Journal: bioRxiv

Article Title: Unleashed condensation by recurrent mutations of an epigenetic regulator promotes cancer

doi: 10.64898/2026.02.18.706652

Figure Lengend Snippet: (a) Analysis of amino acid enrichment for ASXL1 (1-645). By Composition Profiler, using SwissProt 51 dataset as background. (b) Net charge per residue for ASXL1 (1-645). (c) Diagram of ASXL1 (1-590) showing regions for BAP1 and BRD4 binding. Each short red vertical line indicates an Arg. (d) Summary of (1-590) mutants that we designed and their effects on three indicated properties. (e) co-IP for FLAG in 293T cells transfected with FLAG-(1-590) (various mutants)-EGFP, and blotting for indicated proteins.

Article Snippet: Antibodies for the following proteins were used: BAP1 (Cell Signaling, #13187, RRID: AB_2798143), ASXL1 N-terminal (Abcam, ab228009, No RRID but used in literature , , and its staining pattern completely overlaps with GFP signal in our FmEGFP-ASXL1 Y591X cells), ASXL1 C-terminal (Abcam, ab50817, RRID:AB_867743), BRD4 (Abcam, ab128874, RRID:AB_11145462), Ser2 phosphorylated RNA-Pol II (Abcam, ab193468, RRID:AB_2905557), HP1α (Cell Signaling, #2616, RRID:AB_2070987), H2AK119ub (Cell Signaling, #8240, RRID:AB_10891618), GAPDH (Millipore # MAB374, RRID:AB_2107445, mouse, 1:5000 for immunoblotting), FLAG (Sigma # A2220, RRID: AB_10063035), HA (Cell signaling, #3724, RRID: AB_1549585, Rabbit, 1:1000 for immunoblotting).

Techniques: Residue, Binding Assay, Co-Immunoprecipitation Assay, Transfection

Journal: bioRxiv

Article Title: Unleashed condensation by recurrent mutations of an epigenetic regulator promotes cancer

doi: 10.64898/2026.02.18.706652

Figure Lengend Snippet: (a) Immunostaining for ASXL1 and H2AK119ub in 293T cells transfected as indicated. Transfected cells are circled. (b, f) Percent of transfected cells that showed H2Aub reduction. From 3 biological repeats, each with 187-280 transfected cells (b), or 69-231 transfected cells (f). (c) In vitro H2A deubiquitination using purified BAP1, ASXL1 (1-590) (mEGFP- or mCherry-tagged, in green or red, respectively), and ubH2A-nucleosome, followed by SDS-PAGE and Coomassie staining. ubH2A- (lane 7) and unmodified (lane 8) nucleosomes were loaded to show histone identity. Bottom plot: Percent of ubH2A in total H2A (= ubH2A+H2A) was quantified by Image J, from 3 independent assays. (d) Diagram of Δ(391-426), Δ+eIF IDR , of 1-590. Δ+eIF IDR is the 1-590 construct in which the 391-426 region was replaced by eIF IDR . (e) Representative images of 293T cells expressing indicated constructs.

Article Snippet: Antibodies for the following proteins were used: BAP1 (Cell Signaling, #13187, RRID: AB_2798143), ASXL1 N-terminal (Abcam, ab228009, No RRID but used in literature , , and its staining pattern completely overlaps with GFP signal in our FmEGFP-ASXL1 Y591X cells), ASXL1 C-terminal (Abcam, ab50817, RRID:AB_867743), BRD4 (Abcam, ab128874, RRID:AB_11145462), Ser2 phosphorylated RNA-Pol II (Abcam, ab193468, RRID:AB_2905557), HP1α (Cell Signaling, #2616, RRID:AB_2070987), H2AK119ub (Cell Signaling, #8240, RRID:AB_10891618), GAPDH (Millipore # MAB374, RRID:AB_2107445, mouse, 1:5000 for immunoblotting), FLAG (Sigma # A2220, RRID: AB_10063035), HA (Cell signaling, #3724, RRID: AB_1549585, Rabbit, 1:1000 for immunoblotting).

