Journal: Nature cell biology

Article Title: Pluripotency transcription factors and Tet1/2 maintain Brd4-independent stem cell identity

doi: 10.1038/s41556-018-0086-3

Figure Lengend Snippet: ( a ) Gene set enrichment plot showing that genes associated with high H3K9ac and H3K27ac are enriched for two independently defined pluripotency gene sets: Muller Plurinet (genes involved in the protein-protein network shared by diverse pluripotent cell types ) and Wong ESC Core (genes coordinately upregulated in mouse and human ESCs ). Data are derived from a single ChIP-Seq experiment . P values are calculated based on 1000 permutations by the GSEA algorithm and was not adjusted for multiple comparisons. ( b ) 2i increases acetylation at key pluripotency genes. H3K27ac (left) and H3K9ac (right) at enhancer (enh) or promoters of indicated genes as assessed by ChIP-qPCR. ( c ) ChIP-seq meta profile for Brd4 binding in ESCs cultured in S/L or S/L+2i. The metaprofile is centered on the midpoint of all Brd4 ChIP-seq peaks. ( d ) Brd4 ChIP-qPCR illustrating Brd4 binding in ESCs cultured in S/L (left) or S/L+2i (right) treated with DMSO (vehicle) or 500 nM JQ1 for 24 h. ( b,d ) Bars represent mean of n=3 technical replicates from one IP.

Article Snippet: Previously described guide RNA (gRNA) sequences targeting Brd4 or a nongenic region on mouse chromosome 8 (ch8) were cloned into the pCas9 (BB)2A-GFP (pX458, Addgene plasmid number Plasmid #48138), as previously described .

Techniques: Derivative Assay, ChIP-sequencing, ChIP-qPCR, Binding Assay, Cell Culture

Journal: Nature cell biology

Article Title: Pluripotency transcription factors and Tet1/2 maintain Brd4-independent stem cell identity

doi: 10.1038/s41556-018-0086-3

Figure Lengend Snippet: ( a ) Quantification of colony formation assay of cells cultured in S/L (left) or S/L+2i (right) transfected with Cas9 and the indicated sgRNA against a nongenic region of chromosome 8 (ch8, control) or exon 3 (ex3) or exon 4 (ex4) of Brd4 . Dotted line represents average of ch8 controls. Each bar represents quantification of a single well of a six-well plate; transfected samples were seeded in duplicate. ( b ) Alkaline phosphatase staining of colonies formed from single cells of clonal ESC lines edited with sgRNA against chromosome 8 (ch8) or Brd4 exon 3 and cultured in S/L or S/L+2i. One representative well of a six-well plate is shown. ( c ) Quantification of colony formation assay shown in ( b ). ( d ) Western blot depicting Brd4 levels in ESCs expressing doxycycline (dox)-inducible hairpins against Renilla (shRen) or Brd4 (shBrd4). Cells were cultured with or without dox for 48 h prior to harvest. Actin is used as a loading control. Western blot was performed two independent times. ( e ) Brightfield images of ESCs expressing shBrd4-2 cultured for 48 h with or without doxycycline (dox). ( f ) Population doublings of cells cultured for 72 h in doxycycline relative to controls grown without dox. ( g ) Alkaline phosphatase staining of colonies formed from single cells expressing the indicated hairpins grown in the presence or absence of doxycycline (dox) and cultured in S/L or S/L+2i. One representative well of a six-well plate is shown. All bars represent mean ±SEM ( c ) or ±SD ( f ) of n=3 independent samples. ****, P < 0.0001 by 2-way ANOVA with Sidak’s multiple comparisons post test (shBrd4-1, P = 7.2e-10; shBrd4-2, P = 4.6e-8). Scale bar, 100 μm.

Article Snippet: Previously described guide RNA (gRNA) sequences targeting Brd4 or a nongenic region on mouse chromosome 8 (ch8) were cloned into the pCas9 (BB)2A-GFP (pX458, Addgene plasmid number Plasmid #48138), as previously described .

Techniques: Colony Assay, Cell Culture, Transfection, Control, Staining, Western Blot, Expressing

Journal: Nature cell biology

Article Title: Pluripotency transcription factors and Tet1/2 maintain Brd4-independent stem cell identity

