human brd4 bd1 domain  (Addgene inc)

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: Cell Reports Medicine

Article Title: A triple-action PROTAC for wild-type p53 cancer therapy

doi: 10.1016/j.xcrm.2025.102467

Figure Lengend Snippet: Design and in vitro activity of TAPTAC1 (A) A chimera composed of a stapled p53 peptide and a small molecule BET protein inhibitor was designed to achieve triple-action targeting of HDM2, HDMX, and BET proteins to maximally restore p53 while hijacking HDM2 to degrade an oncogenic driver (BET proteins) instead of a tumor suppressor (p53). (B) Chemical composition of TAPTAC1, which links the BET inhibitor JQ1 to the stapled p53 peptide via a lysine-βAla moiety installed at position 25 of the p53 transactivation helix. (C) MD simulations demonstrate the assembly of a ternary complex between TAPTAC1 and the respective JQ1- and p53-binding domains of BRD4 and HDM2. (D) TAPTAC1 effectively generated ternary complexes (green) between the BD1 domain of BRD4 and HDM2 (left) and HDMX (right). Control elution profiles are shown for the individual proteins alone, including BRD4 BD1 (cyan), HDM2 (red), and HDMX (orange), and their combinations, including BRD4 BD1 and HDM2 (purple) and BRD4 BD1 and HDMX (brown). Each SEC experiment was repeated twice using independent preparations of proteins with similar results. (E) An in vitro ubiquitylation assay demonstrated the natural selectivity of HDM2 for p53, as evidenced by time-dependent laddering of p53 but not BRD4 BD1-BD2 (left 4 lanes). In the presence of TAPTAC1, the primary target of HDM2 is switched from p53 to BRD4 BD1-BD2 , which exhibits newfound laddering at the expense of p53 (right 4 lanes). Ubiquitylation assays were repeated three times with independent preparations of proteins and reagents with similar results. See also and .

Article Snippet: The human BRD4 BD1 domain (residues 48–168) in the pNIC28Bsa4 vector (Addgene) was expressed in BL21(DE3) E. coli in LB medium containing 50 mg/mL kanamycin.

Techniques: In Vitro, Activity Assay, Binding Assay, Generated, Control, Ubiquitin Assay

Journal: Cell Reports Medicine

Article Title: A triple-action PROTAC for wild-type p53 cancer therapy

doi: 10.1016/j.xcrm.2025.102467

Figure Lengend Snippet: Potent and selective cytotoxicity of TAPTAC1 correlates with degradation of BET proteins and reactivation of the p53 pathway (A) To assess the relative potency and selectivity of TAPTAC1, we compared its anti-cancer activity in culture with A1874, a 2-in-1 PROTAC comprising the selective HDM2 inhibitor RG7388 and JQ1, and the TAPTAC1 F19A point mutant, which abrogates interaction of the stapled p53 peptide component of the chimera with HDM2 and HDMX. (B–D) In SJSA-1 cells that have genetic amplification of HDM2 but little to no HDMX expression, TAPTAC1 exhibits marginally increased potency compared to A1874, as assessed by viability assay (B). In SJSA-X cells, engineered to express HDMX, the dual-targeting capability of TAPTAC1 results in markedly enhanced potency compared to A1874 (C). In Saos-2 cells, which lack p53, no p53/HDM2/HDMX-based activity or selectivity is evident, consistent with the compounds functioning similarly based on residual BET inhibitor activity alone (D). In each cell line, F19A point mutagenesis likewise abrogates the p53-dependent activity of TAPTAC1, further highlighting its specificity of action. Data are mean ± SEM for experiments performed in technical quadruplicate and repeated three times using independent cultures with similar results. (E) Upon treatment of SJSA-X cells with 100 nM TAPTAC1, we observed prompt degradation of BRD4 within 8 h, coinciding with time-dependent upregulation of p53, which peaked at 12 h and triggered both a surge in p21 and counter-elevation of HDM2 and HDMX by 24 h. The reduction of p53 levels observed between 12 and 24 h is consistent with the characteristic negative feedback loop of the p53 pathway. Time-dependent western blot analyses were performed twice using independent SJSA-X cell cultures and compound treatment. NT, no treatment. (F) Quantitative proteomics revealed that TAPTAC1 treatment (1 μM, 24 h) of SJSA-X cells caused a striking reduction of BET protein levels (cyan, e.g., BRD2-4) and marked upregulation of p53 pathway proteins (red, e.g., p53 [TP53], p21 [CDKN1A], HDM2 [MDM2], and HDMX [MDM4]). Quantitative proteomic analysis was performed using three biological replicates representing independent cultures and treatment. ∗, isoform. See also , , and .

Article Snippet: The human BRD4 BD1 domain (residues 48–168) in the pNIC28Bsa4 vector (Addgene) was expressed in BL21(DE3) E. coli in LB medium containing 50 mg/mL kanamycin.

