Journal: bioRxiv

Article Title: Chromatin modifiers KMT2D, BAF, and p300 are required for de novo binding of transcription factors on enhancers

doi: 10.64898/2026.01.29.702555

Figure Lengend Snippet: (A) Schematic of the 4-OHT-inducible MyoD-ER system. (B) MyoD expression in preadipocytes and C2C12 myoblasts was determined using RNA-Seq (n = 1). RPKM values indicate gene expression levels. (C) Western blot (WB) analysis of nuclear extracts from preadipocytes expressing MyoD-ER-T7 and treated with 4-OHT. Antibodies used were indicated on the right. BRG1 was used as a loading control. (D-I) MyoD-ER-T7 expressing preadipocytes were treated with 4-OHT for 1 hour (h), followed by CUT&RUN analysis. (D) Bar chart showing ARID1A (an exclusive subunit of BAF), KMT2D, and p300 binding status on induced MyoD sites. (E) Box plots displaying the normalized MyoD read counts in subgroups defined in (D). (F) Bar chart showing ARID1A (BAF), KMT2D, and p300 binding on 38,732 MyoD + enhancers defined in (D) prior to 4-OHT treatment. (G-H) Box plots showing the normalized MyoD read counts (G) and HOMER de novo motif analysis (H) on BAF-KMT2D-p300 prebound or de novo sites defined in (F). Statistical significance was determined using a two-sided, unpaired Mann Whitney test. (I) Genome browser view of MyoD binding sites around Maged1 and Cap2 loci.

Article Snippet: BRG1 Inhibitor BRM014 (#HY-119374) and 5Ph-IAA (#HY-134653) were from MedChemExpress (MCE) and used at 1μM.

Techniques: Expressing, RNA Sequencing, Gene Expression, Western Blot, Control, Binding Assay, MANN-WHITNEY

Journal: bioRxiv

Article Title: Chromatin modifiers KMT2D, BAF, and p300 are required for de novo binding of transcription factors on enhancers

doi: 10.64898/2026.01.29.702555

Figure Lengend Snippet: (A) Sanger sequencing results verifying the integration of 132-bp AID sequence after the start codon (ATG) of Kmt2d . (B) Breeding scheme for generating Kmt2c f/f ; Kmt2d AID/AID ; Cre-ER mice. (C) Diagram illustrating the workflow to generate Kmt2c -/- ; Kmt2d AID/AID preadipocytes expressing OsTIR1-F74G-Myc and MyoD-ER-T7. (D) WB of nuclear extracts confirming KMT2C knockout. Antibodies used were indicated on the right. BRG1 was a loading control. (E) WB of nuclear extracts confirming the depletion of KMT2D in Kmt2d AID/AID ; MyoD-ER-T7 preadipocytes upon 5Ph-IAA treatment for various time durations. Antibodies used were indicated on the right. RbBP5 was a loading control.

Article Snippet: BRG1 Inhibitor BRM014 (#HY-119374) and 5Ph-IAA (#HY-134653) were from MedChemExpress (MCE) and used at 1μM.

Techniques: Sequencing, Expressing, Knock-Out, Control

Journal: bioRxiv

Article Title: Chromatin modifiers KMT2D, BAF, and p300 are required for de novo binding of transcription factors on enhancers

doi: 10.64898/2026.01.29.702555

Figure Lengend Snippet: Kmt2d AID/AID ; MyoD-ER-T7 preadipocytes were pretreated with BRG1 inhibitor BRM014 (BRG1i) for 1h, and then 4-OHT was added for 1h to induce MyoD nuclear translocation. Cells were harvested for WB, CUT&RUN, and ATAC-seq. (A) WB of nuclear extracts for KMT2D, p300, and BAF subunits BRG1 and ARID1A. (B) Pie chart illustrating BAF binding status on 38,732 MyoD + enhancers. (C) Heat maps for CUT&RUN of ARID1A (BAF), T7 (MyoD), KMT2D, and p300 on BAF pre-bound and de novo BAF binding sites. (D-E) Chromatin accessibility determined by ATAC-seq signals on MyoD + enhancers. Chromatin accessibility status on BAF prebound sites (D) or de novo BAF binding sites (E) is shown in pie charts ( upper panels ). Average profiles of normalized ATAC-seq reads on constitutively open and MyoD-dependent opening sites are shown in lower panels .