doi: 10.1038/s41556-018-0086-3

Figure Lengend Snippet: ( a ) qRT-PCR on key pluripotency genes in ESCs cultured in S/L or S/L+2i treated with DMSO (veh) or 500 nM JQ1 for 24h. ( b ) qRT-PCR on key pluripotency genes in S/L or S/L+2i-cultured ESCs expressing dox-inducible hairpins against Renilla (shRen) or Brd4 (shBrd4) and treated with dox for 48 h. Shown are levels of Nanog and Esrrb in cells with dox relative to control S/L+shRen cells without dox. ( c ) Heat map shows expression of pluripotency associated genes measured by RNA-Seq in ESCs cultured in S/L or S/L+2i with vehicle (veh, DMSO) or 500 nM JQ1 for 72 h. Color scale represents Log 2 relative to mean expression level. ( d ) Nanog binding to key pluripotency loci in ESCs cultured in S/L or S/L+2i treated with DMSO or 500 nM JQ1 for 24h, assessed by ChIP-qPCR. Bar represents mean of n=3 technical replicates from one IP. ( e ) ATAC-Seq meta profile of chromatin accessibility in ESCs cultured in S/L (left) or S/L+2i (right) with DMSO or 500 nM JQ1. Data from 2 independent replicates are shown. ( f ) At baseline, 82% of Oct4, Sox2, Nanog (OSN) binding sites have an ATAC-Seq peak above background. OSN sites make up 60% (4,250/7,084) of the peaks that maintain accessibility despite JQ1 treatment in S/L+2i cultured ESCs but only 17% (93/545) of peaks lost in both conditions. ( g ) ChIP-seq meta profile for Med1 binding in ESCs cultured in S/L or S/L+2i treated 500 nM JQ1. ( h ) Box plot shows relative Med1 binding at n=635 genes that are downregulated > 2 fold, FDR < 5% by JQ1 in ESCs cultured in S/L but not in ESCs cultured in S/L+2i. Values from ChIP-Seq experiment shown in (g) with 1 ChIP per condition. Box, 25-75 th percentile; bar, median; whiskers, 5-95 th percentile. ****, P < 1e-15; ns, P = 0.27 by 2-way ANOVA with Tukey’s multiple comparisons post test. See . ( i ) Sites that gain Med1 binding in S/L+2i+JQ1 relative to S/L+JQ1 are mostly OSN binding sites. Data presented as mean ± SD of n=3 independent samples ( a , b ).

Article Snippet: Previously described guide RNA (gRNA) sequences targeting Brd4 or a nongenic region on mouse chromosome 8 (ch8) were cloned into the pCas9 (BB)2A-GFP (pX458, Addgene plasmid number Plasmid #48138), as previously described .

Techniques: Quantitative RT-PCR, Cell Culture, Expressing, Control, RNA Sequencing, Binding Assay, ChIP-qPCR, ChIP-sequencing

Journal: Molecular cell

Article Title: ZFX mediates non-canonical oncogenic functions of the androgen receptor splice variant 7 (AR-V7) in castrate-resistant prostate cancer

doi: 10.1016/j.molcel.2018.08.029

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Specific antibodies used in ChIP-Seq include those against the C-terminus of full-length AR (Santa Cruz Biotechnology C-19; catalog # sc-815X; AR-FL specific) or AR-V7 (Precision Antibody catalog # AG10008; AR-V7 specific), the pan-AR antibodies that recognize the N-terminus of AR (Santa Cruz, AR N20; sc-816), BRD4 (Bethyl catalog # A301–985A100), HA tag (Abcam; 9110), ZFX (Thermo Fisher; catalog# PA5–34376) and anti-ZFX serum ( Chen et al., 2008 ) as a kind gift of Dr. Huck Hui Ng.

Techniques: Immunoprecipitation, Virus, Recombinant, Electron Microscopy, Transfection, Protease Inhibitor, cDNA Synthesis, SYBR Green Assay, Purification, RNA Library Preparation, Multiplex Assay, Proliferation Assay, Mutagenesis, Magnetic Beads, Western Blot, Stripping, Membrane, shRNA, Knockdown, Control, Construct, Plasmid Preparation, Software

Journal: Journal of translational medicine

Article Title: Innovative evaluation of selinexor and JQ1 synergy in leukemia therapy via C-MYC inhibition.

doi: 10.1186/s12967-025-06525-z

Figure Lengend Snippet: Fig. 3 Selinexor and JQ1 regulated the expression of C-MYC. (A-B) Expression levels of C-MYC at different concentrations of Selinexor and JQ1 in AML cells, respectively in cells MV4-11 and THP-1 at 24 h (A) and 48 h (B). (C) Detect the IC50 concentration of Selinexor in MV4-11 cells with scramble or BRD4- SgRNA. (D) Cell viability of MV4-11 cells with scramble or BRD4-SgRNA treated with different doses of Selinexor. (E) Expression levels of BRD4 and C-MYC proteins in MV4-11 cells with scramble or BRD4-SgRNA. (F) Detect the IC50 concentration of Selinexor in cells THP-1 cells with scramble or BRD4-SgRNA. (G) Cell viability of THP-1 cells with scramble or BRD4-SgRNA treated with different doses of Selinexor. (H) Expression levels of BRD4 and C-MYC proteins in THP-1 cells with scramble or BRD4-SgRNA. (I) Protein expression levels of C-MYC in MOLM13 cells transfected with vector or overexpressed C-MYC plasmid. (J) Cell viability of MOLM13 cells with scramble or C-MYC treated with different doses of Selinexor and JQ1. (K-L) RNA expression of C-MYC in MV4-11 (K) and MOLM13 (L) cells treated with different doses of Selinexor(50nM) and JQ1(50nM). All experiments were performed with at least three independent replicates

Article Snippet: LentiGuide-Puro plasmid targeting BRD4 and packaging vector pSPAX2 (12260, Addgene), pMD2G (12259, Addgene) were cotransfected into 293T cells to produce sgRNA lentivirus.

