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    Bethyl p300
    Screening of epigenetic bifunctionals yields <t>CBP/p300</t> ligand GNE-781 as the strongest transactivator. A) Chemical architecture of a representative aTAG showing the FKBP F36V ligand (AP1867), the activator ligand (purple) and a linker; lower table summarizes the 13 aTAG molecules synthesized, the chromatin effector they recruit, reporter EC 50 values (note: for biphasic curves such as aTAG-5/aTAG-6, this represents the interpolated dose with maximal activation) and maximal activation (fold-change vs. untreated at a single dose) from 10-point dose titration in an IRF1 reporter system (48h treatment); U2OS reporter system stably expressing 1) a plasmid containing 4 repeats of the IRF1 consensus sequence upstream of firefly luciferase, 2) a plasmid containing doxycycline-inducible IRF1-FKBP F36V -mCherry in pLIX403. B) Scatter-plot of maximal activation versus potency for all compounds after 48h treatment. C) Chemical structures of linker series tested for GNE-781 bifunctionals. D) Extended dose curves of AP1867-GNE781 bifunctionals or binders alone (GNE-781, mAP1867-CONHEt) in IRF1 reporter system (normalized FLuc luminescence at 48 hours). E) Extended dose curves of aTAG-2 with binder competition in IRF1 reporter system (normalized FLuc luminescence at 48 hours). F-G) Normalized NLuc luminescence from NanoBiT experiment in 293T cells, titrated with aTAG-2 ± competitor or binder alone for 24 hours. NanoBiT constructs used were FKBP F36V -LgBiT and SmBiT fused to the C-terminus of either the CBP (F) or p300 (G) bromodomain.
    P300, supplied by Bethyl, used in various techniques. Bioz Stars score: 93/100, based on 60 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/a300+358a/p300+Antibody/bio_rxiv__64898__2026__03__17__712509-257-25-26
    Average 93 stars, based on 60 article reviews
    p300 - by Bioz Stars, 2026-11
    93/100 stars

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    1) Product Images from "An epigenetic bifunctional that toggles between transactivation and repression"

    Article Title: An epigenetic bifunctional that toggles between transactivation and repression

    Journal: bioRxiv

    doi: 10.64898/2026.03.17.712509

    Screening of epigenetic bifunctionals yields CBP/p300 ligand GNE-781 as the strongest transactivator. A) Chemical architecture of a representative aTAG showing the FKBP F36V ligand (AP1867), the activator ligand (purple) and a linker; lower table summarizes the 13 aTAG molecules synthesized, the chromatin effector they recruit, reporter EC 50 values (note: for biphasic curves such as aTAG-5/aTAG-6, this represents the interpolated dose with maximal activation) and maximal activation (fold-change vs. untreated at a single dose) from 10-point dose titration in an IRF1 reporter system (48h treatment); U2OS reporter system stably expressing 1) a plasmid containing 4 repeats of the IRF1 consensus sequence upstream of firefly luciferase, 2) a plasmid containing doxycycline-inducible IRF1-FKBP F36V -mCherry in pLIX403. B) Scatter-plot of maximal activation versus potency for all compounds after 48h treatment. C) Chemical structures of linker series tested for GNE-781 bifunctionals. D) Extended dose curves of AP1867-GNE781 bifunctionals or binders alone (GNE-781, mAP1867-CONHEt) in IRF1 reporter system (normalized FLuc luminescence at 48 hours). E) Extended dose curves of aTAG-2 with binder competition in IRF1 reporter system (normalized FLuc luminescence at 48 hours). F-G) Normalized NLuc luminescence from NanoBiT experiment in 293T cells, titrated with aTAG-2 ± competitor or binder alone for 24 hours. NanoBiT constructs used were FKBP F36V -LgBiT and SmBiT fused to the C-terminus of either the CBP (F) or p300 (G) bromodomain.
    Figure Legend Snippet: Screening of epigenetic bifunctionals yields CBP/p300 ligand GNE-781 as the strongest transactivator. A) Chemical architecture of a representative aTAG showing the FKBP F36V ligand (AP1867), the activator ligand (purple) and a linker; lower table summarizes the 13 aTAG molecules synthesized, the chromatin effector they recruit, reporter EC 50 values (note: for biphasic curves such as aTAG-5/aTAG-6, this represents the interpolated dose with maximal activation) and maximal activation (fold-change vs. untreated at a single dose) from 10-point dose titration in an IRF1 reporter system (48h treatment); U2OS reporter system stably expressing 1) a plasmid containing 4 repeats of the IRF1 consensus sequence upstream of firefly luciferase, 2) a plasmid containing doxycycline-inducible IRF1-FKBP F36V -mCherry in pLIX403. B) Scatter-plot of maximal activation versus potency for all compounds after 48h treatment. C) Chemical structures of linker series tested for GNE-781 bifunctionals. D) Extended dose curves of AP1867-GNE781 bifunctionals or binders alone (GNE-781, mAP1867-CONHEt) in IRF1 reporter system (normalized FLuc luminescence at 48 hours). E) Extended dose curves of aTAG-2 with binder competition in IRF1 reporter system (normalized FLuc luminescence at 48 hours). F-G) Normalized NLuc luminescence from NanoBiT experiment in 293T cells, titrated with aTAG-2 ± competitor or binder alone for 24 hours. NanoBiT constructs used were FKBP F36V -LgBiT and SmBiT fused to the C-terminus of either the CBP (F) or p300 (G) bromodomain.

    Techniques Used: Synthesized, Activation Assay, Titration, Stable Transfection, Expressing, Plasmid Preparation, Sequencing, Luciferase, Construct

    aTAG-2 replaces p300 with CBP at EWS/FLI bound sites and degrades FKBP F36V -EWS/FLI in a E1/proteasome-dependent manner. A) FKBP F36V -HA-EWS/FLI EWS502 cells treated with DMSO, aTAG-2, or binders (mAP1867-CONHEt + GNE-781) at 10 nM for 16 hours were profiled by CBP, p300, and HA ChIP-seq. P300 and CBP occupancy heatmaps at Riggi et al. EWS/FLI bound sites are shown. B) Overlap between unique genes with consensus CBP ChIP-seq peaks gained by aTAG-2 (gained both vs. DMSO and vs. binders), and consensus p300 ChIP-seq peaks lost by aTAG-2. P -value calculated by hypergeometric test. C-D) Enrichr gene ontology analysis (C) and Homer motif analysis (D) of dual CBP-gained and p300-lost peaks/unique genes. E) Western blot for HA-tagged EWS/FLI in FKBP F36V -HA-EWS/FLI EWS502 cells treated with aTAG-2 at 10 nM for 3 hours in the presence of inhibitors (1 μM, except MG-132 at 10 μM; TAK-243: UBA1 inhibitor, TAK-981: SUMOylation inhibitor, MG-132: proteasome inhibitor, lanes are from the same blot & exposure). F) Western blot for HA-tagged EWS/FLI in FKBP F36V -HA-EWS/FLI EWS502 cells treated with aTAG-2 at 10 nM for 1 hour following TUBE2 (ubiquitin) pulldown, in the presence of MG-132 (10 μM). Total ubiquitin loading control is shown in . G) Model for aTAG-2 mechanism of action, where it can induce transactivation, partial target degradation, and inhibit p300/CBP through a RIPTAC-like mechanism depending on context.
    Figure Legend Snippet: aTAG-2 replaces p300 with CBP at EWS/FLI bound sites and degrades FKBP F36V -EWS/FLI in a E1/proteasome-dependent manner. A) FKBP F36V -HA-EWS/FLI EWS502 cells treated with DMSO, aTAG-2, or binders (mAP1867-CONHEt + GNE-781) at 10 nM for 16 hours were profiled by CBP, p300, and HA ChIP-seq. P300 and CBP occupancy heatmaps at Riggi et al. EWS/FLI bound sites are shown. B) Overlap between unique genes with consensus CBP ChIP-seq peaks gained by aTAG-2 (gained both vs. DMSO and vs. binders), and consensus p300 ChIP-seq peaks lost by aTAG-2. P -value calculated by hypergeometric test. C-D) Enrichr gene ontology analysis (C) and Homer motif analysis (D) of dual CBP-gained and p300-lost peaks/unique genes. E) Western blot for HA-tagged EWS/FLI in FKBP F36V -HA-EWS/FLI EWS502 cells treated with aTAG-2 at 10 nM for 3 hours in the presence of inhibitors (1 μM, except MG-132 at 10 μM; TAK-243: UBA1 inhibitor, TAK-981: SUMOylation inhibitor, MG-132: proteasome inhibitor, lanes are from the same blot & exposure). F) Western blot for HA-tagged EWS/FLI in FKBP F36V -HA-EWS/FLI EWS502 cells treated with aTAG-2 at 10 nM for 1 hour following TUBE2 (ubiquitin) pulldown, in the presence of MG-132 (10 μM). Total ubiquitin loading control is shown in . G) Model for aTAG-2 mechanism of action, where it can induce transactivation, partial target degradation, and inhibit p300/CBP through a RIPTAC-like mechanism depending on context.

    Techniques Used: ChIP-sequencing, Western Blot, Ubiquitin Proteomics, Control

    aTAG-2 decreases chromatin accessibility at p300 and EWS/FLI targets. A) FKBP F36V -HA-EWS/FLI EWS502 cells treated with DMSO, aTAG-2, or binders (mAP1867-CONHEt + GNE-781) at 5 nM for 16 hours and profiled by ATAC-seq. Chromatin accessibility changes at selected EWS/FLI targets are shown ( NR0B1, CCND1, FCGRT, GSTM4 ). B) HOMER motif analysis of consensus ATAC-seq peaks lost by aTAG-2 (lost in aTAG-2 vs. DMSO and aTAG-2 vs. binders comparisons). C) Enrichr gene ontology analysis on unique genes corresponding to consensus aTAG-2 lost ATAC-seq peaks (ChEA gene set database). D) Genomic location of consensus aTAG-2 lost ATAC-seq peaks or all ATAC-seq peaks (see Methods ) as reference. E) Overlap of FKBP F36V -HA-EWS/FLI peaks with p300 peaks (MACS2-based peak calling) in DMSO-treated FKBP F36V -HA-EWS/FLI EWS502 cells. P -value calculated by hypergeometric test. ATAC-seq signal heatmaps for HA/p300 co-bound peaks and p300-only bound peaks are shown. HA-only bound peaks are shown in .
    Figure Legend Snippet: aTAG-2 decreases chromatin accessibility at p300 and EWS/FLI targets. A) FKBP F36V -HA-EWS/FLI EWS502 cells treated with DMSO, aTAG-2, or binders (mAP1867-CONHEt + GNE-781) at 5 nM for 16 hours and profiled by ATAC-seq. Chromatin accessibility changes at selected EWS/FLI targets are shown ( NR0B1, CCND1, FCGRT, GSTM4 ). B) HOMER motif analysis of consensus ATAC-seq peaks lost by aTAG-2 (lost in aTAG-2 vs. DMSO and aTAG-2 vs. binders comparisons). C) Enrichr gene ontology analysis on unique genes corresponding to consensus aTAG-2 lost ATAC-seq peaks (ChEA gene set database). D) Genomic location of consensus aTAG-2 lost ATAC-seq peaks or all ATAC-seq peaks (see Methods ) as reference. E) Overlap of FKBP F36V -HA-EWS/FLI peaks with p300 peaks (MACS2-based peak calling) in DMSO-treated FKBP F36V -HA-EWS/FLI EWS502 cells. P -value calculated by hypergeometric test. ATAC-seq signal heatmaps for HA/p300 co-bound peaks and p300-only bound peaks are shown. HA-only bound peaks are shown in .

