brd4 protac  (TargetMol)

Journal: Journal of Cell Communication and Signaling

Article Title: Mechanistic role of GNE‐987 targeting BRD4‐HCP5 axis in pediatric T‐cell acute lymphoblastic leukemia

doi: 10.1002/ccs3.70063

Figure Lengend Snippet: Identification of Key eRNA regulated by BRD4. (A) Volcano plot of differential expression between high and low BRD4 expression groups. (B) GO enrichment analysis of differentially expressed genes. (C) KEGG enrichment analysis of differentially expressed genes. (D) Venn diagram showing the intersection of differentially expressed genes and eRNA. (E) Correlation analysis between BRD4 and HLA Complex P5, STK3, PAAF1, and PINLYP expression. BRD4, Bromodomain‐containing protein 4.

Article Snippet: The experimental group was treated with the BRD4 inhibitor GNE‐987 (HY‐129937A, MCE) at a final concentration of 20 nM, whereas the control group received an equal volume of DMSO (472301, Sigma‐Aldrich).

Techniques: Quantitative Proteomics, Expressing

Journal: Journal of Cell Communication and Signaling

Article Title: Mechanistic role of GNE‐987 targeting BRD4‐HCP5 axis in pediatric T‐cell acute lymphoblastic leukemia

doi: 10.1002/ccs3.70063

Figure Lengend Snippet: BRD4 inhibitor significantly promotes HCP5 super‐enhancer activity and expression. (A) Reverse transcription‐quantitative polymerase chain reaction analysis of HCP5 mRNA expression levels in acute myeloid leukemia cell lines (THP‐1 and HL‐60) treated with the BRD4 inhibitor GNE‐987. (B) Western Blot analysis showing BRD4 expression levels in different groups. (C) Representative agarose gel electrophoresis image and statistical quantification of ChIP‐qPCR products. Compared to the Ctrl group, * p < 0.05, ** p < 0.01. All cell experiments were repeated three times. BRD4, Bromodomain‐containing protein 4; HCP5, HLA Complex P5.

Article Snippet: The experimental group was treated with the BRD4 inhibitor GNE‐987 (HY‐129937A, MCE) at a final concentration of 20 nM, whereas the control group received an equal volume of DMSO (472301, Sigma‐Aldrich).

Techniques: Activity Assay, Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Western Blot, Agarose Gel Electrophoresis, ChIP-qPCR

Journal: Journal of Cell Communication and Signaling

Article Title: Mechanistic role of GNE‐987 targeting BRD4‐HCP5 axis in pediatric T‐cell acute lymphoblastic leukemia

doi: 10.1002/ccs3.70063

Figure Lengend Snippet: Bromodomain‐containing protein 4 Inhibitor GNE‐987 Significantly Inhibits acute myeloid leukemia Cell Proliferation and Migration and Induces Apoptosis. (A) CCK‐8 assay measuring cell proliferation in each group at 0, 12, 24, 36, 48, 60, and 72 h, with absorbance detected at OD450. (B) Representative images of Live and Dead staining for each group, with a bar chart depicting the statistical analysis of cell death ratios. Bar = 50 μm. (C) Colony formation assay for each group, with a bar chart showing the statistical analysis of colony numbers. (D) Flow cytometry analysis of apoptosis levels in each group, with a bar chart depicting the statistical analysis of apoptosis rates. Compared with the Ctrl group, * p < 0.05, ** p < 0.01. All cell experiments were repeated three times.

Article Snippet: The experimental group was treated with the BRD4 inhibitor GNE‐987 (HY‐129937A, MCE) at a final concentration of 20 nM, whereas the control group received an equal volume of DMSO (472301, Sigma‐Aldrich).

