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: Chromatin modifiers KMT2D, BAF, and p300 are required for de novo binding of transcription factors on enhancers

doi: 10.64898/2026.01.29.702555

Figure Lengend Snippet: (A) Schematic of the 4-OHT-inducible MyoD-ER system. (B) MyoD expression in preadipocytes and C2C12 myoblasts was determined using RNA-Seq (n = 1). RPKM values indicate gene expression levels. (C) Western blot (WB) analysis of nuclear extracts from preadipocytes expressing MyoD-ER-T7 and treated with 4-OHT. Antibodies used were indicated on the right. BRG1 was used as a loading control. (D-I) MyoD-ER-T7 expressing preadipocytes were treated with 4-OHT for 1 hour (h), followed by CUT&RUN analysis. (D) Bar chart showing ARID1A (an exclusive subunit of BAF), KMT2D, and p300 binding status on induced MyoD sites. (E) Box plots displaying the normalized MyoD read counts in subgroups defined in (D). (F) Bar chart showing ARID1A (BAF), KMT2D, and p300 binding on 38,732 MyoD + enhancers defined in (D) prior to 4-OHT treatment. (G-H) Box plots showing the normalized MyoD read counts (G) and HOMER de novo motif analysis (H) on BAF-KMT2D-p300 prebound or de novo sites defined in (F). Statistical significance was determined using a two-sided, unpaired Mann Whitney test. (I) Genome browser view of MyoD binding sites around Maged1 and Cap2 loci.

Article Snippet: PROTAC p300/CBP degrader dCBP-1 (#HY-134582) from MCE was used at 250nM. p300/CBP inhibitor A-485 (#6887) was from Tocris Bioscience and used at 3μM. (Z)-4-Hydroxytamoxifen (4-OHT) (#H7904) and Dexamethasone (DEX) (#D4902) were from Millipore-Sigma and used at 400nM and 100nM, respectively.

Techniques: Expressing, RNA Sequencing, Gene Expression, Western Blot, Control, Binding Assay, MANN-WHITNEY

Journal: bioRxiv

Article Title: Chromatin modifiers KMT2D, BAF, and p300 are required for de novo binding of transcription factors on enhancers

doi: 10.64898/2026.01.29.702555

Figure Lengend Snippet: (A) Schematic for generating the knockin allele encoding AID-tagged KMT2D. (B-F) Kmt2d AID/AID ; MyoD-ER-T7 preadipocytes were pretreated with 5Ph-IAA (ΔKMT2D) for 2h, and then 4-OHT was added for 1h to induce MyoD nuclear translocation. Cells were harvested for WB and CUT&RUN analysis. (B) WB of nuclear extracts for KMT2D, ARID1A (BAF), p300, and UTX. Antibodies used were indicated on the right. RbBP5 was the loading control. (C) Pie chart illustrating KMT2D binding status on 38,732 MyoD + enhancers. (D) Heat maps for CUT&RUN of KMT2D, T7 (MyoD), ARID1A (BAF), and p300 on KMT2D prebound or de novo KMT2D binding sites with >2-fold depletion of KMT2D as defined in (C). (E-F) Heat maps ( left panel) for CUT&RUN data on 12,068 MyoD + enhancers with de novo KMT2D binding, further categorized based on BAF binding (E) or p300 binding (F) before and after 4-OHT treatment. All heat maps spanned ± 3kb around MyoD binding sites, and sites were ranked by the intensity of MyoD (T7) in the 4OHT-treated control. Box plots ( right panel ) showing fold changes of BAF binding intensity (E) or p300 binding intensity (F) between KMT2D-depleted (ΔKMT2D) and control samples. Statistical significance was determined using a one-sided Wilcoxon signed-rank test.

Article Snippet: PROTAC p300/CBP degrader dCBP-1 (#HY-134582) from MCE was used at 250nM. p300/CBP inhibitor A-485 (#6887) was from Tocris Bioscience and used at 3μM. (Z)-4-Hydroxytamoxifen (4-OHT) (#H7904) and Dexamethasone (DEX) (#D4902) were from Millipore-Sigma and used at 400nM and 100nM, respectively.

