Journal: bioRxiv

Article Title: BRD4 represses developmental and neuronal genes through interactions with polycomb complexes

doi: 10.64898/2026.01.31.702994

Figure Lengend Snippet: a ) Dot plots showing log2 fold enrichment of BRD proteins in the proximal interactome (Turbo-ID) for PRC1 and PRC2 proteins from mouse embryonic stem cells (mESCs), data from . The size of the circle represents the log2 fold enrichment in BRD4 IP relative to IgG control. b ) Like (a) but for enrichment of PRC proteins in BRD4 immunoprecipitation from K562 cells, data from , . The size of the circle represents the t-test difference between the BRD4 IP and the IgG control. c) Immunoblots of endogenous BRD4 IP in H9 hESCs using antibodies that recognise both short and long BRD4 isoforms, with antibodies detecting RING1B, CBX7, CBX4, H3K27ac, H3K23ac, H3K27me3, along with reverse IP with RING1B and MGA antibodies followed by immunoblots for BRD4 and H3K27me3. d ) Immunoblots of GFP-trap co-immunoprecipitation of GFP-BRD4 long isoform (GFP-BRD4L) with Flag-tagged E2F6 and L3MBTL2, HA-tagged EED and EZH2. Immunoblots for β-ACTIN served as controls, e ) Heatmap of CUT&Tag for BRD4, EED, H3K23ac and ChIP-seq data for H3K14ac and RING1B, at active (H3K4me3+), bivalent (H3K4me3+/H3K27me3+) and PRC2 repressed promoters (H3K27me3+). f ) AlphaScreen counts titration of BRD4-BD1 and -BD2 interaction with H3K14ac/23ac showing that only BRD4-BD2 interacts with H3K14ac/23ac. Normalized average alpha counts of three replicates were set relative to the highest WT. g) Immunoblots of biotinylated H3K14/K23ac pulldown for N-terminal His-FLAG tagged BRD4 (N-terminal 412 amino acids), in the presence of increasing concentration of iBET-BD2 (iBD2).

Article Snippet: 1 μg of biotinylated histone H3K14ac/H3K23ac peptide (Cayman Chemicals, Cat. 27520-250ug-CAY) was incubated with 10 μL of streptavidin magnetic beads (Invitrogen 656-01) in 300 μL of binding buffer (50 mM Tris, pH 7.5, 200 mM NaCl and 0.1% NP-40, proteinase inhibitor cocktail) and rotated at room temperature for 30 min. At the same time, FLAG-His tagged BRD4 N -terminal domain containing BD1 and BD2 (E49-E460) (MedChemExpress Cat# HY-P7846), inhibitor of iBET-BD2 (Cayman Chemical Cat# CAY31766), or DMSO were added to the binding buffer on ice.

Techniques: Control, Immunoprecipitation, Western Blot, ChIP-sequencing, Amplified Luminescent Proximity Homogenous Assay, Titration, Concentration Assay

Journal: bioRxiv

Article Title: BRD4 represses developmental and neuronal genes through interactions with polycomb complexes

doi: 10.64898/2026.01.31.702994

Figure Lengend Snippet: a ) Heatmap showing BRD4 signal (CPM) for WT and BRD4 BD2 mut1 at protein-coding genes and active enhancers of hESCs. b ) Scatter plot comparing log2 fold change (log2 FC) values for BRD4 BD2-Mut1/WT (X-axis) against BRD4 dTAG/DMSO (Y-axis) conditions. GSEA GO-biological process enrichment lists for genes that are commonly up (red) and down (blue) regulated in both conditions (right). c ) Representative genome browser snapshot displaying signals for RNA-seq WT, BRD4-mutant1, DMSO and dTAGV-1 along with MAX, BRD4, H3K27me3 and H3K4me3. For CUT&Tag (BRD2,3,4, H3K4me3, H3K27me3) and CUT&Run (EED, ser5 Pol-II), the signal is compared as CPM and MAX as ChIP-seq signal from ChIP-atlas. d) Heatmaps displaying H3K27me3 and H3K4me3 ChIP-seq signals along with RNA-seq normalized counts at bivalent genes in WT-H9 and H9-derived BRD4 BD2 mut1 neurons. e ) MA plot illustrating differential gene expression in BRD4 BD2 mut1 compared to WT neurons. Significantly up- and down-regulated bivalent and non-bivalent genes are highlighted in red and blue, respectively. The number of differentially expressed genes with a log2 fold change of 1 and an adjusted p-value of <0.05 is indicated (right). f ) Genome browser tracks showing ChIP-seq data for bivalent histone modifications (H3K4me3 and H3K27me3), fold change over input and RNA-seq (RPKM) for neuronal genes.