Techniques: Migration, CCK-8 Assay, Staining, Colony Assay, Flow Cytometry

Journal: Journal of Cell Communication and Signaling

Article Title: Mechanistic role of GNE‐987 targeting BRD4‐HCP5 axis in pediatric T‐cell acute lymphoblastic leukemia

doi: 10.1002/ccs3.70063

Figure Lengend Snippet: Silencing of HLA Complex P5 Reverses the Inhibitory Effect of GNE‐987 on acute myeloid leukemia Cell Viability. (A) Reverse transcription‐quantitative polymerase chain reaction results verify the silencing efficiency of sh‐HCP5. (B) CCK8 assay measuring cell proliferation at 0, 12, 24, 36, 48, 60, and 72 h, with absorbance detected at OD450. (C) Representative images of Live and Dead staining in each group and a bar chart showing the death ratio; scale bar = 50 μm. (D) Colony formation assay and statistical graph of colony numbers for each group. (E) Flow cytometry analysis of apoptosis levels and a statistical graph of apoptosis rates in each group. For panel A, compared with the sh‐NC group, * p < 0.05, ** p < 0.01. For panels B‐E, compared with the Ctrl group, * p < 0.05, ** p < 0.01; compared with the GNE‐987+sh‐NC group, # p < 0.05, ## p < 0.01. All cell experiments were repeated three times.

Article Snippet: The experimental group was treated with the BRD4 inhibitor GNE‐987 (HY‐129937A, MCE) at a final concentration of 20 nM, whereas the control group received an equal volume of DMSO (472301, Sigma‐Aldrich).

Techniques: Reverse Transcription, Real-time Polymerase Chain Reaction, CCK-8 Assay, Staining, Colony Assay, Flow Cytometry

Journal: Journal of Cell Communication and Signaling

Article Title: Mechanistic role of GNE‐987 targeting BRD4‐HCP5 axis in pediatric T‐cell acute lymphoblastic leukemia

doi: 10.1002/ccs3.70063

Figure Lengend Snippet: BRD4 Inhibitor GNE‐987 Promotes HCP5 Transcription and Inhibits acute myeloid leukemia Progression. (A) Gross appearance of mouse spleens and statistical graph of spleen length measurements, bar = 1 cm. (B) H&E staining of bone marrow, spleen, and liver tissues from each group of mice. (C) Western blot analysis of BRD4 protein expression in mouse spleen tissue and corresponding grayscale quantification; (D) reverse transcription‐quantitative polymerase chain reaction analysis of HCP5 mRNA levels in spleen tissue across groups; (E) flow cytometry analysis of human CD3+ cells in mice's peripheral blood and bone marrow. (F) Immunofluorescence staining of the mouse spleen for localization and quantification of Ki67 and GRP78; scale bar = 50 μm. Compared with the ctrl group, * p < 0.05, ** p < 0.01; compared with the GNE‐987+sh‐NC group, # p < 0.05, ## p < 0.01. Each group included six mice. BRD4, Bromodomain‐containing protein 4; HCP5, HLA Complex P5.

Article Snippet: The experimental group was treated with the BRD4 inhibitor GNE‐987 (HY‐129937A, MCE) at a final concentration of 20 nM, whereas the control group received an equal volume of DMSO (472301, Sigma‐Aldrich).

Techniques: Staining, Western Blot, Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Flow Cytometry, Immunofluorescence

Journal: Journal of Cell Communication and Signaling

Article Title: Mechanistic role of GNE‐987 targeting BRD4‐HCP5 axis in pediatric T‐cell acute lymphoblastic leukemia

doi: 10.1002/ccs3.70063

Figure Lengend Snippet: BRD4 inhibitor GNE‐987 activates the HLA Complex P5 super‐enhancer to downregulate BRD4 and inhibit T‐cell Acute Lymphoblastic Leukemia cell proliferation. BRD4, Bromodomain‐containing protein 4.

Article Snippet: The experimental group was treated with the BRD4 inhibitor GNE‐987 (HY‐129937A, MCE) at a final concentration of 20 nM, whereas the control group received an equal volume of DMSO (472301, Sigma‐Aldrich).

Techniques:

Journal: PLOS One

Article Title: SHP2 improved Late-onset fetal growth restriction via modulating ROS/BRD4/PI3K/YAP/PIGF signaling induced angiogenesis

doi: 10.1371/journal.pone.0342649

Figure Lengend Snippet: A: Boxplot of sample normalization for the FGR dataset GSE147776 ; B: Volcano plot of differentially expressed genes for the FGR dataset GSE147776 ; C: Heatmap of differentially expressed gene clustering for the FGR dataset GSE147776 ; Da: GO enrichment bubble plot of DEGs; Db: GO enrichment bar plot of DEGs; Ea: KEGG enrichment bubble plot of DEGs; Eb: KEGG enrichment lollipop plot of DEGs; F: Scatter plot of BRD4 and KEAP1 correlation; G: Scatter plot of KEAP1 and Nrf2 correlation; H: Heatmap of molecular interactions among BRD4, NOX4, NOX2, Nrf2, SHP2, P53, VEGFR, PI3K, CREB, JNK, and P38; I: Lollipop plot of YAP and effector factor correlations (CREB, VEGF, VEGFR2, SOX9, OCT4, SOX2, Ki-67, Nrf2).