Techniques: Immunostaining, Transfection, In Vitro, Purification, SDS Page, Staining, Construct, Expressing

Journal: bioRxiv

Article Title: Unleashed condensation by recurrent mutations of an epigenetic regulator promotes cancer

doi: 10.64898/2026.02.18.706652

Figure Lengend Snippet: (a) 293T cells expressing indicated EGFP-tagged ASXL1 G646Wfs*12 , its 25RA mutant, or BAP1-mCherry. (b) 293T cells co-expressing indicated EGFP-tagged ASXL1 G646Wfs*12 or its 25RA mutant with BAP1-mCherry. (c) In vitro condensation of 1.67 µM of EGFP-ASXL1 (1-590) or mCherry-BAP1 individually and mixed.

Article Snippet: Antibodies for the following proteins were used: BAP1 (Cell Signaling, #13187, RRID: AB_2798143), ASXL1 N-terminal (Abcam, ab228009, No RRID but used in literature , , and its staining pattern completely overlaps with GFP signal in our FmEGFP-ASXL1 Y591X cells), ASXL1 C-terminal (Abcam, ab50817, RRID:AB_867743), BRD4 (Abcam, ab128874, RRID:AB_11145462), Ser2 phosphorylated RNA-Pol II (Abcam, ab193468, RRID:AB_2905557), HP1α (Cell Signaling, #2616, RRID:AB_2070987), H2AK119ub (Cell Signaling, #8240, RRID:AB_10891618), GAPDH (Millipore # MAB374, RRID:AB_2107445, mouse, 1:5000 for immunoblotting), FLAG (Sigma # A2220, RRID: AB_10063035), HA (Cell signaling, #3724, RRID: AB_1549585, Rabbit, 1:1000 for immunoblotting).

Techniques: Expressing, Mutagenesis, In Vitro

Journal: bioRxiv

Article Title: Unleashed condensation by recurrent mutations of an epigenetic regulator promotes cancer

doi: 10.64898/2026.02.18.706652

Figure Lengend Snippet: All panels are based on RNA-seq analysis from . (a) Dot plots for fold changes of all genes in cells co-transduced with BAP1 and the indicated ASXL1 constructs. (b) Volcano plots of genes differentially expressed in cells co-transduced with BAP1 and the indicated ASXL1 constructs. DEGs (Probability > = 0.85) are in red (higher is 10RA than in 1-590) or blue (lower in 10RA than in 1-590). (c) Venn diagrams showing overlap between genes that, compared to co-transduction with BAP1 and 1-590, were downregulated by BAP1 and control vector (salmon), and genes that were downregulated by BAP1 and (1-590) 10RA (cyan) in each repeat (Probability > = 0.85). P values by Fisher’s exact test. (d-f) The most significantly enriched Biological Processes (d), tissue/cell-specific gene signatures (e), and transcriptional regulatory networks (f), for the 297 genes that were upregulated by ASXL1 (1-590) but less efficiently by 25RA in both repeats, as defined in . By Metascape. P values by two-sided hypergeometric test. (g) Expression levels (normalized read counts in RNA-seq) of indicated myeloid inflammatory response genes in in cells co-transduced with BAP1 and the indicated ASXL1 constructs.

Article Snippet: Antibodies for the following proteins were used: BAP1 (Cell Signaling, #13187, RRID: AB_2798143), ASXL1 N-terminal (Abcam, ab228009, No RRID but used in literature , , and its staining pattern completely overlaps with GFP signal in our FmEGFP-ASXL1 Y591X cells), ASXL1 C-terminal (Abcam, ab50817, RRID:AB_867743), BRD4 (Abcam, ab128874, RRID:AB_11145462), Ser2 phosphorylated RNA-Pol II (Abcam, ab193468, RRID:AB_2905557), HP1α (Cell Signaling, #2616, RRID:AB_2070987), H2AK119ub (Cell Signaling, #8240, RRID:AB_10891618), GAPDH (Millipore # MAB374, RRID:AB_2107445, mouse, 1:5000 for immunoblotting), FLAG (Sigma # A2220, RRID: AB_10063035), HA (Cell signaling, #3724, RRID: AB_1549585, Rabbit, 1:1000 for immunoblotting).

