zinc finger domain 2b baz2b inhibitor baz2 icr  (Tocris)

Journal: EMBO Reports

Article Title: BAZ2A‐mediated repression via H3K14ac‐marked enhancers promotes prostate cancer stem cells

doi: 10.15252/embr.202153014

Figure Lengend Snippet: Heatmap profiles of BAZ2A‐bound regions in PC3 cells and the corresponding signals of input, BAZ2A‐bromodomain (BRD) mutant (Y1775F), H3K14ac, and H3K27ac. Data were ranked to BAZ2A binding in PC3 cells. Validation of ChIPseq results by anti‐HA ChIP‐qPCR of PC3 wild‐type (WT) and a PC3 cell line that expresses endogenous BAZ2A with a FLAG‐HA tag (PC3‐H/F‐BAZ2A). GAPDH promoter represents a region not bound by BAZ2A. Average values of three independent experiments. Data were normalized to input and to AOX1 levels. Statistical significance ( P ‐values) was calculated using two‐tailed t ‐test (*< 0.05, **< 0.001, ns: not significant). Error bars represent SD. Wiggle tracks displaying BAZ2A‐bound regions, H3K14ac, H3K27ac, and input at AOX1 , EVX2 , and GAPDH . Pearson correlation heat map of BAZ2A‐bound regions and histone marks. Data for BAZ2A, H3K4me3, H3K27me3, H3K27ac, and H3K14ac are from this work. H3K9me3, H3K9me2, and H3K4me1 were obtained from ENCODE. Images of histone peptide arrays probed with 10 nM of recombinant BAZ2A‐BRD wild‐type (GST‐BAZ2A‐BRD wt ) and mutants (GST‐BAZ2A‐BRD Y1830F or GST‐BAZ2A‐BRD N1873L ). Visualization of binding was performed by incubation with anti‐GST antibodies and imaged on Odyssey Infrared Imaging System. Blue circles mark peptides recognized by BAZ2A‐BRD (i.e., H3K14ac), whereas black circles show some of the acetylated peptides not recognized by BAZ2A‐BRD (H3K27ac and H3K9ac). Right panel shows heatmap of the relative binding intensity of BAZ2A‐BRD against modified histone peptides. Binding intensity was calculated as average of fold change from peptides signal over background controls of two different arrays. BAZ2A read coverage quantification at ± 1 Kb from peak summit of BAZ2A‐bound regions identified in PC3 cells and the corresponding reads for BAZ2A‐BRD mutant (Y1775F) in PC3 cells. Statistical significance ( P ‐values) was calculated using two‐tailed t ‐test (***< 0.001). Regions bound by BAZ2A in PC3 cells are enriched in H3K14ac. Read coverage quantification for H3K14ac levels at regions bound by BAZ2A in PC3 cells (BAZ2Awt and BAZ2A‐BRD mutant [Y1775F]) at ± 1 Kb from BAZ2A peak summits. Statistical significance ( P ‐values) was calculated using two‐tailed t ‐test (***< 0.001). Data information: Boxplots in Fig 1 show the median and quartiles with the whiskers extending to the most extreme data point within 1.5 times the interquartile range.

Article Snippet: PC3 cells were treated with 1 μM of BAZ2A‐BRD inhibitors BAZ2‐ICR (SML1276, Sigma) and GSK2801 (SML0768, Sigma), EZH2 inhibitor GSK126 (S7061‐5MG, Lubio), BET inhibitors JQ1 (SML1524, Sigma), or DMSO as control.

