Journal: Nature Communications

Article Title: Targeted protein degradation reveals BET bromodomains as the cellular target of Hedgehog pathway inhibitor-1

doi: 10.1038/s41467-023-39657-1

Figure Lengend Snippet: a Principal component analysis of 5343 quantified proteins clearly separates all sample groups. b Volcano plot of log2(fold change) to significance shows the potential direct targets of HPP-9. Blue circles represent all hits overlapping between replicates and red circles with text labels are the most significant hits. c HPP-9 dose-dependently degrades the BET bromodomain proteins, as shown by western blot analysis. Representative blot of three independent experiments. d , e All HPPs were profiled for their ability to degrade BRD2/3/4 by western blot, which revealed ( e ) a good correlation between Hedgehog pathway inhibition (Fig. , replotted for clarity) and BET bromodomain degradation. Mean ± SEM of three independent experiments is plotted. f Representative immunoblot of a competition experiment between HPP-9 and HPI-1, hydroxythalidomide, and MG-132. Three independent experiments. g HPP-9 is able to degrade BET proteins in medulloblastoma cells MB55 and MB56. Representative immunoblot (MB56) of N = 2 independent experiments for each cell line. Normalized band signal intensities are shown underneath the blots. Source data are provided as a Source Data file.

Article Snippet: Taqman primers used: Mm00494654_m1 mGli1; Mm01293117_m1 mGli2; Mm00436026_m1 mPtch1; Mm00437762_m1 mB2m; Mm99999915_g1 Gapdh; Mm01271171_g1 mBrd2

Techniques: Western Blot, Inhibition

Journal: Nature Communications

Article Title: Targeted protein degradation reveals BET bromodomains as the cellular target of Hedgehog pathway inhibitor-1

doi: 10.1038/s41467-023-39657-1

Figure Lengend Snippet: a Representative micrographs of SHH-GFP cells treated with DMSO or 20 μM of HPI-1 for 27 h. Scalebar 25 μm. b High-content microscopy dose-response curve for cells treated with increasing concentrations of HPI-1 and probed for nuclear BRD2. Representative curve from N = 3 independent experiments, with n = 9 images analyzed per condition. c qPCR analysis of Brd2 mRNA levels from cells treated with 5 μM HPI-1, 1 μM HPP-9 or JQ1 for 27 h in the presence of ShhN. Data from N independent experiments, as indicated in the bars, performed in duplicate. One-way Brown–Forsythe and Welch ANOVA, Dunnett T3 test. d The affinity of HPI-1 and HPP-9 to the indicated bromodomains (BromoKd, Eurofins discovery). (1,2) indicates which of the two bromodomain subunits were included. e Proposed binding mode for HPI-1. The interacting residues and HPI-1 are modeled in stick, while BRD2(2) is shown in cartoon representation. The dashed yellow lines represent H-bonds while the π-π stacking is shown as a purple dashed line. f Possible physical model of the ternary complex. BRD2(2) (green) and CRBN (gray) are shown in a surface representation, while HPP-9 (orange) is shown as spheres. Source data are provided as a Source Data file.

Article Snippet: Taqman primers used: Mm00494654_m1 mGli1; Mm01293117_m1 mGli2; Mm00436026_m1 mPtch1; Mm00437762_m1 mB2m; Mm99999915_g1 Gapdh; Mm01271171_g1 mBrd2

Techniques: Microscopy, Binding Assay

Journal: Nature Communications

Article Title: Targeted protein degradation reveals BET bromodomains as the cellular target of Hedgehog pathway inhibitor-1

doi: 10.1038/s41467-023-39657-1

Figure Lengend Snippet: SHH-GFP cells were treated with 1 μM of HPP-9 or dBet6 for 27 h and probed for GLI1, BRD2, BRD3, and BRD4 by western blot ( a ) and analyzed by fluorescence microscopy ( b , c ). Representative micrographs showing the nuclear localization of GFP, BRD2, BRD3, and BRD4 are shown in ( b ), and the signal is quantified in ( c ). Scalebar 25 μm. Data shown is the mean from N independent experiments as indicated in or above the bars. At least seven images were analyzed per condition in each experiment. All compounds were used at 1 μM. Two-way ANOVA. d Full dose-response curves were measured by high-content microscopy to determine the pDC 50 values for HPP-9 and dBet6. Each dot is an individual image ( n is at least 7 images/experiment, N = 3, 4 independent experiments as indicated in the table under the graphs, n.a. not active). e BRD2 and BRD4 binding to Gli1 and Gli2 loci was measured by ChIP-qPCR using two primer pairs per locus. Representative data of two independent experiments. f Volcano plot showing differentially expressed genes in SHH-GFP cells upon 8 h of HPP-9 treatment (+ShhN) compared to DMSO + ShhN. Two independent experiments, p adj < 0.05 and 1.5x fold change. Exact p values can be found in Supplementary Data and were determined using the Wald test. The adjusted p values were corrected for multiple testing, using the Benjamin–Hochberg correction. Source data are provided as a Source Data file.

Article Snippet: Taqman primers used: Mm00494654_m1 mGli1; Mm01293117_m1 mGli2; Mm00436026_m1 mPtch1; Mm00437762_m1 mB2m; Mm99999915_g1 Gapdh; Mm01271171_g1 mBrd2

Techniques: Western Blot, Fluorescence, Microscopy, Binding Assay, ChIP-qPCR

Journal: Frontiers in Pharmacology

Article Title: Pharmacologic Targeting of BET Proteins Attenuates Hyperuricemic Nephropathy in Rats

doi: 10.3389/fphar.2021.636154

Figure Lengend Snippet: I-BET151 reduces Brd4 levels in the kidney of HN rats. (A) A rat model of HN was established and treated with I-BET151 as indicated in “Material and Methods.” After 3 weeks, kidneys were taken for immunoblot analysis for Brd2, Brd3, Brd4 or GAPDH. Expression levels of Brd2 (B) , Brd3 (C) , Brd4 (D) were quantified by densitometry analysis and then normalized with GAPDH. (E) Photomicrographs (original magnification, ×400) illustrate immunohistochemical Brd4 staining of kidney tissues. (F) Brd4 staining graphic presentation of quantitative data. Data are represented as the mean ± SEM. * p < 0.05; ** p < 0.01.

Article Snippet: Brd2 antibody was purchased from Boster Biological Technology (Wuhan, China), OAT1 and OAT3 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA), I-BET151 was purchased from Target Mol (MA, United States).

Techniques: Western Blot, Expressing, Immunohistochemical staining, Staining

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