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

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

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

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

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: 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