Techniques: Mutagenesis, Binding Assay, Two Tailed Test, Recombinant, Incubation, Imaging, Modification

Journal: EMBO Reports

Article Title: BAZ2A‐mediated repression via H3K14ac‐marked enhancers promotes prostate cancer stem cells

doi: 10.15252/embr.202153014

Figure Lengend Snippet: A Schematic representation of the strategy to generate PC3 cell line with FLAG‐HA (F/H) tag insertion at BAZ2A locus by CRISPR/Cas9 method. Lower panel. PCR genotyping to measure the insertion of F/H sequences into BAZ2A locus. Arrows represent the primers used for PCR genotyping. B Immunoblot of PC3 wild‐type (wt) cells and the PC3 cell line expressing endogenous BAZ2A with F/H tag showing comparable levels of BAZ2A. SNF2h and STAT1 serve as protein loading control. C Representative protein gel showing the purity of recombinant GST‐BAZ2A‐BRD wild‐type [WT]) and mutants (GST‐BAZ2A‐BRD Y1830F or GST‐BAZ2A‐BRD N1873L ). Proteins are visualized by Coomassie staining. D Western blot showing equal expression of ectopically expressed HA‐BAZ2Awt and HA‐BAZ2A‐BRD mutant (Y1775F) in transfected PC3 cells and endogenous levels of BAZ2A in untransfected PC3 cells. Whole cell lysates from equivalent amounts of cells were analyzed. Nucleolin is shown as a protein loading control. E, F Average density plots of ChIPseq read counts of BAZ2A (BAZ2Awt and BAZ2A‐BRD mutant, Y1775F) and H3K14ac at ± 1 Kb from BAZ2A peak summits in PC3 cells. Source data are available online for this figure.

Article Snippet: PC3 cells were treated with 1 μM of BAZ2A‐BRD inhibitors BAZ2‐ICR (SML1276, Sigma) and GSK2801 (SML0768, Sigma), EZH2 inhibitor GSK126 (S7061‐5MG, Lubio), BET inhibitors JQ1 (SML1524, Sigma), or DMSO as control.

Techniques: CRISPR, Western Blot, Expressing, Recombinant, Staining, Mutagenesis, Transfection

Journal: EMBO Reports

Article Title: BAZ2A‐mediated repression via H3K14ac‐marked enhancers promotes prostate cancer stem cells

doi: 10.15252/embr.202153014

Figure Lengend Snippet: The H3K14ac histone acetyltransferases EP300 , KAT2A , and KAT6A are highly expressed in metastatic prostate cancer tissues. Boxplots showing expression profiles from Gene Expression Omnibus (GEO) data sets GSE6919 (gene expression microarray data) (Yu et al , ; Chandran et al , ). *< 0.05, **< 0.01, **** P < 0.0001, two‐tailed Student's t‐ test; ns, not significant. Boxplots show the median and quartiles with the whiskers extending to the most extreme data point within 1.5 times the interquartile range. Wiggle tracks showing RNAseq expression profiles of EP300 , KAT2A , and KAT6A in PC3 cells. KAT2A is not expressed in PC3 cells due to deletion of the entire locus. Scatter plot showing the expression of KAT2A and KAT6A vs BAZ2A in two large cohorts of metastatic and primary PCa. Data were from (Cancer Genome Atlas Research Network, ; Robinson et al , ). Boxplots showing expression profiles of AOX1 , MANB2 , DMPK , and DDB1 from GEO data sets GSE6919 (gene expression microarray data) (Yu et al , ; Chandran et al , ). **< 0.01, **** P < 0.0001, two‐tailed Student's t‐ test; ns, not significant. Boxplots show the median and quartiles with the whiskers extending to the most extreme data point within 1.5 times the interquartile range. Boxplots showing expression of BAZ2A‐bound C2‐enhancer genes KRT8, EVC , ITPKB , SH3BP4 , and RASD1 in dedifferentiated and aggressive primary tumors scored with Gleason 9 and 10 and indolent tumors assigned with Gleason 6. Data are from TCGA_PRAD (Cancer Genome Atlas Research Network, ). Statistical significance ( P ‐values) was calculated using two‐tailed t ‐test (*< 0.05, ***< 0.001). Boxplots show the median and quartiles with the whiskers extending to the most extreme data point within 1.5 times the interquartile range.