Techniques: Knock-In, Translocation Assay, Control, Binding Assay

Journal: bioRxiv

Article Title: Chromatin modifiers KMT2D, BAF, and p300 are required for de novo binding of transcription factors on enhancers

doi: 10.64898/2026.01.29.702555

Figure Lengend Snippet: Kmt2d AID/AID ; MyoD-ER-T7 preadipocytes were pretreated with BRG1 inhibitor BRM014 (BRG1i) for 1h, and then 4-OHT was added for 1h to induce MyoD nuclear translocation. Cells were harvested for WB, CUT&RUN, and ATAC-seq. (A) WB of nuclear extracts for KMT2D, p300, and BAF subunits BRG1 and ARID1A. (B) Pie chart illustrating BAF binding status on 38,732 MyoD + enhancers. (C) Heat maps for CUT&RUN of ARID1A (BAF), T7 (MyoD), KMT2D, and p300 on BAF pre-bound and de novo BAF binding sites. (D-E) Chromatin accessibility determined by ATAC-seq signals on MyoD + enhancers. Chromatin accessibility status on BAF prebound sites (D) or de novo BAF binding sites (E) is shown in pie charts ( upper panels ). Average profiles of normalized ATAC-seq reads on constitutively open and MyoD-dependent opening sites are shown in lower panels .

Article Snippet: PROTAC p300/CBP degrader dCBP-1 (#HY-134582) from MCE was used at 250nM. p300/CBP inhibitor A-485 (#6887) was from Tocris Bioscience and used at 3μM. (Z)-4-Hydroxytamoxifen (4-OHT) (#H7904) and Dexamethasone (DEX) (#D4902) were from Millipore-Sigma and used at 400nM and 100nM, respectively.

Techniques: Translocation Assay, Binding Assay

Journal: bioRxiv

Article Title: Chromatin modifiers KMT2D, BAF, and p300 are required for de novo binding of transcription factors on enhancers

doi: 10.64898/2026.01.29.702555

Figure Lengend Snippet: (A-D) Kmt2d AID/AID ; MyoD-ER-T7 preadipocytes were pretreated with p300/CBP inhibitor A-485 (p300i) for 1h, and then 4-OHT was added for 1h to induce MyoD nuclear translocation. Cells were collected for WB, CUT&RUN, and ATAC-seq. (A) WB of nuclear extracts for p300, KMT2D, ARID1A (BAF) and histone extracts for H3K27ac. RbBP5 and H3 serve as loading controls. (B) Pie chart illustrating p300 binding status on 38,732 MyoD + enhancers. (C) Heat maps for CUT&RUN of p300, T7 (MyoD), ARID1A (BAF), and KMT2D on p300 prebound and de novo p300 binding sites. Heat maps spanned ± 3kb around MyoD binding sites, and sites were ranked by the intensity of T7 (MyoD) in the 4OHT-treated control. (D) Average profiles of normalized ATAC-seq reads on 9,031 de novo p300 binding sites with or without p300i treatment. (E-F) Kmt2d AID/AID ; MyoD-ER-T7 preadipocytes were pretreated with p300/CBP degrader dCBP-1 (p300-deg) for 3h or p300i for 1h. Then, 4-OHT was added for 1h to induce MyoD nuclear translocation. (E) WB of nuclear extracts for p300 or histone extracts for H3K27ac. (F) Violin plot illustrating changes in binding of T7 (MyoD), p300, ARID1A (BAF), and KMT2D upon p300i or p300-deg treatment. The analysis was performed on MyoD + enhancers with >2-fold reduced p300 binding upon p300-deg. Statistical significance was determined using a one-sided Wilcoxon signed-rank test. ****p < 0.0001.

Article Snippet: PROTAC p300/CBP degrader dCBP-1 (#HY-134582) from MCE was used at 250nM. p300/CBP inhibitor A-485 (#6887) was from Tocris Bioscience and used at 3μM. (Z)-4-Hydroxytamoxifen (4-OHT) (#H7904) and Dexamethasone (DEX) (#D4902) were from Millipore-Sigma and used at 400nM and 100nM, respectively.