    Techniques Used:

    aTAG-2 induces targeted chromatin accessibility changes. FKBP F36V -HA-EWS/FLI EWS502 cells treated with DMSO, aTAG-2, or binders (mAP1867-CONHEt + GNE-781) at 5 nM for 16 hours and profiled by ATAC-seq. A) GGAA repeat length in consensus aTAG-2 lost peaks that are EWS/FLI bound (Riggi et al. EWS/FLI bound enhancers ) (left) or EWS/FLI-bound ATAC stable peaks (right). B) Enrichr gene ontology analysis on unique genes corresponding to top: consensus aTAG-2 lost ATAC-seq peaks, bottom: consensus aTAG-2 gained ATAC-seq peaks (GEO gene set database). C) Venn diagram of unique genes corresponding to consensus aTAG-2 lost/gained ATAC-seq peaks and EWS/FLI target gene sets from Kinsey et al. (upregulated or downregulated). P -values calculated by hypergeometric test. D) ATAC-seq heatmap across HA-bound, p300-unbound peaks are shown (determined using venn diagram in ).
    Figure Legend Snippet: aTAG-2 induces targeted chromatin accessibility changes. FKBP F36V -HA-EWS/FLI EWS502 cells treated with DMSO, aTAG-2, or binders (mAP1867-CONHEt + GNE-781) at 5 nM for 16 hours and profiled by ATAC-seq. A) GGAA repeat length in consensus aTAG-2 lost peaks that are EWS/FLI bound (Riggi et al. EWS/FLI bound enhancers ) (left) or EWS/FLI-bound ATAC stable peaks (right). B) Enrichr gene ontology analysis on unique genes corresponding to top: consensus aTAG-2 lost ATAC-seq peaks, bottom: consensus aTAG-2 gained ATAC-seq peaks (GEO gene set database). C) Venn diagram of unique genes corresponding to consensus aTAG-2 lost/gained ATAC-seq peaks and EWS/FLI target gene sets from Kinsey et al. (upregulated or downregulated). P -values calculated by hypergeometric test. D) ATAC-seq heatmap across HA-bound, p300-unbound peaks are shown (determined using venn diagram in ).

    Techniques Used:

    aTAG-2 replaces p300 with CBP at EWS/FLI bound sites. A) FKBP F36V -HA-EWS/FLI EWS502 cells treated with DMSO, aTAG-2, or binders (mAP1867-CONHEt + GNE-781) at 10 nM for 16 hours and profiled by CBP, p300, and HA ChIP-seq. P300 and CBP occupancy heatmaps at consensus aTAG-2 HA-EWS/FLI lost regions are shown (N=551, csaw-based differential binding). B) IGV tracks showing changes in ATAC, CBP, p300, and HA occupancy at the best characterized EWS/FLI target gene NR0B1 .
    Figure Legend Snippet: aTAG-2 replaces p300 with CBP at EWS/FLI bound sites. A) FKBP F36V -HA-EWS/FLI EWS502 cells treated with DMSO, aTAG-2, or binders (mAP1867-CONHEt + GNE-781) at 10 nM for 16 hours and profiled by CBP, p300, and HA ChIP-seq. P300 and CBP occupancy heatmaps at consensus aTAG-2 HA-EWS/FLI lost regions are shown (N=551, csaw-based differential binding). B) IGV tracks showing changes in ATAC, CBP, p300, and HA occupancy at the best characterized EWS/FLI target gene NR0B1 .

    Techniques Used: ChIP-sequencing, Binding Assay

    aTAG-2 degrades FKBP F36V -HA-tagged EWS/FLI on chromatin. A) FKBP F36V -HA-EWS/FLI EWS502 cells treated with DMSO, aTAG-2, or binders (mAP1867-CONHEt + GNE-781) at 10 nM for 16 hours and profiled by HA ChIP-seq. The number of HA peaks in MACS2-based peak calling is plotted between the conditions. B) Gene set enrichment analysis (Enrichr, GEO database) on unique genes corresponding to aTAG-2 HA-EWS/FLI consensus lost regions (N=551, csaw-based differential binding). C) Western blot for total ubiquitin (FK2) in FKBP F36V -HA-EWS/FLI EWS502 cells treated with aTAG-2 at 10 nM for 1 hour following TUBE2 (ubiquitin) pulldown, in the presence of MG-132 (10 μM). Loading control for . D) Western blot for HA-tagged EWS/FLI in FKBP F36V -HA-EWS/FLI EWS502 cells treated with aTAG-2 at 10 nM for 3 hours in the presence of p300/CBP histone acetyl transferase inhibitor A-485 (10 μM). E) Western blot for HA-tagged EWS/FLI and TRIM8 in FKBP F36V -HA-EWS/FLI EWS502 cells ± dox-inducible TRIM8 sgRNAs treated with aTAG-2 at 10 nM for 16 hours.
    Figure Legend Snippet: aTAG-2 degrades FKBP F36V -HA-tagged EWS/FLI on chromatin. A) FKBP F36V -HA-EWS/FLI EWS502 cells treated with DMSO, aTAG-2, or binders (mAP1867-CONHEt + GNE-781) at 10 nM for 16 hours and profiled by HA ChIP-seq. The number of HA peaks in MACS2-based peak calling is plotted between the conditions. B) Gene set enrichment analysis (Enrichr, GEO database) on unique genes corresponding to aTAG-2 HA-EWS/FLI consensus lost regions (N=551, csaw-based differential binding). C) Western blot for total ubiquitin (FK2) in FKBP F36V -HA-EWS/FLI EWS502 cells treated with aTAG-2 at 10 nM for 1 hour following TUBE2 (ubiquitin) pulldown, in the presence of MG-132 (10 μM). Loading control for . D) Western blot for HA-tagged EWS/FLI in FKBP F36V -HA-EWS/FLI EWS502 cells treated with aTAG-2 at 10 nM for 3 hours in the presence of p300/CBP histone acetyl transferase inhibitor A-485 (10 μM). E) Western blot for HA-tagged EWS/FLI and TRIM8 in FKBP F36V -HA-EWS/FLI EWS502 cells ± dox-inducible TRIM8 sgRNAs treated with aTAG-2 at 10 nM for 16 hours.

    Techniques Used: ChIP-sequencing, Binding Assay, Western Blot, Ubiquitin Proteomics, Control

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    Article Snippet: Membranes were imaged using the Odyssey ® CLx Imaging System and ImageJ was used for quantification, utilizing GAPDH as internal loading control. .. The following primary antibodies were used for immunoblotting in this study: monoclonal anti-E2 (B9) 1:500 , anti-TopBP1 1:1,000 (Bethyl, catalog no. A300-111A), anti-SIRT1 antibody 1:1,000 (Sigma, catalog no. 07-131), anti-p300 1:2,000 (Bethyl, catalog no. A300-358A), anti-Top1 1:200 (Santa Cruz; catalog no. sc-32736), anti-p53 1:1,000 (Thermo Fisher, catalog no. PA5-27822), anti-Acetyl-p53 (K382) 1:1,000 (Cell Signaling Technology, catalog no. 2525), anti-Cyclin B1 (D5C10) XP 1:1,000 (Cell Signaling Technology, catalog no. 4138) and anti-Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) 1:10,000 (Santa Cruz; catalog no. sc-47724). ..

    Virus:

    Article Title: Pbrm1 Steers Mesenchymal Stromal Cell Osteolineage Differentiation by Integrating PBAF-Dependent Chromatin Remodeling and BMP/TGF-β Signaling.
    Article Snippet: .. REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Anti-Pbrm1 Bethyl Laboratories Cat# A301-591A; RRID: AB_1078808 Anti-Arid2 Bethyl Laboratories Cat# A302-230A; RRID: AB_1731041 Anti-Brd7 Santa Cruz Cat# sc-376180; RRID: AB_10989389 Anti-Arid1a Milipore Cat# 04-080; RRID: AB_673052 Anti-Smarca4 Milipore Cat# 07-478; RRID: AB_11214441 Anti-pSmad1/5/8 Cell Signaling Cat# 13820; RRID: AB_2493181 Anti-Total Smad1 Cell Signaling Cat# 9743; RRID: AB_2107780) Anti-pSmad 2/3 Cell Signaling Cat# 8828; RRID: AB_2631089 Anti-Total Smad 2/3 Cell Signaling Cat# 8685; RRID: AB_10889933 Anti-phospho –p44/42 MAPK (pErk1/2) Cell Signaling Cat# 4370; RRID: AB_2315112 Anti- Total p44/42 MAPK (Erk1/2) Cell Signaling Cat# 4695; RRID: AB_390779 Anti-b-actin Milipore Cat# MABT825; RRID: AB_2571580 Anti-BmprIb Abcam Cat# ab175385; RRID: AB_2827412 Anti-BmprII Abcam Cat# ab124463; RRID: AB_11001003 Anti-TgfbrII Abcam Cat# ab186838; RRID: AB_2728775 Anti-H3K27ac Abcam Cat# ab4729; RRID: AB_2118291 Anti-p300 Bethyl Laboratories Cat# A300-358A; RRID: AB_185565 Anti-Smarcc1 Santa Cruz Cat# sc-9746; RRID: AB_671099 Anti-Smarcb1 Bethyl Laboratories Cat# A301-087A; RRID: AB_2191714 Anti-Ezh1 Milipore Cat# ABE281; RRID: AB_2827410 Anti-Suz12 Cell Signaling Cat #3737S; RRID: AB_2196850 APC Annexin- V BD PharMingen Cat# 550474 Mouse IgG1 Cell Signaling Cat# 5415; RRID: AB_10829607 Rabbit IgG Bethyl laboratories Cat# P120-101; RRID: AB_479829 Donkey Anti Rabbit IgG Alexa Fluor 568 Thermo (Invitrogen) Cat# A10042; RRID: AB_2534017 Donkey Anti Mouse IgG Alexa Fluor 488 Molecular probes (Invitrogen) Cat# A-21202; RRID: AB_141607 Goat Anti Rabbit IgG DyLight 488 Bethyl laboratories Cat# A120-201D2; RRID: AB_10634085 Bacterial and Virus Strains DH5a competent E. coli Thermo Fisher Cat#18265017 Biological Samples Human Bone Marrow Derived MSC Lonza Cat# PT-2501 Chemicals, Peptides, and Recombinant Proteins rm BMP-2 PeproTech Cat# 120-02 rh BMP-4/7 R&D Systems Cat# 3727-BP/CF rh TGF-b3 PeproTech Cat# 100-36E rh SCF PeproTech Cat# 300-07 rh FLT-3 PeproTech Cat# 300-19 rh TPO PeproTech Cat# 300-18 rh CXCL12 PeproTech Cat# 300-28A rh TGF-b1 PeproTech Cat# 100-21 Dexamethasone Sigma Cat# D4902 Ascorbic Acid Sigma Cat# A4544 Beta glycerol Phosphate Sigma Cat# 50020 (Continued on next page) e1 Cell Reports 31, 107570, April 28, 2020 .. REAGENT or RESOURCE SOURCE IDENTIFIER NBT-BCIP Sigma Cat# B5655 Alizarin Res S Sigma Cat# A5533 Indomethacin Sigma Cat# I7378 Insulin Sigma Cat# I2643 Oil Red O Sigma Cat# O0625 Hematoxylin Mayer’s Solution Loba Chemie Cat# 0402700100 Alcian Blue Sigma Cat# B8438 Doxycyclin Calbiochem Cat# 324385 Puromycin GIBCO Cat# A11138-03 G418 Calbiochem Cat# 5.09290.0002 RIPA Cell Signaling Technology Cat# 9806 TRIzol Thermo Cat# 15596018 Lipofectamine 3000 Thermo Cat# L3000-015 DnaseI Roche Cat# 04716728001 RnaseA Sigma Cat# R6513 Proteinase K Sigma Cat# P2308 Protein A/G agarose beads Thermo Cat# 20421 IGEPAL CA-630 Sigma Cat# I8896 Nextera Tn5 Transposase Illumina Cat# 15028212 NEBNext High-Fidelity 2x PCR Master Mix NEB Cat# M0541S ApoI NEB Cat# R3566 7-aminoactinomycin D Thermo (Molecular Probes) Cat# A1310 Ficoll/ Lymphoprep Stem cell Technology Cat# 07851 Methylcellulose Stem Cell Technology Cat# H4034 Critical Commercial Assays Column-free human CD34 progenitor isolation kit Stem Cell Technology Cat# 18066A CellTrace CFSE Cell Proliferation Kit Thermo (Molecular Probes) Cat# 34554 TaqMan Reverse Transcription Reagents Applied Biosystems Cat# N8080234 QIAGEN PCR purification kit QIAGEN Cat# 28104 TruSeqChIP Sample Prep Kit Illumina Cat# IP-202-1012-Set A Cat# IP-202-1024- Set B PureLink RNA mini kit Ambion Cat# 12183018A Ribo-Zero rRNA removal kit Illumina Cat# 20037135 Cxcl12 ELISA kit R&D Cat# MCX120 Scf.

    Derivative Assay:

    Article Title: Pbrm1 Steers Mesenchymal Stromal Cell Osteolineage Differentiation by Integrating PBAF-Dependent Chromatin Remodeling and BMP/TGF-β Signaling.
    Article Snippet: .. REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Anti-Pbrm1 Bethyl Laboratories Cat# A301-591A; RRID: AB_1078808 Anti-Arid2 Bethyl Laboratories Cat# A302-230A; RRID: AB_1731041 Anti-Brd7 Santa Cruz Cat# sc-376180; RRID: AB_10989389 Anti-Arid1a Milipore Cat# 04-080; RRID: AB_673052 Anti-Smarca4 Milipore Cat# 07-478; RRID: AB_11214441 Anti-pSmad1/5/8 Cell Signaling Cat# 13820; RRID: AB_2493181 Anti-Total Smad1 Cell Signaling Cat# 9743; RRID: AB_2107780) Anti-pSmad 2/3 Cell Signaling Cat# 8828; RRID: AB_2631089 Anti-Total Smad 2/3 Cell Signaling Cat# 8685; RRID: AB_10889933 Anti-phospho –p44/42 MAPK (pErk1/2) Cell Signaling Cat# 4370; RRID: AB_2315112 Anti- Total p44/42 MAPK (Erk1/2) Cell Signaling Cat# 4695; RRID: AB_390779 Anti-b-actin Milipore Cat# MABT825; RRID: AB_2571580 Anti-BmprIb Abcam Cat# ab175385; RRID: AB_2827412 Anti-BmprII Abcam Cat# ab124463; RRID: AB_11001003 Anti-TgfbrII Abcam Cat# ab186838; RRID: AB_2728775 Anti-H3K27ac Abcam Cat# ab4729; RRID: AB_2118291 Anti-p300 Bethyl Laboratories Cat# A300-358A; RRID: AB_185565 Anti-Smarcc1 Santa Cruz Cat# sc-9746; RRID: AB_671099 Anti-Smarcb1 Bethyl Laboratories Cat# A301-087A; RRID: AB_2191714 Anti-Ezh1 Milipore Cat# ABE281; RRID: AB_2827410 Anti-Suz12 Cell Signaling Cat #3737S; RRID: AB_2196850 APC Annexin- V BD PharMingen Cat# 550474 Mouse IgG1 Cell Signaling Cat# 5415; RRID: AB_10829607 Rabbit IgG Bethyl laboratories Cat# P120-101; RRID: AB_479829 Donkey Anti Rabbit IgG Alexa Fluor 568 Thermo (Invitrogen) Cat# A10042; RRID: AB_2534017 Donkey Anti Mouse IgG Alexa Fluor 488 Molecular probes (Invitrogen) Cat# A-21202; RRID: AB_141607 Goat Anti Rabbit IgG DyLight 488 Bethyl laboratories Cat# A120-201D2; RRID: AB_10634085 Bacterial and Virus Strains DH5a competent E. coli Thermo Fisher Cat#18265017 Biological Samples Human Bone Marrow Derived MSC Lonza Cat# PT-2501 Chemicals, Peptides, and Recombinant Proteins rm BMP-2 PeproTech Cat# 120-02 rh BMP-4/7 R&D Systems Cat# 3727-BP/CF rh TGF-b3 PeproTech Cat# 100-36E rh SCF PeproTech Cat# 300-07 rh FLT-3 PeproTech Cat# 300-19 rh TPO PeproTech Cat# 300-18 rh CXCL12 PeproTech Cat# 300-28A rh TGF-b1 PeproTech Cat# 100-21 Dexamethasone Sigma Cat# D4902 Ascorbic Acid Sigma Cat# A4544 Beta glycerol Phosphate Sigma Cat# 50020 (Continued on next page) e1 Cell Reports 31, 107570, April 28, 2020 .. REAGENT or RESOURCE SOURCE IDENTIFIER NBT-BCIP Sigma Cat# B5655 Alizarin Res S Sigma Cat# A5533 Indomethacin Sigma Cat# I7378 Insulin Sigma Cat# I2643 Oil Red O Sigma Cat# O0625 Hematoxylin Mayer’s Solution Loba Chemie Cat# 0402700100 Alcian Blue Sigma Cat# B8438 Doxycyclin Calbiochem Cat# 324385 Puromycin GIBCO Cat# A11138-03 G418 Calbiochem Cat# 5.09290.0002 RIPA Cell Signaling Technology Cat# 9806 TRIzol Thermo Cat# 15596018 Lipofectamine 3000 Thermo Cat# L3000-015 DnaseI Roche Cat# 04716728001 RnaseA Sigma Cat# R6513 Proteinase K Sigma Cat# P2308 Protein A/G agarose beads Thermo Cat# 20421 IGEPAL CA-630 Sigma Cat# I8896 Nextera Tn5 Transposase Illumina Cat# 15028212 NEBNext High-Fidelity 2x PCR Master Mix NEB Cat# M0541S ApoI NEB Cat# R3566 7-aminoactinomycin D Thermo (Molecular Probes) Cat# A1310 Ficoll/ Lymphoprep Stem cell Technology Cat# 07851 Methylcellulose Stem Cell Technology Cat# H4034 Critical Commercial Assays Column-free human CD34 progenitor isolation kit Stem Cell Technology Cat# 18066A CellTrace CFSE Cell Proliferation Kit Thermo (Molecular Probes) Cat# 34554 TaqMan Reverse Transcription Reagents Applied Biosystems Cat# N8080234 QIAGEN PCR purification kit QIAGEN Cat# 28104 TruSeqChIP Sample Prep Kit Illumina Cat# IP-202-1012-Set A Cat# IP-202-1024- Set B PureLink RNA mini kit Ambion Cat# 12183018A Ribo-Zero rRNA removal kit Illumina Cat# 20037135 Cxcl12 ELISA kit R&D Cat# MCX120 Scf.



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    Screening of epigenetic bifunctionals yields CBP/p300 ligand GNE-781 as the strongest transactivator. A) Chemical architecture of a representative aTAG showing the FKBP F36V ligand (AP1867), the activator ligand (purple) and a linker; lower table summarizes the 13 aTAG molecules synthesized, the chromatin effector they recruit, reporter EC 50 values (note: for biphasic curves such as aTAG-5/aTAG-6, this represents the interpolated dose with maximal activation) and maximal activation (fold-change vs. untreated at a single dose) from 10-point dose titration in an IRF1 reporter system (48h treatment); U2OS reporter system stably expressing 1) a plasmid containing 4 repeats of the IRF1 consensus sequence upstream of firefly luciferase, 2) a plasmid containing doxycycline-inducible IRF1-FKBP F36V -mCherry in pLIX403. B) Scatter-plot of maximal activation versus potency for all compounds after 48h treatment. C) Chemical structures of linker series tested for GNE-781 bifunctionals. D) Extended dose curves of AP1867-GNE781 bifunctionals or binders alone (GNE-781, mAP1867-CONHEt) in IRF1 reporter system (normalized FLuc luminescence at 48 hours). E) Extended dose curves of aTAG-2 with binder competition in IRF1 reporter system (normalized FLuc luminescence at 48 hours). F-G) Normalized NLuc luminescence from NanoBiT experiment in 293T cells, titrated with aTAG-2 ± competitor or binder alone for 24 hours. NanoBiT constructs used were FKBP F36V -LgBiT and SmBiT fused to the C-terminus of either the CBP (F) or p300 (G) bromodomain.