Article Snippet: 1 μg of biotinylated histone H3K14ac/H3K23ac peptide (Cayman Chemicals, Cat. 27520-250ug-CAY) was incubated with 10 μL of streptavidin magnetic beads (Invitrogen 656-01) in 300 μL of binding buffer (50 mM Tris, pH 7.5, 200 mM NaCl and 0.1% NP-40, proteinase inhibitor cocktail) and rotated at room temperature for 30 min. At the same time, FLAG-His tagged BRD4 N -terminal domain containing BD1 and BD2 (E49-E460) (MedChemExpress Cat# HY-P7846), inhibitor of iBET-BD2 (Cayman Chemical Cat# CAY31766), or DMSO were added to the binding buffer on ice.

Techniques: RNA Sequencing, ChIP-sequencing, Derivative Assay, Gene Expression

Journal: bioRxiv

Article Title: BRD4 represses developmental and neuronal genes through interactions with polycomb complexes

doi: 10.64898/2026.01.31.702994

Figure Lengend Snippet: a) Schematic representation of the protocol used to generate unguided neuronal organoids (UNOs), with images of UNO WT at 5,8, and 41 days. b ) Immunofluorescence images of UNOs at day 41 stained for markers of neuronal progenitor (SOX2), post-mitotic early neurons (TUJ1), scale bars: 100 μm. c ) MA plot for RNA-seq data illustrating differentially expressed genes in day 41 UNOs following 20 hours of BRD4 PROTAC (ZxH) treatment (n=3 independent organoids). d) Geneontology (GO) enrichment analyses of up- and down-regulated genes. e ) Genome browser tracks for normalized reads at TSS for pseudo bulk scCUT&Tag and bulk RNA-seq for immediate early genes (IEGs) upon 20 h BRD4 PROTAC in UNOs (data from (c)). f) UMAP plots stratified by genotype show the annotated cell lineages: WT, BRD4 BD2 mut2, and BRD4 BD2 mut3. Cell clusters are identified by colour, illustrating the contribution of each genotype to specific lineages, such as Glutamatergic, GABAnergic, optic vesicle, and RPE. g) Stacked bar charts for 41-day and 63-day UNOs, detailing the percentage of cells for each annotated cell type across the WT, BRD4 BD2 mut2, and BRD4 BD2 mut3 UNOs. h) Representative bright-field microscopy images of 41-day UNOs, Scale bar=1mm (rest of the images in source file). i) Dot plots showing the average expression level (Z scores) and percentage of cells expressed in Glutamatergic, Diencephalic-1(pink in UMAP), and Diencephalic-2(blue in UMAP), and G2M clusters for bivalent genes that showed significant differential expression in the scRNA-seq data in BRD4-BD2 mut1 and BRD4-BD2 mut2 UNOs.

Article Snippet: 1 μg of biotinylated histone H3K14ac/H3K23ac peptide (Cayman Chemicals, Cat. 27520-250ug-CAY) was incubated with 10 μL of streptavidin magnetic beads (Invitrogen 656-01) in 300 μL of binding buffer (50 mM Tris, pH 7.5, 200 mM NaCl and 0.1% NP-40, proteinase inhibitor cocktail) and rotated at room temperature for 30 min. At the same time, FLAG-His tagged BRD4 N -terminal domain containing BD1 and BD2 (E49-E460) (MedChemExpress Cat# HY-P7846), inhibitor of iBET-BD2 (Cayman Chemical Cat# CAY31766), or DMSO were added to the binding buffer on ice.

Techniques: Immunofluorescence, Staining, RNA Sequencing, Microscopy, Expressing, Quantitative Proteomics

Journal: bioRxiv

Article Title: BRD4 represses developmental and neuronal genes through interactions with polycomb complexes

doi: 10.64898/2026.01.31.702994

Figure Lengend Snippet: a) UMAP plots show the distribution of single-cell ATAC sequencing (scATAC-seq) data clustered by genotypes WT and BRD4 BD2 mut2 and annotated by cell lineage for WT and BRD4 BD2 mut2. b ) Z-scores (high scores in red and low scores are in blue) showing top transcription factor motifs enriched at Diencephalic, Glutamatergic, G2M and GABAnergic lineages across scATACseq peaks, which are gained in BRD4 BD2 mut 2 UNO compared to WT control. The complete list of enriched TFs is in the source data table.

Article Snippet: 1 μg of biotinylated histone H3K14ac/H3K23ac peptide (Cayman Chemicals, Cat. 27520-250ug-CAY) was incubated with 10 μL of streptavidin magnetic beads (Invitrogen 656-01) in 300 μL of binding buffer (50 mM Tris, pH 7.5, 200 mM NaCl and 0.1% NP-40, proteinase inhibitor cocktail) and rotated at room temperature for 30 min. At the same time, FLAG-His tagged BRD4 N -terminal domain containing BD1 and BD2 (E49-E460) (MedChemExpress Cat# HY-P7846), inhibitor of iBET-BD2 (Cayman Chemical Cat# CAY31766), or DMSO were added to the binding buffer on ice.