Techniques: Translocation Assay, Binding Assay, Control

Journal: bioRxiv

Article Title: Chromatin modifiers KMT2D, BAF, and p300 are required for de novo binding of transcription factors on enhancers

doi: 10.64898/2026.01.29.702555

Figure Lengend Snippet: (A) C2C12 myoblasts were subjected to 2h or 24h of myogenesis and 2h of p300 inhibition, with A-485 (p300i) applied for 2h prior to experiments. (B) WB of histone extracts for H3K27ac. H3 serves as the loading control. (C-D) Homer motif analysis (C) and heat maps for ChIP-seq of MyoD and CUT&RUN of p300 (D) on 718 de novo MyoD + p300 + sites after 2h of differentiation. (E-F) Homer motif analysis (E) and heat maps for ChIP-seq of MyoD and CUT&RUN of p300 (F) on 3,075 de novo MyoD + p300 + sites after 24h of differentiation.

Article Snippet: PROTAC p300/CBP degrader dCBP-1 (#HY-134582) from MCE was used at 250nM. p300/CBP inhibitor A-485 (#6887) was from Tocris Bioscience and used at 3μM. (Z)-4-Hydroxytamoxifen (4-OHT) (#H7904) and Dexamethasone (DEX) (#D4902) were from Millipore-Sigma and used at 400nM and 100nM, respectively.

Techniques: Inhibition, Control, ChIP-sequencing

Journal: bioRxiv

Article Title: Chromatin modifiers KMT2D, BAF, and p300 are required for de novo binding of transcription factors on enhancers

doi: 10.64898/2026.01.29.702555

Figure Lengend Snippet: (A) Pie chart illustrating KMT2D protein depletion on 4,578 MyoD + enhancers with de novo binding of KMT2D, BAF, and p300 defined . (B) Bar graphs illustrating effects of ΔKMT2D, BRG1i, and p300i on MyoD + enhancers with de novo binding of KMT2D, BAF, and p300. (C) A model depicting the interdependent relationship between myogenic TF MyoD and chromatin modifiers KMT2D, BAF, p300 on enhancers. (D) Box plots showing the normalized MyoD read counts on 3620 MyoD-bound enhancers, grouped by the presence of MyoD binding decrease upon chromatin modifier interventions (MyoD binding dependency). (E) HOMER de novo motif analysis on BAF-KMT2D-p300 dependent and independent sites defined in (A). (F-G) Heat maps (F) and corresponding box plots (G) illustrating changes in binding of T7 (MyoD), KMT2D, ARID1A (BAF), and p300 on 509 BAF-KMT2D-p300 independent MyoD + enhancers defined in Heat maps spanned ± 3kb around MyoD binding sites, and sites were ranked by the intensity of T7 (MyoD) in the 4OHT control. Statistical significance was determined using a one-sided Wilcoxon signed-rank test. n.s. not significant; **p < 0.01; ****p < 0.0001.

Article Snippet: PROTAC p300/CBP degrader dCBP-1 (#HY-134582) from MCE was used at 250nM. p300/CBP inhibitor A-485 (#6887) was from Tocris Bioscience and used at 3μM. (Z)-4-Hydroxytamoxifen (4-OHT) (#H7904) and Dexamethasone (DEX) (#D4902) were from Millipore-Sigma and used at 400nM and 100nM, respectively.

Techniques: Binding Assay, Control

Journal: bioRxiv

Article Title: Chromatin modifiers KMT2D, BAF, and p300 are required for de novo binding of transcription factors on enhancers

doi: 10.64898/2026.01.29.702555

Figure Lengend Snippet: (A-C) Heat maps for CUT&RUN data on MyoD + enhancers with de novo KMT2D binding and with intact MyoD signals upon ΔKMT2D (A) , on MyoD + enhancers with de novo BAF binding and with intact MyoD signals upon BRG1i (B) , and on MyoD + enhancers with de novo p300 binding and with intact MyoD signals upon p300i. All heat maps spanned ± 3kb around MyoD binding sites, and sites were ranked by the intensity of T7 (MyoD) in the 4OHT-treated control.