    Journal: bioRxiv

    Article Title: An epigenetic bifunctional that toggles between transactivation and repression

    doi: 10.64898/2026.03.17.712509

    Figure Lengend Snippet: Screening of epigenetic bifunctionals yields CBP/p300 ligand GNE-781 as the strongest transactivator. A) Chemical architecture of a representative aTAG showing the FKBP F36V ligand (AP1867), the activator ligand (purple) and a linker; lower table summarizes the 13 aTAG molecules synthesized, the chromatin effector they recruit, reporter EC 50 values (note: for biphasic curves such as aTAG-5/aTAG-6, this represents the interpolated dose with maximal activation) and maximal activation (fold-change vs. untreated at a single dose) from 10-point dose titration in an IRF1 reporter system (48h treatment); U2OS reporter system stably expressing 1) a plasmid containing 4 repeats of the IRF1 consensus sequence upstream of firefly luciferase, 2) a plasmid containing doxycycline-inducible IRF1-FKBP F36V -mCherry in pLIX403. B) Scatter-plot of maximal activation versus potency for all compounds after 48h treatment. C) Chemical structures of linker series tested for GNE-781 bifunctionals. D) Extended dose curves of AP1867-GNE781 bifunctionals or binders alone (GNE-781, mAP1867-CONHEt) in IRF1 reporter system (normalized FLuc luminescence at 48 hours). E) Extended dose curves of aTAG-2 with binder competition in IRF1 reporter system (normalized FLuc luminescence at 48 hours). F-G) Normalized NLuc luminescence from NanoBiT experiment in 293T cells, titrated with aTAG-2 ± competitor or binder alone for 24 hours. NanoBiT constructs used were FKBP F36V -LgBiT and SmBiT fused to the C-terminus of either the CBP (F) or p300 (G) bromodomain.

    Article Snippet: Dynabeads Protein A (50 μL) were washed 3 times with TBS-T then pre-incubated with 10 μg of antibody (anti-HA tag antibody - ChIP Grade (ab9110), P300 (Bethyl, A300-358A), CBP (Bethyl, A300-362A)) at 4C for 3 hours in TBS-T (500 μL).

    Techniques: Synthesized, Activation Assay, Titration, Stable Transfection, Expressing, Plasmid Preparation, Sequencing, Luciferase, Construct

    aTAG-2 replaces p300 with CBP at EWS/FLI bound sites and degrades FKBP F36V -EWS/FLI in a E1/proteasome-dependent manner. A) FKBP F36V -HA-EWS/FLI EWS502 cells treated with DMSO, aTAG-2, or binders (mAP1867-CONHEt + GNE-781) at 10 nM for 16 hours were profiled by CBP, p300, and HA ChIP-seq. P300 and CBP occupancy heatmaps at Riggi et al. EWS/FLI bound sites are shown. B) Overlap between unique genes with consensus CBP ChIP-seq peaks gained by aTAG-2 (gained both vs. DMSO and vs. binders), and consensus p300 ChIP-seq peaks lost by aTAG-2. P -value calculated by hypergeometric test. C-D) Enrichr gene ontology analysis (C) and Homer motif analysis (D) of dual CBP-gained and p300-lost peaks/unique genes. E) Western blot for HA-tagged EWS/FLI in FKBP F36V -HA-EWS/FLI EWS502 cells treated with aTAG-2 at 10 nM for 3 hours in the presence of inhibitors (1 μM, except MG-132 at 10 μM; TAK-243: UBA1 inhibitor, TAK-981: SUMOylation inhibitor, MG-132: proteasome inhibitor, lanes are from the same blot & exposure). F) Western blot for HA-tagged EWS/FLI in FKBP F36V -HA-EWS/FLI EWS502 cells treated with aTAG-2 at 10 nM for 1 hour following TUBE2 (ubiquitin) pulldown, in the presence of MG-132 (10 μM). Total ubiquitin loading control is shown in . G) Model for aTAG-2 mechanism of action, where it can induce transactivation, partial target degradation, and inhibit p300/CBP through a RIPTAC-like mechanism depending on context.

    Journal: bioRxiv

    Article Title: An epigenetic bifunctional that toggles between transactivation and repression

    doi: 10.64898/2026.03.17.712509

    Figure Lengend Snippet: aTAG-2 replaces p300 with CBP at EWS/FLI bound sites and degrades FKBP F36V -EWS/FLI in a E1/proteasome-dependent manner. A) FKBP F36V -HA-EWS/FLI EWS502 cells treated with DMSO, aTAG-2, or binders (mAP1867-CONHEt + GNE-781) at 10 nM for 16 hours were profiled by CBP, p300, and HA ChIP-seq. P300 and CBP occupancy heatmaps at Riggi et al. EWS/FLI bound sites are shown. B) Overlap between unique genes with consensus CBP ChIP-seq peaks gained by aTAG-2 (gained both vs. DMSO and vs. binders), and consensus p300 ChIP-seq peaks lost by aTAG-2. P -value calculated by hypergeometric test. C-D) Enrichr gene ontology analysis (C) and Homer motif analysis (D) of dual CBP-gained and p300-lost peaks/unique genes. E) Western blot for HA-tagged EWS/FLI in FKBP F36V -HA-EWS/FLI EWS502 cells treated with aTAG-2 at 10 nM for 3 hours in the presence of inhibitors (1 μM, except MG-132 at 10 μM; TAK-243: UBA1 inhibitor, TAK-981: SUMOylation inhibitor, MG-132: proteasome inhibitor, lanes are from the same blot & exposure). F) Western blot for HA-tagged EWS/FLI in FKBP F36V -HA-EWS/FLI EWS502 cells treated with aTAG-2 at 10 nM for 1 hour following TUBE2 (ubiquitin) pulldown, in the presence of MG-132 (10 μM). Total ubiquitin loading control is shown in . G) Model for aTAG-2 mechanism of action, where it can induce transactivation, partial target degradation, and inhibit p300/CBP through a RIPTAC-like mechanism depending on context.

    Article Snippet: Dynabeads Protein A (50 μL) were washed 3 times with TBS-T then pre-incubated with 10 μg of antibody (anti-HA tag antibody - ChIP Grade (ab9110), P300 (Bethyl, A300-358A), CBP (Bethyl, A300-362A)) at 4C for 3 hours in TBS-T (500 μL).

    Techniques: ChIP-sequencing, Western Blot, Ubiquitin Proteomics, Control

    aTAG-2 replaces p300 with CBP at EWS/FLI bound sites. A) FKBP F36V -HA-EWS/FLI EWS502 cells treated with DMSO, aTAG-2, or binders (mAP1867-CONHEt + GNE-781) at 10 nM for 16 hours and profiled by CBP, p300, and HA ChIP-seq. P300 and CBP occupancy heatmaps at consensus aTAG-2 HA-EWS/FLI lost regions are shown (N=551, csaw-based differential binding). B) IGV tracks showing changes in ATAC, CBP, p300, and HA occupancy at the best characterized EWS/FLI target gene NR0B1 .

    Journal: bioRxiv

    Article Title: An epigenetic bifunctional that toggles between transactivation and repression

    doi: 10.64898/2026.03.17.712509

    Figure Lengend Snippet: aTAG-2 replaces p300 with CBP at EWS/FLI bound sites. A) FKBP F36V -HA-EWS/FLI EWS502 cells treated with DMSO, aTAG-2, or binders (mAP1867-CONHEt + GNE-781) at 10 nM for 16 hours and profiled by CBP, p300, and HA ChIP-seq. P300 and CBP occupancy heatmaps at consensus aTAG-2 HA-EWS/FLI lost regions are shown (N=551, csaw-based differential binding). B) IGV tracks showing changes in ATAC, CBP, p300, and HA occupancy at the best characterized EWS/FLI target gene NR0B1 .

    Article Snippet: Dynabeads Protein A (50 μL) were washed 3 times with TBS-T then pre-incubated with 10 μg of antibody (anti-HA tag antibody - ChIP Grade (ab9110), P300 (Bethyl, A300-358A), CBP (Bethyl, A300-362A)) at 4C for 3 hours in TBS-T (500 μL).

    Techniques: ChIP-sequencing, Binding Assay

    aTAG-2 degrades FKBP F36V -HA-tagged EWS/FLI on chromatin. A) FKBP F36V -HA-EWS/FLI EWS502 cells treated with DMSO, aTAG-2, or binders (mAP1867-CONHEt + GNE-781) at 10 nM for 16 hours and profiled by HA ChIP-seq. The number of HA peaks in MACS2-based peak calling is plotted between the conditions. B) Gene set enrichment analysis (Enrichr, GEO database) on unique genes corresponding to aTAG-2 HA-EWS/FLI consensus lost regions (N=551, csaw-based differential binding). C) Western blot for total ubiquitin (FK2) in FKBP F36V -HA-EWS/FLI EWS502 cells treated with aTAG-2 at 10 nM for 1 hour following TUBE2 (ubiquitin) pulldown, in the presence of MG-132 (10 μM). Loading control for . D) Western blot for HA-tagged EWS/FLI in FKBP F36V -HA-EWS/FLI EWS502 cells treated with aTAG-2 at 10 nM for 3 hours in the presence of p300/CBP histone acetyl transferase inhibitor A-485 (10 μM). E) Western blot for HA-tagged EWS/FLI and TRIM8 in FKBP F36V -HA-EWS/FLI EWS502 cells ± dox-inducible TRIM8 sgRNAs treated with aTAG-2 at 10 nM for 16 hours.

    Journal: bioRxiv

    Article Title: An epigenetic bifunctional that toggles between transactivation and repression

    doi: 10.64898/2026.03.17.712509

    Figure Lengend Snippet: aTAG-2 degrades FKBP F36V -HA-tagged EWS/FLI on chromatin. A) FKBP F36V -HA-EWS/FLI EWS502 cells treated with DMSO, aTAG-2, or binders (mAP1867-CONHEt + GNE-781) at 10 nM for 16 hours and profiled by HA ChIP-seq. The number of HA peaks in MACS2-based peak calling is plotted between the conditions. B) Gene set enrichment analysis (Enrichr, GEO database) on unique genes corresponding to aTAG-2 HA-EWS/FLI consensus lost regions (N=551, csaw-based differential binding). C) Western blot for total ubiquitin (FK2) in FKBP F36V -HA-EWS/FLI EWS502 cells treated with aTAG-2 at 10 nM for 1 hour following TUBE2 (ubiquitin) pulldown, in the presence of MG-132 (10 μM). Loading control for . D) Western blot for HA-tagged EWS/FLI in FKBP F36V -HA-EWS/FLI EWS502 cells treated with aTAG-2 at 10 nM for 3 hours in the presence of p300/CBP histone acetyl transferase inhibitor A-485 (10 μM). E) Western blot for HA-tagged EWS/FLI and TRIM8 in FKBP F36V -HA-EWS/FLI EWS502 cells ± dox-inducible TRIM8 sgRNAs treated with aTAG-2 at 10 nM for 16 hours.

