Journal: Nature

Article Title: BRD8 maintains glioblastoma by epigenetic reprogramming of the p53 network

doi: 10.1038/s41586-022-05551-x

Figure Lengend Snippet: a, Volcano plot of differentially expressed genes after BRD8 depletion. Fold change was calculated by averaging fold changes of two independent sgRNAs of BRD8 compared with sgNeg. Each sgRNA has two biological replicates. b, GSEA analysis showing significantly enriched p53-related proliferation and aging signatures after BRD8 depletion. c and d, RT-qPCR assay of proliferation-related genes after BRD8 depletion in A382WT (c) and patient-derived primary GBM cells (d). Plotted is the mean ± s.d. (n = 3 biologically independent samples). P values were calculated using two-tailed unpaired Student’s t-tests. e, Propidium Iodide (PI) staining and flow cytometry assays of A382WT cells after BRD8 loss (n = 3 biologically independent samples). f, SA-β-gal assay in A382WT cells. Data shown represents three independent results. Scale bar, 50 μm. g, GSEA plot of apoptosis signature after BRD8 depletion in A382WT cells. h, Annexin V staining and flow cytometry assay in A382WT cells. Plotted is the mean ± s.d. (n = 3 biologically independent samples). P values were calculated using two-tailed unpaired Student’s t-tests. i, RT-qPCR assay of apoptosis-related genes after BRD8 loss in A382WT cells. Plotted is the mean ± s.d. (n = 3 biologically independent samples). P values were calculated using two-tailed unpaired Student’s t-tests. j, RT-qPCR assay of CDKN1A expression in sgNeg or sgBRD8–2 expressing U251R273H and U118R213Q cells. Plotted is the mean ± s.d. (n = 3 biologically independent samples). P values were calculated using two-tailed unpaired Student’s t-tests. k, Western blot validating p53 knockdown by four different sgRNAs targeting TP53 in A382WT cells. Data shown represents three independent results. l, Western blot showing depletion efficiency of BRD8 and induction of p53 and p21 in Ctrl or p53-deficient A382WT cells. Data shown represents three independent results. HSC70 serves as a loading control. m, RT-qPCR assays of indicated cell cycle and senescence-related genes after transduction of sgNeg or sgBRD8–1 and sgBRD8–2 in Ctrl and p53-deficient A382WT cells. Plotted is the mean ± s.d. (n = 3 biologically independent samples). P values were calculated using two-tailed unpaired Student’s t-tests. n, SA-β-gal assays in Ctrl or p53-deficient A382WT cells after BRD8 loss. Data shown represents three independent results. Scale bar, 100 μM.

Article Snippet: Individual sgRNAs were cloned into lentiviral expression vectors with an optimized sgRNA scaffold backbone: either LRG2.1 (U6-sgRNA-GFP, Addgene, 108098) or LRNeo2.1 for targeting human genes, or LRG (Addgene, 65656) for targeting mouse genes.

Techniques: Quantitative RT-PCR, Derivative Assay, Two Tailed Test, Staining, Flow Cytometry, Expressing, Western Blot, Knockdown, Control, Transduction

Journal: Nature

Article Title: BRD8 maintains glioblastoma by epigenetic reprogramming of the p53 network