Article Snippet: PROTAC p300/CBP degrader dCBP-1 (#HY-134582) from MCE was used at 250nM. p300/CBP inhibitor A-485 (#6887) was from Tocris Bioscience and used at 3μM. (Z)-4-Hydroxytamoxifen (4-OHT) (#H7904) and Dexamethasone (DEX) (#D4902) were from Millipore-Sigma and used at 400nM and 100nM, respectively.

Techniques: Binding Assay, Control

Journal: bioRxiv

Article Title: Chromatin modifiers KMT2D, BAF, and p300 are required for de novo binding of transcription factors on enhancers

doi: 10.64898/2026.01.29.702555

Figure Lengend Snippet: Kmt2d AID/AID preadipocytes were pretreated with 5Ph-IAA (ΔKMT2D) for 2h, BRG1i or p300i for 1h, and then 100nM DEX was added for 1h to induce GR nuclear translocation. Cells were collected for WB and ChIP-seq. (A) WB of nuclear extracts for KMT2D, ARID1A, and GR and histone extracts for H3K27ac. RbBP5 and H3 serve as loading controls. (B) Bar chart showing ARID1A (BAF), KMT2D, and p300 binding on 4,097 GR + enhancers with >2-fold KMT2D depletion, prior to DEX treatment. (C-D) Box plots showing the normalized GR read counts (C) and HOMER de novo motif analysis (D) on GR + enhancers with BAF-KMT2D-p300 prebound or de novo sites defined in (B). (E) Heat maps for ChIP-seq of GR, KMT2D, ARID1A (BAF), and p300 on GR + enhancers with de novo binding of BAF, KMT2D, and p300. Heat maps spanned ± 3kb around GR binding sites, and sites were ranked by the intensity of GR in the DEX-treated control. (F) Box plots illustrating changes in binding of GR, KMT2D, ARID1A (BAF), and p300 on 109 BAF-KMT2D-p300-independent GR + enhancers. Statistical significance was determined using a one-sided Wilcoxon signed-rank test. n.s. not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Article Snippet: PROTAC p300/CBP degrader dCBP-1 (#HY-134582) from MCE was used at 250nM. p300/CBP inhibitor A-485 (#6887) was from Tocris Bioscience and used at 3μM. (Z)-4-Hydroxytamoxifen (4-OHT) (#H7904) and Dexamethasone (DEX) (#D4902) were from Millipore-Sigma and used at 400nM and 100nM, respectively.

Techniques: Translocation Assay, ChIP-sequencing, Binding Assay, Control

Journal: bioRxiv

Article Title: Chromatin modifiers KMT2D, BAF, and p300 are required for de novo binding of transcription factors on enhancers

doi: 10.64898/2026.01.29.702555

Figure Lengend Snippet: (A) The reported sequential relationships of TF and chromatin modifier enrichment on enhancers established with stable knockout and continuous TF expression models. (B) The interdependent relationships between TF and chromatin modifiers KMT2D, BAF, p300 and among chromatin modifiers on enhancers established with acute interventions of chromatin modifiers and inducible nuclear translocation of TFs.

Article Snippet: PROTAC p300/CBP degrader dCBP-1 (#HY-134582) from MCE was used at 250nM. p300/CBP inhibitor A-485 (#6887) was from Tocris Bioscience and used at 3μM. (Z)-4-Hydroxytamoxifen (4-OHT) (#H7904) and Dexamethasone (DEX) (#D4902) were from Millipore-Sigma and used at 400nM and 100nM, respectively.