Techniques: RNA Sequencing, Transduction, Construct, Control, Plasmid Preparation, Expressing

Journal: bioRxiv

Article Title: Unleashed condensation by recurrent mutations of an epigenetic regulator promotes cancer

doi: 10.64898/2026.02.18.706652

Figure Lengend Snippet: All panels are based on RNA-seq analysis from , but focused on transposable elements (TEs). (a) Heatmap showing relative expression levels of 103 differentially expressed TEs in cells co-transduced with BAP1 and the indicated ASXL1 (1-590) constructs, including 62 and 41 TEs that were upregulated and downregulated by 1-590 than control, respectively. TEs in pink boxes were upregulated by co-transduction with BAP1 and 1-590 compared to BAP1 and control vector, but were upregulated less efficiently by BAP1 and (1-590) 10RA. (b) Percent of TE family types in total TEs and that were up- or down-regulated by co-transduction with BAP1 and 1-590 compared to BAP1 and control vector. Retrotransposons include ERVs/LTRs, LINEs, and SINEs. DNA-based transposons are also shown. (c) Volcano plots for total ERVs, ERVKs, and total LINE/SINEs that were differentially expressed in cells co-transduced with BAP1 and 1-590 over co-transduced with BAP1 and control vector. The red dots in each plot represent the labeled type of TEs, and the grey dots represent other types of TEs in each plot. (d) Expression of representative ERVs in cells co-transduced with BAP1 and the indicated ASXL1 (1-590) constructs, as shown in Integrated genome viewer.

Article Snippet: Antibodies for the following proteins were used: BAP1 (Cell Signaling, #13187, RRID: AB_2798143), ASXL1 N-terminal (Abcam, ab228009, No RRID but used in literature , , and its staining pattern completely overlaps with GFP signal in our FmEGFP-ASXL1 Y591X cells), ASXL1 C-terminal (Abcam, ab50817, RRID:AB_867743), BRD4 (Abcam, ab128874, RRID:AB_11145462), Ser2 phosphorylated RNA-Pol II (Abcam, ab193468, RRID:AB_2905557), HP1α (Cell Signaling, #2616, RRID:AB_2070987), H2AK119ub (Cell Signaling, #8240, RRID:AB_10891618), GAPDH (Millipore # MAB374, RRID:AB_2107445, mouse, 1:5000 for immunoblotting), FLAG (Sigma # A2220, RRID: AB_10063035), HA (Cell signaling, #3724, RRID: AB_1549585, Rabbit, 1:1000 for immunoblotting).

Techniques: RNA Sequencing, Expressing, Transduction, Construct, Control, Plasmid Preparation, Labeling

Journal: bioRxiv

Article Title: Unleashed condensation by recurrent mutations of an epigenetic regulator promotes cancer

doi: 10.64898/2026.02.18.706652

Figure Lengend Snippet: (a, b) Assays on cells from the mice transplanted with BM transduced with using NRAS G12V and vector control, ASXL1 (1-590), or its 10RA mutant, as indicated. (a) Counts of various types of blood cells as indicated in the peripheral blood of the recipient mice at 9 months after transplant. Each dot is a mouse. WBC, white blood cells. NE, neutrophils. LY, lymphocytes. MO, monocytes. EO, eosinophils. BA, basophils. RBC, red blood cells. Hb, hemoglobin. Data are median (horizontal line), 25–75th percentiles (box) and 1.5 times the interquartile range recorded (whiskers), and the lack dashed line indicates average. (b) Representative flow cytometry analysis on c-Kit and CD11b of BM from moribund mice. (c, d) Non-competitive transplant assays to show BM repopulation. Following the experimental scheme in (c), chimerism (by percentages of CD45.1 + and CD45.2 + cells) in the peripheral blood 29 weeks after transplant was analyzed by flow cytometry (d). From 4, 3, 3 mice that received 1-590, or (1-590) 10RA or 25RA, respectively (all with BAP1). Representative flow analyses are shown on the right.