    Article Snippet: Dynabeads Protein A (50 μL) were washed 3 times with TBS-T then pre-incubated with 10 μg of antibody (anti-HA tag antibody - ChIP Grade (ab9110), P300 (Bethyl, A300-358A), CBP (Bethyl, A300-362A)) at 4C for 3 hours in TBS-T (500 μL).

    Techniques: ChIP-sequencing, Binding Assay, Western Blot, Ubiquitin Proteomics, Control

    Screening of epigenetic bifunctionals yields CBP/p300 ligand GNE-781 as the strongest transactivator. A) Chemical architecture of a representative aTAG showing the FKBP F36V ligand (AP1867), the activator ligand (purple) and a linker; lower table summarizes the 13 aTAG molecules synthesized, the chromatin effector they recruit, reporter EC 50 values (note: for biphasic curves such as aTAG-5/aTAG-6, this represents the interpolated dose with maximal activation) and maximal activation (fold-change vs. untreated at a single dose) from 10-point dose titration in an IRF1 reporter system (48h treatment); U2OS reporter system stably expressing 1) a plasmid containing 4 repeats of the IRF1 consensus sequence upstream of firefly luciferase, 2) a plasmid containing doxycycline-inducible IRF1-FKBP F36V -mCherry in pLIX403. B) Scatter-plot of maximal activation versus potency for all compounds after 48h treatment. C) Chemical structures of linker series tested for GNE-781 bifunctionals. D) Extended dose curves of AP1867-GNE781 bifunctionals or binders alone (GNE-781, mAP1867-CONHEt) in IRF1 reporter system (normalized FLuc luminescence at 48 hours). E) Extended dose curves of aTAG-2 with binder competition in IRF1 reporter system (normalized FLuc luminescence at 48 hours). F-G) Normalized NLuc luminescence from NanoBiT experiment in 293T cells, titrated with aTAG-2 ± competitor or binder alone for 24 hours. NanoBiT constructs used were FKBP F36V -LgBiT and SmBiT fused to the C-terminus of either the CBP (F) or p300 (G) bromodomain.

    Journal: bioRxiv

    Article Title: An epigenetic bifunctional that toggles between transactivation and repression

    doi: 10.64898/2026.03.17.712509

    Figure Lengend Snippet: Screening of epigenetic bifunctionals yields CBP/p300 ligand GNE-781 as the strongest transactivator. A) Chemical architecture of a representative aTAG showing the FKBP F36V ligand (AP1867), the activator ligand (purple) and a linker; lower table summarizes the 13 aTAG molecules synthesized, the chromatin effector they recruit, reporter EC 50 values (note: for biphasic curves such as aTAG-5/aTAG-6, this represents the interpolated dose with maximal activation) and maximal activation (fold-change vs. untreated at a single dose) from 10-point dose titration in an IRF1 reporter system (48h treatment); U2OS reporter system stably expressing 1) a plasmid containing 4 repeats of the IRF1 consensus sequence upstream of firefly luciferase, 2) a plasmid containing doxycycline-inducible IRF1-FKBP F36V -mCherry in pLIX403. B) Scatter-plot of maximal activation versus potency for all compounds after 48h treatment. C) Chemical structures of linker series tested for GNE-781 bifunctionals. D) Extended dose curves of AP1867-GNE781 bifunctionals or binders alone (GNE-781, mAP1867-CONHEt) in IRF1 reporter system (normalized FLuc luminescence at 48 hours). E) Extended dose curves of aTAG-2 with binder competition in IRF1 reporter system (normalized FLuc luminescence at 48 hours). F-G) Normalized NLuc luminescence from NanoBiT experiment in 293T cells, titrated with aTAG-2 ± competitor or binder alone for 24 hours. NanoBiT constructs used were FKBP F36V -LgBiT and SmBiT fused to the C-terminus of either the CBP (F) or p300 (G) bromodomain.

    Article Snippet: Dynabeads Protein A (50 μL) were washed 3 times with TBS-T then pre-incubated with 10 μg of antibody (anti-HA tag antibody - ChIP Grade (ab9110), P300 (Bethyl, A300-358A), CBP (Bethyl, A300-362A)) at 4C for 3 hours in TBS-T (500 μL).

    Techniques: Synthesized, Activation Assay, Titration, Stable Transfection, Expressing, Plasmid Preparation, Sequencing, Luciferase, Construct

    aTAG-2 replaces p300 with CBP at EWS/FLI bound sites and degrades FKBP F36V -EWS/FLI in a E1/proteasome-dependent manner. A) FKBP F36V -HA-EWS/FLI EWS502 cells treated with DMSO, aTAG-2, or binders (mAP1867-CONHEt + GNE-781) at 10 nM for 16 hours were profiled by CBP, p300, and HA ChIP-seq. P300 and CBP occupancy heatmaps at Riggi et al. EWS/FLI bound sites are shown. B) Overlap between unique genes with consensus CBP ChIP-seq peaks gained by aTAG-2 (gained both vs. DMSO and vs. binders), and consensus p300 ChIP-seq peaks lost by aTAG-2. P -value calculated by hypergeometric test. C-D) Enrichr gene ontology analysis (C) and Homer motif analysis (D) of dual CBP-gained and p300-lost peaks/unique genes. E) Western blot for HA-tagged EWS/FLI in FKBP F36V -HA-EWS/FLI EWS502 cells treated with aTAG-2 at 10 nM for 3 hours in the presence of inhibitors (1 μM, except MG-132 at 10 μM; TAK-243: UBA1 inhibitor, TAK-981: SUMOylation inhibitor, MG-132: proteasome inhibitor, lanes are from the same blot & exposure). F) Western blot for HA-tagged EWS/FLI in FKBP F36V -HA-EWS/FLI EWS502 cells treated with aTAG-2 at 10 nM for 1 hour following TUBE2 (ubiquitin) pulldown, in the presence of MG-132 (10 μM). Total ubiquitin loading control is shown in . G) Model for aTAG-2 mechanism of action, where it can induce transactivation, partial target degradation, and inhibit p300/CBP through a RIPTAC-like mechanism depending on context.

    Journal: bioRxiv

    Article Title: An epigenetic bifunctional that toggles between transactivation and repression

    doi: 10.64898/2026.03.17.712509

    Figure Lengend Snippet: aTAG-2 replaces p300 with CBP at EWS/FLI bound sites and degrades FKBP F36V -EWS/FLI in a E1/proteasome-dependent manner. A) FKBP F36V -HA-EWS/FLI EWS502 cells treated with DMSO, aTAG-2, or binders (mAP1867-CONHEt + GNE-781) at 10 nM for 16 hours were profiled by CBP, p300, and HA ChIP-seq. P300 and CBP occupancy heatmaps at Riggi et al. EWS/FLI bound sites are shown. B) Overlap between unique genes with consensus CBP ChIP-seq peaks gained by aTAG-2 (gained both vs. DMSO and vs. binders), and consensus p300 ChIP-seq peaks lost by aTAG-2. P -value calculated by hypergeometric test. C-D) Enrichr gene ontology analysis (C) and Homer motif analysis (D) of dual CBP-gained and p300-lost peaks/unique genes. E) Western blot for HA-tagged EWS/FLI in FKBP F36V -HA-EWS/FLI EWS502 cells treated with aTAG-2 at 10 nM for 3 hours in the presence of inhibitors (1 μM, except MG-132 at 10 μM; TAK-243: UBA1 inhibitor, TAK-981: SUMOylation inhibitor, MG-132: proteasome inhibitor, lanes are from the same blot & exposure). F) Western blot for HA-tagged EWS/FLI in FKBP F36V -HA-EWS/FLI EWS502 cells treated with aTAG-2 at 10 nM for 1 hour following TUBE2 (ubiquitin) pulldown, in the presence of MG-132 (10 μM). Total ubiquitin loading control is shown in . G) Model for aTAG-2 mechanism of action, where it can induce transactivation, partial target degradation, and inhibit p300/CBP through a RIPTAC-like mechanism depending on context.

    Article Snippet: Dynabeads Protein A (50 μL) were washed 3 times with TBS-T then pre-incubated with 10 μg of antibody (anti-HA tag antibody - ChIP Grade (ab9110), P300 (Bethyl, A300-358A), CBP (Bethyl, A300-362A)) at 4C for 3 hours in TBS-T (500 μL).

    Techniques: ChIP-sequencing, Western Blot, Ubiquitin Proteomics, Control

    aTAG-2 decreases chromatin accessibility at p300 and EWS/FLI targets. A) FKBP F36V -HA-EWS/FLI EWS502 cells treated with DMSO, aTAG-2, or binders (mAP1867-CONHEt + GNE-781) at 5 nM for 16 hours and profiled by ATAC-seq. Chromatin accessibility changes at selected EWS/FLI targets are shown ( NR0B1, CCND1, FCGRT, GSTM4 ). B) HOMER motif analysis of consensus ATAC-seq peaks lost by aTAG-2 (lost in aTAG-2 vs. DMSO and aTAG-2 vs. binders comparisons). C) Enrichr gene ontology analysis on unique genes corresponding to consensus aTAG-2 lost ATAC-seq peaks (ChEA gene set database). D) Genomic location of consensus aTAG-2 lost ATAC-seq peaks or all ATAC-seq peaks (see Methods ) as reference. E) Overlap of FKBP F36V -HA-EWS/FLI peaks with p300 peaks (MACS2-based peak calling) in DMSO-treated FKBP F36V -HA-EWS/FLI EWS502 cells. P -value calculated by hypergeometric test. ATAC-seq signal heatmaps for HA/p300 co-bound peaks and p300-only bound peaks are shown. HA-only bound peaks are shown in .

    Journal: bioRxiv

    Article Title: An epigenetic bifunctional that toggles between transactivation and repression

    doi: 10.64898/2026.03.17.712509

    Figure Lengend Snippet: aTAG-2 decreases chromatin accessibility at p300 and EWS/FLI targets. A) FKBP F36V -HA-EWS/FLI EWS502 cells treated with DMSO, aTAG-2, or binders (mAP1867-CONHEt + GNE-781) at 5 nM for 16 hours and profiled by ATAC-seq. Chromatin accessibility changes at selected EWS/FLI targets are shown ( NR0B1, CCND1, FCGRT, GSTM4 ). B) HOMER motif analysis of consensus ATAC-seq peaks lost by aTAG-2 (lost in aTAG-2 vs. DMSO and aTAG-2 vs. binders comparisons). C) Enrichr gene ontology analysis on unique genes corresponding to consensus aTAG-2 lost ATAC-seq peaks (ChEA gene set database). D) Genomic location of consensus aTAG-2 lost ATAC-seq peaks or all ATAC-seq peaks (see Methods ) as reference. E) Overlap of FKBP F36V -HA-EWS/FLI peaks with p300 peaks (MACS2-based peak calling) in DMSO-treated FKBP F36V -HA-EWS/FLI EWS502 cells. P -value calculated by hypergeometric test. ATAC-seq signal heatmaps for HA/p300 co-bound peaks and p300-only bound peaks are shown. HA-only bound peaks are shown in .