Article Snippet: BRD4 inhibitor JQ-1, SHP2 inhibitor PHPS1, PI3K agonist 740 Y-P, and YAP inhibitor Verteporfin were purchased from MCE.

Techniques:

Journal: PLOS One

Article Title: SHP2 improved Late-onset fetal growth restriction via modulating ROS/BRD4/PI3K/YAP/PIGF signaling induced angiogenesis

doi: 10.1371/journal.pone.0342649

Figure Lengend Snippet: A: Western blot analysis of NOX4, nuclear BRD4, p-SHP2, p-PI3K, nuclear YAP, Nrf2, and PIGF protein expressions in EPCs in the following six groups: NC group, Model group, Model + JQ-1 group, Model + JQ-1 + PHPS1 group, Model + JQ-1 + PHPS1 + 740Y-P group, and Model + JQ-1 + PHPS1 + 740Y-P + Verteporfin group, GAPDH as the control protein; B: Statistical analysis of relative protein expression levels. N = 3; Data are expressed as mean ± standard deviation; Different lowercase letters on the same column indicate significant differences between groups at P < 0.05, while different uppercase letters indicate significant differences at P < 0.01.

Article Snippet: BRD4 inhibitor JQ-1, SHP2 inhibitor PHPS1, PI3K agonist 740 Y-P, and YAP inhibitor Verteporfin were purchased from MCE.

Techniques: Western Blot, Control, Expressing, Standard Deviation

Journal: PLOS One

Article Title: SHP2 improved Late-onset fetal growth restriction via modulating ROS/BRD4/PI3K/YAP/PIGF signaling induced angiogenesis

doi: 10.1371/journal.pone.0342649

Figure Lengend Snippet: A: Western blot analysis of VEGF and HIF1α protein expressions in EPCs in the following six groups: NC group, Model group, Model + JQ-1 group, Model + JQ-1 + PHPS1 group, Model + JQ-1 + PHPS1 + 740Y-P group, and Model + JQ-1 + PHPS1 + 740Y-P + Verteporfin group, along with statistical analysis of relative protein expression levels. GAPDH as the control protein; B: Tube formation assay measuring vascular intersections, total vessel length, and vessel branch length, along with statistical analysis of number of neovessels visual field. N = 3; Data are expressed as mean ± standard deviation; Different lowercase letters on the same column indicate significant differences between groups at P < 0.05, while different uppercase letters indicate significant differences at P < 0.01.

Article Snippet: BRD4 inhibitor JQ-1, SHP2 inhibitor PHPS1, PI3K agonist 740 Y-P, and YAP inhibitor Verteporfin were purchased from MCE.

Techniques: Western Blot, Expressing, Control, Tube Formation Assay, Standard Deviation

Journal: PLOS One

Article Title: SHP2 improved Late-onset fetal growth restriction via modulating ROS/BRD4/PI3K/YAP/PIGF signaling induced angiogenesis

doi: 10.1371/journal.pone.0342649

Figure Lengend Snippet: A: Western blot analysis of OCT4, SOX2, and C-Myc protein expressions in EPCs in the following six groups: NC group, Model group, Model + JQ-1 group, Model + JQ-1 + PHPS1 group, Model + JQ-1 + PHPS1 + 740Y-P group, and Model + JQ-1 + PHPS1 + 740Y-P + Verteporfin group, along with statistical analysis of relative protein expression levels. GAPDH as the control protein; B: Colony formation assay observing EPCs proliferation, along with statistical analysis of number of cloned cells; C: Flow cytometry analysis of the cell cycle, along with statistical analysis of cell cycle distribution; D: Flow cytometry analysis of cell apoptosis, along with statistical analysis of apoptosis rate. N = 3; Data are expressed as mean ± standard deviation; Different lowercase letters on the same column indicate significant differences between groups at P < 0.05, while different uppercase letters indicate significant differences at P < 0.01.