Article Snippet: BRG1 Inhibitor BRM014 (#HY-119374) and 5Ph-IAA (#HY-134653) were from MedChemExpress (MCE) and used at 1μM.

Techniques: Translocation Assay, Binding Assay

Journal: bioRxiv

Article Title: BAF complex-independent gene activation by SS18::SSX

doi: 10.64898/2026.01.26.701739

Figure Lengend Snippet: (A) Western Blot of HSSY-II whole cell extracts treated for 72h with DMSO or 500nM ACBI1 revealed using SMARCA4, SMARCA2, SMARCC1, SS18::SSX or β-actin antibodies. n = 1. (B) Volcano plots of mass spectrometry data following SS18::SSX pull down in HSSY-II synovial sarcoma cells treated for 72h with DMSO or 500nM ACBI1, normalised to interactome of DMSO treated cells. Data represents log2 fold change enrichment plotted on the x-axis and -log10 (adjusted p value) plotted on the y-axis. Blue/Red dots indicate lost/gained interactors upon ACBI1 treatment. Black dots indicate BAF members. n = 4 biological replicates. (C) List of BAF members present in interactome dataset and their associated log2 fold change value in ACBI1 SS18::SSX pull downs normalised to DMSO. Listed members are shown as black dots in (B). (D) Viability of 4 synovial sarcoma cell lines (Yamato-SS, SYO-1, CME-1, HSSY-II) and an osteosarcoma cell line (fusion negative control) KHOS-240S treated with various concentrations of ACBI1. The symbols represent the mean ± S.E.M of n = 3 biological replicates, the solid line represent the nonlinear curve fitting and its associated IC50. (E) Western Blot of HSSY-II-Cas9 whole cell extracts expressing a safe sgRNA as control or with double guides targeting SSX + a control region (sgSSX/SAFE), SMARCA2 + SMARCA4 (sgSMARCA2/4) or SMARCC1 + SMARCC2 (sgSMARCC1/2) for 7 days revealed using SMARCA4, SMARCA2, SMARCC1, SMARCC2, SS18::SSX or GAPDH antibodies. Representative of n = 2 biological replicates. (F) Cell competition assay performed in the osteosarcoma cell line KHOS-240S-Cas9 or in the 4 synovial sarcoma lines expressing Cas9 expressing a safe sgRNA as control or double guides targeting SSX + a control region (sgSSX/SAFE), SMARCA2 + SMARCA4 (sgSMARCA2/4) or SMARCC1 + SMARCC2 (sgSMARCC1/2). Data represents the mean ± S.E.M of n = 2 biological replicates.

Article Snippet: The SMARCA2/SMARCA4 degrader ACBI1 (2375564-55-7) and the EP300/CREBBP degrader dCBP-1 (2484739-25-3) were purchased from MedChemExpress, resuspended in dimethyl sulfoxide (DMSO) and kept at −80°C.

Techniques: Western Blot, Mass Spectrometry, Negative Control, Expressing, Control, Competitive Binding Assay

Journal: bioRxiv

Article Title: BAF complex-independent gene activation by SS18::SSX

doi: 10.64898/2026.01.26.701739

Figure Lengend Snippet: (A) Western Blot of KHOS-240S-Cas9 whole cell extracts expressing a safe sgRNA as control or with double guides targeting SSX + a control region (sgSSX/SAFE), SMARCA2 + SMARCA4 (sgSMARCA2/4) or SMARCC1 + SMARCC2 (sgSMARCC1/2) revealed using SMARCA4, SMARCA2, SMARCC1, SMARCC2 or GAPDH antibodies. (B) Tracking of Indels by Decomposition (TIDE) assay displaying the percentage of aberrant sequences after Cas9 editing for guides targeting SMARCA2, SMARCA4, SMARCC1 and SMARCC2 versus the wild-type sequence (sgSAFE guide) in Yamato-SS cell line.

Article Snippet: The SMARCA2/SMARCA4 degrader ACBI1 (2375564-55-7) and the EP300/CREBBP degrader dCBP-1 (2484739-25-3) were purchased from MedChemExpress, resuspended in dimethyl sulfoxide (DMSO) and kept at −80°C.

