Journal: bioRxiv

Article Title: Histone H2BK120 ubiquitination modulates PRC1 activity and H2AK119ub deposition on nucleosomes

doi: 10.64898/2026.02.20.706939

Figure Lengend Snippet: a , Schematic of the domain organizations of the human cPRC1 complex. Abbreviations of domain names: Chromo, chromodomain; ATHL, AT-hook like; ABM, acidic-patch-binding motif; Pc, Polycomb box; RAWUL, ring finger and WD40 associated ubiquitin-like; PEST, proline, glutamic acid, serine and threonine rich; HD1, homology domain 1; FCS, zinc finger with a characteristic phenylalanine–cysteine–serine sequence motif; SAM, sterile alpha motif. b , In vitro ubiquitination assays of RNF2-BMI1 with unmodified or H2BK120ub-modified nucleosomes, visualized by Coomassie blue staining. The bottom row represents the ratio of the band intensity of H2A-ub (including H2A-ub and H2A-ub2) to that of H3, normalized to lane 5 (BMI1-NCP H2BK120ub , set to 1.00). The experiment was repeated at least three times with similar results c , In vitro ubiquitination assays of RNF20-RNF40 with unmodified or H2AK119ub-modified nucleosomes, analyzed by Western blot. The bottom row represents the ratio of the band intensity of H2B-ub to that of H3, normalized to lane 1 (RNF20-RNF40-NCP H2AK119ub , set to 1.00). The experiment was repeated at least three times with similar results d , Overall structure of cPRC1-E2∼ub-NCP H2BK120ub complex. Cryo-EM density map (upper panel) and atomic model (lower panel) of cPRC1-E2∼ub-NCP H2BK120ub complex are shown from two orthogonal views. The cryo-EM map is segmented according to the components of the cPRC1-E2∼ub-NCP H2BK120ub complex. The color scheme of the cPRC1-E2∼ub-NCP H2BK120ub complex is the same as depicted in (a), and the 147-bp DNA chains are shown in light and dark gray, respectively. Ub, ubiquitin; E2, UbcH5c. e , Detailed view of the recognition interface between H2BK120ub and BMI1. f , In vitro ubiquitination assays of wild-type and mutant cPRC1 complexes with unmodified or H2BK120ub-modified nucleosomes, analyzed by Coomassie blue staining (top) and Western blot (bottom). The middle row represents the ratio of the band intensity of H2A-ub (including H2A-ub and H2A-ub2) to that of H3, normalized to lane 2 (BMI1 WT -NCP H2BK120ub , set to 1.00). The experiment was repeated at least three times with similar results.

Article Snippet: 10 μg of H2BK120ub antibodies (Cell Signaling Technology, Cat. #5546S; Active Motif, Cat. #39623) were added into each sample and rocked at 4 L overnight.

Techniques: Binding Assay, Ubiquitin Proteomics, Sequencing, Sterility, In Vitro, Modification, Staining, Western Blot, Cryo-EM Sample Prep, Mutagenesis

Journal: bioRxiv

Article Title: Histone H2BK120 ubiquitination modulates PRC1 activity and H2AK119ub deposition on nucleosomes

doi: 10.64898/2026.02.20.706939

Figure Lengend Snippet: a , Schematic of the domain organizations of the human ncPRC1.1 ternary complex. Abbreviations of domain names: NZF, Npl4-type zinc finger; RID, Ring1B-interacting domain; RAWUL, ring finger and WD40 associated ubiquitin-like. b , In vitro ubiquitination assays of ncPRC1.1 and ncPRC1.6 with unmodified or H2BK120ub-modified nucleosomes, analyzed by Coomassie blue staining (top) and Western blot (bottom). The middle row represents the ratio of the band intensity of H2A-ub (including H2A-ub, H2A-ub2, and H2A-ub3) to that of H3, normalized to lane 2 (RNF2-PCGF1-NCP H2BK120ub , set to 1.00). The experiment was repeated at least three times with similar results. c , Overall structure of human ncPRC1.1-E2∼ub-NCP H2BK120ub complex. Cryo-EM density map (upper panel) and atomic model (lower panel) of ncPRC1.1-E2∼ub-NCP H2BK120ub complex are shown from two orthogonal views. The cryo-EM map is segmented according to the components of ncPRC1.1-E2∼ub-NCP H2BK120ub complex. The color scheme of ncPRC1.1-E2∼ub-NCP H2BK120ub complex is the same as depicted in (a), and the 147-bp DNA chains are shown in light and dark gray, respectively. Ub, ubiquitin; E2, UbcH5c. d , Structural comparison of cPRC1-E2∼ub-NCP H2BK120ub and ncPRC1.1-E2∼ub-NCP H2BK120ub . e , Detailed view of the recognition interface between H2BK120ub and PCGF1.