Article Snippet: Antibodies for the following proteins were used: BAP1 (Cell Signaling, #13187, RRID: AB_2798143), ASXL1 N-terminal (Abcam, ab228009, No RRID but used in literature , , and its staining pattern completely overlaps with GFP signal in our FmEGFP-ASXL1 Y591X cells), ASXL1 C-terminal (Abcam, ab50817, RRID:AB_867743), BRD4 (Abcam, ab128874, RRID:AB_11145462), Ser2 phosphorylated RNA-Pol II (Abcam, ab193468, RRID:AB_2905557), HP1α (Cell Signaling, #2616, RRID:AB_2070987), H2AK119ub (Cell Signaling, #8240, RRID:AB_10891618), GAPDH (Millipore # MAB374, RRID:AB_2107445, mouse, 1:5000 for immunoblotting), FLAG (Sigma # A2220, RRID: AB_10063035), HA (Cell signaling, #3724, RRID: AB_1549585, Rabbit, 1:1000 for immunoblotting).

Techniques: Transduction, Plasmid Preparation, Control, Mutagenesis, Flow Cytometry

Journal: bioRxiv

Article Title: Unleashed condensation by recurrent mutations of an epigenetic regulator promotes cancer

doi: 10.64898/2026.02.18.706652

Figure Lengend Snippet: (a) Volcano plots of ERVs and LINE/SINEs differentially expressed in cells co-transduced with BAP1 and the indicated ASXL1 (1-1067) constructs. The red dots in each plot represent the labeled type of TEs, and the grey dots represent other types of TEs in each plot. (b) Heatmap for the relative expression levels for the 72 TEs that were expressed more highly by ASXL1 (1-1067) 36EQ than by 1-1067 in RNA-seq analyses from both before and after colony formation. (c) Venn diagrams showing the overlap between the TEs that were expressed more highly by transduction of ASXL1 (1-1067) 36EQ (salmon) than by 1-1067 and TEs that were expressed more highly by ASXL1 (1-1067) 34DN (cyan) than by 1-1067. P values by two-sided Fisher’s exact test. (d) Venn diagrams showing the overlap between the TEs (salmon) that were expressed more highly by transduction of ASXL1 (1-1067) 36EQ or 34DN than by 1-1067, from before colony RNA-seq, and the TEs (cyan) that were induced less efficiently by ASXL1 (1-590) 10RA than 1-590, from RNA-seq in . P values by two-sided Fisher’s exact test.

Article Snippet: Antibodies for the following proteins were used: BAP1 (Cell Signaling, #13187, RRID: AB_2798143), ASXL1 N-terminal (Abcam, ab228009, No RRID but used in literature , , and its staining pattern completely overlaps with GFP signal in our FmEGFP-ASXL1 Y591X cells), ASXL1 C-terminal (Abcam, ab50817, RRID:AB_867743), BRD4 (Abcam, ab128874, RRID:AB_11145462), Ser2 phosphorylated RNA-Pol II (Abcam, ab193468, RRID:AB_2905557), HP1α (Cell Signaling, #2616, RRID:AB_2070987), H2AK119ub (Cell Signaling, #8240, RRID:AB_10891618), GAPDH (Millipore # MAB374, RRID:AB_2107445, mouse, 1:5000 for immunoblotting), FLAG (Sigma # A2220, RRID: AB_10063035), HA (Cell signaling, #3724, RRID: AB_1549585, Rabbit, 1:1000 for immunoblotting).