    Article Snippet: Dynabeads Protein A (50 μL) were washed 3 times with TBS-T then pre-incubated with 10 μg of antibody (anti-HA tag antibody - ChIP Grade (ab9110), P300 (Bethyl, A300-358A), CBP (Bethyl, A300-362A)) at 4C for 3 hours in TBS-T (500 μL).

    Techniques:

    aTAG-2 induces targeted chromatin accessibility changes. FKBP F36V -HA-EWS/FLI EWS502 cells treated with DMSO, aTAG-2, or binders (mAP1867-CONHEt + GNE-781) at 5 nM for 16 hours and profiled by ATAC-seq. A) GGAA repeat length in consensus aTAG-2 lost peaks that are EWS/FLI bound (Riggi et al. EWS/FLI bound enhancers ) (left) or EWS/FLI-bound ATAC stable peaks (right). B) Enrichr gene ontology analysis on unique genes corresponding to top: consensus aTAG-2 lost ATAC-seq peaks, bottom: consensus aTAG-2 gained ATAC-seq peaks (GEO gene set database). C) Venn diagram of unique genes corresponding to consensus aTAG-2 lost/gained ATAC-seq peaks and EWS/FLI target gene sets from Kinsey et al. (upregulated or downregulated). P -values calculated by hypergeometric test. D) ATAC-seq heatmap across HA-bound, p300-unbound peaks are shown (determined using venn diagram in ).

    Journal: bioRxiv

    Article Title: An epigenetic bifunctional that toggles between transactivation and repression

    doi: 10.64898/2026.03.17.712509

    Figure Lengend Snippet: aTAG-2 induces targeted chromatin accessibility changes. FKBP F36V -HA-EWS/FLI EWS502 cells treated with DMSO, aTAG-2, or binders (mAP1867-CONHEt + GNE-781) at 5 nM for 16 hours and profiled by ATAC-seq. A) GGAA repeat length in consensus aTAG-2 lost peaks that are EWS/FLI bound (Riggi et al. EWS/FLI bound enhancers ) (left) or EWS/FLI-bound ATAC stable peaks (right). B) Enrichr gene ontology analysis on unique genes corresponding to top: consensus aTAG-2 lost ATAC-seq peaks, bottom: consensus aTAG-2 gained ATAC-seq peaks (GEO gene set database). C) Venn diagram of unique genes corresponding to consensus aTAG-2 lost/gained ATAC-seq peaks and EWS/FLI target gene sets from Kinsey et al. (upregulated or downregulated). P -values calculated by hypergeometric test. D) ATAC-seq heatmap across HA-bound, p300-unbound peaks are shown (determined using venn diagram in ).

    Article Snippet: Dynabeads Protein A (50 μL) were washed 3 times with TBS-T then pre-incubated with 10 μg of antibody (anti-HA tag antibody - ChIP Grade (ab9110), P300 (Bethyl, A300-358A), CBP (Bethyl, A300-362A)) at 4C for 3 hours in TBS-T (500 μL).

    Techniques:

    aTAG-2 replaces p300 with CBP at EWS/FLI bound sites. A) FKBP F36V -HA-EWS/FLI EWS502 cells treated with DMSO, aTAG-2, or binders (mAP1867-CONHEt + GNE-781) at 10 nM for 16 hours and profiled by CBP, p300, and HA ChIP-seq. P300 and CBP occupancy heatmaps at consensus aTAG-2 HA-EWS/FLI lost regions are shown (N=551, csaw-based differential binding). B) IGV tracks showing changes in ATAC, CBP, p300, and HA occupancy at the best characterized EWS/FLI target gene NR0B1 .

    Journal: bioRxiv

    Article Title: An epigenetic bifunctional that toggles between transactivation and repression

    doi: 10.64898/2026.03.17.712509

    Figure Lengend Snippet: aTAG-2 replaces p300 with CBP at EWS/FLI bound sites. A) FKBP F36V -HA-EWS/FLI EWS502 cells treated with DMSO, aTAG-2, or binders (mAP1867-CONHEt + GNE-781) at 10 nM for 16 hours and profiled by CBP, p300, and HA ChIP-seq. P300 and CBP occupancy heatmaps at consensus aTAG-2 HA-EWS/FLI lost regions are shown (N=551, csaw-based differential binding). B) IGV tracks showing changes in ATAC, CBP, p300, and HA occupancy at the best characterized EWS/FLI target gene NR0B1 .

    Article Snippet: Dynabeads Protein A (50 μL) were washed 3 times with TBS-T then pre-incubated with 10 μg of antibody (anti-HA tag antibody - ChIP Grade (ab9110), P300 (Bethyl, A300-358A), CBP (Bethyl, A300-362A)) at 4C for 3 hours in TBS-T (500 μL).

    Techniques: ChIP-sequencing, Binding Assay

    aTAG-2 degrades FKBP F36V -HA-tagged EWS/FLI on chromatin. A) FKBP F36V -HA-EWS/FLI EWS502 cells treated with DMSO, aTAG-2, or binders (mAP1867-CONHEt + GNE-781) at 10 nM for 16 hours and profiled by HA ChIP-seq. The number of HA peaks in MACS2-based peak calling is plotted between the conditions. B) Gene set enrichment analysis (Enrichr, GEO database) on unique genes corresponding to aTAG-2 HA-EWS/FLI consensus lost regions (N=551, csaw-based differential binding). C) Western blot for total ubiquitin (FK2) in FKBP F36V -HA-EWS/FLI EWS502 cells treated with aTAG-2 at 10 nM for 1 hour following TUBE2 (ubiquitin) pulldown, in the presence of MG-132 (10 μM). Loading control for . D) Western blot for HA-tagged EWS/FLI in FKBP F36V -HA-EWS/FLI EWS502 cells treated with aTAG-2 at 10 nM for 3 hours in the presence of p300/CBP histone acetyl transferase inhibitor A-485 (10 μM). E) Western blot for HA-tagged EWS/FLI and TRIM8 in FKBP F36V -HA-EWS/FLI EWS502 cells ± dox-inducible TRIM8 sgRNAs treated with aTAG-2 at 10 nM for 16 hours.

    Journal: bioRxiv

    Article Title: An epigenetic bifunctional that toggles between transactivation and repression

    doi: 10.64898/2026.03.17.712509

    Figure Lengend Snippet: aTAG-2 degrades FKBP F36V -HA-tagged EWS/FLI on chromatin. A) FKBP F36V -HA-EWS/FLI EWS502 cells treated with DMSO, aTAG-2, or binders (mAP1867-CONHEt + GNE-781) at 10 nM for 16 hours and profiled by HA ChIP-seq. The number of HA peaks in MACS2-based peak calling is plotted between the conditions. B) Gene set enrichment analysis (Enrichr, GEO database) on unique genes corresponding to aTAG-2 HA-EWS/FLI consensus lost regions (N=551, csaw-based differential binding). C) Western blot for total ubiquitin (FK2) in FKBP F36V -HA-EWS/FLI EWS502 cells treated with aTAG-2 at 10 nM for 1 hour following TUBE2 (ubiquitin) pulldown, in the presence of MG-132 (10 μM). Loading control for . D) Western blot for HA-tagged EWS/FLI in FKBP F36V -HA-EWS/FLI EWS502 cells treated with aTAG-2 at 10 nM for 3 hours in the presence of p300/CBP histone acetyl transferase inhibitor A-485 (10 μM). E) Western blot for HA-tagged EWS/FLI and TRIM8 in FKBP F36V -HA-EWS/FLI EWS502 cells ± dox-inducible TRIM8 sgRNAs treated with aTAG-2 at 10 nM for 16 hours.

    Article Snippet: Dynabeads Protein A (50 μL) were washed 3 times with TBS-T then pre-incubated with 10 μg of antibody (anti-HA tag antibody - ChIP Grade (ab9110), P300 (Bethyl, A300-358A), CBP (Bethyl, A300-362A)) at 4C for 3 hours in TBS-T (500 μL).

    Techniques: ChIP-sequencing, Binding Assay, Western Blot, Ubiquitin Proteomics, Control

    Screening of epigenetic bifunctionals yields CBP/p300 ligand GNE-781 as the strongest transactivator. A) Chemical architecture of a representative aTAG showing the FKBP F36V ligand (AP1867), the activator ligand (purple) and a linker; lower table summarizes the 13 aTAG molecules synthesized, the chromatin effector they recruit, reporter EC 50 values (note: for biphasic curves such as aTAG-5/aTAG-6, this represents the interpolated dose with maximal activation) and maximal activation (fold-change vs. untreated at a single dose) from 10-point dose titration in an IRF1 reporter system (48h treatment); U2OS reporter system stably expressing 1) a plasmid containing 4 repeats of the IRF1 consensus sequence upstream of firefly luciferase, 2) a plasmid containing doxycycline-inducible IRF1-FKBP F36V -mCherry in pLIX403. B) Scatter-plot of maximal activation versus potency for all compounds after 48h treatment. C) Chemical structures of linker series tested for GNE-781 bifunctionals. D) Extended dose curves of AP1867-GNE781 bifunctionals or binders alone (GNE-781, mAP1867-CONHEt) in IRF1 reporter system (normalized FLuc luminescence at 48 hours). E) Extended dose curves of aTAG-2 with binder competition in IRF1 reporter system (normalized FLuc luminescence at 48 hours). F-G) Normalized NLuc luminescence from NanoBiT experiment in 293T cells, titrated with aTAG-2 ± competitor or binder alone for 24 hours. NanoBiT constructs used were FKBP F36V -LgBiT and SmBiT fused to the C-terminus of either the CBP (F) or p300 (G) bromodomain.

