Journal: bioRxiv

Article Title: Functional remodeling of the parasubthalamic nucleus drives alcohol drinking escalation in dependence

doi: 10.64898/2026.04.30.722112

Figure Lengend Snippet: A. Experimental strategy for the measure of neuronal activation in the PSTN during withdrawal following a history of voluntary alcohol drinking combined, or not, with chronic intermittent alcohol vapor inhalation. BL, baseline; PV, post-vapor. B. Representative images of c-Fos immunoreactivity in the PSTN during early withdrawal. C. Alcohol intoxication reduced, while early withdrawal increased the number of c-Fos+ cells in the PSTN. Air-2BC vs. CIE-2BC: **, p<0.01; ***, p<0.001; ****, p<0.0001. D. Experimental strategy for the expression of designer receptors in PSTN neurons active in early withdrawal from CIE and their subsequent chemogenetic re-activation or inhibition. E. Alcohol intake escalation prior to 4-OHT administration. BL vs. PV4: *, p<0.05; ****, p<0.0001. F. Re-activating the early withdrawal PSTN ensemble reduced alcohol intake. Pairwise comparisons: **, p<0.01; ****, p<0.0001. G. Labeling of the early withdrawal PSTN ensemble by mCherry. H. Experimental strategy for the expression of designer receptors in PSTN neurons active in late withdrawal from CIE and their subsequent chemogenetic re-activation or inhibition. I. Alcohol intake escalation prior to 4-OHT administration. Air-2BC vs. CIE-2BC: **, p<0.01. J. Inhibiting the late withdrawal PSTN ensemble increased alcohol intake in Air-2BC but reduced it in CIE-2BC mice. Pairwise comparisons: **, p<0.01; ***, p<0.001; ****, p<0.0001. K. Re-activating the late withdrawal PSTN ensemble reduced alcohol intake in Air-2BC but increased it in CIE-2BC mice. Pairwise comparisons: *, p<0.05; ****, p<0.0001. L. Labeling of the late withdrawal PSTN ensemble by mCherry and mCitrine. See also Figure S1 and Table S2.

Article Snippet: 4-hydroxytamoxifen (4-OHT) was obtained from Hello Bio (HB6040) and dissolved in 200 proof ethanol at 20 mg/mL with 30-min agitation at 37°C.

Techniques: Activation Assay, Expressing, Inhibition, Labeling

Journal: Cell Death & Disease

Article Title: The EHMT2-MBLAC2 axis suppresses ribosomal DNA transcription in response to nucleolar DNA damage

doi: 10.1038/s41419-026-08616-1

Figure Lengend Snippet: A Schematic illustration of 45S rDNA repeats with I- Ppo I endonuclease targeting sequence. Cells were treated with 4-OHT and Shield-1 to induce the expression of I- Ppo I endonuclease leading to the introduction of DSBs into 28S rDNA arrays and the subsequent suppression of rDNA transcription. B The formation of EHMT2 nucleolar caps in HeLa I- Ppo I cells following rDNA DSBs induction. HeLa I-Ppo I cells transfected with EGFP-EHMT2 plasmid were treated with 1 μM Shield-1 and 2 μM 4-OHT for 4 h to induce rDNA DSBs before fixation. Fixed cells were labeled with 53BP1 or γH2AX. Nuclei were counterstained with DAPI. Enlarged images depict the details of the dashed circle labeled nuclei. Quantification of relative signal intensities of EGFP-EHMT2, 53BP1 and γH2AX caps was performed by ImageJ using the ROI tool. The white lines in the enlargements of the representative images indicate the lines for quantification. The edge of the indicated nucleolar caps was labeled with dotted circle. C Analysis of nucleolar transcription activity by 5-Ethynyl Uridine (EU) incorporation assay in EHMT2-deficient HeLa I- Ppo I cells following rDNA DSBs induction. Cells transfected with control (siCTR) or two independent EHMT2-targeted siRNAs were induced for rDNA DSBs for 4 h. Cells were subsequently cultured in medium supplemented with EU for 1 h before fixation. Fixed cells were labeled with EU, nucleolar marker Fibrillarin (FBL) and DSB marker γH2AX. Quantification of relative EU nucleolar intensity from three independent experiments is shown in Tukey boxplots. D HeLa I- Ppo I cells transduced with control gRNA (CTR gRNA) and two EHMT2 gRNAs (EHMT2 KO1 and EHMT2 KO2) were subjected to EU incorporation assay as described in ( C ). Quantification of relative EU nucleolar intensity from three independent experiments is shown in Tukey boxplots. E siRNA-mediated EHMT2 knockdown efficiency was measured by Western blotting. F CRISPR/Cas9-mediated EHMT2 knockout efficiency was measured by immunoblotting. G HeLa I- Ppo I cells transduced with control gRNA (CTR gRNA) and two EHMT2 gRNAs (EHMT2 KO1 and EHMT2 KO2) were treated with 1 μM Shield-1 and 2 μM 4-OHT to induce rDNA DSBs for 3 h, and left to rest on ice for 10 minutes, then were further treated with 1 μM of Shield-1 and 2 μM of 4-OHT solution for 1 h. Quantification of 45S pre-rRNA fold change is shown and data were from four independent experiments. Bars represent mean ± SEM; ns not significant; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: At 24 h after seeding, cells were treated with Shield-1 (Selleck, #S3469, 1 μM) and 4-OHT (Selleck, #S8956, 2 μM) for 5, 10 or 20 min to induce rDNA DSBs.