Techniques: Activity Assay, Mutagenesis, Amplification, Expressing, Viability Assay, Western Blot, Quantitative Proteomics

Journal: Cell Reports Medicine

Article Title: A triple-action PROTAC for wild-type p53 cancer therapy

doi: 10.1016/j.xcrm.2025.102467

Figure Lengend Snippet: Pharmacologic profile and therapeutic efficacy of TAPTAC1 in a mouse model of osteosarcoma (A) TAPTAC1 was incubated in mouse or human plasma, and the relative amount of intact compound was measured over time by mass spectrometry, revealing notable compound stability. Compound levels were tracked over time (6 time points each) in an individual sample of mouse or human plasma. (B and C) Mice ( n = 3/arm) received 3 mg/kg of TAPTAC1 by intravenous (IV) or intraperitoneal (IP) injection (B) or 3 or 10 mg/kg TAPTAC1 by IV injection (C), followed by serial blood withdrawal for mass spectrometry quantitation of compound. Calculated parameters for IP (3 mg/kg) and IV (3 mg/kg and 10 mg/kg) administration, respectively, included T 1/2 5.33, 5.01, and 4.71 h; C max 0.94, 2.1, and 31.2 μM; and AUC last 8.3, 10.3, and 61.7 μM∗hr. Plotted pharmacokinetic data are mean ± SEM for experiments performed in three mice per arm. (D) Treatment of NSG mice bearing SJSA-X osteosarcoma tumors (mean tumor volume ±SD of 401 ± 53mm 3 on treatment day 1) with 10 mg/kg TAPTAC1 IV daily (qd) resulted in marked shrinkage of tumors as compared to the unabetted tumor growth observed in vehicle-treated mice. Plotted data are mean tumor volume ±SEM as measured daily ( n = 5/arm). Statistical analysis using a longitudinal mixed-effects model revealed a significant difference in tumor volume trajectories between the treated and vehicle groups ( p < 0.001). (E) Photographs of tumors removed postmortem on treatment day 7 from each of three mice treated with either vehicle or TAPTAC1 (10 mg/kg/day IV) revealed the striking anti-tumor effect of TAPTAC1. (F) Quantitative proteomics (TMTpro 18-plex) of the tumor specimens demonstrated marked downregulation of BRD4 and persistent upregulation of HDMX (MDM4), in TAPTAC1- vs. vehicle-treated mice at day 7, in addition to changes that reflect replacement of tumor with host connective tissue. Quantitative proteomic analysis was performed using three biological replicates ( n = 3 tumor specimens) per treatment arm. (G) The in vivo efficacy experiment was repeated with reduced dosing to 3.0, 1.0, and 0.3 mg/kg/day IV (mean tumor volume ±SD of 216 ± 47 mm 3 on treatment day 1) and demonstrated dose-responsive anti-tumor activity and associated prolongation of survival. Data are mean tumor volume ±SEM as measured daily ( n = 5/arm). Statistical analysis using a longitudinal mixed-effects model revealed a significant difference in tumor volume trajectories between the 3 mg/kg dosing arm and vehicle group (vehicle vs. TAPTAC1 at: 0.3 mg/kg, p = 0.651; 1 mg/kg, p = 0.072; 3 mg/kg, p < 0.001). (H–J) Lowering the TAPTAC1 dosing interval to 3 mg/kg twice weekly (biw) also suppressed SJSA-X tumor growth relative to vehicle throughout the month-long treatment period (longitudinal mixed-effects model, vehicle vs. TAPTAC1 at 3 mg/kg BIW, p < 0.0001) (H), with no significant difference between the groups in animal weight trajectory ( p = 0.07) and marked prolongation of survival for TAPTAC1-treated mice (log rank test, p = 0.00033). The study was conducted with n = 6 vehicle-treated mice and n = 7 TAPTAC1-treated mice. See also , , and .

Article Snippet: The human BRD4 BD1 domain (residues 48–168) in the pNIC28Bsa4 vector (Addgene) was expressed in BL21(DE3) E. coli in LB medium containing 50 mg/mL kanamycin.

Techniques: Drug discovery, Incubation, Clinical Proteomics, Mass Spectrometry, Injection, IV Injection, Quantitation Assay, Quantitative Proteomics, In Vivo, Activity Assay