Article Snippet: BRD4 inhibitor JQ-1, SHP2 inhibitor PHPS1, PI3K agonist 740 Y-P, and YAP inhibitor Verteporfin were purchased from MCE.

Techniques: Western Blot, Expressing, Control, Colony Assay, Clone Assay, Flow Cytometry, Standard Deviation

Journal: PLOS One

Article Title: SHP2 improved Late-onset fetal growth restriction via modulating ROS/BRD4/PI3K/YAP/PIGF signaling induced angiogenesis

doi: 10.1371/journal.pone.0342649

Figure Lengend Snippet: SHP2 induces endothelial progenitor cell activation by regulating ROS/BRD4 and PI3K/YAP/PIGF, improving late-onset fetal growth restriction.

Article Snippet: BRD4 inhibitor JQ-1, SHP2 inhibitor PHPS1, PI3K agonist 740 Y-P, and YAP inhibitor Verteporfin were purchased from MCE.

Techniques: Activation 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 and b ) Immunoblots showing depletion of BRD4-L and BRD4-S after 4 hours of ZxH-3-26 treatment (ZxH, BRD4-specific PROTAC) (a); dTAGV-1 and dTAG13 mediated BRD4 degradation in BRD4-dTAG hESCs (b), BRD3 and β-ACTIN serve as controls. c ) Time-course heatmap of RNA-seq data (4 hours, 8 hours, 20 hours) of PROTAC treatment and 20 hours of dTAGV-1 treatment in BRD4-dTAG hESCs comparing log2fold change values across four k-means clusters (C1–C4) based on differential expression levels, indicating similar directional changes at least in two of the ZxH treatment time points (left). Heatmaps of CUT&Tag counts per million reads (CPM) signal for short and long isoforms of BRD4 (Diagenode and Abcam antibodies) (middle). Enrichment of GO biological processes of the genes in the four clusters (right). d ) Genome-browser visualization of CUT&Tag for BRD4 performed using two antibodies, along with average RNAseq signal (n=3 replicates), performed 8 hours after DMSO and ZxH treatment in H9 hESCs at representative neuronal and developmental genes, along with known BRD4 target gene MYC. e ) Percentage peak overlap for BRD4, EED, RAD21, NIPBL, serine-5 phosphorylated RNA Pol II (RNA-Pol II s5p), H3K27ac, H3K4me3, and H3K27me3 across 15 ChromHMM states in H9-hESCs.

Article Snippet: RNA-seq libraries upon BRD4 PROTAC (TargetMol Chemicals, Cat. # T17297, ZxH-3-26) in H9 hESCs, and dTAGV-1 treatment in H9 hESC-BRD4-dTAG lines were generated using NEBNext ® Poly(A) mRNA Magnetic Isolation Module (NEB, Cat.# E7490L) followed by NEBNext ® Ultra TM II RNA (NEB, Cat.# E7770L).

Techniques: Western Blot, RNA Sequencing, Quantitative Proteomics, RNA sequencing

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) Pairwise peak intersection for chromatin modifications. Values indicate the fraction of overlap between peak-sets. Horizontal comparison shows the percentage of overlap between each peak set on the X-axis, with peak sets compared on the Y-axis, and vice versa. b ) Heatmaps of CUT&Tag counts per million reads (CPM) signal for BRD4 (BRD4, Diagenode antibody), BRD4(Abcam antibody), H3K27me3, H3K4me3, H3K27ac, CUT&RUN for BRD2, BRD3, EED and EZH2, ChIPseq data for PRC1.6 components (PCGF6, MAX, MYC), along with PRC1 component (CBX8 and RING1B). Clustered based on enrichment of PRC1.6 components, active (H3K4me3), bivalent (H3K27me3+ & H3K4me3+), and other gene promoters. c ) Venn diagrams and Metascape functional annotations (below) of upregulated (left, in purple) and downregulated (right, in purple) genes following 8 hours of ZxH-mediated BRD4 degradation and in two PCGF6 knockout human pluripotent stem cell lines (data from Lan et.al. 2022). d ) Similar to (b), but clustering based on commonly upregulated genes (clusters 1-3). Upregulated gene promoters are categorized by their bivalent or active chromatin modifications. e ) Genome-browser visualization of BRD4, MAX, and bivalent histone modifications, along with average TTseq signal (n=3 replicates), performed 1 hour after DMSO and dTAGV-1 treatment in BRD4-dTAG hESCs (Western blotting showing BRD4 degradation in ).