Techniques: Sequencing, Expressing, Transfection, Plasmid Preparation, Labeling, Activity Assay, Control, Cell Culture, Marker, Transduction, Knockdown, Western Blot, CRISPR, Knock-Out

Journal: Cell Death & Disease

Article Title: The EHMT2-MBLAC2 axis suppresses ribosomal DNA transcription in response to nucleolar DNA damage

doi: 10.1038/s41419-026-08616-1

Figure Lengend Snippet: A HeLa I- Ppo I cells lenti-virally transduced with control gRNA (CTR gRNA) and EHMT2-targeting gRNAs (EHMT2 KO1 and EHMT2 KO2) were induced with Shield-1 and 4-OHT for 4 h to introduce DSBs into rDNAs. Cells were subjected to neutral comet assay at 6 h and 24 h after recovery from 4 h-rDNA DSBs induction. Relative tail moment from three independent experiments were analyzed and plotted. B The schematic illustration depicts the workflow of micronuclei counting in response to rDNA DSBs. C Representative images depict the micronuclei derived from undamaged and I- Ppo I induced cells treated with ATM or EHMT2 inhibitors. Quantification shows the percentage of cells with micronuclei. Data from three independent experiments were quantified. White arrowheads indicate the locations of micronuclei. D Schematic diagram represents 45S rDNA repeats. I- Ppo I targeted sequence is shown. The labels and the coverage indicated by the arrowheads suggest the primers used in the following quantification of rDNA copy number in I- Ppo I survival cells. E The flow diagram describes the procedure of quantification of rDNA copy number in I- Ppo I survivor cells. F The relative rDNA copy number in EHMT2-deficient GES1 I- Ppo I survival cells was measured and quantified as depicted in ( G ). Data were derived from three independent experiments. G CRISPR–Cas9-mediated EHMT2 knockout (KO) efficiency was validated by Western blotting. H Clonogenic survival of EHMT2 KO HeLa I- Ppo I cells were quantified upon I- Ppo I activation. Briefly, rDNA DSBs in cells were induced with Shield-1 and 4-OHT for 5, 10, or 20 min. Cells were then washed with PBS twice and were cultured to grow for ten days before subjecting to Coomassie blue staining. Bars represent mean ± SEM; ns not significant; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: At 24 h after seeding, cells were treated with Shield-1 (Selleck, #S3469, 1 μM) and 4-OHT (Selleck, #S8956, 2 μM) for 5, 10 or 20 min to induce rDNA DSBs.