Techniques: Transduction, Construct, Labeling, Expressing, RNA Sequencing

Journal: bioRxiv

Article Title: BAP1 loss impairs Non-Homologous End Joining DNA repair promoting genomic instability

doi: 10.64898/2026.02.17.706330

Figure Lengend Snippet: (A) Frequency of BAP1 genetic alterations in tumors from the BAP1 cancer predisposition syndrome and in pan-cancer cohorts of metastatic tumors. Patient cohorts were retrieved from QIMR Oncotarget 2016 and TCGA Firehose Legacy (UM), TCGA PanCancer Atlas (mesothelioma and kidney renal clear cell carcinoma), MSK Hepatology 2021 (intrahepatic cholangiocarcinoma), and MSK Cell 2021 (pan-cancer) datasets via the cBioPortal platform. Data are shown as bar plots. (B, C) Kaplan–Meier survival curves for overall survival (B) and disease-free survival (C) in UM patients with BAP1 wild-type (green) or BAP1-mutated (red) tumors. P-values are indicated. Data were retrieved from QIMR Oncotarget 2016 and TCGA Firehose Legacy. (D, E) Violin plots showing tumor basal diameter, tumor thickness (D), aneuploidy score, fraction of genome altered and microsatellite instability (MSI) score (E) in BAP1 wild-type versus BAP1-mutated UM patients. Orange line: median. **p < 0 . 01, *p < 0 . 05, ns: not significant . P-values were calculated using an unpaired t-test. (F) Western blot analysis of BAP1 silenced MP41 cells. (G) Western blot analysis of γH2AX in MP41 cells following BAP1 silencing. MP41 cells were treated with bleomycin (50 µg/ml, 24 h) and collected at the indicated recovery time points. (H, I) Representative immunofluorescence images of pan-nuclear γH2AX+ MP41 cells (red) at the indicated recovery time points after bleomycin treatment upon BAP1 silencing. DAPI stains DNA (blue) (H). Violin plot quantifying the percentage of pan-nuclear γH2AX+ cells per field across 3 independent experiments (I). Each dot represents a field. Red line: median. Scale bar: 10 µm. ***p < 0 . 001, **p < 0 . 01, *p < 0 . 05, ns: not significant . P-values were determined by one-way ANOVA with Tukey’s correction.

Article Snippet: The following primary antibodies were incubated overnight at 4°C: rabbit BAP1 (D7W70) mAB (Cell Signaling Technology, 13271S), mouse Anti-β-Actin (Sigma Aldrich, A1978-200UL), mouse BRCA1 (D-9) monoclonal IgG (Santa Cruz Biotechnology, sc-6954), mouse monoclonal anti-GAPDH, clone GAPDH-71.1 (Sigma Aldrich, G8795-200UL), mouse Anti-phospho-Histone H2A.X (Ser139), clone JBW301 (Merck/Sigma, 05-636-I), rabbit H3 (D1H2) mAb (Cell Signaling, # 4499), Ubiquityl-Histone H2A (Lys119) (D27C4) XP® Rabbit mAb (Cell Signaling, #8240) and anti-Histone H2A (Abcam, ab18255).

Techniques: Western Blot, Immunofluorescence

Journal: bioRxiv

Article Title: BAP1 loss impairs Non-Homologous End Joining DNA repair promoting genomic instability

doi: 10.64898/2026.02.17.706330

Figure Lengend Snippet: (A) Western blot analysis of H2AK119ub levels upon BAP1 depletion in G1 cells. MP41 were irradiated (5 Gy) at 6 hours from Nocodazole release and collected 3 hours post IR (B, C) Scatter dot plots quantifying RPA foci (green in C) during recovery from bleomycin treatment in MP41 cells depleted of BAP1. Representative immunofluorescence images are shown in B. DAPI stains DNA. Scale bar: 10 µm. Red line: mean. ****p < 0.0001, **p < 0.01, ns: not significant. P-values by one-way ANOVA with Tukey’s correction (D–F) Scatter dot plots quantifying RPA foci in Cyclin A2+ cells (panel D), Cyclin A2– cells (G1 phase, panel E) in MP41 cells depleted of BAP1. Representative immunofluorescence images are shown in F. Red line: mean. *p < 0.05, **p < 0.01, ns: not significant. P-values by one-way ANOVA with Tukey’s correction.

