p300  (MedChemExpress)

Journal: bioRxiv

Article Title: CpG island density predicts CBP/p300 dependency across 3D chromatin clusters

doi: 10.64898/2026.05.04.722036

Figure Lengend Snippet: a. Overview of Pol2 HiChIP derived clusters. b. Clusters ranked by the percentage of their loop anchors overlapping CpG islands, broken into high, medium and low CpG content. c. Overlap of cluster features, including promoter-promoter loops, p300 and PAX3-FOXO1 ChIP-seq peak overlap, and target gene categories (Core Regulatory Transcription Factors and Housekeeping gene), at clusters ranked by percent CpG islands at anchors. d. Percentage of CpG islands at loop anchors within clusters, separated by gene category. e. Total Pol2 contacts per million (CPM) signal among clusters grouped by CpG content. f. Gene expression from RNA-seq (log2 transcripts per million, TPM) among clusters grouped by CpG content. g. Percentage of promoter-promoter loops among clusters grouped by CpG content. h. Frequency of PAX3-FOXO1 binding at loop anchors in clusters grouped by CpG content. i. Frequency of p300 binding at loop anchors in clusters grouped by CpG content. j. Gene expression changes in RMS cells after dual CRISPR mediated knockout of CBP and EP300, for genes in clusters grouped by CpG content. k. GAPDH and MYOD1 gene regulatory clusters, shown with Pol2 HiChIP clustering, CpG island abundance, P3F and p300 binding, H3K27ac and Pol2 ChIP-seq levels, and RNA-seq, revealing distinct cluster features. l. Model of CpG rich Housekeeping clusters vs. p300 rich Core Regulatory clusters.

Article Snippet: In 2022, a team at GlaxoSmithKline identified proline-based p300 inhibitors from a DNA-encoded library.

Techniques: HiChIP, Derivative Assay, ChIP-sequencing, Gene Expression, RNA Sequencing, Binding Assay, CRISPR, Knock-Out

Journal: bioRxiv

Article Title: CpG island density predicts CBP/p300 dependency across 3D chromatin clusters

doi: 10.64898/2026.05.04.722036

Figure Lengend Snippet: a. Molecular structure of IHK-44. b. IHK-44 docked to the HAT domain of p300. c-d. Dose-response ( c ) and time-course ( d ) Western blot of RH4 cells after IHK-44 treatment. Dose-response samples were treated for 6 h each. Time-course samples were treated at 100 nM each. H2B and H3 are shown as loading controls. e. Heatmaps of HBK16ac and H2BK20ac ChIP-seq shows that p300-bound enhancers are downregulated after 100 nM IHK-44 treatment over a 6 h time-course treatment period.

Article Snippet: In 2022, a team at GlaxoSmithKline identified proline-based p300 inhibitors from a DNA-encoded library.

Techniques: Western Blot, ChIP-sequencing

Journal: bioRxiv

Article Title: CpG island density predicts CBP/p300 dependency across 3D chromatin clusters

doi: 10.64898/2026.05.04.722036

Figure Lengend Snippet: a. Schematic of CHESS-DIA methodology. b. Violin plots of normalized protein abundance levels of CBP (left) and p300 (right) across nuclear compartments. c. Box plots of protein abundance levels between CBP (top) and p300 (bottom) in euchromatin fractions from RH4 cells treated with DMSO or increasing concentrations of IHK-44 (100 nM, 125 nM, 150 nM). Box plots represent median and quartiles, whiskers representing 1.5 x IQR. Statistical significance determined using a two-tailed Welch’s T-test. d. Box plots of protein abundance levels between PAX3-FOXO1, MYOD1, AND MYOG in euchromatin fractions from RH4 cells treated with DMSO or increasing concentrations of IHK-44 (100 nM, 125 nM, 150 nM). Box plots represent median and quartiles, whiskers representing 1.5 x IQR. Statistical significance determined using a two-tailed Welch’s T-test. e. Scatter plot comparing changes between RNA and protein abundance in RH4 cells treated with DMSO or 100 nM IHK-44 for 6 h. Lineage-specific TFs are highlighted and labeled in red, housekeeping genes in blue, other genes in gray.

Article Snippet: In 2022, a team at GlaxoSmithKline identified proline-based p300 inhibitors from a DNA-encoded library.