Article Snippet: RNA-seq libraries upon BRD4 PROTAC (TargetMol Chemicals, Cat. # T17297, ZxH-3-26) in H9 hESCs, and dTAGV-1 treatment in H9 hESC-BRD4-dTAG lines were generated using NEBNext ® Poly(A) mRNA Magnetic Isolation Module (NEB, Cat.# E7490L) followed by NEBNext ® Ultra TM II RNA (NEB, Cat.# E7770L).

Techniques: Comparison, Functional Assay, Knock-Out, Western Blot

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: RNA-seq libraries upon BRD4 PROTAC (TargetMol Chemicals, Cat. # T17297, ZxH-3-26) in H9 hESCs, and dTAGV-1 treatment in H9 hESC-BRD4-dTAG lines were generated using NEBNext ® Poly(A) mRNA Magnetic Isolation Module (NEB, Cat.# E7490L) followed by NEBNext ® Ultra TM II RNA (NEB, Cat.# E7770L).

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: RNA-seq libraries upon BRD4 PROTAC (TargetMol Chemicals, Cat. # T17297, ZxH-3-26) in H9 hESCs, and dTAGV-1 treatment in H9 hESC-BRD4-dTAG lines were generated using NEBNext ® Poly(A) mRNA Magnetic Isolation Module (NEB, Cat.# E7490L) followed by NEBNext ® Ultra TM II RNA (NEB, Cat.# E7770L).

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: RNA-seq libraries upon BRD4 PROTAC (TargetMol Chemicals, Cat. # T17297, ZxH-3-26) in H9 hESCs, and dTAGV-1 treatment in H9 hESC-BRD4-dTAG lines were generated using NEBNext ® Poly(A) mRNA Magnetic Isolation Module (NEB, Cat.# E7490L) followed by NEBNext ® Ultra TM II RNA (NEB, Cat.# E7770L).

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: RNA-seq libraries upon BRD4 PROTAC (TargetMol Chemicals, Cat. # T17297, ZxH-3-26) in H9 hESCs, and dTAGV-1 treatment in H9 hESC-BRD4-dTAG lines were generated using NEBNext ® Poly(A) mRNA Magnetic Isolation Module (NEB, Cat.# E7490L) followed by NEBNext ® Ultra TM II RNA (NEB, Cat.# E7770L).

Techniques: Single Cell, Sequencing, Control

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: Nucleic Acids Research

Article Title: E2F1 K117 methylation by SETD6 disrupts BRD4–E2F1 binding and modulates E2F1 chromatin binding and gene regulation in prostate cancer cells

doi: 10.1093/nar/gkaf1513

Figure Lengend Snippet: E2F1-BRD4 binding is lost with K117 monomethylated E2F1 in vitro . ( A ) Crystal structure of the human BRD4 BD1 (sandy-brown) in complex with an acetylated K117ac/K120ac E2F1 peptide (blue) (PDB 6ULS) showing the key interaction of K117ac with BD1. ( B ) Coomassie BB stained 12% SDS gel of the purified GST tagged truncated BRD4 (2-477 aa) protein including BD1 and BD2 (BD1/2) as well as the purified BD1 domain (2-220 aa). The GST-tagged BRD4 proteins are marked with asterisks. ( C ) Binding of the GST-BRD4 BD1/2 to modified E2F1 peptides. 15 aa long E2F1 peptides with different combinations of unmodified, acetylated, and methylated K117 and K120 were synthesized on peptide SPOT arrays. The sequence of each peptide is listed in the table. Peptide arrays were incubated with 5 nM GST-BRD4 BD1/2 and binding was detected using a GST-specific antibody. The bar diagram shows the binding of E2F1-BRD4 to K117ac/K120ac and K117me/K120ac observed in three independent experiments. The bars represent the averages. The P- value was determined by two flanked t ‐test with equal variance. ( D ) Same as in panel (C), but GST-BRD4 BD1 was used. Additional data are provdied in .

Article Snippet: PLA Duolink assays were performed according to the manufacturer’s instructions (Sigma) using antibodies against BRD4 (Bethyl, A700-004), E2F1 (SantaCruz, SC-251) and Flag (Sigma, F1804) overnight at 4°C.

