brd2  (Proteintech)

Journal: Nucleic Acids Research

Article Title: INO80/SWR remodelers regulate Pol II transcription through BRD2 and chromatin landscape

doi: 10.1093/nar/gkaf892

Figure Lengend Snippet: INO80/SWR remodelers regulate Pol II transcription via H2A.Zac reader BRD2. ( A ). Radar plot showing the coefficient from GLM model (see method for detail). ( B ). The bar chart displaying the coefficients of the top 15 factors with the largest absolute coefficient from the GLM model. ( C ). Some factors selected from the GLM model are enriched in ChIP-MS of INO80, P400, and SRCAP. ( D ). Top: A schematic diagram illustrating the Pol II-TurboID system in mESCs upon INO80, P400, and SRCAP degradation. Bottom: Changes in the interaction of acetyllysine reader proteins, acetyltransferases, and demethylases with Pol II identified by Turbo-ID MS upon INO80, P400, or SRCAP degradation. The color bar indicates log2 fold change. ( E ). Meta-analysis showing the average values of TT-seq and BRD2 ChIP-Seq signals, for direct target genes and all active genes before and after 1 h of INO80, P400, and SRCAP degradation. Blue indicates pre-degradation, and red indicates post-degradation. Box plots display the log2 fold changes of TT-seq and BRD2 ChIP-Seq signals for direct target genes ( n = 192, 584, 1 278 for INO80, P400, and SRCAP, respectively) and all genes ( n = 12 108). Red indicates direct target genes, and grey indicates all genes. Statistical analysis was determined using the Wilcoxon test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. The log2 fold change values were displayed above the box plot.

Article Snippet: 1 μl GFP abs (ABcam, ab290), 1 μg RPB1 NTD abs (CST, 14958S),1 μl BRD2 abs (Proteintech, 22236–1-AP), and equal isotype IgG control for each ChIP reaction, respectively.

Techniques: ChIP-sequencing

Journal: Nucleic Acids Research

Article Title: INO80/SWR remodelers regulate Pol II transcription through BRD2 and chromatin landscape

doi: 10.1093/nar/gkaf892

Figure Lengend Snippet: P400 and SRCAP help maintain H2A.Zac chromatin occupancy. INO80 appears to utilize an H2A.Zac-independent mechanism to regulate BRD2 and regulate the occupancy of P400 and SRCAP. ( A ). Western blot showing total H2A.Z acetylation levels at different time points of INO80, P400, and SRCAP degradation. Note: The Western blot results have been repeated three times, consistently showing a similar trend. ( B ). Meta-analysis showing the average values of H2A.Z, H2A.Z acetylation, and H3.3 CUT&Tag signals, for direct target genes and all active genes before and after 1 h of INO80, P400, and SRCAP degradation. Blue indicates pre-degradation, and red indicates post-degradation. Box plots display the log2 fold changes of H2A.Z, H2A.Z acetylation, and H3.3 CUT&Tag signals for direct target genes and all genes. Direct target genes are shown in different colours (red, blue, or yellow, depending on the experimental condition), whereas all active genes are shown in grey. Statistical analysis was determined using the Wilcoxon test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. ( C ). MA-plots illustrating the log2 fold changes following the degradation of one INO80/SWR protein, as identified by CUT&Tag analysis of the remaining two proteins. The red dots indicate the genes with significantly changed signals identified by DESeq2 (FDR < 0.05). ( D ). Left: Schematic representation of the cross-regulation experiment. CUT&Tag assays were performed for the other two remodelers after degrading one remodeler. Right: Heatmap showing cross-regulation experimental results, with arrows originating from the degradation target and pointing to the target observed for changes. The color bar indicates log2 fold change. The starting point of each arrow represents the degraded protein, while the endpoint of the arrow indicates the protein subjected to CUT&Tag sequencing. The heatmap colors reflect the changes in sequencing signals across all active genes, providing a visual representation of the impact of degrading one remodeler on the occupancy of the others. ( E ). Left three boxplots showing the log2 fold change of P400 or SRCAP, H2A.Z, and H2A.Zac CUT&Tag signal upon the degradation of INO80 at three gene clusters. Right two boxplots showing the CUT&Tag signal of INO80, P400, or SRCAP at three gene clusters. The genes are classified into maximal ( n = 2573 and 661 for P400 and SRCAP target respectively), moderate ( n = 2572 and 660 for P400 and SRCAP target, respectively) and minimal ( n = 2573 and 661 for P400 and SRCAP target, respectively) changes according to the log2 fold change of P400 or SRCAP CUT&Tag signals upon the degradation of INO80. Statistical analysis was determined using the Wilcoxon test.