Techniques: Quantitative Proteomics, Two Tailed Test, Labeling

Journal: bioRxiv

Article Title: CpG island density predicts CBP/p300 dependency across 3D chromatin clusters

doi: 10.64898/2026.05.04.722036

Figure Lengend Snippet: a. Dose-response curves showing relative cell viability of FP-RMS (RH4), FN-RMS (RD), and normal myoblast (KM155C25DIST) cells after 72 h treatment with IHK-44. Values represent mean ± SEM of n = 4 technical replicates. b. Growth curves of % confluence over time (h) in FP-RMS (RH4, RH5), FN-RMS (CTR), and normal myoblast (HSMM) cell lines treated with DMSO or 185 nM IHK-44. Values represent mean ± SEM of n = 3 technical replicates. c. Heatmap summarizing IC 50 values (nM) for CBP/p300 bromodomain inhibitors (CCS1477, GNE-049), CBP/p300 HAT domain inhibitors (A-485, IHK-44), p300 degrader (JQAD1), and CBP/p300 degraders (dCBP-1, CBPD-409) across sensitive and resistant RMS cell lines. d. Bar plots summarizing cell-cycle arrest of RH4 cells treated with increasing doses (10, 100, 1000 nM) of IHK-44 for 72 h. e. Bar plots summarizing apoptosis Annexin V/PI staining of RH4 cells treated with increasing doses (10, 100, 1000 nM) of IHK-44 for 72 h. f. IC 50 values across 958 cell lines treated with IHK-44 from the PRISM screen grouped by primary disease. Box plot represents median and quartiles, whiskers showing 1.5 × IQR. Fusion-positive RMS is highlighted and labeled in red. Fusion-negative RMS in highlighted and labeled in red. g. Correlation between IHK-44 efficacy (log2.AUC) and other compounds in the DepMap Repurposing dataset. A-485 is highlighted and labeled in red. h. Correlation between IHK-44 efficacy (log2.AUC) and gene dependency scores in the DepMap CHRONOS dataset. p300 is highlighted and labeled in red. i. Western blots of non-treated histone acetylation levels in a panel of sensitive and resistant RMS cell lines. H2B and H3 are shown as loading controls

Article Snippet: In 2022, a team at GlaxoSmithKline identified proline-based p300 inhibitors from a DNA-encoded library.

Techniques: Staining, Labeling, Western Blot

Journal: bioRxiv

Article Title: CpG island density predicts CBP/p300 dependency across 3D chromatin clusters

doi: 10.64898/2026.05.04.722036

Figure Lengend Snippet: a. Design of a Random Forest machine learning model for predicting response to CBP/p300 inhibition at 6 hours, considering a mix of gene features, followed by SHAP permutation to learn the relative importance of each feature to predict responsiveness. b. The ROC (true positive versus false positive) curves for classification models from each gene feature group, and the combination of all features. Each line is colored according to the feature colors in ( a. ). AUC, area under the curve. c. The precision-recall curves for classification models from each gene feature group, and the combined all feature performance. Each line is colored according to the feature colors in ( a. ). AP, Average Precision. d. SHAP feature importance analysis colored by feature group across 100 regression runs. Each boxplot is colored according to the feature group colors in ( a. ).

Article Snippet: In 2022, a team at GlaxoSmithKline identified proline-based p300 inhibitors from a DNA-encoded library.

Techniques: Inhibition

Journal: Advanced Science

Article Title: Identification of A p300–SP1–BRD4 Transcriptional Axis as a Key Driver of AR Hyperactivation in Polycystic Ovarian Syndrome