Techniques: Binding Assay, In Vitro, Staining, SDS-Gel, Purification, Modification, Methylation, Synthesized, Sequencing, Incubation

Journal: Nucleic Acids Research

Article Title: E2F1 K117 methylation by SETD6 disrupts BRD4–E2F1 binding and modulates E2F1 chromatin binding and gene regulation in prostate cancer cells

doi: 10.1093/nar/gkaf1513

Figure Lengend Snippet: E2F1-BRD4 binding is lost with K117 monomethylated E2F1 in cells. ( A ) GFP-tagged BRD4 (2-477) and Flag-E2F1 (2-437) were transfected into DU145 SETD6 WT and KO cells. The GFP-tagged BRD4 was purified by GFP-trap and analyzed by western-blot with an anti-GFP antibody. Co-purification of Flag-E2F1 was determined by anti-Flag antibody. Equal loading of cell lysate isolated from transfected DU145 SETD6 WT or KO was verified by western-blot analysis against β-actin, GFP, and Flag. ( B ) GFP-tagged BRD4 (2-477) and Flag-E2F1 (2-437) WT or K117R were transfected into in DU145 SETD6 KO cells. Some of the transfected cells were treated with JQ1-Bromodomain-Kac binding inhibitor (5 µM) or DMSO as control. GPF-trap and western-blot analysis was conducted as in panel A. (C–E) Interaction of BRD4 and E2F1 investigated by PLA. All experiments were conducted in DU145 cells. Exemplary microscopy images are shown. Scale bar: 10 µm. PLA signal quantification (PLA dots per nucleus, AU) for each sample is shown on the right. Statistical analysis was performed using Student’s t -test in GraphPad (**** P < .0001). ( C ) Interaction of endogenous BRD4 and Flag-E2F1 in the absence and the presence of the SAHA deacetylase inhibitor (20 µM) for 5 h (Flag-E2F1). Negative control (Neg) refers to reaction conducted without addition of Flag primary antibody. The interaction of BRD4 and E2F1 was detected and it was shown to be stimulated by increasing acetylation levels after SAHA treatment. Number of analyzed cells: 183, 132, 223. ( D ) Detection of the interaction of endogenous BRD4 and endogenous E2F1 in the presence of 40 µM SAHA for 5 h in DU145 cells (Control) and SETD6 KO cells (KO1 and KO2). Negative control (Negative) refers to reaction conducted without addition of E2F1 primary antibody. Number of analyzed cells: 67, 236, 104, 91. ( E ) Interaction of endogenous BRD4 and endogenous E2F1 in the presence of 40 µM SAHA for 5 h in DU145 cells and with overexpression of GFP (GFP empty) or GFP-SETD6 (GFP-SETD6). Negative control (Negative) refers to reaction conducted without addition of E2F1 primary antibody. Number of analyzed GFP positive cells: 19, 18, 28.

Article Snippet: PLA Duolink assays were performed according to the manufacturer’s instructions (Sigma) using antibodies against BRD4 (Bethyl, A700-004), E2F1 (SantaCruz, SC-251) and Flag (Sigma, F1804) overnight at 4°C.

Techniques: Binding Assay, Transfection, Purification, Western Blot, Copurification, Isolation, Control, Microscopy, Histone Deacetylase Assay, Negative Control, Over Expression

Journal: Nucleic Acids Research

Article Title: E2F1 K117 methylation by SETD6 disrupts BRD4–E2F1 binding and modulates E2F1 chromatin binding and gene regulation in prostate cancer cells

doi: 10.1093/nar/gkaf1513

Figure Lengend Snippet: E2F1-BRD4 co-occurence is observed in SETD6 KO but not SETD6 WT cells. ( A ) Heatmap of RPKM-normalized E2F1 ChIP-seq signals at E2F1 peaks (±0.8 kb) showing differential chromatin binding of E2F1 in SETD6 WT and KO cells stably expressing Flag-E2F1. The third heatmap shows BRD4 Chip-seq signals in a prostate cancer cell line (SRR1170714) using the same clustering. See also . ( B ) Example browser views showing ChIP-seq of BRD4 (SRR1170714, green) and E2F1 in SETD6 WT and KO cells. See also for additional examples. ( C ) Correlation analysis of E2F1 binding in SETD6 WT and KO cells with the literature BRD4 chromatin binding profile used in panel (A). E2F1 and BRD4 signals were determined in the E2F1 peak regions shown in panel (A) and their correlation was determined. ( D ) Bar-graph showing the slope of the correlation line of BRD4 and E2F1 binding signals in SETD6 WT or KO cells determined using three BRD4 ChIP-seq data sets (datasets SRR1170714, SRR5467129, and SRR5467130). The corresponding analyses are shown in panel (C) and . P -value determined by two-flanked t -test assuming equal variance.

