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