distribution of RCSs identified with PARalyzer relative to genomic features and their distribution. The number of features in each set is indicated at the top. (b) EZH2 CLIP tags mapping to two representative protein-coding genes. An EZH2 ChIP-seq track is shown, with the scale indicated to the right in reads per 10 million mapped (RP10M). Red bars show RCSs called by PARalyzer. Rep1–4, biological replicate. (c) Density profile of RCSs over the 250 unique RefSeq transcripts with the most RCSs, each divided in 100 bins. TSS, transcription start site; TTS, transcription termination site. The 10 kb upstream and downstream are included, each divided in 50 200 nts bins. The distribution of RNA-seq reads (gray dashed line) is shown for comparison. (d) Same as c but for CLIP tags (after duplicate removal) and comparing tags containing the T>C transition (solid black line) with tags not containing the mutation (dashed gray line). (e) Distribution of CLIP tags and RNA-seq reads on the first 50 bp of first exons (E1), first introns (i1), last introns (in) and last exons (En) from RefSeq transcripts with at least two exons. The bars represent the mean percentage of total reads mapping to these features from 2 (RNA-seq) or 4 (CLIP-seq) biological replicates + numerical range. (f) PAR-CLIP as in Fig. 1d after chasing the 4-SU with uridine for the indicated time. Autoradiography (top) and HA blot (bottom) demonstrating equal protein loading.
Figure 3
Figure 3
EZH2-bound nascent RNAs originate from PRC2+ and H3K27me3- promoters. (a) Bar plot for the –log10 of the P-value of the top 15 most enriched GO terms in genes producing ezRNAs, as determined by the hypergeometric distribution. (b) Density profile of raw CLIP tags (after duplicate removal and repeat masking) relative to the top 20,000 EZH2 ChIP enriched regions (ERs). As a control, the density of the same CLIP tags was calculated on 20,000 random genomic regions of comparable size (dashed gray line). (c) The heatmap (bottom) shows EZH2 densities at all unique RefSeq TSSs ± 2.5 kb, sorted by EZH2 occupancy. The distribution of ezRNA-producing TSSs within this heatmap is indicated by individual bars (middle) and a density plot (top, black line) and compared to their density over a randomly permutated heatmap (top, dashed gray line). Data was from 2 biological replicates. (d) Same as c but for H3K27me3. The heatmap shows the average of 3 biological replicates. (e) Heatmaps for H3K4me3 and H3K36me3 occupancy comparing the 1,108 genes producing ezRNAs and a random gene set. Data were obtained from GSM590111 and GSM590119. (f) PRC2 occupancy in 5 kb windows at TSSs from different position along the EZH2 gradient in the heatmap (top). The y axis represents reads per 10 million mapped (RP10M) and the scale is indicated by the number to the left of each profile. In all cases the input was scaled at the highest magnification for meaningful comparison.
Figure 4
Figure 4
Decreased levels of H3K27me3 at ezRNA+ genes. (a) Density profiles for H3K27me3 in E14 mESCs (left panel) spanning 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 (middle panel). Log-converted RPKM values for the two sets of transcripts are shown in the violin plot (right panel). The plots show the average of 3 (H3K27me3), or 2 (EZH2, RNA-seq) biological replicates. (b) Same as a, but control genes were selected by equalizing RPKM levels on a transcript-by-transcript basis. (c) H3K27me3 density plot calculated as in a for V6.5 ESCs (left) and MEFs (right). Data were obtained from GSE12241.
Figure 5
Figure 5
PRC2 senses transcriptional activity at ESC promoters. (a) In ESCs, PRC2 binds to a majority of TSSs in mouse ESCs, independent of their transcriptional state, sampling their activity. (b) Regulation of PRC2 activity occurs during the “sensing” phase, when PRC2 binds to nascent ezRNAs that somehow impede the deposition of H3K27me3 on chromatin. This could happen either by (I) inhibition of PRC2 activity, possibly dependent on additional in vivo factors, or (II) eviction of PRC2 from chromatin or of other factors required for H3K27me3 deposition. This allows the cell to continue expressing genes for which activating transcription factors are present, despite the constant presence of PRC2. (c) During differentiation, lineage specific transcriptional repressors are upregulated and silence transcriptional activity at selected target genes. Lacking the inhibitory ezRNAs, PRC2 initiates deposition of H3K27me3, establishing the nucleating conditions for the formation of facultative heterochromatin. (d) By the time the differentiation program is completed, a positive feedback loop of H3K27me3 deposition, EED binding, PRC2 recruitment, and more H3K27me3 deposition gives rise to stable and self-perpetuating facultative heterochromatin, which maintains transcriptional repression even after the original transcription factor ceases to be expressed.
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Comment in

  • Poly-combing the genome for RNA.
    Goff LA, Rinn JL. Goff LA, et al. Nat Struct Mol Biol. 2013 Dec;20(12):1344-6. doi: 10.1038/nsmb.2728. Nat Struct Mol Biol. 2013. PMID: 24304912

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