doi: 10.1038/s41586-022-05551-x

Figure Lengend Snippet: a, Heatmap of the log2(fold change) of sgRNA abundance in the indicated panel of cancer cell lines ranked by GBM-dependent and p53-dependent potency. b, Kaplan–Meier plot of mice that had been orthotopically transplanted with U87WT cells expressing the sgRNAs sgNeg or sgBRD8 into the brains of recipient mice. P value was calculated by log-rank (Mantel–Cox) test. c, Cellular competition-based GFP dropout assays of sgNeg, sgCDK1, sgBRD8–1 and sgBRD8–2 in control (Ctrl) or p53-depleted (sgTP53–1, sgTP53–2, sgTP53–3 or sgTP53–4) A382WT cells. A GFP reporter is linked to sgRNA expression, and the percentage of independent replicates (normalized to P0, n = 3 biologically independent samples) is shown as the mean ± s.d. at the indicated time points. P0 refers to day 3 after infection. d, GSEA plots of the upregulated gene signature after BRD8 depletion (sgBRD8 up) and p53 induction by doxycycline (P53OE up) in p53-overexpressing (P53OE) and BRD8-depleted A382WT cells (sgBRD8) compared with controls (Ctrl or sgNeg), respectively. Two biological replicates were analysed for each sample. NES, normalized enrichment score. e, Density plots showing BRD8, p53, H3K4me3 and H3K27ac enrichment surrounding the summit of high-confidence BRD8 peaks in A382WT cells, ranked by BRD8 peak intensity. f, Venn diagram showing integrated RNA-seq and ChIP-seq analysis of significantly upregulated genes after BRD8 depletion (sgBRD8 up) and p53 induction (P53OE up). g, GFP dropout assays of sgNeg, sgCDK1 and sgBRD8–2 in control (Ctrl) or p21-depleted (sgCDKN1A-2 or sgCDKN1A-3) A382WT cells. Plotted is the mean ± s.d. at the indicated time points (normalized to P0, n = 3 biologically independent samples). P values were calculated using two-tailed unpaired Student’s t-tests. h, CDKN1A expression in TCGA Firehouse GBM samples divided into three subgroups on the basis of TP53 status and BRD8 expression level. P values were calculated using two-tailed unpaired Student’s t-tests.

Article Snippet: Individual sgRNAs were cloned into lentiviral expression vectors with an optimized sgRNA scaffold backbone: either LRG2.1 (U6-sgRNA-GFP, Addgene, 108098) or LRNeo2.1 for targeting human genes, or LRG (Addgene, 65656) for targeting mouse genes.

Techniques: Expressing, Control, Infection, RNA Sequencing, ChIP-sequencing, Two Tailed Test

Journal: Nature

Article Title: BRD8 maintains glioblastoma by epigenetic reprogramming of the p53 network

doi: 10.1038/s41586-022-05551-x

Figure Lengend Snippet: a, Summary of the sgRNAs used in this study targeting BRD8 exons encoding the bromodomain (sgBRD8–1 and sgBRD8–2) or introns (sgIntron-1 and sgIntron2) flanking the exons encoding the bromodomain. b, Western blots showing BRD8 depletion by two independent sgRNAs in the indicated cells. Data shown represents two independent results. sgNeg is a negative control. HSC70 serves as loading control. c, Cellular competition-based GFP dropout assays of individual sgRNAs targeting BRD8 and MDM2 along with sgNeg and a positive control sgRNA targeting the replication protein CDK1 (sgCDK1). Plotted is the percentage of independent GFP positive cells (normalized to P0, n = 3 biologically independent samples) at the indicated time points. P0 refers to day 3 after infection. d, Western blot showing BRD8 levels after targeting its introns by two independent sgRNAs (sgIntron-1 and sgIntron-2) (top) and GFP dropout assays along with sgNeg and sgCDK1 in A382WT cells (bottom). Plotted is the mean ± s.d. (normalized to P0, n = 3 biologically independent samples). e, Western blots showing BRD8 knockdown in A382WT cells transduced with two individual shRNAs targeting BRD8 (shBRD8–1 and shBRD8–2) (top) and GFP dropout assays in A382WT with shRluc as a negative control and shRPA3 as a positive control (bottom). Plotted is the mean ± s.d. (normalized to P0, n = 3 biologically independent samples). f, Design of CRISPR-resistant BRD8 cDNAs against sgBRD8–1 (BRD8 CR-1) or sgBRD8–2 (BRD8 CR-2). g, Western blots showing overexpression of CRISPR-resistant BRD8 cDNAs (BRD8 CR-1 and BRD8 CR-2) in the two indicated TP53WT GBM cells with empty vector as control (Ctrl). h and i, GFP dropout assays of sgNeg, sgCDK1, sgBRD8–1 and sgBRD8–2 in indicated TP53WT GBM cells expressing Ctrl or CRISPR-resistant BRD8 cDNAs (BRD8 CR-1 or BRD8 CR-2). Plotted is the mean ± s.d. (normalized to P0, n = 3 biologically independent samples). j, MTT-based proliferation assays in primary GBM patient cells, xenograft cells, and immortalized brain-derived neural stem cells (BNSC). Plotted is the mean ± s.d. (normalized to sgNeg, n = 3 biologically independent samples). P values were calculated using two-tailed unpaired Student’s t-tests. k, The top panel depicts the Aka luciferase-expressing construct used in orthotopic brain transplantation assays for noninvasive imaging in vivo. Bottom left: in vitro validation of luciferase activity in cultured cells using D-luc or AkaLuc-HCL substrates in empty control (Ctrl), Fluc-expressing (Fluc), and AkaLuc-expressing (AkaLuc) cells. Bottom right: Bioluminescence imaging of NOD SCID mice that had orthotopic brain transplantation of AkaLuc-expressing U87WT cells transduced with sgNeg or sgBRD8–2 (see correlating Kaplan-Meier survival plot, Fig. 1b). l and m, Kaplan-Meier survival plot of recipient mice that had been orthotopically transplanted with A382WT (l) or U251R273H (m) GBM cells expressing sgNeg or sgBRD8 into the brains of recipient mice. P value was calculated by Log-rank (Mantel-Cox) test.