doi: 10.1002/advs.202518185

Figure Lengend Snippet: PCOS ovary exhibits abnormal AR activation, p300 upregulation and histone acetylation. Three‐week‐old female C57BL/6J mice were subcutaneously implanted with silicone tubes (1 cm) containing dehydroepiandrosterone (DHEA) for 35 days to establish a PCOS model, the equal‐length empty silicone tubes served as the control (Ctrl) (ten mice in each group). (a) Representative photomicrographs of ovarian sections (Hematoxylin‐eosin and Masson trichrome staining) from Ctrl and DHEA‐treated mice. Corpora lutea were indicated by asterisk, and preantral follicles and the collagen depositions were indicated by arrows. (b) Quantitation of (a). Data were presented as Box‐and‐whisker plots with data points and as means ± SEM, n = 6. *p < 0.05 , Student's t‐test. (c) Western blotting. Ovarian tissue homogenates from Ctrl and DHEA‐treated mice were assayed for p300, AR, α‐SMA and Collagen I (Col1α). Two representative samples from each group were shown. GAPDH served as loading control. Quantifications were shown on the right side. Data were presented as means ± SEM, n = 6. *p < 0.05 , Student's t‐test. (d) Volcano plot of gene expression profile from the database [GEO Dataset Accession Number: GSE293353 , GSE277906 , GSE155489 , GSE138518 ], comprising RNA‐seq data ovarian granulosa cells of 15 women diagnosed with PCOS and 15 healthy control women. The number and position of genes statistic‐significantly decreased (1926, blue), no difference (14952, gray) or increased (548, red) including EP300 (Log2 (flod‐change) = 0.903643) and AR (Log2(fold‐change) = 0.544997), green point was marked. (e) Representative photomicrographs of ovarian sections from Ctrl and DHEA‐treated mice stained for p300 and AR by immunohistochemistry (IHC) staining. Positively‐stained granulosa cells were indicated by arrows. (f) Western blotting. Ovarian tissue homogenates were examined for Pan‐Acetyl‐Lysine (Pan‐Ac). Two representative samples from each group were shown. H3 served as loading control. Quantifications of histone 3 and histone acetylation were on the right side. Data were presented as means ± SEM, n = 6. *p < 0.05 , Student's t ‐test. (g) RT‐PCR of ovarian tissues from Ctrl and DHEA‐treated mice (n = 6) for Ar mRNA. Beta‐actin gene Actb served as internal control. Two samples from each group were shown. Quantifications were on the upper side. Data were presented as means ± SEM. *p < 0.05 , Student's t ‐test.

Article Snippet: Treatments include the p300‐selective inhibitors C646 (10 u m , HY‐13823, MCE, USA) and A‐485 (0, 0.1, 0.5, 1, 5, 10 u m , HY‐107455, MCE, USA), p300 agonist CTPB (5 u m , HY‐124960, MCE, USA), and the SP1‐selective inhibitor Plicamycin (Plica, 100 n m , HY‐A0122).

Techniques: Activation Assay, Control, Staining, Quantitation Assay, Whisker Assay, Western Blot, Gene Expression, RNA Sequencing, Immunohistochemistry, Reverse Transcription Polymerase Chain Reaction

Journal: Advanced Science

Article Title: Identification of A p300–SP1–BRD4 Transcriptional Axis as a Key Driver of AR Hyperactivation in Polycystic Ovarian Syndrome

doi: 10.1002/advs.202518185

Figure Lengend Snippet: p300‐driven H3K18ac and H3K27ac upregulates AR transcription in granulosa cells of mouse PCOS ovaries. (a) Western blotting. Ovarian tissue homogenates from Ctrl and DHEA‐treated mice were examined for p300, CBP, Gcn5, Tip60 and SRC‐3. GAPDH served as loading control. Two representative samples from each group were shown. Quantifications were shown on the right side. Data were presented as means ± SEM, n = 6. *p < 0.05 , Student's t‐test. (b) Western blotting. Ovarian tissue homogenates were assayed for H4 acetylation (H4K16ac, H4K12ac, H3K18ac, H3K27ac) and H4 (the left panel), and H3 acetylation (H4K8ac, H4K5ac, H3K14ac, H3K9ac) and H3 (the right panel). Quantifications were shown. Data were presented as means ± SEM, n = 6. *p < 0.05 , Student's t‐test. (c) Representative photomicrographs of ovarian sections from Ctrl and DHEA‐treated mice stained for p300 (red), BRD4 (green) and AR (magenta) by multiplex immunofluorescence (mIF) staining. Cell nuclei were stained with DAPI. (d) Quantifications of (c). The percentage of each fluorescent positive area relative to the DAPI area. (e) Primary granulosa cells (GCs) were treated with CTPB (5 µ m , 24 h). The cell lysates were assayed for p300 and AR. Quantifications were shown on the lower side. Data were presented as means ± SEM, n = 3, *p < 0.05 , Student's t ‐test. (f) Primary granulosa cells (GCs) were treated with DHEA (25 µ m , 48 h) followed by C646 (10 µ m , 24 h) or A‐485 (1 µ m , 24 h) treatment, and then cell lysates were assayed for p300, AR, H3K18ac, H3K27ac, H4K16ac, H4K12ac, H3, H4, and GAPDH. (g) Quantification of (e). Data were presented as means ± SEM, n = 4, *P < 0.05 , two‐way ANOVA followed by Tukey's post‐hoc test. (h) Luciferase assay. HEK293T cells were transfected with an AR promoter‐luciferase reporter AR‐luc plus a renilla luciferase reporter, and then treated with DHEA (25 µ m , 48 h) with or without C646 (10 µ m , 24 h). Cell lysates were assayed for luciferase activities. The relative luciferase activities of fold changes were presented. Data were presented as means ± SEM, n = 4, *P < 0.05 , one‐way ANOVA followed by Tukey's post‐hoc test.