Techniques: Western Blot, Expressing, Control, Sequencing

Journal: bioRxiv

Article Title: BAF complex-independent gene activation by SS18::SSX

doi: 10.64898/2026.01.26.701739

Figure Lengend Snippet: (A) RNA-seq heatmap showing the 1,000 most variable genes with a cutoff z-score of 4 from HSSY-II cells treated for 72h with DMSO or 500nM ACBI1 or of HSSY-II-Cas9 cells expressing a safe sgRNA as control or with double guides targeting SSX + a control region (sgSSX/SAFE), SMARCA2 + SMARCA4 (sgSMARCA2/4) or SMARCC1 + SMARCC2 (sgSMARCC1/2). n=2 biological replicates. (B) Log2-transformed fold change of FPKM values in HSSY-II RNA-seq relative to DMSO or sgSAFE over selected SS18::SSX target genes. Data represents the mean. (C) qRT-PCR displaying log2-transformed fold change of mRNA levels normalised by GAPDH in Yamato-SS cells treated for 72h with DMSO or 500nM ACBI1 or of Yamato-SS Cas9 cells expressing sgSAFE, (sgSSX/SAFE), (sgSMARCA2/4) or (sgSMARCC1/2) relative to DMSO or sgSAFE for 7 days. Data represents the mean of n=2 biological replicates. (D) Left: heatmaps for SMARCA4 and SS18::SSX1 (endogenously HA tagged) ChIP-seq from Banito et al., 2018 over SMARCA4 peaks (n = 20,278). Rows correspond to ±5-kb regions across the midpoint of each SMARCA4-enriched region, ranked by increasing signal. Right: ATAC-seq heatmaps displaying log2-transformed fold change of ACBI1 treated cells over DMSO after 24h or 72h of treatment and of sgSSX/SAFE expressing cells over sgSAFE cells. n=2 biological replicates. (E) Scatter plot of log2-transformed fold change of FPKM values in HSSY-II RNA-seq relative to DMSO or sgSAFE over genes associated with SMARCA4 occupancy and showing <-1.5 fold down-regulation upon sgSSX/SAFE knockout. Biological replicates are combined using their mean. Bold line represents median and dotted line represents -1.5 cutoff. p-values represent two-tailed Wilcoxon test. (F) Gene tracks for SMARCA4, SS18::SSX1 ChIP-seq, Log2-Fold changes of ATAC-seq or Log2-Fold changes of RNA-seq at loci from (E) such as MAFA , MNX1 , SKOR1 , FGF8 , WNT7B , BMP7 and ONECUT3 .

Article Snippet: The SMARCA2/SMARCA4 degrader ACBI1 (2375564-55-7) and the EP300/CREBBP degrader dCBP-1 (2484739-25-3) were purchased from MedChemExpress, resuspended in dimethyl sulfoxide (DMSO) and kept at −80°C.

Techniques: RNA Sequencing, Expressing, Control, Transformation Assay, Quantitative RT-PCR, ChIP-sequencing, Knock-Out, Two Tailed Test

Journal: bioRxiv

Article Title: BAF complex-independent gene activation by SS18::SSX

doi: 10.64898/2026.01.26.701739

Figure Lengend Snippet: (A) Gene set enrichment analysis (GSEA) comparing differentially expressed genes in the ACBI1, sgSMARCA2/4 or sgSMARCC1/2 RNAseq data in HSSY-II to the top 500 genes downregulated in sgSSX. (B) qRT-PCR displaying log2-transformed fold change of mRNA levels normalised by GAPDH in CME-1-Cas9 cells expressing sgSAFE, (sgSSX/SAFE), (sgSMARCA2/4) or (sgSMARCC1/2) for 7 days relative to sgSAFE. Data represents the mean of n = 2 biological replicates. (C)(D) qRT-PCR displaying log2-transformed fold change of mRNA levels normalised by GAPDH in CME-1 (C) or SYO-1 (D) cells treated for 72h with DMSO or 500nM ACBI1 relative to DMSO. Data represents the mean of n = 2 biological replicates. (E) Left: heatmaps for SMARCA4 and SS18::SSX1 (endogenously HA tagged) ChIP-seq from Banito et al., 2018 over T2T hs1 Refseq promoters (n = 20,844). Rows correspond to ±5-kb regions across the midpoint of each SMARCA4-enriched region, ranked by increasing signal. Right: ATAC-seq heatmaps displaying log2-transformed fold change of ACBI1 treated cells over DMSO after 24h or 72h of treatment and of sgSSX/SAFE expressing cells over sgSAFE cells.