Article Snippet: 1 μl GFP abs (ABcam, ab290), 1 μg RPB1 NTD abs (CST, 14958S),1 μl BRD2 abs (Proteintech, 22236–1-AP), and equal isotype IgG control for each ChIP reaction, respectively.

Techniques: Western Blot, Sequencing

Journal: Nucleic Acids Research

Article Title: INO80/SWR remodelers regulate Pol II transcription through BRD2 and chromatin landscape

doi: 10.1093/nar/gkaf892

Figure Lengend Snippet: A regulatory circuit coordinates BRD2 occupancy, INO80/SWR chromatin remodeler function, and H2A.Z modification states. ( A ). Co-immunoprecipitation (Co-IP) assay. Left: Lysate of INO80, P400, SRCAP GFP tag degron cells were immunoprecipitated with antibodies against either BRD2 or rabbit IgG. 5% of cell lysate without antibodies was loaded as an input. Western blot analyses of anti-GFP antibody revealed a strong interaction between INO80, P400, SRCAP and BRD2. Western blot using antibodies against DMAP1, INTS5, and H2A.Zac to confirm their interactions with BRD2. Right: Lysate of INO80, P400, SRCAP GFP tag degron cells were immunoprecipitated with antibodies against either GFP (“INO80” lane, “P400” lane, “SRCAP” lane) or rabbit IgG. Western blot analyses of anti-GFP antibody revealed interaction between P400, SRCAP, and INTS5. For BRD2, chromatin was cross-linked, sheared, and immunoprecipitated using an antibody against GFP. Normal IgG and input chromatin served as negative and positive controls, respectively. ( B ). TurboID-Western Blot assay. Western blot showing BRD2 and INTS5-RPB1 associations are reduced after the INO80, P400 and SRCAP degradation. INO80/SWR remodelers (GFP-tagged) do not appear to interact with RPB1 (TurboID-tagged). ( C ). Volcano plots showing the differentially expressed genes identified by TT-seq experiment after 1 h of BRD2 degradation. The differentially expressed genes were identified using DESeq2 software (FDR < 0.05, absolute fold change > 1.5). ( D ). Scatter plot showing the log 2 fold change log 2 (FC) in TT-seq signals upon BRD2 versus INO80/SWR remodeler degradation. Genes exhibiting significant downregulation (log 2 (FC) < 0 in both conditions) are highlighted in blue, indicating coregulated transcriptional targets. ( E and F ). The MA plots show the INO80, P400, SRCAP, H2A.Z, and H2A.Zac CUT &Tag signal changes at TSS regions after degradation of BRD2. Regions with increased or decreased binding were identified by DESeq2 software (FDR < 0.05) and marked red. ( G ). Proposed model of INO80/SWR remodelers regulating gene transcription activation in mammalian cells.

Article Snippet: 1 μl GFP abs (ABcam, ab290), 1 μg RPB1 NTD abs (CST, 14958S),1 μl BRD2 abs (Proteintech, 22236–1-AP), and equal isotype IgG control for each ChIP reaction, respectively.

Techniques: Modification, Co-Immunoprecipitation Assay, Immunoprecipitation, Western Blot, Software, Binding Assay, Activation Assay

Journal: Frontiers in Immunology

Article Title: A novel carboxamide bromodomain inhibitor attenuates osteoarthritis via epigenetic repression of NF-κB and MAPK signaling

doi: 10.3389/fimmu.2025.1633334

Figure Lengend Snippet: NCD selectively binds to BRD2 (A) Schematic representation of the DNA-encoded library (DEL) screening process. Purified target proteins were incubated with the DEL standard library. DNA-tagged compounds that bound to the target were isolated, amplified by PCR, and identified using next-generation sequencing. (B, C) Schematic and quantitative results of the TR-FRET assay using NCD. A lower TR-FRET signal indicates stronger binding to the bromodomain (BD), as it prevents the probe from binding to the ligand. NCD exhibited selective binding, with reduced TR-FRET signals observed for BRD2, indicating high affinity. (D, E) Schematic and results of the binding kinetics assay using a biosensor. Binding affinities (KD values) of NCD for the bromodomains of BRD2–4 were measured. Lower KD values indicate stronger binding affinity, with NCD showing the highest affinity for BRD2.

Article Snippet: TR-FRET assays were conducted using kits specific for BRD2 (BD1 + BD2), BRD3 (BD1 + BD2), and BRD4 (BD1 + BD2) obtained from BPS Bioscience (San Diego, CA, USA).

Techniques: Purification, Incubation, Isolation, Amplification, Next-Generation Sequencing, Binding Assay