Article Snippet: PLA Duolink assays were performed according to the manufacturer’s instructions (Sigma) using antibodies against BRD4 (Bethyl, A700-004), E2F1 (SantaCruz, SC-251) and Flag (Sigma, F1804) overnight at 4°C.

Techniques: ChIP-sequencing, Binding Assay, Stable Transfection, Expressing

Journal: Nucleic Acids Research

Article Title: E2F1 K117 methylation by SETD6 disrupts BRD4–E2F1 binding and modulates E2F1 chromatin binding and gene regulation in prostate cancer cells

doi: 10.1093/nar/gkaf1513

Figure Lengend Snippet: Promoter and enhancer binding of BRD4 at genes preferentially bound by E2F1 and upregulated in SETD6 WT or KO context. ( A ) Average of aggregated BRD4 signals at promoter and enhancer elements of genes preferentially bound by E2F1 and upregulated in SETD6 WT or KO context. Note stronger binding in SETD6 KO cells. P -value determined by two-flanked t -test assuming equal variance. ( B ) Representative genome browser views showing co-occupancy of BRD4 and E2F1 at five genomic regions in SETD6 KO cells: C6orf226, TBCC, RPL21, RPL38, and MYC. ChIP-seq tracks were visualized using IGV (version 2.13.1) displaying BRD4 (SRR1170714, green), E2F1 in SETD6 WT (blue), and E2F1 in SETD6 KO (red) DU145 cells. ( C ) ChIP from SETD6 WT and KO DU145 cells performed using a BRD4-specific antibody to enrich BRD4-bound chromatin fragments. IgG was used as a negative control to assess the specificity of the immunoprecipitation. BRD4 occupancy was evaluated by qPCR at the same loci as shown in panel (B). Two independent biological replicates with three technical repeats were performed. Statistical significance was determined using a two-tailed t -test assuming equal variance. The negative controls RPL21 and RPL28 did not yield a detectable signal. Note the elevated BRD4 binding in SETD6 KO context. ( D ) RT-qPCR analysis of the relative expression of the five target genes shown in panel (B) in untreated SETD6 WT and KO DU145 cells (control) as well as after addition of DMSO and JQ1. Note the strong effect of JQ1 on gene expression in SETD6 KO cells.

Article Snippet: PLA Duolink assays were performed according to the manufacturer’s instructions (Sigma) using antibodies against BRD4 (Bethyl, A700-004), E2F1 (SantaCruz, SC-251) and Flag (Sigma, F1804) overnight at 4°C.

Techniques: Binding Assay, ChIP-sequencing, Negative Control, Immunoprecipitation, Two Tailed Test, Quantitative RT-PCR, Expressing, Control, Gene Expression

Journal: Nucleic Acids Research

Article Title: E2F1 K117 methylation by SETD6 disrupts BRD4–E2F1 binding and modulates E2F1 chromatin binding and gene regulation in prostate cancer cells

doi: 10.1093/nar/gkaf1513

Figure Lengend Snippet: Summary of the results of this study. SETD6 monomethylates E2F1 at K117. This methylation disrupts the E2F1–BRD4 interaction leading to different target loci being bound by both factors. In the absence of K117 methylation, E2F1 is acetylated at K117 and K120 leading to BRD4 binding and a concerted engagement of both protein at genomic target sites. As a consequence, methylated and unmethylated E2F1 regulates distinct gene sets in prostate cancer cells.

Article Snippet: PLA Duolink assays were performed according to the manufacturer’s instructions (Sigma) using antibodies against BRD4 (Bethyl, A700-004), E2F1 (SantaCruz, SC-251) and Flag (Sigma, F1804) overnight at 4°C.