Techniques: Sequencing, Control

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: The BRD4 inhibitor JQ1 augments the antitumor efficacy of abemaciclib in preclinical models of gastric carcinoma

doi: 10.1186/s13046-023-02615-2

Figure Lengend Snippet: JQ1 is a promising candidate in combination with ABE. A Ranked synergy models of ABE and screened epigenetic drugs, including JQ1, tazemetostat, vorinostat, 5-azacitidine, SETDB1-TTD-IN-1 and UNC669. Synergy scores were labelled to the top of each model. B Dependency score of CDK4 and BRD4 for tumor cells across different cancer types. Score < 0 represents essential genes and Score < -1 represents super essential genes. C Transcriptional expression level of BRD4 between tumor and normal tissues in TCGA-STAD cohort. D Immunohistochemistry (IHC) staining of BRD4 in GC patients from The Human Protein Atlas. E CUT&Tag-seq binding enrichment of H3K27ac in AGS cells treated with DMSO or ABE. F Pie plot of the genomic distribution of H3K27ac peaks in ABE-treated AGS cells. G Gene tracks depicting H3K27ac signals at the BRD4 locus. The signal from ABE and DMSO was normalized at same level. H Western blotting images showing the protein level of BRD4 protein level after ABE treatment. GAPDH was used as a loading control. I Quantification of the protein level of BRD4 in AGS cells after ABE treatment for 24 h using ImageJ software. The data are presented as the mean ± SEM of three replicates. J Enrichment analysis of top1000 upregulated H3K27ac peaks in ABE-treated AGS cells. K Gene tracks depicting H3K27ac signals at the SMAD3, VCL, SNAI1 and RAC1 locus. The signals from ABE and DMSO were normalized at same level

Article Snippet: D Immunohistochemistry (IHC) staining of BRD4 in GC patients from The Human Protein Atlas.

Techniques: Expressing, Immunohistochemistry, Binding Assay, Western Blot, Control, Software

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: The BRD4 inhibitor JQ1 augments the antitumor efficacy of abemaciclib in preclinical models of gastric carcinoma

doi: 10.1186/s13046-023-02615-2

Figure Lengend Snippet: CDK4/6 and BRD4 Inhibition Specifically induce cell senescence and DNA damage in vitro. A , C , E Immunofluorescence staining of the DNA damage marker γH2AX (scale bar = 25 μm), DNA senescence marker p21 and p53 (scale bar = 50 μm ) in AGS cell line upon the treatment of the indicted agents for 24 h. B , D , F Quantification of the positive cell ratio and mean fluorescence intensity using QuPath software. The data are presented as the mean ± SEM of three replicates. G Western blotting images showing the protein level of Phospho-Rb (Ser807/811), γH2AX, p53 and p21 in AGS cells from the indicted treatment cohort. GAPDH was used as a loading control. H Quantification of the protein level of Phospho-Rb (Ser807/811), γH2AX, p53 and p21 in AGS cells from the indicted treatment for 24 h using imageJ software. The data are presented as the mean ± SEM of three replicates. Significance of each group was compared with DMSO group

Article Snippet: D Immunohistochemistry (IHC) staining of BRD4 in GC patients from The Human Protein Atlas.

Techniques: Inhibition, In Vitro, Immunofluorescence, Staining, Marker, Fluorescence, Software, Western Blot, Control

Journal: Cancers

Article Title: The Prognostic Role of BRD4 Expression in High-Grade Serous Ovarian Cancer

doi: 10.3390/cancers16111962

Figure Lengend Snippet: Overall survival at 12 months according to BRD4 gene expression.

Article Snippet: For the identification of BRD4 protein, KIT Human BRD4 (Bromodomain-containing protein 4 Elisa Kit) (Assay Genie, London, UK) was used.

Techniques: Expressing

Journal: Cancers

Article Title: The Prognostic Role of BRD4 Expression in High-Grade Serous Ovarian Cancer

doi: 10.3390/cancers16111962

Figure Lengend Snippet: Kaplan–Meier curves of OS at 12 months of patients with low BRD4 expression (red line) versus intermediate/high gene expression (black line).

Article Snippet: For the identification of BRD4 protein, KIT Human BRD4 (Bromodomain-containing protein 4 Elisa Kit) (Assay Genie, London, UK) was used.