Article Snippet: Individual sgRNAs were cloned into lentiviral expression vectors with an optimized sgRNA scaffold backbone: either LRG2.1 (U6-sgRNA-GFP, Addgene, 108098) or LRNeo2.1 for targeting human genes, or LRG (Addgene, 65656) for targeting mouse genes.

Techniques: Western Blot, Negative Control, Control, Positive Control, Infection, Knockdown, Transduction, CRISPR, Over Expression, Plasmid Preparation, Expressing, Derivative Assay, Two Tailed Test, Luciferase, Construct, Transplantation Assay, Imaging, In Vivo, In Vitro, Biomarker Discovery, Activity Assay, Cell Culture

Journal: Nature

Article Title: BRD8 maintains glioblastoma by epigenetic reprogramming of the p53 network

doi: 10.1038/s41586-022-05551-x

Figure Lengend Snippet: a, Western blot and silver staining of proteins immunoprecipitated by anti-Flag antibody in Flag-tagged BRD8-overexpressing or empty vector control (Ctrl) A382WT cells. Data shown represents three independent results. b, Summary of peptides detected by IP-MS in Flag-tagged BRD8 or Ctrl immunoprecipitates for 17 subunits of the EP400 complex. c, IP and western blots showing validation of core subunits of the EP400 complex in BRD8 immunoprecipitates. Data shown represents two independent results. HSC70 serves as loading control. d, Summary of specific and shared components of the EP400 and SRCAP complexes. Proteins pulled down by BRD8 are shown in dark blue text. e, RT-qPCR showing expression levels of EP400 and SRCAP in A382WT GBM cells. Plotted represents three biological replicates. f, Summary of GSEA analysis using P53OE up signature (genes significantly upregulated after p53 induction by doxycycline) after individual depletion of multiple components of the EP400 complex. Two independent sgRNAs were used for targeting each individual subunit, and sgNeg was used as control. g, Summary of GSEA analysis upon depletion of indicated components of the EP400 complex using the sgBRD8 up signature (genes significantly upregulated after BRD8 depletion by two independent sgRNAs and each sgRNA has two biological replicates. h, Schematic of the EP400 complex that occupies p53 target loci in GBM, with BRD8 being the most specific regulator.

Article Snippet: Individual sgRNAs were cloned into lentiviral expression vectors with an optimized sgRNA scaffold backbone: either LRG2.1 (U6-sgRNA-GFP, Addgene, 108098) or LRNeo2.1 for targeting human genes, or LRG (Addgene, 65656) for targeting mouse genes.

Techniques: Western Blot, Silver Staining, Immunoprecipitation, Plasmid Preparation, Control, Protein-Protein interactions, Biomarker Discovery, Quantitative RT-PCR, Expressing

Journal: Nature

Article Title: BRD8 maintains glioblastoma by epigenetic reprogramming of the p53 network