Techniques: Transduction, Control, Introduce, Neutral Comet Assay, Derivative Assay, Sequencing, CRISPR, Knock-Out, Western Blot, Activation Assay, Cell Culture, Staining

Journal: Cell Death & Disease

Article Title: The EHMT2-MBLAC2 axis suppresses ribosomal DNA transcription in response to nucleolar DNA damage

doi: 10.1038/s41419-026-08616-1

Figure Lengend Snippet: A Schematic diagram of 4D-FastDIA quantitative proteomics analysis of EHMT2 substrates. B Principal Component Analysis (PCA) of samples from EHMT2 gRNA CTR and EHMT2 KO2 HeLa I- Ppo I cells. C Differentially expressed proteins in the EHMT2 gRNA CTR and EHMT2 KO2 HeLa I- Ppo I cells were classified for subcellular structural mapping. D Heatmaps of protein expression levels in HeLa I- Ppo I cells after rDNA DSBs showing differences between EHMT2 gRNA CTR and EHMT2 KO2 HeLa I- Ppo I cells. E Volcano plot showing the fold changes of all identified proteins and P -value in the cells between EHMT2 gRNA CTR and EHMT2 KO2 groups. F shRNA-based validation screen for EHMT2 targets involved in rDNA DSB-induced transcriptional suppression. HeLa I- Ppo I cells were infected with the indicated lenti-shRNA viruses. rDNA DSBs in cells were then induced with Shield-1 and 4-OHT and were subsequently subjected to EU incorporation assay. Relative nucleolar EU intensity was quantified from three independent experiments. G The knockdown efficiencies of the targeted shRNAs were validated by RT-qPCR. Data were from three independent experiments. Bars represent mean ± SEM; ns not significant; ** P < 0.01; **** P < 0.0001.

Article Snippet: At 24 h after seeding, cells were treated with Shield-1 (Selleck, #S3469, 1 μM) and 4-OHT (Selleck, #S8956, 2 μM) for 5, 10 or 20 min to induce rDNA DSBs.

Techniques: Quantitative Proteomics, Expressing, shRNA, Biomarker Discovery, Infection, Knockdown, Quantitative RT-PCR

Journal: Cell Death & Disease

Article Title: The EHMT2-MBLAC2 axis suppresses ribosomal DNA transcription in response to nucleolar DNA damage

