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: BAF complex-independent gene activation by SS18::SSX

doi: 10.64898/2026.01.26.701739

Figure Lengend Snippet: (A) Left: Immunofluorescence of HEK293T cells expressing wild-type SS18::SSX1 (FL) treated for 72h with either 500nM ACBI1 or 500nM dCBP- 1 (cyan) stained for H2AK119ub1 (H2Aub) (magenta). Images are representative of n = 2 biological replicates. Scale bar, 5 μm. Right: profile of fluorescence intensity of FL, treated for 72h with either 500nM ACBI1 or 500nM dCBP-1 over H2Aub or eGFP foci. Data represent the mean ± S.E.M of n = 2 biological replicates. For each replicate and condition, > 10 foci where profiled. R² indicates Pearson correlation coefficient. (B) Left: Immunofluorescence of HEK293T cells expressing eGFP or wild-type SS18::SSX1, either untreated (FL) (cyan) or treated for 72h with 500nM ACBI1 or 500nM dCBP-1 stained for SMARCC1 (magenta). Images are representative of n = 2 biological replicates. Scale bar, 5 μm. Right: Profile of fluorescence intensity. Data represent the mean ± S.E.M of n = 2 biological replicates. For each replicate, > 10 foci where profiled. R² indicates Pearson correlation coefficient.

Article Snippet: The SMARCA2/SMARCA4 degrader ACBI1 (2375564-55-7) and the EP300/CREBBP degrader dCBP-1 (2484739-25-3) were purchased from MedChemExpress, resuspended in dimethyl sulfoxide (DMSO) and kept at −80°C.

Techniques: Immunofluorescence, Expressing, Staining, Fluorescence

Journal: bioRxiv

Article Title: BAF complex-independent gene activation by SS18::SSX

doi: 10.64898/2026.01.26.701739

Figure Lengend Snippet: (A) Left: Immunofluorescence of HEK293T cells expressing eGFP or wild-type SS18::SSX1, either untreated (FL) (cyan) or treated for 72h with 500nM ACBI1 or 500nM dCBP-1 stained for EP300 (magenta). Images are representative of n = 2 biological replicates. Scale bar, 5 μm. Right: Profile of fluorescence intensity. Data represent the mean ± S.E.M of n = 2 biological replicates. For each replicate, > 10 foci where profiled. R² indicates Pearson correlation coefficient. (B ) Western Blot of KHOS-240S whole cell extracts treated for 24h with DMSO, 500nM or 1uM dCBP- 1 revealed using EP300, CREBBP or β-actin antibodies. (C) RNA-seq heatmap showing the 1,000 most variable genes with a cutoff z-score of 4 from KHOS-240S cells expressing eGFP or SS18::SSX1 (FL) treated for 72h with either DMSO, 500nM ACBI1 or 500nM dCBP-1. n = 2 biological replicates. ( D) Log2-transformed fold change of FPKM values in KHOS-240S RNA-seq relative to eGFP over selected SS18::SSX target genes. Data represents the mean. (E) Scatter plot of log2-transformed fold change of FPKM values in KHOS-240S RNA-seq relative to eGFP over genes up-regulated by SS18::SSX1 expression and showing >2 fold up-regulation. Biological replicates are combined using their mean. Bold line represents median and dotted line represents cutoff of 2. (F) RNA-seq heatmap showing the 1,000 most variable genes with a cutoff z-score of 4 from wild-type hMSC or cells expressing eGFP-SSX1, eGFP-SS18::SSX1 isoform1, eGFP-SS18::SSX1 isoform2, eGFP-EWSR1::SSX1 or eGFP- EPC1::SSX1 treated for 48h with 500nM dCBP-1. n = 2 biological replicates. (G) Top: Immunofluorescence of HEK293T cells expressing wild-type eGFP-SSX1, eGFP-SS18::SSX1 isoform1, eGFP-SS18::SSX1 isoform2, eGFP-EWSR1::SSX1 or eGFP-EPC1::SSX1(cyan) stained for EP300 (magenta). Images are representative of n = 2 biological replicates. Scale bar, 5 μm. Bottom: profile of fluorescence intensity of eGFP constructs over SSX-rich eGFP foci. Data represent the mean ± S.E.M of n = 2 biological replicates. For each replicate and condition, > 10 foci where profiled. R² and r indicate Pearson correlation coefficients. (H) Scatter plot of log2-transformed fold change of CPM values in hMSC RNA-seq relative to untreated hMSC over the union genes up-regulated by SS18::SSX1 isoform1 (log2FC >2) and up-regulated by EPC1::SSX1 (log2FC >1). Biological replicates are combined using their mean. Bold line represents median. (I) Log2-transformed fold change of CPM values in hMSC RNA-seq relative to untreated cells over selected SSX target genes. Data represents the mean. (J) qRT-PCR displaying log2- transformed fold change of mRNA levels normalised by GAPDH in HSSY-II, SYO-1, CME-1 and Yamato-SS cells treated for 72h with 500nM dCBP-1 relative to DMSO. Data represents the mean of n = 2 biological replicates. (K) Viability of 4 synovial sarcoma cell lines (Yamato-SS, SYO-1, CME-1, HSSY-II) and KHOS-240S osteosarcoma cells treated with various concentrations of dCBP-1. The symbols represent the mean ± S.E.M of n = 3 biological replicates, the solid line represent the nonlinear curve fitting and its associated IC50. p-value represents extra sum-of- squares F test between KHOS-240S and SYO-1 nonlinear regression curves.