    Journal: bioRxiv

    Article Title: An epigenetic bifunctional that toggles between transactivation and repression

    doi: 10.64898/2026.03.17.712509

    Figure Lengend Snippet: Screening of epigenetic bifunctionals yields CBP/p300 ligand GNE-781 as the strongest transactivator. A) Chemical architecture of a representative aTAG showing the FKBP F36V ligand (AP1867), the activator ligand (purple) and a linker; lower table summarizes the 13 aTAG molecules synthesized, the chromatin effector they recruit, reporter EC 50 values (note: for biphasic curves such as aTAG-5/aTAG-6, this represents the interpolated dose with maximal activation) and maximal activation (fold-change vs. untreated at a single dose) from 10-point dose titration in an IRF1 reporter system (48h treatment); U2OS reporter system stably expressing 1) a plasmid containing 4 repeats of the IRF1 consensus sequence upstream of firefly luciferase, 2) a plasmid containing doxycycline-inducible IRF1-FKBP F36V -mCherry in pLIX403. B) Scatter-plot of maximal activation versus potency for all compounds after 48h treatment. C) Chemical structures of linker series tested for GNE-781 bifunctionals. D) Extended dose curves of AP1867-GNE781 bifunctionals or binders alone (GNE-781, mAP1867-CONHEt) in IRF1 reporter system (normalized FLuc luminescence at 48 hours). E) Extended dose curves of aTAG-2 with binder competition in IRF1 reporter system (normalized FLuc luminescence at 48 hours). F-G) Normalized NLuc luminescence from NanoBiT experiment in 293T cells, titrated with aTAG-2 ± competitor or binder alone for 24 hours. NanoBiT constructs used were FKBP F36V -LgBiT and SmBiT fused to the C-terminus of either the CBP (F) or p300 (G) bromodomain.

    Article Snippet: Dynabeads Protein A (50 μL) were washed 3 times with TBS-T then pre-incubated with 10 μg of antibody (anti-HA tag antibody - ChIP Grade (ab9110), P300 (Bethyl, A300-358A), CBP (Bethyl, A300-362A)) at 4C for 3 hours in TBS-T (500 μL).

    Techniques: Synthesized, Activation Assay, Titration, Stable Transfection, Expressing, Plasmid Preparation, Sequencing, Luciferase, Construct

    aTAG-2 replaces p300 with CBP at EWS/FLI bound sites and degrades FKBP F36V -EWS/FLI in a E1/proteasome-dependent manner. A) FKBP F36V -HA-EWS/FLI EWS502 cells treated with DMSO, aTAG-2, or binders (mAP1867-CONHEt + GNE-781) at 10 nM for 16 hours were profiled by CBP, p300, and HA ChIP-seq. P300 and CBP occupancy heatmaps at Riggi et al. EWS/FLI bound sites are shown. B) Overlap between unique genes with consensus CBP ChIP-seq peaks gained by aTAG-2 (gained both vs. DMSO and vs. binders), and consensus p300 ChIP-seq peaks lost by aTAG-2. P -value calculated by hypergeometric test. C-D) Enrichr gene ontology analysis (C) and Homer motif analysis (D) of dual CBP-gained and p300-lost peaks/unique genes. E) Western blot for HA-tagged EWS/FLI in FKBP F36V -HA-EWS/FLI EWS502 cells treated with aTAG-2 at 10 nM for 3 hours in the presence of inhibitors (1 μM, except MG-132 at 10 μM; TAK-243: UBA1 inhibitor, TAK-981: SUMOylation inhibitor, MG-132: proteasome inhibitor, lanes are from the same blot & exposure). F) Western blot for HA-tagged EWS/FLI in FKBP F36V -HA-EWS/FLI EWS502 cells treated with aTAG-2 at 10 nM for 1 hour following TUBE2 (ubiquitin) pulldown, in the presence of MG-132 (10 μM). Total ubiquitin loading control is shown in . G) Model for aTAG-2 mechanism of action, where it can induce transactivation, partial target degradation, and inhibit p300/CBP through a RIPTAC-like mechanism depending on context.

    Journal: bioRxiv

    Article Title: An epigenetic bifunctional that toggles between transactivation and repression

    doi: 10.64898/2026.03.17.712509

    Figure Lengend Snippet: aTAG-2 replaces p300 with CBP at EWS/FLI bound sites and degrades FKBP F36V -EWS/FLI in a E1/proteasome-dependent manner. A) FKBP F36V -HA-EWS/FLI EWS502 cells treated with DMSO, aTAG-2, or binders (mAP1867-CONHEt + GNE-781) at 10 nM for 16 hours were profiled by CBP, p300, and HA ChIP-seq. P300 and CBP occupancy heatmaps at Riggi et al. EWS/FLI bound sites are shown. B) Overlap between unique genes with consensus CBP ChIP-seq peaks gained by aTAG-2 (gained both vs. DMSO and vs. binders), and consensus p300 ChIP-seq peaks lost by aTAG-2. P -value calculated by hypergeometric test. C-D) Enrichr gene ontology analysis (C) and Homer motif analysis (D) of dual CBP-gained and p300-lost peaks/unique genes. E) Western blot for HA-tagged EWS/FLI in FKBP F36V -HA-EWS/FLI EWS502 cells treated with aTAG-2 at 10 nM for 3 hours in the presence of inhibitors (1 μM, except MG-132 at 10 μM; TAK-243: UBA1 inhibitor, TAK-981: SUMOylation inhibitor, MG-132: proteasome inhibitor, lanes are from the same blot & exposure). F) Western blot for HA-tagged EWS/FLI in FKBP F36V -HA-EWS/FLI EWS502 cells treated with aTAG-2 at 10 nM for 1 hour following TUBE2 (ubiquitin) pulldown, in the presence of MG-132 (10 μM). Total ubiquitin loading control is shown in . G) Model for aTAG-2 mechanism of action, where it can induce transactivation, partial target degradation, and inhibit p300/CBP through a RIPTAC-like mechanism depending on context.

    Article Snippet: Dynabeads Protein A (50 μL) were washed 3 times with TBS-T then pre-incubated with 10 μg of antibody (anti-HA tag antibody - ChIP Grade (ab9110), P300 (Bethyl, A300-358A), CBP (Bethyl, A300-362A)) at 4C for 3 hours in TBS-T (500 μL).

    Techniques: ChIP-sequencing, Western Blot, Ubiquitin Proteomics, Control

    aTAG-2 replaces p300 with CBP at EWS/FLI bound sites. A) FKBP F36V -HA-EWS/FLI EWS502 cells treated with DMSO, aTAG-2, or binders (mAP1867-CONHEt + GNE-781) at 10 nM for 16 hours and profiled by CBP, p300, and HA ChIP-seq. P300 and CBP occupancy heatmaps at consensus aTAG-2 HA-EWS/FLI lost regions are shown (N=551, csaw-based differential binding). B) IGV tracks showing changes in ATAC, CBP, p300, and HA occupancy at the best characterized EWS/FLI target gene NR0B1 .

    Journal: bioRxiv

    Article Title: An epigenetic bifunctional that toggles between transactivation and repression

    doi: 10.64898/2026.03.17.712509

    Figure Lengend Snippet: aTAG-2 replaces p300 with CBP at EWS/FLI bound sites. A) FKBP F36V -HA-EWS/FLI EWS502 cells treated with DMSO, aTAG-2, or binders (mAP1867-CONHEt + GNE-781) at 10 nM for 16 hours and profiled by CBP, p300, and HA ChIP-seq. P300 and CBP occupancy heatmaps at consensus aTAG-2 HA-EWS/FLI lost regions are shown (N=551, csaw-based differential binding). B) IGV tracks showing changes in ATAC, CBP, p300, and HA occupancy at the best characterized EWS/FLI target gene NR0B1 .

    Article Snippet: Dynabeads Protein A (50 μL) were washed 3 times with TBS-T then pre-incubated with 10 μg of antibody (anti-HA tag antibody - ChIP Grade (ab9110), P300 (Bethyl, A300-358A), CBP (Bethyl, A300-362A)) at 4C for 3 hours in TBS-T (500 μL).

    Techniques: ChIP-sequencing, Binding Assay

    aTAG-2 degrades FKBP F36V -HA-tagged EWS/FLI on chromatin. A) FKBP F36V -HA-EWS/FLI EWS502 cells treated with DMSO, aTAG-2, or binders (mAP1867-CONHEt + GNE-781) at 10 nM for 16 hours and profiled by HA ChIP-seq. The number of HA peaks in MACS2-based peak calling is plotted between the conditions. B) Gene set enrichment analysis (Enrichr, GEO database) on unique genes corresponding to aTAG-2 HA-EWS/FLI consensus lost regions (N=551, csaw-based differential binding). C) Western blot for total ubiquitin (FK2) in FKBP F36V -HA-EWS/FLI EWS502 cells treated with aTAG-2 at 10 nM for 1 hour following TUBE2 (ubiquitin) pulldown, in the presence of MG-132 (10 μM). Loading control for . D) Western blot for HA-tagged EWS/FLI in FKBP F36V -HA-EWS/FLI EWS502 cells treated with aTAG-2 at 10 nM for 3 hours in the presence of p300/CBP histone acetyl transferase inhibitor A-485 (10 μM). E) Western blot for HA-tagged EWS/FLI and TRIM8 in FKBP F36V -HA-EWS/FLI EWS502 cells ± dox-inducible TRIM8 sgRNAs treated with aTAG-2 at 10 nM for 16 hours.

    Journal: bioRxiv

    Article Title: An epigenetic bifunctional that toggles between transactivation and repression

    doi: 10.64898/2026.03.17.712509

    Figure Lengend Snippet: aTAG-2 degrades FKBP F36V -HA-tagged EWS/FLI on chromatin. A) FKBP F36V -HA-EWS/FLI EWS502 cells treated with DMSO, aTAG-2, or binders (mAP1867-CONHEt + GNE-781) at 10 nM for 16 hours and profiled by HA ChIP-seq. The number of HA peaks in MACS2-based peak calling is plotted between the conditions. B) Gene set enrichment analysis (Enrichr, GEO database) on unique genes corresponding to aTAG-2 HA-EWS/FLI consensus lost regions (N=551, csaw-based differential binding). C) Western blot for total ubiquitin (FK2) in FKBP F36V -HA-EWS/FLI EWS502 cells treated with aTAG-2 at 10 nM for 1 hour following TUBE2 (ubiquitin) pulldown, in the presence of MG-132 (10 μM). Loading control for . D) Western blot for HA-tagged EWS/FLI in FKBP F36V -HA-EWS/FLI EWS502 cells treated with aTAG-2 at 10 nM for 3 hours in the presence of p300/CBP histone acetyl transferase inhibitor A-485 (10 μM). E) Western blot for HA-tagged EWS/FLI and TRIM8 in FKBP F36V -HA-EWS/FLI EWS502 cells ± dox-inducible TRIM8 sgRNAs treated with aTAG-2 at 10 nM for 16 hours.

    Article Snippet: Dynabeads Protein A (50 μL) were washed 3 times with TBS-T then pre-incubated with 10 μg of antibody (anti-HA tag antibody - ChIP Grade (ab9110), P300 (Bethyl, A300-358A), CBP (Bethyl, A300-362A)) at 4C for 3 hours in TBS-T (500 μL).