Article Snippet: PC3 cells were treated with 1 μM of BAZ2A‐BRD inhibitors BAZ2‐ICR (SML1276, Sigma) and GSK2801 (SML0768, Sigma), EZH2 inhibitor GSK126 (S7061‐5MG, Lubio), BET inhibitors JQ1 (SML1524, Sigma), or DMSO as control.

Techniques: Expressing, Microarray, Two Tailed Test

Journal: EMBO Reports

Article Title: BAZ2A‐mediated repression via H3K14ac‐marked enhancers promotes prostate cancer stem cells

doi: 10.15252/embr.202153014

Figure Lengend Snippet: Scatter plot showing the expression of EP300 and BAZ2A in two large cohorts of metastatic and primary PCa. Data were from Cancer Genome Atlas Research Network ; Robinson et al . EP300 acetylates H3K14 in PC3 cells. Western blot showing H3K14ac levels in PC3 cells treated for 3 days without and with the selective catalytic EP300 inhibitor A‐485 (10 μM). Histone H3 serves as protein loading control. Genomic annotations of BAZ2A‐bound regions in PC3 cells. Volcano plot showing fold change (log 2 values) in transcript levels of PC3 cells upon BAZ2A knockdown. Gene expression values of two replicates were averaged and selected for 1.5‐fold changes and P < 0.05. Table showing expression changes in genes with BAZ2A‐bound promoter upregulated upon BAZ2A‐KD in PC3 cells in metastatic PCa vs normal prostate tissue and in PC3 cells depleted of EP300 via siRNA. qRT–PCR showing increased expression levels of genes with BAZ2A‐bound promoter and downregulated in metastatic tumors ( AOX1 , MANB2 , DMPK , and DDB1 —see (E)) in PC3 cells treated with siRNA‐EP300. Data information: Data were from three independent experiments. Values were normalized to GAPDH mRNA. Statistical significance ( P ‐values) was calculated using two‐tailed t ‐test (*<0.05, ****< 0.0001). Error bars represent SD. Source data are available online for this figure.

Article Snippet: PC3 cells were treated with 1 μM of BAZ2A‐BRD inhibitors BAZ2‐ICR (SML1276, Sigma) and GSK2801 (SML0768, Sigma), EZH2 inhibitor GSK126 (S7061‐5MG, Lubio), BET inhibitors JQ1 (SML1524, Sigma), or DMSO as control.

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Two Tailed Test

Journal: EMBO Reports

Article Title: BAZ2A‐mediated repression via H3K14ac‐marked enhancers promotes prostate cancer stem cells

doi: 10.15252/embr.202153014

Figure Lengend Snippet: BAZ2A binds to a class of inactive enhancers marked by H3K14ac. Heat maps of BAZ2A, H3K14ac, H3K27ac, H3K4me1, and DNAseq signal at annotated intergenic enhancers are shown. Enhancer regions were clustered in 4 groups based on the presence (+) or absence (−) of H3K27ac and H3K4me1. C1 (H3K27ac + /H3K4me1 + ), C2 (H3K27ac + /H3K4me1 − ), C3 (H3K27ac − /H3K4me1 + ), and C4 (H3K27ac − /H3K4me1 − ). Inactive enhancers (C2) are enriched in H3K14ac and BAZ2A levels. Read coverage at enhancer classes C1 and C2 for BAZ2A, H3K14ac, H3K27ac, H3K4me1, and H3K27me3 at ± 1 Kb from annotated enhancer regions. Statistical significance ( P ‐values) was calculated using two‐tailed t ‐test (***< 0.001). Boxplots show the median and quartiles with the whiskers extending to the most extreme data point within 1.5 times the interquartile range. Pie charts showing the number of genes in the nearest linear genome proximity to BAZ2A‐bound C2 enhancers and their expression changes upon BAZ2A‐KD in PC3 cells. Wiggle tracks showing RNA levels in PC3 cells treated with siRNA‐BAZ2A and occupancy of BAZ2A, H3K14ac, H3K27ac, and H3K4me1 at C2 enhancer and its nearest gene CXCR4 . Top 10 biological process gene ontology (GO) terms as determined using DAVID 6.8 for genes in the nearest linear proximity to BAZ2A‐bound C2 enhancers that are differentially expressed upon BAZ2A‐KD in PC3 cells. Heatmap showing expression changes in BAZ2A‐bound C2‐enhancer genes that were both significantly upregulated upon BAZ2A‐KD and lower expressed in Gleason 9 and 10 tumors (advanced) compared with Gleason 6 tumors (indolent). Values from tumors were from Cancer Genome Atlas Research Network . GO terms associated with the genes listed in (F). Genes labeled in bold were analyzed in (H). qRT–PCR showing increased expression levels of BAZ2A‐bound C2‐enhancer genes in PC3 cells treated with siRNA‐EP300. Data information: Data were from three independent experiments. Downregulation of EP300 expression is shown in Fig . Values were normalized to GAPDH mRNA. Statistical significance ( P ‐values) was calculated using two‐tailed t ‐test (*< 0.05, **< 0.001). Error bars represent SD.