Techniques: Knock-Out, Expressing, Translocation Assay

Journal: Science Advances

Article Title: Lactate derived from cancer-associated fibroblasts promotes alternative splicing and castration resistance in prostate cancer

doi: 10.1126/sciadv.ady5324

Figure Lengend Snippet: ( A ) Immunofluorescence staining and fluorescence intensity quantification of A − CAFs and A + CAFs showing the expression of APCDD1 (green) and COL1A1 (red). Scale bars: 50 μm. ( B and C ) Schematic of the Boyden chamber coculture system and colony formation assays used to indirectly coculture C4-2 and LNCaP cells with A + CAFs or A − CAFs with or without ENZ treatment. Quantification of colonies is shown on the right ( n = 3). ( D ) Images of PDOs cocultured with A + CAFs or A − CAFs, with and without ENZ treatment. Quantification of organoid size is shown on the right ( n = 30). Scale bars: 100 μm. ( E ) Schematic of in vivo experiments. C4-2 cells were co-injected with A + CAFs or A − CAFs into castrated NSG mice, followed by ENZ treatment. Tumor growth analysis confirms that A + CAFs significantly enhanced tumor growth under ENZ treatment ( n = 5 for each group). ( F ) CCK-8 assay showing increased cell viability in C4-2 cells treated with A + CTM, but not A − CTM, under ENZ treatment. ( G and H ) Gene ontology enrichment and gene set enrichment analysis of differentially expressed genes in C4-2 cells treated with A + CTM versus A − CTM, highlighting RNA splicing–related pathways ( n = 5 for each group). ( I ) RT-PCR analysis of AR and AR-V7 expression in C4-2 cells treated with A + CTM, showing an increase in AR-V7 levels. ( J ) CCK-8 assay showing the reversal of A + CTM-induced ENZ resistance in C4-2 cells treated with an AR-V7 PROTAC. Data represent the means ± SD. Statistical significance was determined by a two-tailed unpaired t test [(B) to (D)] and two-way ANOVA [(E), (F), and (J)] followed by a multiple comparisons test. ** P < 0.01, and *** P < 0.001. ns, not significant.

Article Snippet: C4-2 and LNCaP cells were plated in 96-well plates at a density of 3000 cells per well and treated with A + CTM, A − CTM, NALA (L7022, Sigma-Aldrich), l -lactate (L1750, Sigma-Aldrich), PROTAC AR-V7 degrader-1 (HY-145479, MedChem Express), or ENZ.

Techniques: Immunofluorescence, Staining, Fluorescence, Expressing, In Vivo, Injection, CCK-8 Assay, Reverse Transcription Polymerase Chain Reaction, Two Tailed Test

Journal: Science Advances

Article Title: Lactate derived from cancer-associated fibroblasts promotes alternative splicing and castration resistance in prostate cancer

doi: 10.1126/sciadv.ady5324

Figure Lengend Snippet: ( A to C ) AR-V7 expression and ENZ resistance after heat inactivation, proteinase K (PK), DNase, or ribonuclease (RNase) treatment with A + CTM in C4-2 cells. ( D ) Heatmap showing untargeted metabolomic profiling of A + CTM and A − CTM ( n = 6 for each group). ( E ) KEGG pathway analysis of differentially abundant metabolites in A + CTM versus A − CTM. ( F ) Principal components analysis (PCA) of targeted metabolomics data from A + CTM and A − CTM ( n = 6 for each group). ( G ) Heatmap of key metabolites detected in A + CTM and A − CTM. ( H ) Gene set enrichment analysis in A + CAFs. ( I ) Volcano plot of metabolomic data comparing A + CAFs and A − CAFs ( n = 6 for each group). ( J ) Quantification of relative lactate levels in the conditioned medium from A + CAFs, A − CAFs, C4-2 cells, and controls. h, hours. ( K ) Colony formation assays of C4-2 cells treated with DMSO or lactate (LAC) with and without ENZ treatment ( n = 3). ( L ) AR and AR-V7 expression in C4-2 cells treated with DMSO or lactate. ( M ) Tumor growth of C4-2 cells injected into castrated NSG mice treated with PBS or lactate with or without ENZ ( n = 5 for each group). ( N ) Images of PDOs treated with lactate or DMSO with or without ENZ. Quantification of organoid size is shown on the right ( n = 30). Scale bars: 100 μm. ( O and P ) Schematic and colony formation assay of medium change experiments in the coculture system of A + CAFs and C4-2 cells with or without ENZ. The medium was changed every 24 or 72 hours to modulate lactate concentration within the medium ( n = 3). Data represent the means ± SD. Statistical significance was determined by a two-tailed unpaired t test and two-way ANOVA followed by a multiple comparisons test. ** P < 0.01, *** P < 0.001. All experiments were independently replicated three times in the laboratory ( n = 3) to ensure biological reproducibility.

