Journal: Molecular cell

Article Title: Unbalanced chromatin binding of Polycomb complexes drives neurodevelopmental disorders

doi: 10.1016/j.molcel.2026.01.023

Figure Lengend Snippet: (A) Strategy to generate Rnf2 WT/R70H ESCs by homologous recombination. (B) DEG from WT and two clones of Rnf2 WT/R70H ESCs (log 2 fold > 2, q < 0.01). n = 2 independent experimental replicates. (C) GO of upregulated genes in Rnf2 WT/R70H ESCs. (D) Heatmaps of Ring1b, H3K27me3, and H2AK119ub ChIP-seq (average signal of two independent experimental replicates) in WT and clone #1 of Rnf2 WT/R70H ESCs. (E) Strategy to generate HA and FLAG-tagged Rnf2 alleles by CRISPR-Cas9 in WT and Rnf2 WT/R70H ESCs. (F) Normalized Ring1b WT and Ring1b R70H Cut&Run signals in WT and Rnf2 WT/R70H ESCs. Signal was generated from two biological replicates from two independent WT and Rnf2 WT/R70H clones. HA and FLAG Cut&Run signals were merged (average of 4 replicates) to avoid potential bias from the HA and FLAG antibodies’ efficiency. (G) Anti-FLAG IPs in Rnf2 HA-WT/FLAG-R70H and Rnf2 FLAG-WT/HA-R70H ESCs followed by LC-MS/MS in three independent experimental replicates. Results are normalized to IgG as a negative control. Volcano plot shows proteins enriched or weakened in FLAG-Ring1b R70H compared with FLAG-Ring1b WT from Rnf2 WT/R70H ESCs. (H) Heatmaps of Cbx7 and Pcgf2, Rybp, Mtf2/Pcl2, and Jarid2 ChIP-seq (average signal of two independent experimental replicates) in WT and clone #1 of Rnf2 WT/R70H ESCs. (I) Genome browser screenshots of ChIP-seq from (H). (J) Mutabind2 scores upon the human RING1B R70H variant vs. full length and lacking their IDR, PCGF1-6 using AlphaFold and ColabFold. (K) Full-length Pcgf2 or lacking the IDR used in (L). (L) Anti-HA IPs followed by WBs against HA, Phc1, and Ring1b in WT and Rnf2 WT/R70H ESCs expressing HA-Pcgf2 WT or HA-Pcgf2 ΔIDR . (M) Model of PRC1/2 recruitment in Rnf2 WT/R70H ESCs. See also .

Article Snippet: JARID2 (D6M9X) (ChIP-seq) , Cell Signaling Technology , Cat# 13594, RRID:AB_2798269.

Techniques: Homologous Recombination, Clone Assay, ChIP-sequencing, CRISPR, Generated, Liquid Chromatography with Mass Spectroscopy, Negative Control, Variant Assay, Expressing

Journal: Nature Communications

Article Title: EPOP restricts PRC2.1 targeting to chromatin by directly modulating enzyme complex dimerization

doi: 10.1038/s41467-025-68280-5

Figure Lengend Snippet: a Domain structures of the proteins captured in the crystal structure. Domain names are summarized below. SUZ12(N), N-terminal domain of SUZ12, contains ZnB, zinc finger-binding helix; WDB1, WD40-binding domain 1; C2, C2 domain; Zn, zinc finger domain; WDB2, WD40-binding domain 2. RBBP4 contains NT, N-terminal domain; WD40, WD40 domain. PHF19(RC), reversed chromodomain of PHF19. EPOP(C), C-terminal domain of EPOP. Subdomains or secondary structures of the PHF19(RC) and EPOP(C) are shown in brackets. (DS), dimer stabilization helix; (SC), short connecting helix; (C2B), C2-binding domain; (CT), C-terminal tail; (SH), short helix; (L1), loop 1; (βH), β hairpin; (L2), loop 2. The domain structures are color-coded based on proteins, except the DS helix of PHF19(RC), which is disordered in the structure and colored in gray. b 2F o F c electron density map of the EPOP fragment, contoured at 1.0σ, is shown in gray. The anomalous signal of the L325M mutant contoured at 10.0σ is shown in gold. c Sequence alignment of human EPOP, human SKDA1, and Drosophila Corto. Residues deleted in the EPOP D5 mutant are indicated by a dotted box. EPOP residues interacting with SUZ12 are indicated by blue discs, and proline residues contributing to the shape complementarity by blue squares. The secondary structure of the EPOP(C) is shown above the sequence alignment. d Cartoon representation of the overall structure. The SUZ12(N)–RBBP4–PHF19(RC)–EPOP(C) heterotetrameric complex adopts a dimeric structural architecture in the crystal lattice. Protein domains are labeled and color-coded. The two protomers are distinguished by the prime sign. e Overall structure in a different view with the rotation matrix relative to ( d ) indicated. f Close-up view of the EPOP binding interface. SUZ12 and RBBP4 domains are highlighted as transparent surfaces. Interacting residues are shown as sticks. Hydrogen bonds are indicated by black dotted lines. All proline residues from the EPOP fragment are also shown as sticks. g Structural alignment to PRC2.2. The current structure is aligned to a PRC2.2 subcomplex containing AEBP2 and JARID2 fragments (PDBs 5WAI and 6WKR). SUZ12 and RBBP4 are displayed as surfaces. Protein domains are labeled and color-coded. AEBP2 domains: C2B, C2-binding domain; CC, central connecting helix. JARID2 domain: TR, transrepression domain. Only the EPOP(C) domain from the current structure is shown for clarity. h Structural alignment to PRC2.1. The current structure is aligned to a PRC2.1 subcomplex containing the PHF19(RC) domain (PDB 6NQ3). SUZ12 and RBBP4 are displayed as surfaces. Protein domains are labeled and color-coded. Only one protomer of the dimer and the EPOP(C) domain from the current structure are shown for clarity. i Schematic model of the dimer disruption mechanism. The domain-swapped dimer is shown. The transient PRC2 core dimer is locked by PHF19 in PRC2.1 PHF19 . EPOP unlocks the PRC2.1 PHF19 dimer by partially displacing PHF19. The schematic is color-coded based on the crystal structures, with the gray disc representing PRC2 subunits and domains not involved in EPOP-mediated regulation.

Article Snippet: Antibodies used in this study are listed below: SUZ12 antibody (CST, Cat. # 3737S), MTF2 antibody (Proteintech, Cat. # 162081AP), EPOP antibody (Active Motif, Cat. # 61753), JARID2 antibody (CST, Cat. # 13594S), AEBP2 antibody (CST, Cat. # 14129S), Elongin B antibody (Abcam, Cat. # ab168836), EZH2 antibody (CST, Cat. # 5246S), FLAG antibody (Sigma, Cat. # F1804), Myc antibody (CST, Cat. # 2276S), H3 antibody (CST, Cat. # 9715S), H3K27me3 antibody (CST, Cat. # 9733S), β-Tubulin antibody (CST, Cat. # 2128S).

Techniques: Binding Assay, Mutagenesis, Sequencing, Labeling, Disruption