Techniques: Expressing, Gene Expression

Journal: Cancers

Article Title: The Prognostic Role of BRD4 Expression in High-Grade Serous Ovarian Cancer

doi: 10.3390/cancers16111962

Figure Lengend Snippet: Kaplan–Meier curves of OS at 24 months of patients with low BRD4 expression (red line) versus intermediate/high gene expression (black line).

Article Snippet: For the identification of BRD4 protein, KIT Human BRD4 (Bromodomain-containing protein 4 Elisa Kit) (Assay Genie, London, UK) was used.

Techniques: Expressing, Gene Expression

Journal: Cancers

Article Title: The Prognostic Role of BRD4 Expression in High-Grade Serous Ovarian Cancer

doi: 10.3390/cancers16111962

Figure Lengend Snippet: Kaplan–Meier curves of PFS at 12 months of patients with low BRD4 expression (red line) versus intermediate/high gene expression (black line).

Article Snippet: For the identification of BRD4 protein, KIT Human BRD4 (Bromodomain-containing protein 4 Elisa Kit) (Assay Genie, London, UK) was used.

Techniques: Expressing, Gene Expression

Journal: Cancers

Article Title: The Prognostic Role of BRD4 Expression in High-Grade Serous Ovarian Cancer

doi: 10.3390/cancers16111962

Figure Lengend Snippet: Kaplan–Meier curves of PFS at 24 months of patients with low BRD4 expression (red line) versus intermediate/high gene expression (black line).

Article Snippet: For the identification of BRD4 protein, KIT Human BRD4 (Bromodomain-containing protein 4 Elisa Kit) (Assay Genie, London, UK) was used.

Techniques: Expressing, Gene Expression

Journal: Cancers

Article Title: The Prognostic Role of BRD4 Expression in High-Grade Serous Ovarian Cancer

doi: 10.3390/cancers16111962

Figure Lengend Snippet: Kaplan–Meier curves of overall survival (OS) at 12 months in patients with low (blue line), intermediate (green line) and high (yellow line) BRD4 protein expression.

Article Snippet: For the identification of BRD4 protein, KIT Human BRD4 (Bromodomain-containing protein 4 Elisa Kit) (Assay Genie, London, UK) was used.

Techniques: Expressing

Journal: Cancers

Article Title: The Prognostic Role of BRD4 Expression in High-Grade Serous Ovarian Cancer

doi: 10.3390/cancers16111962

Figure Lengend Snippet: Kaplan–Meier curves of overall survival (OS) at 12 months in patients with intermediate/low (blue line) and high (green line) BRD4 protein expression.

Article Snippet: For the identification of BRD4 protein, KIT Human BRD4 (Bromodomain-containing protein 4 Elisa Kit) (Assay Genie, London, UK) was used.

Techniques: Expressing

Journal: Cancers

Article Title: The Prognostic Role of BRD4 Expression in High-Grade Serous Ovarian Cancer

doi: 10.3390/cancers16111962

Figure Lengend Snippet: Kaplan–Meier curves of overall survival (OS) at 24 months in patients with intermediate/low (blue line) and high (green line) BRD4 protein expression.

Article Snippet: For the identification of BRD4 protein, KIT Human BRD4 (Bromodomain-containing protein 4 Elisa Kit) (Assay Genie, London, UK) was used.

Techniques: Expressing

Journal: Cancers

Article Title: The Prognostic Role of BRD4 Expression in High-Grade Serous Ovarian Cancer

doi: 10.3390/cancers16111962

Figure Lengend Snippet: Kaplan–Meier curves of PFS at 12 months in patients with intermediate/low (blue line) and high (green line) BRD4 protein expression.

Article Snippet: For the identification of BRD4 protein, KIT Human BRD4 (Bromodomain-containing protein 4 Elisa Kit) (Assay Genie, London, UK) was used.

Techniques: Expressing

Journal: Cancers

Article Title: The Prognostic Role of BRD4 Expression in High-Grade Serous Ovarian Cancer

doi: 10.3390/cancers16111962

Figure Lengend Snippet: Kaplan–Meier curves of PFS at 24 months in patients with intermediate/low (blue line) and high (green line) BRD4 protein expression.

Article Snippet: For the identification of BRD4 protein, KIT Human BRD4 (Bromodomain-containing protein 4 Elisa Kit) (Assay Genie, London, UK) was used.

Techniques: Expressing

Journal: Cancers

Article Title: The Prognostic Role of BRD4 Expression in High-Grade Serous Ovarian Cancer

doi: 10.3390/cancers16111962

Figure Lengend Snippet: Association between BRD4 gene and protein expression.

Article Snippet: For the identification of BRD4 protein, KIT Human BRD4 (Bromodomain-containing protein 4 Elisa Kit) (Assay Genie, London, UK) was used.

Techniques: Expressing