Article Snippet: 10 μg of H2BK120ub antibodies (Cell Signaling Technology, Cat. #5546S; Active Motif, Cat. #39623) were added into each sample and rocked at 4 L overnight.

Techniques: Ubiquitin Proteomics, In Vitro, Modification, Staining, Western Blot, Cryo-EM Sample Prep, Comparison

Journal: bioRxiv

Article Title: Histone H2BK120 ubiquitination modulates PRC1 activity and H2AK119ub deposition on nucleosomes

doi: 10.64898/2026.02.20.706939

Figure Lengend Snippet: a , Cryo-EM density maps and atomic models of human ncPRC1.4-NCP H2BK120ub&H2AK119ub complex (left), human ncPRC1.4-NCP H2AK119ub complex (middle), human ncPRC1.1-NCP H2BK120ub&H2AK119ub complex (top right), and human ncPRC1.6-NCP H2BK120ub&H2AK119ub complex (bottom right) in the dyad view of nucleosome. b , Detailed view of the recognition interfaces between RYBP and the nucleosome acidic patch (left), and between RYBP and H2AK119ub (right). c , In vitro ubiquitination assays of wild-type and mutant ncPRC1.1 complexes with unmodified nucleosomes, analyzed by Coomassie blue staining (middle) and Western blot (bottom). The upper panel shows the protein purity of wild-type and mutant ncPRC1.1 complexes loaded at a higher concentration than in the ubiquitination assay. The middle row represents the ratio of the band intensity of H2A-ub (including H2A-ub and H2A-ub2) to that of H3, normalized to lane 2 (RNF2-PCGF1 WT -NCP H2BK120ub , set to 1.00). The experiment was repeated at least three times with similar results. w/o, without RYBP.

Article Snippet: 10 μg of H2BK120ub antibodies (Cell Signaling Technology, Cat. #5546S; Active Motif, Cat. #39623) were added into each sample and rocked at 4 L overnight.

Techniques: Cryo-EM Sample Prep, In Vitro, Ubiquitin Proteomics, Mutagenesis, Staining, Western Blot, Concentration Assay

Journal: bioRxiv

Article Title: Histone H2BK120 ubiquitination modulates PRC1 activity and H2AK119ub deposition on nucleosomes

doi: 10.64898/2026.02.20.706939

Figure Lengend Snippet: a , Heatmaps show normalized H2BK120ub, H2AK119ub, Bmi1, H3K4me3, and H3K27me3 signals in WT and Rnf20 KO+Flag-Bmi1 mESCs. Signal differences were calculated by subtracting WT mESC signals from those of Rnf20 -KO+ Flag-Bmi1. Data were plotted across 5 kb windows spanning the TSS to TES of all genes determined by UCSC Genes 2013. Genes were clustered into two groups using K-Means based on WT H2BK120ub and H2AK119ub signals. Heatmaps were sorted by WT H2BK120ub enrichment. TSS, transcription start site. TES, transcription end site. b , Heatmaps of normalized H2BK120ub, H2AK119ub, Bmi1, H3K4me3, and H3K27me3 signals in different gene groups from WT and Rnf20- KO+Flag-Bmi1 mESCs, shown similarly as in . c , Heatmaps (left) show gene expression in different gene groups from WT and Rnf20- KO+Flag-Bmi1 mESCs. Word clouds (medium) illustrate the gene families in each group. GO analysis (right) presents the top four GO terms ranked by P values, with P values calculated using the hypergeometric test. d , Heatmaps (left) display normalized gene expression and a percentage stacked bar plot (right) shows the distribution of average normalized expression in unique_ub, overlap, and unique_me from WT mESCs at differentiation time points of 0, 4, 7, and 14 days. The normalization was applied by rows.

Article Snippet: 10 μg of H2BK120ub antibodies (Cell Signaling Technology, Cat. #5546S; Active Motif, Cat. #39623) were added into each sample and rocked at 4 L overnight.

Techniques: Gene Expression, Expressing

Journal: bioRxiv

Article Title: Histone H2BK120 ubiquitination modulates PRC1 activity and H2AK119ub deposition on nucleosomes

doi: 10.64898/2026.02.20.706939

Figure Lengend Snippet: Direct interaction between H2BK120ub and specific PCGF subunits promotes PRC1 activity while limiting RYBP association with nucleosomes. Co-enrichment of H2BK120ub and H2AK119ub is observed in the gene bodies of a subset of developmental genes.

Article Snippet: 10 μg of H2BK120ub antibodies (Cell Signaling Technology, Cat. #5546S; Active Motif, Cat. #39623) were added into each sample and rocked at 4 L overnight.

Techniques: Activity Assay