Article Snippet: PC3 cells were treated with 1 μM of BAZ2A‐BRD inhibitors BAZ2‐ICR (SML1276, Sigma) and GSK2801 (SML0768, Sigma), EZH2 inhibitor GSK126 (S7061‐5MG, Lubio), BET inhibitors JQ1 (SML1524, Sigma), or DMSO as control.

Techniques: Two Tailed Test, Expressing, Labeling, Quantitative RT-PCR

Journal: EMBO Reports

Article Title: BAZ2A‐mediated repression via H3K14ac‐marked enhancers promotes prostate cancer stem cells

doi: 10.15252/embr.202153014

Figure Lengend Snippet: RNAseq showing differential gene expression between PC3‐CSCs and the parental PC3 cells ( P < 0.05, Log 2 fold change 1). Top 10 biological process gene ontology (GO) terms as determined using DAVID 6.8 for genes downregulated and upregulated in PC3‐CSCs relative to parental PC3 cells. qRT–PCR of genes differentially expressed in PC3‐CSCs compared with PC3 cells showing the upregulation of CSC markers ( ALDH1A2, LGR5 , CD24 ) and downregulation of genes related to developmental processes ( HOXA1, HOXA4, SOX7 ). Data are from two independent experiments. mRNA levels were normalized to GAPDH . BAZ2A is required for the dedifferentiation of PC3 into PC3‐CSCs. Left panel: Representative images from two independent experiments showing the impairment of PC3 cells to form tumorspheres upon stable depletion of BAZ2A. Right panel: BAZ2A mRNA levels in three independent PC3 cell lines stably expressing shRNA‐ BAZ2A were measured by qRT–PCR and normalized to GAPDH mRNA from 2 independent experiments. Left panel: qRT–PCR showing BAZ2A mRNA levels in PC3 cells depleted of endogenous BAZ2A with siRNA specifically targeting human BAZ2A sequences and expressing mouse BAZ2Awt and BAZ2A‐BRD mutant (BAZ2A Y1775F ). Values were normalized to MRPS7 mRNA. Data are from three independent experiments. Right panel: Quantification of tumorspheres from three independent experiments. Statistical significance ( P ‐values) was calculated using two‐tailed t ‐test (*< 0.05; **< 0.01). Error bars represent SD.

Article Snippet: PC3 cells were treated with 1 μM of BAZ2A‐BRD inhibitors BAZ2‐ICR (SML1276, Sigma) and GSK2801 (SML0768, Sigma), EZH2 inhibitor GSK126 (S7061‐5MG, Lubio), BET inhibitors JQ1 (SML1524, Sigma), or DMSO as control.