Techniques: Methylation, Binding Assay

Journal: bioRxiv

Article Title: PROTAC-Driven Protective Therapy increases the therapeutic window of anticancer drugs

doi: 10.64898/2026.01.12.698947

Figure Lengend Snippet: A) Structure of the MDM2-recruiting BRD4 degrader A1874 formed by JQ1 (a BRD4 inhibitor) and Idasanutlin (an MDM2 antagonist). B) BRD4, MDM2, p53 and p21 protein levels in isogenic TP53 WT and KO HCT116 cells treated with 1 µM A1874 for 24 h, assessed by western blot. Vinculin (VLC) was used as loading control. C) MDM2 mRNA expression levels in TP53 WT and TP53 KO HCT116 cells treated with 1 µM A1874 for 24 h assessed by qPCR. Error bars indicate mean ± s.d. (n = 3). D) Representative immunofluorescence images of MDM2 levels in TP53 WT and KO HCT116 cells upon treatment with vehicle or 1 µM A1874 for 24 h. Quantification of MDM2 nuclear intensity from 1600 cells is shown on the right. Statistical significance was determined with unpaired t-tests. **** = p < 0.0001.

Article Snippet: Cells were treated with the following compounds at the indicated doses: BRD4 PROTAC A1874 (HY-114305, MedChemExpress) at 1 μM; PARP1 PROTACs SK-575 (HY-139156, MedChemExpress) at 100 nM and 180055 (HY-170620, MedChemExpress) at 1 μM; and PARP inhibitors Talazoparib (HY-16106, MedChemExpress) and Olaparib (HY-10162, MedChemExpress) at concentrations ranging from 48 μM to 0.02 μM in 3-fold dilutions.

Techniques: Western Blot, Control, Expressing, Immunofluorescence

Journal: bioRxiv

Article Title: PROTAC-Driven Protective Therapy increases the therapeutic window of anticancer drugs

doi: 10.64898/2026.01.12.698947

Figure Lengend Snippet: A) BRD4, MDM2, p53, and p21 protein levels in a panel of cancer cell lines treated with vehicle or 1 µM A1874 for 24 h, assessed by western blot. Vinculin was used as loading control. Left panel displays colon cancer lines, including TP53 WT (HCT116 and RKO) and mutant (DLD1 and HT29) cells. Middle panel displays ovarian cancer cell lines, including TP53 WT (A2780) and mutant (ES2, OVCAR8 and SKOV3) cells. Right panel displays BJ and RPE1 p53-proficient primary cell lines. B) MDM2 mRNA expression levels in the indicated cell lines treated with vehicle or 1 µM A1874 for 24 h, assessed by qPCR. Error bars indicate mean ± s.d. (n = 3). C) Schematic representation of the CRISPRa/dCas9-SAM system. It consists of a dead Cas9 (dCas9) fused to the transcriptional activator VP64. The gRNA targeting MDM2 promoter forms a complex with MS2 (blue) and recruits transcriptional p65 (orange) and HSF1 (red) activators. This induces MDM2 expression from the endogenous locus. D) MDM2 mRNA expression levels in SKOV3 transduced with E.V. or gRNAs targeting MDM2 promoter, assessed by qPCR. Error bars indicate mean ± s.d. (n = 3). E) BRD4, and MDM2 protein levels in empty vector (E.V.)-transduced or MDM2-overexpressing SKOV3 cell lines treated with 1 µM A1874 for 24 h, assessed by western blot. Vinculin was used as loading control. F) MDM2 mRNA expression levels in the indicated cells treated with vehicle or 1 µM A1874 for 24 h, assessed by qPCR. Error bars indicate mean ± s.d. (n = 3).

Article Snippet: Cells were treated with the following compounds at the indicated doses: BRD4 PROTAC A1874 (HY-114305, MedChemExpress) at 1 μM; PARP1 PROTACs SK-575 (HY-139156, MedChemExpress) at 100 nM and 180055 (HY-170620, MedChemExpress) at 1 μM; and PARP inhibitors Talazoparib (HY-16106, MedChemExpress) and Olaparib (HY-10162, MedChemExpress) at concentrations ranging from 48 μM to 0.02 μM in 3-fold dilutions.

Techniques: Western Blot, Control, Mutagenesis, Expressing, Transduction, Plasmid Preparation