Article Snippet: Treatments include the p300‐selective inhibitors C646 (10 u m , HY‐13823, MCE, USA) and A‐485 (0, 0.1, 0.5, 1, 5, 10 u m , HY‐107455, MCE, USA), p300 agonist CTPB (5 u m , HY‐124960, MCE, USA), and the SP1‐selective inhibitor Plicamycin (Plica, 100 n m , HY‐A0122).

Techniques: Western Blot, Control, Staining, Multiplex Assay, Immunofluorescence, Luciferase, Transfection

Journal: Advanced Science

Article Title: Identification of A p300–SP1–BRD4 Transcriptional Axis as a Key Driver of AR Hyperactivation in Polycystic Ovarian Syndrome

doi: 10.1002/advs.202518185

Figure Lengend Snippet: p300 knockout in granulosa cells resists ovarian AR activation and fibrosis in PCOS mice. (a) Generation of conditional Ep300 knockout mice ( Ep300 GC ‐/− ) by crossing Cyp19a1 ‐ Cre with Ep300 fl/fl mice. Ep300 locus in wild‐type mice were represented by boxes (exons 1–31). The positions of Cyp19a1 ‐ Cre genotyping PCR primers F2 and R2 (arrows), and the loxP sites (triangles) in Ep300 fl/fl mice and the genotyping PCR primers F1 and R1 (arrows) were depicted. (b) Agarose gel representation of genotyping of WT, Ep300 fl/− , Ep300 fl/fl and Ep300 GC ‐/− mice. (c) Representative photomicrographs of ovarian sections (Hematoxylin‐eosin and Masson trichrome staining) from Ep300 fl/fl and Ep300 GC ‐/− mice treated with subcutaneous implantation of silicone implants (1 cm) for sustained release of dehydroepiandrosterone (DHEA) for 35 days (n = 6). (d) Quantification of (c). The corpora lutea and collagen deposition were indicated by asterisk or arrows, respectively. Data were presented as Box‐and‐whisker plots with data points and as means ± SEM; n = 6, *p < 0.05 , one‐way ANOVA followed by Tukey's post‐hoc test. (e) Representative photomicrographs of ovarian sections from Ep300 fl/fl and Ep300 GC ‐/− mice treated with DHEA stained for AR by immunohistochemistry (IHC) staining. Positively‐stained granulosa cells were indicated by arrows. (f) Western blotting. The ovarian tissue homogenates were assayed for p300, AR, α‐SMA, Collagen I (Col1α) and GAPDH. Two randomly‐selected samples from each group were shown. Quantifications were shown on the right side. Data were presented as means ± SEM; n = 6, *p < 0.05 , one‐way ANOVA followed by Tukey's post‐hoc test.

Article Snippet: Treatments include the p300‐selective inhibitors C646 (10 u m , HY‐13823, MCE, USA) and A‐485 (0, 0.1, 0.5, 1, 5, 10 u m , HY‐107455, MCE, USA), p300 agonist CTPB (5 u m , HY‐124960, MCE, USA), and the SP1‐selective inhibitor Plicamycin (Plica, 100 n m , HY‐A0122).

Techniques: Knock-Out, Activation Assay, Agarose Gel Electrophoresis, Staining, Whisker Assay, Immunohistochemistry, Western Blot

Journal: Advanced Science

Article Title: Identification of A p300–SP1–BRD4 Transcriptional Axis as a Key Driver of AR Hyperactivation in Polycystic Ovarian Syndrome

