Interactions between JARID2 and noncoding RNAs regulate PRC2 recruitment to chromatin

Abstract

JARID2 is an accessory component of Polycomb repressive complex-2 (PRC2) required for the differentiation of embryonic stem cells (ESCs). A role for JARID2 in the recruitment of PRC2 to target genes silenced during differentiation has been put forward, but the molecular details remain unclear. We identified a 30-amino-acid region of JARID2 that mediates interactions with long noncoding RNAs (lncRNAs) and found that the presence of lncRNAs stimulated JARID2-EZH2 interactions in vitro and JARID2-mediated recruitment of PRC2 to chromatin in vivo. Native and crosslinked RNA immunoprecipitations of JARID2 revealed that Meg3 and other lncRNAs from the imprinted Dlk1-Dio3 locus, an important regulator of development, interacted with PRC2 via JARID2. Lack of MEG3 expression in human induced pluripotent cells altered the chromatin distribution of JARID2, PRC2, and H3K27me3. Our findings show that lncRNAs facilitate JARID2-PRC2 interactions on chromatin and suggest a mechanism by which lncRNAs contribute to PRC2 recruitment.

Figure 1. Identification of the RNA-binding region of JARID2

(A) Domain organization of human JARID2 and scheme of the 6xHis-fused truncations utilized in the mapping experiments. (B) In vitro streptavidin pull-down after incubation of increasing concentrations of the indicated JARID2 recombinant fragments with biotinylated HOTAIR_1–333. Input, 2 μg; titration, 2 and 4 μg. (C) High resolution mapping of the residues of JARID2 necessary for RNA binding in vitro. The two indicated fragments (right) were incubated with HOTAIR_1–333 and assayed as in (B). Input, 2 μg; titration, 1, 2, and 4 μg. See also Figure S1.

Figure 2. JARID2 and EZH2 share interacting lncRNAs in vivo, including Meg3

(A) PAR-CLIP with JARID2 antibodies or IgG in E14 ESC cells. The position of full length JARID2 is indicated. The asterisk marks a prfesumed degradation product. (B) Immunoblot on the same material utilized for the autoradiography in (A). J2, anti-JARID2 antibody. (C) PAR-CLIP (top) and immunoblot for JARID2 (bottom) performed in cells pulsed (+) or not pulsed (-) with 4-SU and stably transfected with an shRNA against Jarid2 or a control shRNA. Extracts were treated with increasing concentration of a cocktail of DNase-free RNase A and T1. (D) PAR-CLIP-seq blot for JARID2. (E) Distribution of JARID2 RCSs identified by PARalyzer in the genome. The stacked columns represent % of total RCSs. “Repeats” include all features listed in the RepMask database. (F) Venn diagram of lncRNAs containing RCSs for JARID2, EZH2, or both. PAR-CLIP data for EZH2 were taken from GSE49433 (Kaneko et al., 2013). (G) Genome browser view of JARID2 and EZH2 CLIP tags (black bars) or RCSs identified by PARalyzer (red bars) mapping to the Meg3 lncRNA. Gene models for Meg3 according to both TROMER and ENSEMBL are shown. Meg3 fragments tested for in vitro binding are indicated at the bottom. See also Figure S2 and Table S1.

Figure 3. The JARID2 RBR mediates interactions of the Meg3 lncRNA with PRC2

(A) In vitro pull-down assay with different fragments of Meg3 using 4 μg of GST-JARID2 119-450 WT or ΔRBR. (B) Quantification of bands shown in (A). (C) RIPs for EZH2 were performed in E14 ESCs stably transfected with an shRNA against Jarid2 (KD) or empty vector (ctrl). Co-precipitated proteins were revealed by western blot. 10% input and IgG lanes are shown as control. (D) RT-qPCR on EZH2 RIPs from control E14 ESCs (white bars) or Jarid2 knockdown E14 ESCs (black bars). Data is shown as % of RIP input. Bars represent the mean of 4 replicates + s.d. (E and F) As in (C) and (D) but Ezh2 was knocked down and the RIP were performed with JARID2 antibodies. (G) Western blots for HA RIPs from nuclear extracts of KH2 transiently transfected with N3-tagged EZH2, EZH2_ΔRBR (top), JARID2, and JARID2_ΔRBR (bottom). I, input; IP, HA immunoprecipitation; FT, flow-through. (H) RT-qPCR normalized to Gapdh levels on HA RIPs described in (E). Bars indicate the mean of 3 biological replicates + s.e.m. *, P < 0.05 by Mann-Whitney U test. See also Figure S3 and S4.

