ha tag  (EpiCypher)

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) RING1 and RNF2 variants (top). Reported variants in ClinVar and cancer-related somatic (COSMIC) mutations in RING1 and RNF2 genes (bottom). Metadome plots (middle) represent the level of predicted intolerance for amino acid change in RING1A and RING1B. For COSMIC, only positions of interest are shown as labels. Circle size represents the number of patients reported. (B) ColabFold predictions of RING1A and RING1B variants in altering interaction with PCGF proteins. (C) WBs of dKO-RING1A/B cells expressing HA-tagged WT and mutant RING1A and RING1B. Vinculin and histone H3 served as loading controls. n = 3 independent experimental replicates. (D) Possible mechanisms of deleterious variants that result in a decrease or absence of H2AK119ub. (E) Partial protein sequence alignments of a subset of RING1B homologs. The conserved RING1B-R70 residue corresponds to C. elegans R181 and is indicated by a star. Conserved zinc-coordinating residues, blue ; required for stabilizing the E2 enzyme-E3 ligase interaction in mammals, red ; required for binding to the nucleosome in mammals, green predicted to be important for the RING1B:PCGF4 interaction, magenta 47; and predicted to mediate β sheet interactions, cyan. * indicates identical residues, and : and. indicate residues with strongly and weakly similar physicochemical properties, respectively. The secondary structure of SPAT-3 and H. sapiens RING1B is shown below. (F) WBs of H2AK119ub in the indicated genotypes. The dilution factor is 1:3. The spat-3(mgw26) allele is a full deletion of the spat-3 coding region. Quantification of H2AK119ub and SPAT-3 isoform A is normalized to loading controls (histone H3/actin) and shown relative to the sample indicated by an asterisk. ND, not detectable. (G) WBs in dKO-RING1A/B cells stably expressing HA-RING1B WT or HA-RING1B R70H . Vinculin and histone H2A and H3 served as fractionation controls. n = 3 independent experimental replicates. (H) Normalized H3K27me3 Cut&Run signal (two independent experimental replicates) in cells treated with 1 μM of vehicle (DMSO) or GSK343 for 72 h. See also and .

Article Snippet: HA-Tag (CUT&RUN) , EpiCypher , Cat# 13-2010, RRID:AB_3094663.

Techniques: Expressing, Mutagenesis, Sequencing, Residue, Binding Assay, Stable Transfection, Fractionation

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: HA-Tag (CUT&RUN) , EpiCypher , Cat# 13-2010, RRID:AB_3094663.

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

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) Normalized Ring1b WT and Ring1b R70H Cut&Run signals in either WT or Rnf2 WT/R70H NPCs in WT Ring1b peak regions. Two biological replicates from two independent clones. Wilcox test. *** p < 0.001. (B) Normalized H3K27me3 and H2AK119ub Cut&Run signals in either WT or Rnf2 WT/R70H NPCs over all genome. Signal was generated from two biological replicates from two independent clones. Wilcox test. *** p < 0.001. (C) Genome browser screenshots of HA, FLAG, H3K27me3, and H2AK119ub Cut&Run (average signal between replicates) in the cells shown on the left. (D) Anti-FLAG IPs in Rnf2 HA-WT/FLAG-R70H and Rnf2 FLAG-WT/HA-R70H NPCs followed by LC-MS/MS in three independent experimental replicates. Results are normalized to IgG as negative control. Volcano plot shows proteins enriched or weakened in FLAG-Ring1b R70H compared with FLAG-Ring1b WT from Rnf2 WT/R70H NPCs. (E) RNA-seq heatmap of PcG target genes in ESCs that are upregulated in WT NPCs but retained PRC1/2 and are repressed in Rnf2 WT/R70H NPCs. #1 and #2 are two different Rnf2 WT/R70H ESC clones. On the right, GO from each cluster. Deseq2; Wald test (FC > 4), q < 0.05. (F) Simplified genome browser screenshots of Ring1b WT , Ring1b R70H , H3K27me3, and H2AK119ub Cut&Run in WT and Rnf2 WT/R70H NPCs. Ring1b signal in WT NPCs and Ring1b WT and Ring1b R70H signals in Rnf2 WT/R70H NPCs are from merging average signals HA and FLAG Cut&Run two replicates from two clones. (G) Normalized signal of Ring1b WT and Ring1b R70H Cut&Run signals as in (F) around the transcription start site (TSS) of genes from (E). (H) Normalized signal of H3K27me3 and H2AK119ub Cut&Run signals (average from two replicates) as in (F) around the TSS of genes from (E). (I) Normalized ATAC-seq signal (average from two replicates) in WT and Rnf2 WT/R70H ESCs and NPCs around the TSS of genes from (E). See also .

Article Snippet: HA-Tag (CUT&RUN) , EpiCypher , Cat# 13-2010, RRID:AB_3094663.

Techniques: Clone Assay, Generated, Liquid Chromatography with Mass Spectroscopy, Negative Control, RNA Sequencing

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) PCA from ATAC-seq from two independent biological replicates of WT and Rnf2 WT/R70H ESCs and NPCs. (B) Genome browser of ATAC-seq signal (average of two replicates) from WT and Rnf2 WT/R70H ESCs and NPCs. (C) RT-qPCR of pluripotency genes and NPC markers in WT and Rnf2 WT/R70H ESCs and NPCs. n = 3. #1 and #2 represent two clones of Rnf2 WT/R70H ESCs. *** p < 0.005, **** p < 0.001 by ANOVA test. (D) WB of Pax6 in WT and clone #1 of Rnf2 WT/R70H ESCs and NPCs. Vinculin served as a loading control. (E) ATAC-seq peaks reduced in Rnf2 WT/R70H NPCs and HOMER analysis. (F) Normalized expression of genes from (E) in WT and clones #1 and #2 of Rnf2 WT/R70H NPCs. *** p < 0.001. NS, not significant. Wilcox test. (G) ATAC-seq specific peaks in Rnf2 WT/R70H NPCs and HOMER analysis. (H) Normalized expression of genes from (G) in WT and clones #1 and #2 of Rnf2 WT/R70H NPCs. *** p < 0.001. NS, not significant. Wilcox test. (I) ATAC-seq signal in WT and Rnf2 WT/R70H NPCs at Sox2- or Sox3-occupied sites in WT NPCs. Sox2 and Sox3 ChIP from Bergsland et al. (J) Genome browser of ATAC-seq signal from WT and Rnf2 WT/R70H ESCs and NPCs as well as Ring1b WT and Ring1b R70H Cut&Run signal in WT and Rnf2 WT/R70H NPCs. (K) Normalized expression and GO of genes occupied by Ring1b WT and Ring1b R70H and compacted. *** p < 0.001. NS, not significant. Wilcox test. See also .

Article Snippet: HA-Tag (CUT&RUN) , EpiCypher , Cat# 13-2010, RRID:AB_3094663.

Techniques: Quantitative RT-PCR, Clone Assay, Control, Expressing