Journal: bioRxiv

Article Title: Phenotypic screening converges on CDK9 inhibition as a therapeutic strategy in translocation renal cell carcinoma

doi: 10.1101/2025.08.25.672235

Figure Lengend Snippet: (A) Outline of the chromatin displacement assay workflow. (B) Application of chromatin displacement assay to BRD4, using the tool compound JQ1. HEK293T cells were treated with JQ1 dose titration (3 nM to 10 μM). Cells were subject to in situ cell extraction (or not, bottom vs. top rows) and two color IF staining for BRD4 (green) and nucleus (red) was performed. Dose-dependent displacement of BRD4 from chromatin by JQ1 was quantified and plotted for each of 6 individual runs. Data are presented as mean ± SD. (C) Visualization of TFE3 staining and localization in HEK293T cells, with or without Torin1 treatment (which induces nuclear TFE3 localization), and with or without extraction. TFE3 cytoplasmic or nuclear intensity is quantified based on the IF images (mean ± SD, n=6). Note that cytoplasmic signal (white arrow) is dramatically reduced by extraction while nuclear (i.e. chromatin-bound) signal (blue arrow) is not. P -values computed by unpaired t-test; ****P<0.0001. (D) Visualization of ASPL-TFE3 fusion staining and localization in FUUR1 tRCC cells. Cytoplasmic and nuclear staining of the TFE3 fusion with or without extraction was quantified based on the IF images (mean ± SD, n=6) similar to panel (C). Note that TFE3 fusions are constitutively nuclear, as compared with WT TFE3, which shuttles between the cytoplasm and nucleus. P -values computed by unpaired t-test; *P<0.05, ****P<0.0001. (E) Composition of epigenetic modulator (“Epi-Mod”) compound library (n = 121 compounds) used for pilot screening in TFE3 chromatin displacement assay. (F) Replicate-Replicate scatterplot of Epi-Mod library screen using chromatin displacement assay in FUUR1 cells. Select inhibitor classes are highlighted: HDACi, green; HATi, red; HKMTi, blue. (G) Top, IF images showing displacement of ASPL-TFE3 fusion by panobinostat (10 μM) (with extraction condition). Staining: TFE3, green; Nucleus: Red. Quantification of dose-dependent displacement of TFE3, BRD4 and nuclear staining by panobinostat. Data is presented as mean ± SD, n=2.

Article Snippet: For immunofluorescence staining, the fixed cells were blocked (PBS + 1% BSA) for 30 minutes at room temperature and stained with primary antibodies for 1 hour at 37°C at the following dilutions: rabbit anti-human TFE3 antibody (Millipore Sigma #ZRB1272) at 1:1000 and mouse anti-human BRD4 antibody (Santa Cruz Biotechnology #sc518021) at 1:200.

Techniques: Titration, In Situ, Extraction, Staining, Drug discovery

Journal: bioRxiv

Article Title: Phenotypic screening converges on CDK9 inhibition as a therapeutic strategy in translocation renal cell carcinoma

doi: 10.1101/2025.08.25.672235

Figure Lengend Snippet: (A) Screening funnel of 25,000 compounds leading to 4 hits after dose response validation studies. (B) Replicate-Replicate scatterplot for run 4 (out of 5 total runs) of chromatin displacement screening of 25,000 compounds in in FUUR1 cells. Chromatin displacers, red; chromatin retention, green (see methods for hit selection from the 5 separate runs). Primary hits from this run are labeled in red or green; validated hits are named. (C) Dose-dependent increase in nuclear signal of TFE3 with BRD6866 treatment (chromatin retention) in a chromatin displacement assay (CDA) in FUUR1 cells. Nuclear signal (DAPI) as well as nuclear IF signal for TFE3 and BRD4 were quantified and plotted (mean ± SD, n=3). (D) Dose-dependent decrease in nuclear signal of TFE3 with BRD7659 (chromatin displacement) in a CDA in FUUR1 cells. Nuclear signal (DAPI) as well as nuclear IF signal for TFE3 and BRD4 were quantified and plotted (mean ± SD, n=3). (E) Chemical structure of BRD6866 and BRD7659. (F) RNA Seq of UOK109 cells after 16h treatment with BRD6866 at 1μM. Differential transcriptomics analysis using a volcano plot is shown. Antiapoptotic genes with known sensitivity to CDK9 inhibition, MCL1 and XIAP are labeled . RNA Seq schematic created using BioRender.com. (G) Hallmark gene set pathway enrichment analysis upon BRD6866 treatment was compared to two recent studies with CDK9 inhibition (CDK9i) and CDK9 degradation (CDK9d) ( , ). (H) CDK activity profiling assay (Reaction Biology) showing extent of inhibition of various CDK/cyclin pairs by BRD6866 (10 μM). Loss of activity for each CDK and cyclin pairs by BRD6866 was compared to DMSO control. CDK9/cyclin pairs are bolded. The cyclin interacting motif PFTAIRE defines a subgroup of CDKs that does not fall under named categories.

Article Snippet: For immunofluorescence staining, the fixed cells were blocked (PBS + 1% BSA) for 30 minutes at room temperature and stained with primary antibodies for 1 hour at 37°C at the following dilutions: rabbit anti-human TFE3 antibody (Millipore Sigma #ZRB1272) at 1:1000 and mouse anti-human BRD4 antibody (Santa Cruz Biotechnology #sc518021) at 1:200.

Techniques: Biomarker Discovery, Selection, Labeling, RNA Sequencing, Inhibition, Activity Assay, Control