Figure 4. Decreased occupancy of PRC2 at some chromatin targets in MEG3^- cells

(A-C) MA plots for JARID2 (A), EZH2 (B), and H3K27me3 (C) occupancy, as determined by the normalized and input-corrected read densities in MEG3⁺ (above dotted line) vs. MEG3^- (below dotted line) hiPSC lines. Each dot represents an ER in common between at least two hIPSC lines. DBRs with an FDR < 0.1 are displayed in red. (D) Venn diagram for DBRs with FDR < 0.1. (E) RT-qPCR analysis of PRC2 targets in MEG3⁺ and MEG3^- hiPSCs. Bars represent the mean RNA abundance (as % of GAPDH) in the 5 MEG3⁺ and 3 MEG3^- lines tested. *; P < 0.05, as calculated by Mann-Whitney U test. (F) RT-qPCR for Meg3 24 h after transfection of KH2 ESCs with control (white bar) or Meg3 siRNAs. (G and H) ChIP-qPCR for JARID2 (G) or EZH2 (H) with primers mapping to PRC2 peaks near the indicated genes in KH2 ESCs treated with control (white bars) or Meg3 (black bars) siRNAs. Bars represent the mean of 3 replicates + s.e.m. *, P < 0.05 by Mann-Whitney U test. See also Figure S5, S6, S7, Table S2 and S3.

Figure 5. HOTAIR stimulates EZH2-JARID2 interactions via the JARID2 RBR

(A) FLAG-6xHis-tagged recombinant EZH2 (20 pmol) was incubated with 6xHis-tagged JARID2_119–450 WT or ΔRBR (40 pmol) in presence of increasing amounts of HOTAIR_1–333 (0–12 pmol). Pull-down was performed with anti-FLAG beads and proteins revealed by 6xHis immunoblot. (B) Densitometric quantification of the signal for JARID2 shown in (A). WT and ΔRBR lanes were normalized each to lane 1 and lane 6 (no HOTAIR control), respectively. (C-F) In vitro interaction stimulation assays with recombinant 6xHis-tagged JARID2_119–574 and FLAG-6xHis-tagged full-length EZH2 in presence of increasing concentrations of HOTAIR_1–333 (C), a smaller 5′ truncation of HOTAIR (D), a microRNA (E), or double stranded DNA (F). Pulldown was performed with anti-FLAG beads and proteins stained with Coomassie blue. (G) Same as (C-F) using Meg3 fragments.

Figure 6. JARID2 recruits EZH2 to chromatin in an RBR-dependent manner

(A) Western blots for foreskin fibroblasts transduced with lentiviruses expressing JARID2, JARID2_ΔRBR, or mock-transduced. (B and C) ChIP-qPCR with antibodies against JARID2 (B) or EZH2 (C) at known PRC2 chromatin targets in foreskin fibroblasts transduced as in (A). Several primer sets are shown for HOXD8. The primers for MYT1 were designed at a distal location, devoid of PRC2, and serve as a negative control. The ChIP enrichment is normalized against that obtained with the same antibodies in mock-transfected control cells (dotted line). Bars represent mean of 4 technical replicates + s.d (B) or 3 biological replicates + s.e.m (C). *, P < 0.05 by Mann-Whitney U test.

Figure 7. Proposed model for the interplay of lncRNAs, JARID2, and PRC2

(A) At some target genes, the presence of JARID2 by itself (I) is not sufficient for maximum PRC2 recruitment, which requires scaffolding by lncRNAs (II). The presence of both JARID2 and lncRNAs stimulates further recruitment and assembly of PRC2 on chromatin, resulting in increased H3K27me3 (III). The structure of lncRNAs bound to JARID2 (and PRC2) remains to be elucidated and the one shown here is only for the purpose of illustration. (B) In some cases, lncRNAs might contribute to the initial recruitment of JARID2 to chromatin (I). Because JARID2 also binds PRC2 via protein–protein interactions, this results in increased PRC2 recruitment and H3K27 methylation (II).