Nascent RNA interaction keeps PRC2 activity poised and in check

Abstract

Polycomb-repressive complex 2 (PRC2) facilitates the maintenance and inheritance of chromatin domains repressive to transcription through catalysis of methylation of histone H3 at Lys27 (H3K27me2/3). However, through its EZH2 subunit, PRC2 also binds to nascent transcripts from active genes that are devoid of H3K27me2/3 in embryonic stem cells. Here, biochemical analyses indicated that RNA interaction inhibits SET domain-containing proteins, such as PRC2, nonspecifically in vitro. However, CRISPR-mediated truncation of a PRC2-interacting nascent RNA rescued PRC2-mediated deposition of H3K27me2/3. That PRC2 activity is inhibited by interactions with nascent transcripts supports a model in which PRC2 can only mark for repression those genes silenced by transcriptional repressors.

Keywords: EZH2; JARID2; PRC2; chromatin; ncRNA.

Figure 1.

Holo-PRC2 activity is inhibited by RNA in vitro. (A) HKMT assay performed with increasing amounts of RepA (0.2–6.4 pmol) (left panel) or miR17–92 (0.2–1.6 pmol) (right panel) on 4.56 pmol of recombinant nucleosomes using 1 pmol of PRC2 with 2.5 pmol of JARID2 (residues 119–574) and 4 pmol of AEBP2 for 1 h. Results were analyzed by autoradiography using [³H]ENHANCE spray after SDS-PAGE. (B, top) HKMT assay as in A using HOTAIR FL (0.16–1.25 pmol), HOTAIR_1–333 (1–8 pmol), or HOTAIR_1–116 (2.87–23 pmol). (Bottom) RNA structure prediction using the ViennaRNA package. (C,D) HKMT assay performed with increasing amounts of either Poly(G) (1–64 pmol) (C) or Poly(A) (4–64 pmol) (D) on 2.28 pmol of native nucleosomes using 1 pmol of PRC2 with 1.5 pmol of JARID2 (residues 119–574). The assay was incubated for 15 min with 12.5 μM SAM (radiolabeled:cold: 1:45) and analyzed by scintillation counting after SDS-PAGE. (E) Core PRC2 and RNA-binding assays. (F) HKMT assay using 1 pmol of SET9 and recombinant octamers with increasing amounts of HOTAIR_1–309 (1–64 pmol).

Figure 2.

Transcription termination proximal to the TSS results in increased H3K27me3 at the Ntn1 promoter. (A) Schematic of the Ntn1 gene locus. CRISPR-Cas9 cleavage sites (Downstream 1: 2.6 kb downstream from the TSS; Downstream 2: 95.5 kb downstream from the TSS) are shown by scissors. Position of the RT–PCR primer sets used in C are shown by arrows. Positions of ChIP primer sets (#1, #2, #3) used in D are also indicated. (B) PCR genotyping. (C) RT-qPCR normalized to Gapdh levels on wild-type (white bars) and Ntn1 Downstream 2 (gray bars) and Downstream 1 (black bars) mESCs. Bars represents the mean of three biological replicates +SEM. (*) P < 0.05 by Mann-Whitney U-test. (D) ChIP-qPCR for H3K27me3 normalized by H3 on wild-type (white bars) and Ntn1 downstream knockout (KO) (gray bars) and upstream knockout (black bars) mESCs for the genes indicated at the bottom. All ChIP primer sets are targeted to respective promoter regions. Bars represents the mean of three biological replicates +SEM. (*) P < 0.05 by Mann-Whitney U-test. (E) HKMT assay as in Figure 1 using 1–16 pmol of each of the various sized runoff transcripts derived from Ntn1. Values are given as a percentage of enzymatic activity obtained in the absence of RNA.

Figure 3.

JARID2 contacts nascent RNAs. (A) Procedure for obtaining JARID2 PAR-CLIP data as a function of mESC differentiation. (B) Histogram of mapped CLIP tags reads to the whole genome. Three genome categories for read assignments, which are exon, intron, and promoter regions (from the TSS to −3 kb), are shown in sense (black bar) and antisense (gray bar) directions. (C) Schematic of Ntn1, Xkr6, and Foxo3 gene loci. Unique JARID2 PAR-CLIP-tags are shown in bars with blue (plus strand) and dark red (minus strand). (Rep1–3) Biological replicates. Note that there are upstream CLIP tags that originated from Ntn1 alternative transcript ENSMUST00000108674. TSSs are shown by arrows.

Figure 4.

Dynamics of JARID2–nascent RNA interaction correlates with gene expression during mESC differentiation. (A) JARID2 CLIP tag mapping to two representative lncRNAs before and after RA-mediated differentiation in mESCs. (B) MA plots for JARID2–RNA interaction on gene bodies, comparing undifferentiated versus differentiating mESCs. The graph indicates fold change (logFC; Y-axis) versus abundance (logCPM; X-axis) of mapped CLIP tags. Differentially binding genes (DBGs) with an FDR < 0.05 (red dots) and 0.25 < FDR < 0.05 (green dots) are displayed. (C, left) Heat maps for mRNA levels in undifferentiated versus differentiating mESCs, showing up-regulated (gain of JARID2–RNA interaction; FDR > 0.05, logFC > 1) and down-regulated (loss of JARID2–RNA interaction; FDR > 0.05, logFC < −1) DBGs (see Supplemental Table S5). (Right) Differential expression is also displayed with individual bars. Data were obtained from GSE43221 (Kashyap et al. 2013).