doi: 10.1002/advs.202518185

Figure Lengend Snippet: Pharmacological inhibition of p300 alleviates abnormal AR activation and ovarian fibrosis in PCOS mice. Two PCOS models were established in three‐week‐old female C57BL/6J mice by either subcutaneous implantation of dehydroepiandrosterone (DHEA)‐containing silicone tubes (1 cm) for 35 days or by feeding a high‐fat diet (HF, 60% fat) for 3 months. Ten days prior to the completion of the modeling period, mice were injected intraperitoneally with the p300 inhibitor C646 (10 mg/kg) (ten mice in each group). (a) Representative estrous cyclicity of three mice each group during 12 consecutive days. P: proestrus, E: estrus, D/M: diestrus phase/metestrus. (b) Body weight (g) (the upper side) and ovary weight (mg) (the lower side). Data were presented as means ± SEM; n = 10, *p < 0.05 , one‐way ANOVA followed by Tukey's post‐hoc test. (c) Representative photomicrographs of ovarian sections stained by Hematoxylin‐eosin (the upper pictures), Masson trichrome (the middle pictures) or immunohistochemistry of AR (the lower pictures). The corpora lutea were indicated by asterisk, and primordial/primary follicles, collagen deposition and positively‐stained granulosa cells were indicated by arrows. (d) Quantifications of primordial/primary follicles ratio and corpora lutea ratio, ovarian fibrosis staining and AR positively‐stained granulosa cells in (c). Data were presented as Box‐and‐whisker plots with data points. Data were presented as means ± SEM; n = 6, *p < 0.05 , one‐way ANOVA followed by Tukey's post‐hoc test. (e) Western blotting. The ovarian tissue homogenates were assayed for p300, AR, α‐SMA, Collagen I (Col1α), H3K18ac, H3K27ac, H3 and GAPDH. Two randomly‐selected samples from each group were shown. (f) Quantifications of (e). Data were presented as means ± SEM. n = 6, *p < 0.05 , one‐way ANOVA followed by Tukey's post‐hoc test.

Article Snippet: Treatments include the p300‐selective inhibitors C646 (10 u m , HY‐13823, MCE, USA) and A‐485 (0, 0.1, 0.5, 1, 5, 10 u m , HY‐107455, MCE, USA), p300 agonist CTPB (5 u m , HY‐124960, MCE, USA), and the SP1‐selective inhibitor Plicamycin (Plica, 100 n m , HY‐A0122).

Techniques: Inhibition, Activation Assay, Injection, Staining, Immunohistochemistry, Whisker Assay, Western Blot

Journal: Advanced Science

Article Title: Identification of A p300–SP1–BRD4 Transcriptional Axis as a Key Driver of AR Hyperactivation in Polycystic Ovarian Syndrome

doi: 10.1002/advs.202518185

Figure Lengend Snippet: AR transactivation is linked to increased chromatin accessibility and SP1 footprint dynamics. Three‐week‐old female C57BL/6J mice were subcutaneously implanted with silicone tubes (1 cm) containing dehydroepiandrosterone (DHEA) for 35 days to establish a PCOS model, the equal‐length empty silicone tubes served as the control (Ctrl). Ovarian tissues were collected for ATAC‐seq and RNA‐seq analysis (n = 3 in each group). (a) Heatmap of normalized expression (FPKM) for 15 key genes associated with estrogen signaling, cortisol synthesis and secretion, HIF‐1α signaling, and oocyte meiosis in Ctrl and DHEA‐treated mice by RNA‐seq analysis. (b) Open chromatin regions surrounding gene transcription start sites (TSSs) in ovarian tissues from Ctrl and DHEA‐treated mice by ATAC‐seq analysis. (c) Pie chart depicting the peaks annotation identified in the ATAC‐seq analysis. The chart illustrates the distribution of accessible chromatin regions across various functional elements in the genome. (d) Venn diagram illustrating the overlap between gene with increased peaks (ATAC‐seq, p < 0.05 ) and genes with increased expression (RNA‐seq, |log2FC| > 1, p < 0.05 ) in Ctrl vs. DHEA‐treated mice. (e) Peak plot showing the ATAC‐seq peaks (normalized reads) and RNA‐seq mRNA expression (FPKM) at the Ar locus (ChrX: 98147619–98152019) in Ctrl (blue) vs. DHEA‐treated (red) mice. The peaks exhibiting a specific increase upon DHEA treatment are indicated by gray boxes and red asterisk. (f) A heatmap displays the top six transcription factors (TFs) binding to the Ar promoter region in the ATAC‐seq analysis, along with their mRNA expression identified by RNA‐seq analysis, and the predicted TF motifs were shown on the right. (g) Aggregated footprint plot of SP1. The aggregated plot depicts the predicted SP1 binding motifs across 852 total possibilities, comparing the Ctrl and DHEA conditions. The dashed lines indicate the boundaries of the SP1 motif at the Ar promoter. The plot visualizes the change in chromatin accessibility and transcription factor (TF) footprints across the different treatments. (h) Western blotting (the left side) and ChIP assay (the right side). Western blotting assay, the ovarian tissue homogenates from Ctrl and DHEA‐treated mice were assayed for SP1, p300, BRD4 and GAPDH. ChIP assay, the ovarian tissue homogenates were immunoprecipitated with isoform‐matched immunoglobulin or antibodies to SP1, p300 or BRD4, respectively, and then the genomic DNA (Input) and the antibody‐bound DNAs were PCR‐amplified with primers covering the SP1 motif on Ar promoter. The PCR products were analyzed on 1.5% agarose gels. (i) Western blotting. KGN cells were treated with DHEA (25 µ m , 48 h) with or without Plicamycin (Plica, 100 n m , 24 h), and then cell lysates were assayed for SP1, AR and GAPDH. The quantification graph is shown below. Data were presented as means ± SEM. n = 4, *p < 0.05 , one‐way ANOVA followed by Tukey's post‐hoc test.