    Techniques: ChIP-sequencing, Binding Assay, Western Blot, Ubiquitin Proteomics, Control

    ( A ) Venn diagram showing overlap of HOXB13, p300, and HDAC3 binding sites in LNCaP cells. ( B ) Heatmaps showing indicated ChIP-seq intensity centered (±2 kb) around the 4 clusters of overlapping sites identified in A . I, HOXB13-only sites; II, HOXB13 and HDAC3 co-binding sites; III, HOXB13 and p300 co-binding sites; IV, HOXB13, HDAC3, and p300 co-binding sites (HHP). Scale bar: enrichment intensity. ( C ) GO analysis genes near the HOXB13, HDAC3, and p300 co-binding site (region IV in A ), identified by GREAT, with gene regulatory domain definition within 100 kb. P values by binomial test. ( D ) IGV showing HOXB13, p300, HDAC3, and H3K27ac ChIP-seq signal at HOXB13-repressed genes KLK3 (top) and FASN (bottom) loci in LNCaP cells with indicated genetic aberrations. ( E ) IGV showing H3K27ac ChIP-seq signal at HOXB13-repressed genes KLK3 (top) and FASN (bottom) loci in control or HOXB13 -KD LNCaP cells with DMSO or CCS1477 treatment.

    Journal: JCI Insight

    Article Title: Targeting p300 and CBP abolishes HOXB13-loss-induced lipogenesis and tumor metastasis

    doi: 10.1172/jci.insight.195743

    Figure Lengend Snippet: ( A ) Venn diagram showing overlap of HOXB13, p300, and HDAC3 binding sites in LNCaP cells. ( B ) Heatmaps showing indicated ChIP-seq intensity centered (±2 kb) around the 4 clusters of overlapping sites identified in A . I, HOXB13-only sites; II, HOXB13 and HDAC3 co-binding sites; III, HOXB13 and p300 co-binding sites; IV, HOXB13, HDAC3, and p300 co-binding sites (HHP). Scale bar: enrichment intensity. ( C ) GO analysis genes near the HOXB13, HDAC3, and p300 co-binding site (region IV in A ), identified by GREAT, with gene regulatory domain definition within 100 kb. P values by binomial test. ( D ) IGV showing HOXB13, p300, HDAC3, and H3K27ac ChIP-seq signal at HOXB13-repressed genes KLK3 (top) and FASN (bottom) loci in LNCaP cells with indicated genetic aberrations. ( E ) IGV showing H3K27ac ChIP-seq signal at HOXB13-repressed genes KLK3 (top) and FASN (bottom) loci in control or HOXB13 -KD LNCaP cells with DMSO or CCS1477 treatment.

    Article Snippet: The antibodies used in this study include HOXB13 (Cell Signaling Technology, 90944T), PSA (Cell Signaling Technology, 2475T), p300 (Cell Signaling Technology, 54062S), CBP (Cell Signaling Technology, 7389S), FASN (Santa Cruz Biotechnology, sc-48357) for WB application, and p300 (Bethyl, A300-358A) and H3K27ac (Cell Signaling Technology, 8173S) for ChIP application.

    Techniques: Binding Assay, ChIP-sequencing, Control

    ( A ) Heatmap showing shHOXB13-induced genes that are dependent on p300 and CBP in LNCaP cells with HOXB13 KD and/or p300 / CBP KD. HOXB13-repressed genes were identified by DESeq2 with fold change ≥ 1.5 and adjusted P < 0.05. Color bar: z -score. ( B ) GO analysis of the genes identified in A . Top enriched molecular concepts are shown on the y -axis, while the x -axis indicates number of genes in each significant GO term. ( C ) Genome browser showing mRNA expression of HOXB13-target genes KLK3 and FASN in LNCaP cells with indicated treatment. ( D ) Western blot analysis of FASN, PSA, and HOXB13 expression in LNCaP cells with HOXB13 KD and/or p300 / CBP KD. ( E ) Heatmap showing shHOXB13-induced genes that are repressed by CCS1477 in LNCaP cells with HOXB13 KD and/or CCS1477 treatment. Control (sh Ctrl ) or HOXB13 -KD (sh HOXB13 ) cells were subjected to control (DMSO) or CCS1477 (250 nM) treatment for 48 hours, and RNA-seq was performed in triplicate. HOXB13-repressed genes were identified by DESeq2 with fold change ≥ 2 and adjusted P < 0.05. Color bar: z -score. ( F ) Western blot analysis of FASN, PSA, and HOXB13 expression in control or HOXB13 -KD LNCaP cells treated with DMSO or CCS1477. ( G ) GO analysis of the genes identified in E . Top enriched molecular concepts are shown on the y -axis, while the x -axis indicates enrichment significance. Lipid- and cell cycle–related GO terms are highlighted in Red. P values by 1-sided hypergeometric test.

    Journal: JCI Insight

    Article Title: Targeting p300 and CBP abolishes HOXB13-loss-induced lipogenesis and tumor metastasis

    doi: 10.1172/jci.insight.195743

    Figure Lengend Snippet: ( A ) Heatmap showing shHOXB13-induced genes that are dependent on p300 and CBP in LNCaP cells with HOXB13 KD and/or p300 / CBP KD. HOXB13-repressed genes were identified by DESeq2 with fold change ≥ 1.5 and adjusted P < 0.05. Color bar: z -score. ( B ) GO analysis of the genes identified in A . Top enriched molecular concepts are shown on the y -axis, while the x -axis indicates number of genes in each significant GO term. ( C ) Genome browser showing mRNA expression of HOXB13-target genes KLK3 and FASN in LNCaP cells with indicated treatment. ( D ) Western blot analysis of FASN, PSA, and HOXB13 expression in LNCaP cells with HOXB13 KD and/or p300 / CBP KD. ( E ) Heatmap showing shHOXB13-induced genes that are repressed by CCS1477 in LNCaP cells with HOXB13 KD and/or CCS1477 treatment. Control (sh Ctrl ) or HOXB13 -KD (sh HOXB13 ) cells were subjected to control (DMSO) or CCS1477 (250 nM) treatment for 48 hours, and RNA-seq was performed in triplicate. HOXB13-repressed genes were identified by DESeq2 with fold change ≥ 2 and adjusted P < 0.05. Color bar: z -score. ( F ) Western blot analysis of FASN, PSA, and HOXB13 expression in control or HOXB13 -KD LNCaP cells treated with DMSO or CCS1477. ( G ) GO analysis of the genes identified in E . Top enriched molecular concepts are shown on the y -axis, while the x -axis indicates enrichment significance. Lipid- and cell cycle–related GO terms are highlighted in Red. P values by 1-sided hypergeometric test.

    Article Snippet: The antibodies used in this study include HOXB13 (Cell Signaling Technology, 90944T), PSA (Cell Signaling Technology, 2475T), p300 (Cell Signaling Technology, 54062S), CBP (Cell Signaling Technology, 7389S), FASN (Santa Cruz Biotechnology, sc-48357) for WB application, and p300 (Bethyl, A300-358A) and H3K27ac (Cell Signaling Technology, 8173S) for ChIP application.

    Techniques: Expressing, Western Blot, Control, RNA Sequencing

    ( A ) IVIS imaging of intravenous PC-3M xenograft tumors at weeks 0 (left) and 7 (right) after inoculation. Mice ( n = 4 mice for pGIPZ vehicle group, n = 5 mice for the remaining groups) started treatment with CCS1477 on day 10. Heatmap shows IVIS signal intensity color scale. ( B and C ) Representative ex vivo IVIS images ( B , n = 4 mice for pGIPZ vehicle group, n = 5 mice for the remaining groups) and quantifications ( C ) of PC-3M tumor metastasis to the lung and liver. Heatmap shows IVIS signal intensity color scale. Data are mean ± SD. Indicated P values were calculated by unpaired, 2-sided t test. ( D ) Graphical model illustrating HOXB13 as a molecular coordinator balancing HDAC3/NCoR and p300/CBP to fine-tune lipogenic gene expression in PCa. In HOXB13-high PCa cells, HOXB13 recruits the HDAC3-NCoR complex to lipogenic enhancers, catalyzing H3K27ac deacetylation and maintaining lipogenic gene expression at a basal level. In HOXB13-low PCa, this balance is disrupted — reduced recruitment of HDAC3 and NCoR leads to increased p300 and CBP binding and elevated H3K27ac levels at lipogenic enhancers. This results in aberrant activation of lipogenic programs, and induction of EMT-promoting genes such as MMPs.

    Journal: JCI Insight

    Article Title: Targeting p300 and CBP abolishes HOXB13-loss-induced lipogenesis and tumor metastasis

    doi: 10.1172/jci.insight.195743

    Figure Lengend Snippet: ( A ) IVIS imaging of intravenous PC-3M xenograft tumors at weeks 0 (left) and 7 (right) after inoculation. Mice ( n = 4 mice for pGIPZ vehicle group, n = 5 mice for the remaining groups) started treatment with CCS1477 on day 10. Heatmap shows IVIS signal intensity color scale. ( B and C ) Representative ex vivo IVIS images ( B , n = 4 mice for pGIPZ vehicle group, n = 5 mice for the remaining groups) and quantifications ( C ) of PC-3M tumor metastasis to the lung and liver. Heatmap shows IVIS signal intensity color scale. Data are mean ± SD. Indicated P values were calculated by unpaired, 2-sided t test. ( D ) Graphical model illustrating HOXB13 as a molecular coordinator balancing HDAC3/NCoR and p300/CBP to fine-tune lipogenic gene expression in PCa. In HOXB13-high PCa cells, HOXB13 recruits the HDAC3-NCoR complex to lipogenic enhancers, catalyzing H3K27ac deacetylation and maintaining lipogenic gene expression at a basal level. In HOXB13-low PCa, this balance is disrupted — reduced recruitment of HDAC3 and NCoR leads to increased p300 and CBP binding and elevated H3K27ac levels at lipogenic enhancers. This results in aberrant activation of lipogenic programs, and induction of EMT-promoting genes such as MMPs.

    Article Snippet: The antibodies used in this study include HOXB13 (Cell Signaling Technology, 90944T), PSA (Cell Signaling Technology, 2475T), p300 (Cell Signaling Technology, 54062S), CBP (Cell Signaling Technology, 7389S), FASN (Santa Cruz Biotechnology, sc-48357) for WB application, and p300 (Bethyl, A300-358A) and H3K27ac (Cell Signaling Technology, 8173S) for ChIP application.

    Techniques: Imaging, Ex Vivo, Gene Expression, Binding Assay, Activation Assay