Article Snippet: The SMARCA2/SMARCA4 degrader ACBI1 (2375564-55-7) and the EP300/CREBBP degrader dCBP-1 (2484739-25-3) were purchased from MedChemExpress, resuspended in dimethyl sulfoxide (DMSO) and kept at −80°C.

Techniques: Quantitative RT-PCR, Transformation Assay, Expressing, ChIP-sequencing

Journal: bioRxiv

Article Title: BAF complex-independent gene activation by SS18::SSX

doi: 10.64898/2026.01.26.701739

Figure Lengend Snippet: (A) Schematics showing timeline for ACBI1/DMSO treatment and SS18::SSX/EV delivery in KHOS-240S cells. (B) Western Blot of KHOS-240S whole cell extracts after 24h pre-treatment with DMSO or 500nM ACBI1 (Day 0), or at day 2 and 3 following expression of an empty vector (EV) or SS18::SSX1 transgene and continued treatment with DMSO or ACBI1. (C) RNA-seq heatmap showing the 1,000 most variable genes with a cutoff z-score of 2 from KHOS-240S cells pre-treated with DMSO or 500nM ACBI1 for 24h prior to expression of an empty vector (EV) or SS18::SSX1 transgene for additional 72h and co-treatment with DMSO or ACBI1. n=2 biological replicates. Dotted square highlights genes up- regulated by SS18::SSX1 expression (D) Log2-transformed fold change of FPKM values in KHOS-240S RNA-seq relative to EV/DMSO over selected SS18::SSX target genes. Data represents the mean. (E) Scatter plot of log2-transformed fold change of FPKM values in KHOS-240S RNA-seq relative to EV/DMSO over genes up-regulated by SS18::SSX1 expression and showing >1.5 fold up-regulation. Biological replicates are combined using their mean. Bold line represents median and dotted line represents 1.5 cutoff. p-values represent two-tailed Wilcoxon test. (F) Left: heatmaps for SS18::SSX1 (HA tagged-transgene) CUT&RUN from Benabdallah et al., 2023 over SS18::SSX1 peaks (n = 16,687). Rows correspond to ±5-kb regions across the midpoint of each SS18::SSX1-enriched region, ranked by increasing signal. Right: ATAC-seq heatmaps (in greys) of KHOS-240S cells pre-treated with DMSO or 500nM ACBI1 for 24h prior to expression of an empty vector (EV) or SS18::SSX1 transgene for additional 72h and co-treatment with DMSO or ACBI1 or ATAC-seq heatmaps (in blue-red gradient) of comparison using log2- transformed fold change of either ACBI1 relative to DMSO in EV or SS18::SSX1 condition or SS18::SSX relative to EV in DMSO or ACBI1 conditions. n-2 biological replicates. (G) Gene tracks for SS18::SSX1 CUT&RUN, ATAC-seq of KHOS-240S cells pre-treated with DMSO or 500nM ACBI1 for 24h prior to expression of an empty vector (EV) or SS18::SSX1 transgene for additional 72h and co-treatment with DMSO or ACBI1 or Log2-Fold changes of RNA-seq of SS18::SSX relative to EV in DMSO or ACBI1 conditions at SS18::SSX1 target loci in KHOS-240S cells displaying up-regulation upon SS18::SSX transgene expression such as IGF2 , TPPP , MAFA , SHH , HES4 and ONECUT3 .

Article Snippet: The SMARCA2/SMARCA4 degrader ACBI1 (2375564-55-7) and the EP300/CREBBP degrader dCBP-1 (2484739-25-3) were purchased from MedChemExpress, resuspended in dimethyl sulfoxide (DMSO) and kept at −80°C.