doi: 10.1038/s41419-026-08616-1

Figure Lengend Snippet: A The cellular localization of MBLAC2 in HeLa I- Ppo I cells following rDNA DSBs induction. HeLa I- Ppo I cells were transfected with EGFP-MBLAC2 and were treated with Shield-1 and 4-OHT for 4 h. Fixed cells were labeled with anti-53BP1, anti-γH2AX or anti-C23 antibodies. Nuclei were counterstained with DAPI. Enlarged images show the details of the indicated proteins. Quantification of relative signal intensities of EGFP-EHMT2, 53BP1, γH2AX and C23 was performed by ImageJ. The white lines in the enlargements of the representative images indicate the lines for analysis. The edge of the indicated nucleolar caps was labeled with dotted circle. B Analysis of nucleolar transcription activity by EU incorporation assay in MBLAC2-inactivated HeLa I- Ppo I cells following rDNA DSBs induction. Cells transduced with control (shCTR) or two independent MBLAC2-targeted shRNAs were induced for rDNA DSBs for 4 h. EU nucleolar intensity was subsequently determined by EU incorporation assay. At least 200 cells exhibiting well-circumscribed nucleoli were quantitatively assessed across two independent experiments. Quantification of relative EU nucleolar intensity is shown in Tukey boxplots. C Nucleolar EU intensities were analyzed in HeLa I- Ppo I cells treated with the EHMT2 gRNA or MBLAC2 siRNA after I- Ppo I induction. Relative nucleolar EU intensity was quantified from at least two independent experiments. Immunoblot of MBLAC2 and EHMT2 in the HeLa I- Ppo I cells induced with the indicated gRNA or siRNA. D Colony survival of HeLa I- Ppo I cells transfected with CTR siRNA and siRNA targeting MBLAC2 or EHMT2 following I- Ppo I induction, respectively. Cells were induced for rDNA DSBs for 5 min. After washing with PBS twice, cells were allowed to grow for two weeks before harvest and Coomassie blue staining. The relative outgrowth of the colonies between groups were quantified and plotted. The protein expression of MBLAC2 and EHMT2 in HeLa I- Ppo I cells transfected with the indicated siRNAs were examined by immunoblot. E Immunoblot of MBLAC2 and EHMT2 in the HeLa I- Ppo I cells transduced with control gRNA (CTR gRNA) and two EHMT2 gRNAs (EHMT2 KO1 and EHMT2 KO2). F shRNA-mediated MBLAC2 knockdown efficiency was measured by Western blotting. G The EHMT2-MBLAC2 interaction was confirmed by Co-immunoprecipitation (Co-IP). Flag-DYRK1B was used as the positive control. H HeLa I- Ppo I cells transduced with control gRNA (CTR gRNA) and two EHMT2 gRNAs (EHMT2 KO1 and EHMT2 KO2) were treated with cycloheximide for 0, 5, and 10 h. The protein expression of MBLAC2 and EHMT2 in HeLa I- Ppo I cells was examined by immunoblot. I The relative MBLAC2 protein level in HeLa I-PpoI cells treated with cycloheximide as depicted in ( A ) was measured. Data were derived from three independent experiments. J HeLa I-PpoI cells were transduced with control gRNA (CTR gRNA) and two EHMT2 gRNAs (EHMT2 KO1 and EHMT2 KO2). Fold change of MBLAC2 mRNA in I- Ppo I cells treated with cycloheximide was determined by RT-qPCR. Quantification of MBLAC2 mRNA fold change was from three independent experiments. K Proposed working model of EHMT2-MBLAC2 axis in promoting rDNA DSB-induced transcriptional suppression. Bars represent mean ± SEM; ns not significant; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: At 24 h after seeding, cells were treated with Shield-1 (Selleck, #S3469, 1 μM) and 4-OHT (Selleck, #S8956, 2 μM) for 5, 10 or 20 min to induce rDNA DSBs.

Techniques: Transfection, Labeling, Activity Assay, Transduction, Control, Western Blot, Staining, Expressing, shRNA, Knockdown, Immunoprecipitation, Co-Immunoprecipitation Assay, Positive Control, Derivative Assay, Quantitative RT-PCR

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) Cell morphology under microscope at day 5 (D5) of myogenesis. (B) qRT-PCR analysis of myogenic markers Myog and Ckm at D0 and D5 of myogenesis. Data represents mean ± SD, **** p ≤ 0.0001 (C-G) MyoD-ER expressing preadipocytes were treated with 4-OHT during myogenesis. Cells were harvested for CUT&RUN at indicated time points. (C) HOMER motif analysis of induced MyoD binding sites. All regions were used, and only the top 2 motifs are shown. (D) Genome browser view of MyoD binding around the Myog locus. (E) Venn diagrams depicting 36,425 4-OHT-induced MyoD binding sites used for k-means clustering. (F) Heat maps of K-means clustering depicting MyoD binding dynamics on 36,425 sites defined in (E). (G) GO analysis of genes associated with MyoD binding sites in each cluster defined in (F).

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: Microscopy, Quantitative RT-PCR, Expressing, 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) 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) 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) Cell morphology under microscope at day 5 (D5) of myogenesis. (B) qRT-PCR analysis of myogenic markers Myog and Ckm at D0 and D5 of myogenesis. Data represents mean ± SD, **** p ≤ 0.0001 (C-G) MyoD-ER expressing preadipocytes were treated with 4-OHT during myogenesis. Cells were harvested for CUT&RUN at indicated time points. (C) HOMER motif analysis of induced MyoD binding sites. All regions were used, and only the top 2 motifs are shown. (D) Genome browser view of MyoD binding around the Myog locus. (E) Venn diagrams depicting 36,425 4-OHT-induced MyoD binding sites used for k-means clustering. (F) Heat maps of K-means clustering depicting MyoD binding dynamics on 36,425 sites defined in (E). (G) GO analysis of genes associated with MyoD binding sites in each cluster defined in (F).

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: Microscopy, Quantitative RT-PCR, Expressing, 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) 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