Techniques: Expressing, Quantitative RT-PCR, Stable Transfection, shRNA, Mutagenesis, Two Tailed Test

Journal: EMBO Reports

Article Title: BAZ2A‐mediated repression via H3K14ac‐marked enhancers promotes prostate cancer stem cells

doi: 10.15252/embr.202153014

Figure Lengend Snippet: Heatmaps showing the relative BAZ2A‐BRD binding intensity at modified histone peptides upon incubation with BAZ2A‐BRDi BAZ2‐ICR or GSK2801 displayed in Fig . Binding intensity was calculated as average of fold change from peptides signal over background controls of two different arrays. Cell proliferation curves of PC3 cells treated with siRNA‐ BAZ2A . Error bars represent the standard deviation from three independent experiments. Cell proliferation curves of PC3 cells treated with 5 μM BAZ2‐ICR and GSK2801. Error bars represent the standard deviation from three independent experiments. Representative images of tumorspheres generated from PC3 cells treated with BAZ2‐ICR (1 μM) or GSK2801 (1 μM). Half‐maximal effective concentration (EC50) of BAZ2‐ICR. Measurements were performed 5 days after treatment of PC3 cells and initiation of tumorspheres using the indicated BAZ2‐ICR concentration. The amount of tumorspheres was assessed by measurement of cell viability. Error bars represent SD. Values are from three independent experiments. Representative images showing tumorspheres derived from the metastatic PCa cell line DU‐145 treated with DMSO or BAZ2‐ICR (1 μM) inhibitor for 5 days.

Article Snippet: PC3 cells were treated with 1 μM of BAZ2A‐BRD inhibitors BAZ2‐ICR (SML1276, Sigma) and GSK2801 (SML0768, Sigma), EZH2 inhibitor GSK126 (S7061‐5MG, Lubio), BET inhibitors JQ1 (SML1524, Sigma), or DMSO as control.

Techniques: Binding Assay, Modification, Incubation, Standard Deviation, Generated, Concentration Assay, Derivative Assay

Journal: EMBO Reports

Article Title: BAZ2A‐mediated repression via H3K14ac‐marked enhancers promotes prostate cancer stem cells

doi: 10.15252/embr.202153014

Figure Lengend Snippet: Images of histone peptide arrays incubated with 10 nM of recombinant GST‐BAZ2A‐BRD wt in the presence of 50 nM of BAZ2A‐BRDi, BAZ2‐ICR, or GSK2801. Visualization of binding was performed by incubation with anti‐GST antibodies and imaged on Odyssey Infrared Imaging System. For a better visualization of the data, the image of GST‐BAZ2A‐BRD wt without BAZ2A‐BRDi shown in Fig has been included. Blue circles mark peptides recognized by BAZ2A‐BRD (i.e., H3K14ac), whereas black circles show some of the acetylated peptides not recognized by BAZ2A‐BRD (H3K27ac and H3K9ac). qRT–PCR showing increased expression levels of BAZ2A‐regulated genes in PC3 cells treated with DMSO or 1 μM BAZ2‐ICR for 5 days. Values were normalized to GAPDH mRNA levels. Average values of two independent experiments. Gray and black dots represent the values obtained from each single experiment. Western blot showing similar levels of BAZ2A and H3K14ac in PC3 cells treated without or with 1 μM BAZ2‐ICR for 5 days. Histone H3 was used to ensure similar protein levels. Cell proliferation curves of PC3 cells treated with 5, 10, and 50 μM BAZ2‐ICR. Error bars represent the standard deviation from three independent experiments. Representative wiggle tracks from RNAseq of PC3 and PC3‐CSCs of BAZ2A and BAZ2B . BAZ2B depletion in PC3 cells does not affect the dedifferentiation into PC3‐CSCs. Left panel: BAZ2B mRNA levels were measured by qRT–PCR in PC3‐CSCs and normalized to GAPDH mRNA. Right panel: Representative figures showing PC3‐CSCs generated from PC3 cells transfected with siRNA‐control or siRNA‐ BAZ2B . Source data are available online for this figure.