Article Snippet: C4-2 and LNCaP cells were plated in 96-well plates at a density of 3000 cells per well and treated with A + CTM, A − CTM, NALA (L7022, Sigma-Aldrich), l -lactate (L1750, Sigma-Aldrich), PROTAC AR-V7 degrader-1 (HY-145479, MedChem Express), or ENZ.

Techniques: Expressing, Metabolomic, Injection, Colony Assay, Concentration Assay, Two Tailed Test

Journal: Science Advances

Article Title: Lactate derived from cancer-associated fibroblasts promotes alternative splicing and castration resistance in prostate cancer

doi: 10.1126/sciadv.ady5324

Figure Lengend Snippet: ( A ) OCR analysis of C4-2 cells treated with 10 or 20 mM lactate and PBS, followed by mitochondrial stress tests using oligomycin, carbonyl cyanide p -trifluoromethoxyphenylhydrazone (FCCP), and rotenone. Quantification of maximum and basal respiration is shown on the right ( n = 5). ( B ) CCK-8 assay showing cell proliferation of C4-2 cells treated with DMSO or rotenone under continuous ENZ treatment. ( C ) RT-PCR and Western blot analysis of AR and AR-V7 expression in C4-2 cells cultured with A + CTM and treated with DMSO or rotenone. ( D ) Western blot analysis showing pan-lactylation (Pan-Lacyl), pan-ubiquitination (Pan-Ubi), pan-phosphorylation (Pan-Pho), and pan-acetylation (Pan-Ace) in C4-2 cells treated with 1640 or A + CTM. ( E ) Western blot analysis of Pan-Lac in C4-2 cells treated with NALA or DMSO. ( F ) Heatmap and KEGG pathway enrichment analysis of differentially lactylated proteins in C4-2 cells treated with A + CTM compared to A − CTM ( n = 3). ( G ) Volcano plot showing the differentially lactylated proteins in C-2 cells treated with A + CTM compared to A − CTM. ( H ) CCK-8 assay showing cell proliferation of C4-2 cells treated with siSNRPA or siNC, NALA or DMSO, and ENZ. ( I ) RT-PCR analysis of AR and AR-V7 expression in C4-2, LNCaP, and VCaP cells treated with siSNRPA and siNC. Data represent the means ± SD. Statistical significance was determined by a two-way ANOVA followed by a multiple comparisons test. * P < 0.05, ** P < 0.01, and *** P < 0.001. All experiments were independently replicated three times in the laboratory ( n = 3) to ensure biological reproducibility.

Article Snippet: C4-2 and LNCaP cells were plated in 96-well plates at a density of 3000 cells per well and treated with A + CTM, A − CTM, NALA (L7022, Sigma-Aldrich), l -lactate (L1750, Sigma-Aldrich), PROTAC AR-V7 degrader-1 (HY-145479, MedChem Express), or ENZ.

Techniques: CCK-8 Assay, Reverse Transcription Polymerase Chain Reaction, Western Blot, Expressing, Cell Culture, Ubiquitin Proteomics, Phospho-proteomics