Article Snippet: Treatments include the p300‐selective inhibitors C646 (10 u m , HY‐13823, MCE, USA) and A‐485 (0, 0.1, 0.5, 1, 5, 10 u m , HY‐107455, MCE, USA), p300 agonist CTPB (5 u m , HY‐124960, MCE, USA), and the SP1‐selective inhibitor Plicamycin (Plica, 100 n m , HY‐A0122).

Techniques: Control, RNA Sequencing, Expressing, Functional Assay, Binding Assay, Western Blot, Immunoprecipitation, Amplification

Journal: Advanced Science

Article Title: Identification of A p300–SP1–BRD4 Transcriptional Axis as a Key Driver of AR Hyperactivation in Polycystic Ovarian Syndrome

doi: 10.1002/advs.202518185

Figure Lengend Snippet: p300 forms a transcriptional complexes with SP1 and BRD4 to drive AR activation. (a) Scatter plots showing EP300 ‐ AR ( Ep300 ‐ Ar ) and SP1 ‐ AR ( Sp1 ‐ Ar ) expression correlations in human ovarian granulosa cells (the left panel) and murine ovaries (the right panel). (b) ChIP assay. KGN cells were treated with DHEA (25 µ m , 48 h) in presence or absence of C646 (10 µ m , 24 h) or Plicamycin (Pli, 100 n m , 24 h), respectively, and then cell lysates were immunoprecipitated with isoform‐matched immunoglobulin or antibodies to p300, SP1, H3K18ac, or H3K27ac, respectively, and then the genomic DNA (Input) and the antibody‐bound DNAs were PCR‐amplified with primers covering the SP1 motif on AR promoter. The PCR products were analyzed on 1.5% agarose gels. (c) Quantification of (b). Data were presented as Box‐and‐whisker plots with data points and as means ± SEM; n = 4, *P < 0.05 , one‐way ANOVA followed by Tukey's post‐hoc test. (d) Co‐IP assay. KGN cells were treated with DHEA (25 µ m , 48 h) with or without C646 (10 µ m , 24 h), and then cell lysates were immunoprecipitated with isoform‐matched immunoglobulin (Ig) or antibody (IP Ab) to p300, BRD4, or SP1, and then immunoprecipitants were assessed for p300, BRD4, or SP1 by western blotting reciprocally (the upper panel). The non‐IP lysates were assayed for p300, BRD4, SP1, and GAPDH as input controls. (e) Quantification of the Input bands in (d). Data were presented as means ± SEM; n = 3, *P < 0.05 , one‐way ANOVA followed by Tukey's post‐hoc test. (f) Quantification of the IP bands in (d). Data were presented as means ± SEM; n = 3, *P < 0.05 , one‐way ANOVA followed by Tukey's post‐hoc test. (g) KGN cells were treated with DHEA (25 µ m , 48 h) with or without C646 (10 µ m , 24 h), and then were stained for p300 (red), BRD4 (green) and SP1 (magenta) by multiplex immunofluorescence (mIF) staining. The colocalization analysis peak plot is shown below. (h) HEK293T cells were transfected with an AR promoter‐luciferase reporter AR‐luc (GGGGAGGGG) or a mutant AR promoter reporter ARmut‐luc (TAATGTAAT) plus a renilla luciferase reporter for 24 h, in presence or absence of a plasmid overexpressing SP1 (Gv‐SP1). At the same time, cells were treated with or without C646 (10 µ m , 24 h) or Plicamycin (Plica, 100 n m , 24 h), respectively. Then cell lysates were assayed for SP1 and GAPDH (Western blotting) or luciferase activities. The relative luciferase activities of fold changes were presented. Data were presented as means ± SEM; n = 4, *P < 0.05 , two‐way ANOVA followed by Tukey's post‐hoc test.