doi: 10.1038/s41586-022-05551-x

Figure Lengend Snippet: a, Scatter plot depicting the enrichment fold-change of immunoprecipitated proteins using Flag-tagged BRD8 compared to empty vector control in MS assays. b, GFP dropout assays of sgNeg (a negative control), sgCDK1 (a positive control) and two or three independent sgRNAs targeting 17 individual subunits of the EP400 complex in parental (p53WT) or p53-deficient (p53KO) A382 cells. Plotted is the mean ± s.d. of GFP-positive percentages from independent cells (normalized to P0, n = 3 biologically independent samples) at the indicated time points. c, Venn diagram showing overlapping genes located near high-confidence peaks among H2AZ, BRD8 and p53 in A382WT cells. d, Scatter plot depicting enrichment of GO analysis of gene sets with false discovery rate (FDR) of <0.05 using the 544 co-targets of H2AZ, BRD8 and p53 in c. e, Metaprofile of BRD8, p53 and H2AZ occupancy comparing BRD8 and p53 overlapping (OL) peaks with overall peaks (All). f, Metaprofile comparing H2AZ occupancy surrounding the summit of H2AZ peaks in A382WT cells transduced with sgBRD8 relative to sgNeg. H2AZ binding intensity is shown as sequencing depth normalized tag count. g, Venn diagram of overlapping upregulated genes after BRD8 depletion (sgBRD8 up) and p53 activation (p53OE up) (left), and GSEA plot of sgRNA-mediated depletion of H2AZ (sgH2AZ) compared with sgNeg in A382WT cells using the gene signature generated from the left Venn diagram. h, GFP dropout assays of sgNeg, sgCDK1 and sgH2AZ-1 in control (Ctrl) or two p53-deficient (sgTP53–3 and sgTP53–4) A382WT cells. Plotted is the mean ± s.d. at the indicated time points (normalized to P0, n = 3 biologically independent samples). P values were calculated using two-tailed unpaired Student’s t-tests.

Article Snippet: Individual sgRNAs were cloned into lentiviral expression vectors with an optimized sgRNA scaffold backbone: either LRG2.1 (U6-sgRNA-GFP, Addgene, 108098) or LRNeo2.1 for targeting human genes, or LRG (Addgene, 65656) for targeting mouse genes.

Techniques: Immunoprecipitation, Plasmid Preparation, Control, Negative Control, Positive Control, Transduction, Binding Assay, Sequencing, Activation Assay, Generated, Two Tailed Test

Journal: Nature

Article Title: BRD8 maintains glioblastoma by epigenetic reprogramming of the p53 network

doi: 10.1038/s41586-022-05551-x

Figure Lengend Snippet: a, Exon scanning of BRD8 in A382WT cells, with the x axis showing BRD8 codons (NP0006687) along the entire protein with annotation of the conserved domain and the y axis showing the log2(fold change) of each sgRNA. b, GFP dropout assays of sgNeg, sgCDK1, sgBRD8–1 and sgBRD8–2 in A382WT cells expressing Ctrl, CR-2-C3F or BDdel-C3F. A GFP reporter is linked to sgRNA expression. Plotted is the average percentage (normalized to P0, n = 3 biologically independent samples, the mean ± s.d.) at the indicated time points. P0 refers to day 3 after infection. P values were calculated using two-tailed unpaired Student’s t-tests. c, Schematic of the in vitro pull-down assays using recombinant GST-tagged BRD8 proteins and H2AZ peptides. d, In vitro pull-downs and western blots showing interactions of the bromodomain of BRD8 (top) and full-length of BRD8 (bottom) with H2AZ and H2AZac. Data shown represent two independent results. e,f, Meta plots showing chromatin accessibility after depleting BRD8 (e) or H2AZ (f) on BRD8-associated and p53-associated regions. g–i, Schematic of dual luciferase assay using CDKN1A promoter-driven firefly luciferase (g) in H2AZ wild-type (Control) (h) and H2AZ knockout (H2AZKO) (i) A382KO (p53-deficient) cells. Cells were transfected with the CDKN1A promoter-driven firefly construct, together with the indicated cDNAs. Luciferase activity was measured 48 h after transfection and Renilla luciferase activity was used as an internal control for normalization. Plotted is the mean ± s.d. (n = 4 biologically independent samples). P values were calculated using two-tailed unpaired Student’s t-tests. j, Schematic of BRD8 binding to H2AZ using its BD to enforce a compact chromatin state to repress the accessibility of p53 to its cell cycle targets. Targeting the BRD8 BD releases H2AZ and restores p53 tumour suppressive function by establishing more accessible chromatin for p53-mediated transactivation of its targets.

Article Snippet: Individual sgRNAs were cloned into lentiviral expression vectors with an optimized sgRNA scaffold backbone: either LRG2.1 (U6-sgRNA-GFP, Addgene, 108098) or LRNeo2.1 for targeting human genes, or LRG (Addgene, 65656) for targeting mouse genes.

Techniques: Expressing, Infection, Two Tailed Test, In Vitro, Recombinant, Western Blot, Luciferase, Control, Knock-Out, Transfection, Construct, Activity Assay, Binding Assay