Journal: Science Advances

Article Title: Lactate derived from cancer-associated fibroblasts promotes alternative splicing and castration resistance in prostate cancer

doi: 10.1126/sciadv.ady5324

Figure Lengend Snippet: ( A ) Schematic representation of SNRPA protein domains. ( B ) MS analysis of K123 as a lactylation site in SNRPA. ( C and D ) Lactylation in SNRPA K123WT and K123R C4-2 and LNCaP cells treated with NALA or DMSO. ( E ) Dot plot showing transcriptomic analysis of SNRPA K123WT or K123R C4-2 cells under NALA and ENZ treatment ( n = 3 for each group). ( F ) Gene ontology enrichment analysis of differentially expressed genes in K123R and K123WT cells. ( G ) CCK-8 assay showing that NALA treatment reverses ENZ resistance in LNCaP cells expressing K123R SNRPA but not in WT SNRPA-expressing cells. ( H ) AR and AR-V7 expression in C4-2 cells expressing WT or K123R SNRPA under NALA or DMSO treatment. ( I ) Schematic of the AR-V7 minigene construct containing ISEm and ESEm. ( J and K ) RNA pull-down and RIP assay showing increased binding of SNRPA to ESEm in NALA or DMSO-treated C4-2 cells ( n = 3). ( L ) Schematic of the human AR gene. ( M ) ChIP analysis demonstrating the enhanced recruitment of SNRPA to P1-to-P3 regions following NALA treatment ( n = 3). ( N and O ) Co-IP assays showing a direct interaction between SNRPA and AARS1. ( P ) SNRPA K123 lactylation in C4-2 cells after AARS1 knockdown. ( Q ) CCK-8 assay showing that AARS1 knockdown reverses NALA-induced ENZ resistance in C4-2 cells. ( R ) RT-PCR and Western blot analysis of AR and AR-V7 expression in C4-2 cells with AARS1 knockdown. Data represent the means ± SD. Statistical significance was determined by a two-tailed unpaired t test and two-way ANOVA followed by a multiple comparisons test. * P < 0.05, ** P < 0.01, and *** P < 0.001. All experiments were independently replicated three times in the laboratory ( n = 3) to ensure biological reproducibility.

Article Snippet: C4-2 and LNCaP cells were plated in 96-well plates at a density of 3000 cells per well and treated with A + CTM, A − CTM, NALA (L7022, Sigma-Aldrich), l -lactate (L1750, Sigma-Aldrich), PROTAC AR-V7 degrader-1 (HY-145479, MedChem Express), or ENZ.

Techniques: CCK-8 Assay, Expressing, Construct, Binding Assay, Co-Immunoprecipitation Assay, Knockdown, Reverse Transcription Polymerase Chain Reaction, Western Blot, Two Tailed Test

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 SK-575 PROTAC formed by the PARPi Olaparib linked to Thalidomide, a CRBN binder. B) Western blot analysis of PARP1 expression in isogenic CRBN WT and KO KBM7 cells treated with 100 nM SK-575 or Thalidomide for 48h. ACTB was used as loading control. C) Viability assay by MTT performed in CRBN WT or KO KBM7 cells pre-treated with 100 nM SK-575 for 24 h followed by a treatment with both SK-575 and the indicated doses of Olaparib or Talazoparib for another 24 h. Data are normalized to vehicle or SK-575 pre-treated cells and correspond to three biological replicates. IC50s were calculated with non-linear regression analysis and are shown in the figure (µM). Statistical significance was determined with Extra sum-of-squares F test. D) Analysis of percentage of viability as assessed by Annexin V/PI staining using flow cytometry analysis from three biological replicates. Statistical significance was determined with Ordinary one-way ANOVA and Tukey’s multiple comparisons test. E) Representative images of the Annexin V/PI staining. Percentage of cells for each population are shown. F) Western blot showing PARP1 degradation in human bone marrow mononuclear cells treated with 100 nM SK-575 for 48 h. G) Colony-forming cells (CFC) assay of human bone marrow mononuclear cells pre-treated with 100 nM SK-575 24 h before seeding them in MethoCult semi-solid methylcellulose-based media with the indicated doses of Talazoparib with or without 100 nM SK-575. Colonies were quantified after 14 days. Error bars indicate mean ± s.d. (n = 3). Statistical significance was determined with unpaired t-tests. In all the figures * = p < 0.05, and *** = p < 0.001.

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, Expressing, Control, Viability Assay, Staining, Flow Cytometry

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