Article Snippet: Treatments include the p300‐selective inhibitors C646 (10 u m , HY‐13823, MCE, USA) and A‐485 (0, 0.1, 0.5, 1, 5, 10 u m , HY‐107455, MCE, USA), p300 agonist CTPB (5 u m , HY‐124960, MCE, USA), and the SP1‐selective inhibitor Plicamycin (Plica, 100 n m , HY‐A0122).

Techniques: Activation Assay, Expressing, Immunoprecipitation, Amplification, Whisker Assay, Co-Immunoprecipitation Assay, Western Blot, Staining, Multiplex Assay, Immunofluorescence, Transfection, Luciferase, Mutagenesis, Plasmid Preparation

Journal: Advanced Science

Article Title: Identification of A p300–SP1–BRD4 Transcriptional Axis as a Key Driver of AR Hyperactivation in Polycystic Ovarian Syndrome

doi: 10.1002/advs.202518185

Figure Lengend Snippet: AR activation undermines the anti‐fibrosis effects of p300 inhibition in ovaries of PCOS ovaries. Three‐week‐old female C57BL/6J mice were accepted high‐fat diet (HF, 60% fat) for 3 months, and mice receiving Control vehicle (Ctrl) or DHT (1.66 mg/kg, 10 days) were grouped into Control (Ctrl), C646, high‐fat diet (HF), and C646‐treated HF mice (ten mice in each group). (a) Representative microphotographs of ovarian sections stained by Hematoxylin‐eosin (the left panel) and Masson trichrome (the right panel). The corpora lutea and collagen deposition were indicated by asterisk or arrows, respectively. (b) Quantitation of corpora lutea ratio and fibrosis staining of (a). Data were presented as Box‐and‐whisker plots with data points. Data were presented as means ± SEM; n = 6, *p < 0.05 , two‐way ANOVA followed by Tukey's post‐hoc test. (c) Western blotting. The ovarian tissues from the experimental mice in (a) were assayed for p300, AR, Collagen I (Col1α), α‐SMA, and GAPDH. Two randomly‐selected samples from each group were shown. (d) Quantification of (c). Data were presented as means ± SEM. n = 6, *p < 0.05 , two‐way ANOVA followed by Tukey's post‐hoc test. (e) A schematic of sequential p300 induction of histone 3 acetylation, formation of p300/BRD4/SP1 complex and AR transactivation, leading to PCOS. P300 inhibition with A‐485/C646 blocks the process.

Article Snippet: Treatments include the p300‐selective inhibitors C646 (10 u m , HY‐13823, MCE, USA) and A‐485 (0, 0.1, 0.5, 1, 5, 10 u m , HY‐107455, MCE, USA), p300 agonist CTPB (5 u m , HY‐124960, MCE, USA), and the SP1‐selective inhibitor Plicamycin (Plica, 100 n m , HY‐A0122).

Techniques: Activation Assay, Inhibition, Control, Staining, Quantitation Assay, Whisker Assay, Western Blot

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: 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: Journal of Nanobiotechnology

Article Title: CD8a antibody-functionalized biomimetic red blood cell membrane ectosomes delivering C646 reverse CD8⁺ T Cell exhaustion via H3K18la histone delactylation in gastric cardia adenocarcinoma

doi: 10.1186/s12951-025-03957-z

Figure Lengend Snippet: Bioinformatics Analysis Identifies Key Factors in Histone Lactylation Modification. Note: ( A ) Schematic workflow of bioinformatics analysis for identifying key factors; ( B ) Volcano plot of DEGs in tumor tissues and adjacent normal tissues from dataset GSE2685 (Normal = 8, Tumor = 22); ( C ) Heatmap showing the correlation of co-expression module genes with tumor and normal tissues, with each cell displaying the correlation coefficient and p -value; ( D ) Venn diagram illustrating the intersection of Blue module genes, DEGs, CD8 + T cell-related genes, and "Histone lactylation"-related genes; ( E ) Box plot of p300 differential expression; ( F ) The tSNE distribution map of EP300 in various cell types in the scRNA-seq data

Article Snippet: The p300 activator (cholera toxin B subunit, CTB; HY-134964) and p300 inhibitor (C646; HY-13823) were both purchased from MCE (USA); (3) PBS + vector group, CD8a-NVEs@C646 + vector group, and CD8a-NVEs@C646 + PDCD1 group, all treated with 20 mM sodium lactate for 24 h, with CD8a-NVEs@C646 treatment for 48 h. Cells from all groups were harvested for downstream analysis.