Article Snippet: PC3 cells were treated with 1 μM of BAZ2A‐BRD inhibitors BAZ2‐ICR (SML1276, Sigma) and GSK2801 (SML0768, Sigma), EZH2 inhibitor GSK126 (S7061‐5MG, Lubio), BET inhibitors JQ1 (SML1524, Sigma), or DMSO as control.

Techniques: Incubation, Recombinant, Binding Assay, Imaging, Quantitative RT-PCR, Expressing, Western Blot, Standard Deviation, Generated, Transfection

Journal: EMBO Reports

Article Title: BAZ2A‐mediated repression via H3K14ac‐marked enhancers promotes prostate cancer stem cells

doi: 10.15252/embr.202153014

Figure Lengend Snippet: Representative brightfield images of organoids derived from mouse prostate cells treated with DMSO, BAZ2‐ICR (5 μM), GSK2801 (5 μM), or GSK126 (5 μM) inhibitors followed by transduction with shRNA‐control or shRNA‐ Pten . Quantification of shRNA‐control and shRNA‐ Pten organoid morphology upon treatment with DMSO, EZH2 (GSK126), and BAZ2A‐BRD inhibitors (BAZ2‐ICR and GSK2801). Representative images of Ki67‐immunostained mouse prostate organoid sections expressing shRNA‐control and shRNA‐ Pten and treated with DMSO or BAZ2‐ICR (5 μM). Quantification of Ki67 + cells in mouse prostate organoids shown in (C). **< 0.01, **** P < 0.0001, two‐tailed Student's t‐ test; ns, not significant. The median values are represented by a line. Quantification of mouse prostate organoids per field expressing shRNA‐control and shRNA‐ Pten and treated with DMSO, GSK126 (5 μM), BAZ2‐ICR (5 μM), or GSK2801 (5 μM). Values were normalized to shRNA‐control samples. Average values of two independent experiments.

Article Snippet: PC3 cells were treated with 1 μM of BAZ2A‐BRD inhibitors BAZ2‐ICR (SML1276, Sigma) and GSK2801 (SML0768, Sigma), EZH2 inhibitor GSK126 (S7061‐5MG, Lubio), BET inhibitors JQ1 (SML1524, Sigma), or DMSO as control.

Techniques: Derivative Assay, Transduction, shRNA, Expressing, Two Tailed Test

Journal: EMBO Reports

Article Title: BAZ2A‐mediated repression via H3K14ac‐marked enhancers promotes prostate cancer stem cells

doi: 10.15252/embr.202153014

Figure Lengend Snippet: qRT–PCR showing Pten mRNA levels in mouse prostate organoids expressing shRNA‐control and shRNA‐ Pten and treated with DMSO, GSK126 (5 μM), BAZ2‐ICR (5 μM), or GSK2801 (5 μM). Values are from two experiments. Data were normalized to Hprt mRNA and organoids treated with siRNA‐Control.

Article Snippet: PC3 cells were treated with 1 μM of BAZ2A‐BRD inhibitors BAZ2‐ICR (SML1276, Sigma) and GSK2801 (SML0768, Sigma), EZH2 inhibitor GSK126 (S7061‐5MG, Lubio), BET inhibitors JQ1 (SML1524, Sigma), or DMSO as control.

Techniques: Quantitative RT-PCR, Expressing, shRNA

Journal: EMBO Reports

Article Title: BAZ2A‐mediated repression via H3K14ac‐marked enhancers promotes prostate cancer stem cells

doi: 10.15252/embr.202153014

Figure Lengend Snippet: List of primers used in this study.

Article Snippet: PC3 cells were treated with 1 μM of BAZ2A‐BRD inhibitors BAZ2‐ICR (SML1276, Sigma) and GSK2801 (SML0768, Sigma), EZH2 inhibitor GSK126 (S7061‐5MG, Lubio), BET inhibitors JQ1 (SML1524, Sigma), or DMSO as control.

Techniques: Clone Assay