Fig 2: Catalytic activity of polycomb repressive complex 1 (PRC1) is required for aebp2 binding, H3K27me3 establishment, and transcriptional repression of developmental genes.(A) Experimental design of drug treatments to inhibit Rnf2 activity. Embryos at the one-cell stage were added to media containing either PRT4165 (150 μM) or DMSO and raised until 4 hr post fertilization (hpf). (B) PRT4165 treatment of embryos confers bulk loss of H2Aub1 at 4 hpf. Left: Western blot for H2Aub1 in 4 hpf embryos treated with DMSO (vehicle) or 150 μM PRT4165 (Rnf2 inhibitor). Right: Quantification western blot in left panel. (C) Impact of Rnf2 inhibition on Aebp2 genomic localization and H3K27me3. K-means clustering of Aebp2 and H3K27me3 chromatin immunoprecipitation (ChIP)-seq enrichment at all loci with called peaks in any of the datasets plotted. Embryos were treated from the one-cell stage with either DMSO or 150 μM PRT4165 and harvested at 4 hpf for ChIP analysis. H3K27me3 ChIP-seq from untreated embryos at 4.3 hpf is plotted as a comparitor. (D) Impact of Rnf2 inhibition on gene expression. Volcano plot of RNA-seq data from PRT4165-treated vs. untreated embryos (4 hpf). Green and red data points signify transcripts with p-values < 0.01 and at least a 3-fold change (increase or decrease) in expression, respectively. Marquee upregulated genes encoding developmental transcription factors are labelled. (E) Genome browser screenshots of representative developmental genes which, upon Rnf2 inhibition, lose Aebp2 binding and H3K27me3 marking, and become transcriptionally active. Figure 4—source data 1.Uncropped western blots for panel B.

Fig 3: Model for polycomb-mediated establishment of developmental gene silencing during zebrafish embryogenesis.Prior to zygotic genome activation (ZGA), Rnf2-PRC1 is recruited by transcription factors (TFs) to promoters (shown) and enhancers (not shown) of developmental genes bearing Placeholder nucleosomes (H2A.Z(FV), H3K4me1, H3K27ac). Rnf2-PRC1 deposition of H2Aub1 recruits Aebp2-PRC2 to catalyze H3K27me3 addition. H2Aub1 ablation (via Rnf2 inhibition) eliminates Aebp2-PRC2 recruitment and prevents H3K27me3 establishment. Notably, H2Aub1 loss causes precocious transcription of certain developmental genes after ZGA, identifying H2Aub1 as a critical component of silencing at ZGA.

Fig 4: EZHIP mitigates PRC2-cofactors interactions. a Genome-browser representation of SUZ12 enrichment in U2OS WT, EED-/-, and EZHIP-/-. Duplicates are merged, same scale for all tracks. b Immunofluorescence staining of EED and EZHIP (top) or H3K27me2 and EZHIP (bottom) on U2OS, nuclei are stained with DAPI. Representative results. Scale bar, 2 µm. c Left, purification scheme for PRC2. Right, histone methyltransferase (HMT) assay to monitor the enzymatic activity of PRC2 purified from WT (left panel) or EZHIP-/- (middle panel) U2OS cells (titration: 1, 2, 5 × ) on native nucleosomes. Right panel, same assay as previously, but this time titrating recombinant hEZHIP on PRC2-purified from EZHIP-/- U2OS cells (PRC2 quantity 5 × ). The upper panels are autoradiography, and the lower panels are the corresponding SDS-PAGE coomassie staining. Representative image. d Quantification of EZH2-Flag IP through mass spectrometry (iBAQ values). Heatmap representing the Log2-transformed median centered values. Horizontal axis: U2OS WT and EZHIP-/-, n = 3. Vertical axes: PRC2 components. Values are normalized on iBAQ values from untagged U2OS WT and EZHIP-/-. e EZH2-Flag Co-IP from nuclear extracts either WT or EZHIP-/-, and probed with antibodies against EZH2, EED, AEBP2, or JARID2

Fig 5: Suz12 chromatin interactors identified via prS bead ChIP‐SICAP experiment and single injection or wtS bead enrichment followed by HpH fractionation Experimental procedure.Intensity of all PRC2 complex components and streptavidin in ChIP‐SICAP with wtS beads and subjected to HpH fractionation or prS bead and single injection. The error bars show standard deviation of two replicatesDigesting mock beads prepared according to Barshop et al (blue) and prS (red) protocols. Intensity of streptavidin peptides (left) and XICs of the top‐two streptavidin peptides (right).Comparing beads prepared according to Barshop et al with prS beads using a Suz12 ChIP‐SICAP assay. Intensity of streptavidin (left). Number of identified proteins (right).Comparative Suz12 ChIP‐SICAP between 2i and serum. Full MS spectra between 612–618 m/z at retention time: 64–65 min are shown. Specific precursors (m/z: 617.2866 and 612.2821) from Aebp2 isoform 3/4 are detected in the forward experiment (top) and the reverse experiment (bottom).MS/MS spectra indicating the N‐terminal peptide of Aebp2 isoform 3/4 : mDIDSTISSGR.Western blot of whole‐cell lysate of mES cells grown in 2i and serum media. Top: anti‐Aebp2; bottom: anti‐GAPDH. Arrows A and B show two bands detected by Aebp2 antibody. The intensities of band A and B were compared between 2i and serum normalized to GAPDH as the loading control.