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: Nature Communications

Article Title: Identification of a SNAI1 enhancer RNA that drives cancer cell plasticity

doi: 10.1038/s41467-025-58032-w

Figure Lengend Snippet: a The interaction between SNAI1e and BRD4 in MDA‐MB‐231 cells was analyzed by RNA immunoprecipitation (RIP). YTHDC1 served as a negative control. RT-qPCR was performed to detect SNAI1e expression in immunoprecipitants from MDA‐MB‐231 cells. The results are expressed as mean ± SD from three independent experiments. b The interaction between SNAI1e and BRD4 in MDA-MB-231 cells was analyzed by RNA pull-down. Western blotting analysis was performed to detect FLAG expression in whole-cell lysates (Input) and immunoprecipitants (IP). LETS1 and SNAI1e-AS served as negative controls. The RNA amounts used for pull-down were evaluated by agarose gel electrophoresis. c The interaction between SNAI1e truncation mutants and BRD4 in MDA-MB-231 cells was analyzed by RNA pull-down. Western blotting analysis was performed to detect FLAG expression in whole-cell lysates (Input) and immunoprecipitants (IP). The RNA amounts used for pull-down were evaluated by agarose gel electrophoresis. d Schematic representation of full-length (FL) BRD4 and the truncation mutants tested. e The interactions between SNAI1e and BRD4 FL or the truncation mutants in MDA-MB-231 cells were analyzed by RNA pull-down. SNAI1e-AS , antisense SNAI1e ; SNAI1e-S , sense SNAI1e . Western blotting analysis was performed to detect FLAG expression in whole-cell lysates (Input) and immunoprecipitants (IP). The RNA amounts used for pull-down were evaluated by agarose gel electrophoresis. f The direct interaction between SNAI1e and the FLAG-BRD4 BD1/2 recombinant protein was analyzed by in vitro RIP. The results are expressed as mean ± SD from three independent experiments. The FLAG-tagged proteins in immunoprecipitants were evaluated by western blotting. g The direct interaction between SNAI1e and the recombinant FLAG-BRD4 BD1/2 protein was analyzed by in vitro RNA pull-down. Western blotting analysis was performed to detect FLAG expression in whole-cell lysates (Input) and immunoprecipitants (IP). The RNA amounts used for pull-down were evaluated by agarose gel electrophoresis. Significance was calculated by using one-way ANOVA followed by Dunnett’s ( a ) and Tukey’s ( f ) multiple comparisons test. Data are representative of at least three ( b , c , e , g ) independent experiments with similar results. Co.vec empty control vector.

Article Snippet: For in vitro RIP, 10 pmol of in vitro-transcribed SNAI1e was incubated with 2 μg recombinant FLAG-SMURF2 protein (Sigma‒Aldrich; SRP0228) or FLAG-BRD4 BD1/2 recombinant protein (R&D systems; SP-600) for 16 h at 4 °C as described previously .

Techniques: RNA Immunoprecipitation, Negative Control, Quantitative RT-PCR, Expressing, Western Blot, Agarose Gel Electrophoresis, Recombinant, In Vitro, Control, Plasmid Preparation

Journal: Antiviral research

Article Title: Screening of an epigenetic compound library identifies BRD4 as a potential antiviral target for hepatitis B virus covalently closed circular DNA transcription.

doi: 10.1016/j.antiviral.2023.105552

Figure Lengend Snippet: Fig. 8. MS436-mediated epigenetic change of cccDNA minichromosome. (A–D) HepAD38 cells cultured in T75 flask were induced in Tet-free me dium for 10 days for cccDNA establishment, then treated with DMSO or MS436 (10 μM) every other day for 4 days in the presence of 3TC (10 μM). Cells were crosslinked and lysed for further chromatin fragmentation by sonication-based shearing. cccDNA was immunoprecipitated by ChIP-grade non-immune IgG isotype control or antibodies against H3K27ac, H3K9me3, H3K4me3 or BRD4 and detected by qPCR. The enrichment of aforementioned histone PTMs or BRD4 protein on cccDNA was plotted as fold change to NIS IgG control, respectively. Data are shown as mean ± SD (n = 3). **p < 0.01, ***p < 0.001, ns: not significant. (E) The intracellular total BRD4 proteins, including both the long and short isoforms, were detected by western blot. β-actin served as the loading control.

Article Snippet: Human BRD4 siRNA (a pool of 3 unique 27-mer siRNA oligo duplexes) was purchased from OriGene (cat# SR323604).

Techniques: Cell Culture, Sonication, Immunoprecipitation, Control, Western Blot

Journal: Antiviral research

Article Title: Screening of an epigenetic compound library identifies BRD4 as a potential antiviral target for hepatitis B virus covalently closed circular DNA transcription.

doi: 10.1016/j.antiviral.2023.105552

Figure Lengend Snippet: Fig. 11. siRNA knock down of BRD4 reduces HBV transcription. HepG2-NTCP cells in 6-well-plate were transfected with control or BRD4 siRNA for 2 days, followed by HBV infection with 500 vge/cell for 24 h, then a second round of siRNA transfection was con ducted in the virally infected cells. The cells were cultured for another 2 days, (A) cellular BRD4 and β-actin proteins were detected by western blot; the intracellular HBV RNA and cccDNA were analyzed by northern blot and qPCR, respectively. (B) The relative cccDNA levels are plotted as fold change to siControl. The relative levels of HBV mRNA normalized to cccDNA are plotted as fold change to that of siCon trol. Data are shown as mean ± SD, n = 3; ***p < 0.001.

Article Snippet: Human BRD4 siRNA (a pool of 3 unique 27-mer siRNA oligo duplexes) was purchased from OriGene (cat# SR323604).

Techniques: Knockdown, Transfection, Control, Infection, Cell Culture, Western Blot, Northern Blot

Journal: Antiviral research

Article Title: Screening of an epigenetic compound library identifies BRD4 as a potential antiviral target for hepatitis B virus covalently closed circular DNA transcription.

doi: 10.1016/j.antiviral.2023.105552

Figure Lengend Snippet: Fig. 12. Depletion of BRD4 by PROTAC degraders inhibits HBV transcription. (A, C) Chemical structure of dBET1 and MZ-1. (B, D) HepG2-NTCP cell were infected with HBV (500 vge/cell) for 6 days, followed by treatment with (B) dBET1 (10 μM) in the absence or presence of 3TC (10 μM) or (D) MZ-1 (1 μM) and 3TC (10 μM) every other day for 4 days. Cellular BRD4 was detected by western blot, β-actin served as loading control. HBV total RNA and/or cytoplasmic core DNA were detected by northern and Southern blot, respectively.

Article Snippet: Human BRD4 siRNA (a pool of 3 unique 27-mer siRNA oligo duplexes) was purchased from OriGene (cat# SR323604).

Techniques: Infection, Western Blot, Control, Northern Blot, Southern Blot