Article Snippet: The SMARCA2/SMARCA4 degrader ACBI1 (2375564-55-7) and the EP300/CREBBP degrader dCBP-1 (2484739-25-3) were purchased from MedChemExpress, resuspended in dimethyl sulfoxide (DMSO) and kept at −80°C.

Techniques: Immunofluorescence, Expressing, Staining, Fluorescence, Western Blot, RNA Sequencing, Transformation Assay, Construct, Quantitative RT-PCR

Journal: bioRxiv

Article Title: A modular toolbox for in cellulo screening of small molecule inhibitors targeting chromatin reader domains

doi: 10.1101/2025.09.06.674632

Figure Lengend Snippet: (A) Schematic showing how the bromodomains of CBP and p300 are being used as chromatin readers in the Parbit system, relative to their full-length endogenous proteins. (B-E) Immunofluorescence analysis of mESCs showing nuclear localization of CBP and p300 constructs after drug treatment. (B, D) Representative images of mESCs expressing either endogenous CBP-eGFP or p300-eGFP fusions (B) , or synthetic CBP/p300 Acyl-eCR constructs (CBP_BRD.1x/Parbit, p300_BRD.1x/Parbit, or eGFP/Parbit) (D) , with GFP signal in green and nuclear Hoechst staining in magenta. Cells were treated with DMSO, GNE-049, or dCBP-1 at 1 µM for 24 hours. Scale bars: 5 µm. (C, E) Quantification of mean nuclear GFP intensity (arbitrary units) for endogenous fusions and Acyl-eCR constructs (E) . Data represent mean ± SD from 40 cells per condition. (F) Normalized FACS data showing the effects of drug treatments on cells either expressing endogenous proteins fused to eGFP or Acyl-eCR constructs. The percentage represents the GFP signal in treated cells as a ratio of the signal observed in untreated samples of the same cell type, after normalizing for drug-induced autofluorescence in wild-type cells. Drug treatments were performed for 24 hours.

Article Snippet: The CBP/p300 bromodomain inhibitor: GNE-049 (MedChemExpress, HY-108435), CBP/p300 PROTAC: dCBP-1 (MedChemExpress, HY-134582), BRD4 bromodomain inhibitor: (+)-JQ-1 (MedChemExpress, HY-13030), BRD4 PROTAC: ARV-825 (MedChemExpress, HY-16954), BRD9 bromodomain inhibitor: iBRD9 (MedChemExpress, HY-18975), and broad-spectrum bromodomain inhibitor: Bromosporine (MedChemExpress, HY-15815) were dissolved in DMSO and then diluted to 1μM in mESC media for 24-hour treatments, unless stated otherwise.