Article Snippet: The following primary antibodies were incubated overnight at 4°C: rabbit BAP1 (D7W70) mAB (Cell Signaling Technology, 13271S), mouse Anti-β-Actin (Sigma Aldrich, A1978-200UL), mouse BRCA1 (D-9) monoclonal IgG (Santa Cruz Biotechnology, sc-6954), mouse monoclonal anti-GAPDH, clone GAPDH-71.1 (Sigma Aldrich, G8795-200UL), mouse Anti-phospho-Histone H2A.X (Ser139), clone JBW301 (Merck/Sigma, 05-636-I), rabbit H3 (D1H2) mAb (Cell Signaling, # 4499), Ubiquityl-Histone H2A (Lys119) (D27C4) XP® Rabbit mAb (Cell Signaling, #8240) and anti-Histone H2A (Abcam, ab18255).

Techniques: Western Blot, Irradiation, Immunofluorescence

Journal: bioRxiv

Article Title: BAP1 loss impairs Non-Homologous End Joining DNA repair promoting genomic instability

doi: 10.64898/2026.02.17.706330

Figure Lengend Snippet: (A) Representative immunofluorescence images of RPA (green) and Cyclin A2 (red) in BAP1-depleted MP41 cells ± PTC-209 treatment. DAPI stains DNA. Cells were treated with PTC-209 (0.1 µM, 48 h) prior to irradiation and collected at 3 h post-IR. Arrows mark Cyclin A2– (G1 phase) cells with RPA foci. Scale bar: 10 µm. (B, C) Scatter dot plots showing (B) the percentage of RPA+ Cyclin A2– cells and (C) the number of RPA foci per Cyclin A2– cell at 3 h post-IR. Red line: mean. ****p < 0.0001, ***p < 0.001, **p < 0.01, ns: not significant. P-values by one-way ANOVA with Tukey’s correction. (D) Representative immunofluorescence images of 53BP1 (green) in BAP1-depleted MP41 cells ± PTC-209 treatment at 1 and 3 h post-IR. DAPI stains DNA. Scale bar: 10 µm. (E) Scatter dot plot quantifying 53BP1 foci per cell. Red line: median. ****p < 0.0001, **p < 0.01, ns: not significant. P-values by one-way ANOVA with Tukey’s correction. (F) Schematic model. In BAP1-proficient cells, BAP1 deubiquitylates H2AK119ub deposited by PRC1, permitting 53BP1 recruitment and NHEJ execution in G1. In BAP1-depleted cells, H2AK119ub accumulates, blocking 53BP1 loading, and driving aberrant DNA end resection with RPA recruitment in G1, where HR is not available.

Article Snippet: The following primary antibodies were incubated overnight at 4°C: rabbit BAP1 (D7W70) mAB (Cell Signaling Technology, 13271S), mouse Anti-β-Actin (Sigma Aldrich, A1978-200UL), mouse BRCA1 (D-9) monoclonal IgG (Santa Cruz Biotechnology, sc-6954), mouse monoclonal anti-GAPDH, clone GAPDH-71.1 (Sigma Aldrich, G8795-200UL), mouse Anti-phospho-Histone H2A.X (Ser139), clone JBW301 (Merck/Sigma, 05-636-I), rabbit H3 (D1H2) mAb (Cell Signaling, # 4499), Ubiquityl-Histone H2A (Lys119) (D27C4) XP® Rabbit mAb (Cell Signaling, #8240) and anti-Histone H2A (Abcam, ab18255).

Techniques: Immunofluorescence, Irradiation, Blocking Assay