Techniques: Western Blot, Expressing, Plasmid Preparation, RNA Sequencing, Transformation Assay, Two Tailed Test, Comparison

Journal: bioRxiv

Article Title: BAF complex-independent gene activation by SS18::SSX

doi: 10.64898/2026.01.26.701739

Figure Lengend Snippet: (A)(C) Volcano plots of mass spectrometry data following eGFP-SS18::SSX (A) or eGFP-SS18 (C) pull down in ACBI1 treated HEK293T cells normalised to interactome from DMSO treated cells and to eGFP-SSX pull down. Data represents log2 fold change enrichment plotted on the x-axis and -log10 (adjusted p value) plotted on the y-axis. Blue dots indicate lost interactors upon ACBI1 treatment, Black dots indicate BAF members. n = 3 biological replicates. (B)(D) List of BAF members present in interactome dataset and their associated log2 fold change value. Listed members are shown as black dots in (A) and (C). (E) Alphafold predictions scores of SS18 (iso1) with SMARCA4, RBM14 and TAF2. ipTM (interface predicted TM-score) is displayed. (F) Gene ontology enrichment analysis of the significantly enriched proteins from the mT-v5-SS18::SSX2 proteomics in HEK293T cells. Analysis was conducted using STRING (v12). The list of total proteins detected in proteomics was used as the statistical background for the enrichment analysis. p-value < 0.05 and log2 fold change > 1 set as significantly enriched. (G) Left: Immunofluorescence of HEK293T cells expressing eGFP, FL, ΔSNH or ΔQPGY (cyan) stained for H2AK119ub1 (H2Aub, magenta). Images are representative of n = 2 biological replicates. Scale bar, 5 μm Right: Profile of fluorescence intensity of eGFP, FL, ΔSNH or ΔQPGY over H2Aub foci. Data represent the mean ± S.E.M of n = 2 biological replicates. For each replicate and condition, > 10 foci where profiled. R² indicates Pearson correlation coefficient.

Article Snippet: The SMARCA2/SMARCA4 degrader ACBI1 (2375564-55-7) and the EP300/CREBBP degrader dCBP-1 (2484739-25-3) were purchased from MedChemExpress, resuspended in dimethyl sulfoxide (DMSO) and kept at −80°C.

Techniques: Mass Spectrometry, Immunofluorescence, Expressing, Staining, Fluorescence

Journal: bioRxiv

Article Title: BAF complex-independent gene activation by SS18::SSX

doi: 10.64898/2026.01.26.701739

Figure Lengend Snippet: (A) Left: Immunofluorescence of HEK293T cells expressing wild-type SS18::SSX1 (FL) treated for 72h with either 500nM ACBI1 or 500nM dCBP- 1 (cyan) stained for H2AK119ub1 (H2Aub) (magenta). Images are representative of n = 2 biological replicates. Scale bar, 5 μm. Right: profile of fluorescence intensity of FL, treated for 72h with either 500nM ACBI1 or 500nM dCBP-1 over H2Aub or eGFP foci. Data represent the mean ± S.E.M of n = 2 biological replicates. For each replicate and condition, > 10 foci where profiled. R² indicates Pearson correlation coefficient. (B) Left: Immunofluorescence of HEK293T cells expressing eGFP or wild-type SS18::SSX1, either untreated (FL) (cyan) or treated for 72h with 500nM ACBI1 or 500nM dCBP-1 stained for SMARCC1 (magenta). Images are representative of n = 2 biological replicates. Scale bar, 5 μm. Right: Profile of fluorescence intensity. Data represent the mean ± S.E.M of n = 2 biological replicates. For each replicate, > 10 foci where profiled. R² indicates Pearson correlation coefficient.

Article Snippet: The SMARCA2/SMARCA4 degrader ACBI1 (2375564-55-7) and the EP300/CREBBP degrader dCBP-1 (2484739-25-3) were purchased from MedChemExpress, resuspended in dimethyl sulfoxide (DMSO) and kept at −80°C.