Techniques: Immunofluorescence, Construct, Expressing, Staining

Journal: bioRxiv

Article Title: A modular toolbox for in cellulo screening of small molecule inhibitors targeting chromatin reader domains

doi: 10.1101/2025.09.06.674632

Figure Lengend Snippet: (A) Schematic showing how the Parbit expression cassette is used to generate stably expressed Acyl-eCRs in mESCs. RMCE by Cre, followed by a double selection of ganciclovir and puromycin, was applied to generate these constructs at a defined site in the mouse genome. A CAG promoter drives the constitutive expression of the bromodomain of interest, which is fused to a nuclear localization signal (NLS), and an eGFP tag. This construct also fuses a biotin acceptor site to the N-terminus of the protein, which can be biotinylated in vivo by a bacterial BirA ligase. (B) Schematic diagram showing how CBP was endogenously tagged with an eGFP tag. A homology donor construct was generated by cloning a 900 bp upstream and a 1,048 bp (CBP) or 1,068 bp (p300) downstream homology arm flanking a 30 bp flexible GGS linker that was fused to an eGFP tag. This donor construct was co-transfected with a pX330 CRISPR-Cas9 plasmid, which had an sgRNA targeting the C-terminus of the CBP gene. (C-D) Sanger sequencing of genotyping PCR products from the C-terminus of the CBP (C) and p300 (D) loci, confirming the in-frame homologous integration of an eGFP tag. Data shown are from mESC clone #1 for both CBP and p300 tagging. (E) Flow cytometry data showing the eGFP signal from cell lines where either CBP or p300 were endogenously tagged with eGFP. Two clonal replicates for each protein tagging are shown. Cell lines were maintained in culture for more than two weeks to demonstrate stable expression of the eGFP fusion proteins. (F) Western blot of nuclear extracts from cell lines treated with 1 μM dCBP-1 PROTAC for the stated duration. An antibody against GFP was used to probe the eGFP tag on the Acyl-eCR constructs. The CBP_BRD.1x eCR runs at approximately 60 kDa, and the Empty-eGFP construct runs at 33 kDa. Non-specific bands are marked by an asterisk (*). Revert700 Total Protein Stain is used to show equal loading in lanes.

Article Snippet: The CBP/p300 bromodomain inhibitor: GNE-049 (MedChemExpress, HY-108435), CBP/p300 PROTAC: dCBP-1 (MedChemExpress, HY-134582), BRD4 bromodomain inhibitor: (+)-JQ-1 (MedChemExpress, HY-13030), BRD4 PROTAC: ARV-825 (MedChemExpress, HY-16954), BRD9 bromodomain inhibitor: iBRD9 (MedChemExpress, HY-18975), and broad-spectrum bromodomain inhibitor: Bromosporine (MedChemExpress, HY-15815) were dissolved in DMSO and then diluted to 1μM in mESC media for 24-hour treatments, unless stated otherwise.

Techniques: Expressing, Stable Transfection, Selection, Construct, In Vivo, Generated, Cloning, Transfection, CRISPR, Plasmid Preparation, Sequencing, Flow Cytometry, Western Blot, Staining

Journal: bioRxiv

Article Title: A modular toolbox for in cellulo screening of small molecule inhibitors targeting chromatin reader domains

doi: 10.1101/2025.09.06.674632

Figure Lengend Snippet: (A) Raw eGFP fluorescence intensity was measured in untreated cell lines expressing different acyl-eCR constructs after extended passaging to assess stable expression. Each dot represents an independent replicate measurement (n = 6 per construct). Black numbers denote the mean fluorescence intensity for each cell line. The y-axis is shown on a logarithmic scale. (B-C) Correlation between dCBP-1–mediated degradation of Acyl-eCR constructs and the structural similarity of their bromodomains to CBP or p300. Degradation efficiency (y-axis) was measured by flow cytometry as normalized GFP signal (%) after dCBP-1 treatment (data from ; n = 6 independent measurements per construct). Structural similarity to CBP/p300 bromodomains was quantified using (B) TM-score and (C) RMSD (root-mean-square deviation). Each data point represents the mean ± SD of six replicates for a single construct, with a linear regression line and corresponding Pearson correlation coefficient (r) shown.

Article Snippet: The CBP/p300 bromodomain inhibitor: GNE-049 (MedChemExpress, HY-108435), CBP/p300 PROTAC: dCBP-1 (MedChemExpress, HY-134582), BRD4 bromodomain inhibitor: (+)-JQ-1 (MedChemExpress, HY-13030), BRD4 PROTAC: ARV-825 (MedChemExpress, HY-16954), BRD9 bromodomain inhibitor: iBRD9 (MedChemExpress, HY-18975), and broad-spectrum bromodomain inhibitor: Bromosporine (MedChemExpress, HY-15815) were dissolved in DMSO and then diluted to 1μM in mESC media for 24-hour treatments, unless stated otherwise.

Techniques: Fluorescence, Expressing, Construct, Passaging, Flow Cytometry