Techniques: Modification, Expressing, Quantitative Proteomics

Journal: Journal of Nanobiotechnology

Article Title: CD8a antibody-functionalized biomimetic red blood cell membrane ectosomes delivering C646 reverse CD8⁺ T Cell exhaustion via H3K18la histone delactylation in gastric cardia adenocarcinoma

doi: 10.1186/s12951-025-03957-z

Figure Lengend Snippet: p300 Regulates Histone Lactylation in CD8 + T Cells. Note: ( A ) Schematic representation of the experimental design, showing the workflow for detecting CD8 + T cells treated with lactate, p300 inhibitors, or activators. ( B–C ) WB analysis of PKla levels in CD8 + T cells over time (B) and under varying lactate concentrations ( C ). ( D–E ) WB analysis of the time-dependent ( D ) and dose-dependent ( E ) changes in H3K18la and H3K9la expression in CD8 + T cells following lactate treatment. *** p < 0.001, ** p < 0.01, and * p < 0.05 compared to the 0-h or untreated lactate group. ( F ) WB analysis of H3K18la and H3K9la expression in CD8 + T cells following p300 knockdown, activation, or inhibition. ( G ) ELISA detection of IFN-γ levels in the supernatant of CD8 + T cells across different treatment groups. (H) FCM analysis of GZMB expression in CD8 + T cells. ( I ) FCM analysis of CD8 + T cell proliferation. ( J ) LDH release assay showing the cytotoxic effects of CD8 + T cells on MKN-45 and SNU1 cells. In panels ( F–J ), * p < 0.05, ** p < 0.01, and *** p < 0.001 compared to the control group; # p < 0.05, ## p < 0.01, and ### p < 0.001 compared to the Lactate group; & p < 0.01 compared to the Lactate + sh-NC group. All cell-based experiments were performed in triplicate

Article Snippet: The p300 activator (cholera toxin B subunit, CTB; HY-134964) and p300 inhibitor (C646; HY-13823) were both purchased from MCE (USA); (3) PBS + vector group, CD8a-NVEs@C646 + vector group, and CD8a-NVEs@C646 + PDCD1 group, all treated with 20 mM sodium lactate for 24 h, with CD8a-NVEs@C646 treatment for 48 h. Cells from all groups were harvested for downstream analysis.

Techniques: Expressing, Knockdown, Activation Assay, Inhibition, Enzyme-linked Immunosorbent Assay, Lactate Dehydrogenase Assay, Control

Journal: Journal of Nanobiotechnology

Article Title: CD8a antibody-functionalized biomimetic red blood cell membrane ectosomes delivering C646 reverse CD8⁺ T Cell exhaustion via H3K18la histone delactylation in gastric cardia adenocarcinoma

doi: 10.1186/s12951-025-03957-z

Figure Lengend Snippet: CD8a-NVEs@C646 Facilitates Histone Delactylation Modification in CD8 + T Cells. Note: ( A ) Schematic workflow of RNA-seq and ChIP-seq experiments for CD8 + T cells treated with CD8a-NVEs@C646; ( B–C ) ChIP-seq analysis showing signals at TSS regions in PBS-treated (n = 3) and CD8a-NVEs@C646-treated groups (n = 3); ( D ) RNA-seq volcano plot illustrating significantly upregulated and downregulated genes in the CD8a-NVEs@C646-treated group compared to PBS (n = 3); ( E ) Venn diagram of overlapping genes from ChIP-seq and RNA-seq analyses related to CD8 + T cells; ( F ) ChIP analysis of p300 and H3K18la enrichment at the PDCD1 promoter region; ( G ) The protein interaction between p300 and H3K18la was detected by co-IP assay; ( H ) RT-qPCR analysis of PDCD1 mRNA expression in CD8 + T cells following CD8a-NVEs@C646 treatment; ( I ) WB analysis of PDCD1 and H3K18la protein expression levels in CD8 + T cells treated with CD8a-NVEs@C646. * p < 0.05, *** p < 0.001 compared to the PBS or sh-NC group; experiments were conducted in triplicate

Article Snippet: The p300 activator (cholera toxin B subunit, CTB; HY-134964) and p300 inhibitor (C646; HY-13823) were both purchased from MCE (USA); (3) PBS + vector group, CD8a-NVEs@C646 + vector group, and CD8a-NVEs@C646 + PDCD1 group, all treated with 20 mM sodium lactate for 24 h, with CD8a-NVEs@C646 treatment for 48 h. Cells from all groups were harvested for downstream analysis.

Techniques: Modification, RNA Sequencing, ChIP-sequencing, Co-Immunoprecipitation Assay, Quantitative RT-PCR, Expressing