Techniques: Immunofluorescence, Expressing, Staining, Fluorescence

Journal: bioRxiv

Article Title: BAF complex-independent gene activation by SS18::SSX

doi: 10.64898/2026.01.26.701739

Figure Lengend Snippet: (A) Left: Immunofluorescence of HEK293T cells expressing eGFP or wild-type SS18::SSX1, either untreated (FL) (cyan) or treated for 72h with 500nM ACBI1 or 500nM dCBP-1 stained for EP300 (magenta). Images are representative of n = 2 biological replicates. Scale bar, 5 μm. Right: Profile of fluorescence intensity. Data represent the mean ± S.E.M of n = 2 biological replicates. For each replicate, > 10 foci where profiled. R² indicates Pearson correlation coefficient. (B ) Western Blot of KHOS-240S whole cell extracts treated for 24h with DMSO, 500nM or 1uM dCBP- 1 revealed using EP300, CREBBP or β-actin antibodies. (C) RNA-seq heatmap showing the 1,000 most variable genes with a cutoff z-score of 4 from KHOS-240S cells expressing eGFP or SS18::SSX1 (FL) treated for 72h with either DMSO, 500nM ACBI1 or 500nM dCBP-1. n = 2 biological replicates. ( D) Log2-transformed fold change of FPKM values in KHOS-240S RNA-seq relative to eGFP over selected SS18::SSX target genes. Data represents the mean. (E) Scatter plot of log2-transformed fold change of FPKM values in KHOS-240S RNA-seq relative to eGFP over genes up-regulated by SS18::SSX1 expression and showing >2 fold up-regulation. Biological replicates are combined using their mean. Bold line represents median and dotted line represents cutoff of 2. (F) RNA-seq heatmap showing the 1,000 most variable genes with a cutoff z-score of 4 from wild-type hMSC or cells expressing eGFP-SSX1, eGFP-SS18::SSX1 isoform1, eGFP-SS18::SSX1 isoform2, eGFP-EWSR1::SSX1 or eGFP- EPC1::SSX1 treated for 48h with 500nM dCBP-1. n = 2 biological replicates. (G) Top: Immunofluorescence of HEK293T cells expressing wild-type eGFP-SSX1, eGFP-SS18::SSX1 isoform1, eGFP-SS18::SSX1 isoform2, eGFP-EWSR1::SSX1 or eGFP-EPC1::SSX1(cyan) stained for EP300 (magenta). Images are representative of n = 2 biological replicates. Scale bar, 5 μm. Bottom: profile of fluorescence intensity of eGFP constructs over SSX-rich eGFP foci. Data represent the mean ± S.E.M of n = 2 biological replicates. For each replicate and condition, > 10 foci where profiled. R² and r indicate Pearson correlation coefficients. (H) Scatter plot of log2-transformed fold change of CPM values in hMSC RNA-seq relative to untreated hMSC over the union genes up-regulated by SS18::SSX1 isoform1 (log2FC >2) and up-regulated by EPC1::SSX1 (log2FC >1). Biological replicates are combined using their mean. Bold line represents median. (I) Log2-transformed fold change of CPM values in hMSC RNA-seq relative to untreated cells over selected SSX target genes. Data represents the mean. (J) qRT-PCR displaying log2- transformed fold change of mRNA levels normalised by GAPDH in HSSY-II, SYO-1, CME-1 and Yamato-SS cells treated for 72h with 500nM dCBP-1 relative to DMSO. Data represents the mean of n = 2 biological replicates. (K) Viability of 4 synovial sarcoma cell lines (Yamato-SS, SYO-1, CME-1, HSSY-II) and KHOS-240S osteosarcoma cells treated with various concentrations of dCBP-1. The symbols represent the mean ± S.E.M of n = 3 biological replicates, the solid line represent the nonlinear curve fitting and its associated IC50. p-value represents extra sum-of- squares F test between KHOS-240S and SYO-1 nonlinear regression curves.

Article Snippet: The SMARCA2/SMARCA4 degrader ACBI1 (2375564-55-7) and the EP300/CREBBP degrader dCBP-1 (2484739-25-3) were purchased from MedChemExpress, resuspended in dimethyl sulfoxide (DMSO) and kept at −80°C.

Techniques: Immunofluorescence, Expressing, Staining, Fluorescence, Western Blot, RNA Sequencing, Transformation Assay, Construct, Quantitative RT-PCR