Techniques: Expressing, Concentration Assay, Transfection, Plasmid Preparation, RNA Expression

Journal: Nature Communications

Article Title: Intrinsic signaling pathways modulate targeted protein degradation

doi: 10.1038/s41467-024-49519-z

Figure Lengend Snippet: a Scheme of screening using HiBiT-BRD4–expressing cells in ( d , e ). b , c MZ1-dependent decrease of HiBiT luminescence in a HCT116 cell line expressing HiBiT-BRD4. HiBiT-BRD4 cells were treated with the indicated concentration (nM) of MZ1 for 2 h ( b ) or for the indicated number of hours ( c ) before HiBiT luminescence analysis ( n = 5 ( b ) or 4 ( c ), biological replicates). d Heatmap presentation of chemicals that enhance or repress BRD4 degradation ( n = 3, biological replicates). HiBiT-BRD4 cells were treated with candidate chemicals for 6 h and with 30 nM MZ1 for 2 h. e Chemicals that enhance HiBiT-BRD4 degradation in the presence of MZ1. The data in ( d ) are presented to analyze chemicals that enhance/repress the BRD4 degradation in the presence ( y -axis) or absence ( x -axis) of MZ1 ( n = 3, biological replicates). f , g HiBiT-BRD4 cells were treated with PDD, GSK, or luminespib (1, 3, or 10 μM) for 4 h and with 30 nM MZ1 ( f ) or 50 nM dBET6 ( g ) for 2 h ( n = 4 ( f ) or 3 ( g ), biological replicates).

Article Snippet: About 30 μg of sonicated chromatin was incubated with antibodies against BRD4 (Cell Signaling Technology, #13440, clone E2A7X, rabbit monoclonal, 10 μL) at 4 ̊C overnight.

Techniques: Expressing, Concentration Assay

Journal: Nature Communications

Article Title: Intrinsic signaling pathways modulate targeted protein degradation

doi: 10.1038/s41467-024-49519-z

Figure Lengend Snippet: a PDD promotes MZ1-induced degradation of BRD4 and BRD2. HeLa cells were treated with 3 μM PDD and/or 100 nM MZ1 for the indicated number of hours. The lower panel shows the band intensities of the blots from three biological replicates. P values in ANOVA are shown. b PDD promotes BRD4 degradation at a lower concentration than the concentration at which it exhibits cytotoxicity. (i) HiBiT-BRD4 cells were treated with the indicated concentration of PDD (6 h) together with MZ1 (2 h), and BRD4 degradation was quantified ( n = 6, biological replicates). Data were normalized to the MZ1 treatment alone. Asterisk: * P = 0.0003 or ** P < 0.0001 in ANOVA. (ii) The parental HCT116 cells were treated with the indicated concentration of PDD for 3 days, and cell viability was quantified ( n = 5, biological replicates). Asterisk: * P = 0.0003 or ** P < 0.0001 in ANOVA. c HCT116 cells were treated with 3 μM PDD for 6 h, and total RNA was isolated and subjected to RNA-sequencing analysis ( n = 3, biological replicates). d Knockdown of PARG promotes BRD4 degradation. HT1080 cells were transfected with the indicated siRNAs for 3 days, then treated with MZ1 as indicated. e , f PDD promotes BRD4 degradation induced by different PROTACs. HeLa ( e ) or HCT116 ( f ) cells were treated with PDD and/or ARV771 ( e ) or with the CRL4 CRBN -based dBET6 ( f ), as indicated. g , h Targeted degradation of ERα or MEK1 is not affected by PDD. MCF7 ( g ) or HCT116 ( h ) cells were treated with the indicated chemicals. i HiBiT-BRD4 cells were pre-treated with either PDD (4 h) and/or the indicated inhibitors (0.5 h) and then with 30 nM MZ1 for an additional 2 h ( n = 4, biological replicates).

Article Snippet: About 30 μg of sonicated chromatin was incubated with antibodies against BRD4 (Cell Signaling Technology, #13440, clone E2A7X, rabbit monoclonal, 10 μL) at 4 ̊C overnight.

Techniques: Concentration Assay, Isolation, RNA Sequencing, Knockdown, Transfection

Journal: Nature Communications

Article Title: Intrinsic signaling pathways modulate targeted protein degradation

doi: 10.1038/s41467-024-49519-z

Figure Lengend Snippet: a PDD promotes ubiquitylation of BRD4 and BRD2. HeLa cells were treated with the indicated concentration of MZ1 for 1 h. MG132 was added 5 min prior to the treatment with MZ1. The ubiquitylated proteins were purified from the cell lysates using TUBE-conjugated agarose. Input or TUBE-pulldown samples were subjected to western blotting, as indicated. b TUBE pulldown using either K29-, K48-, or K63-specific TUBEs. c BRD4 was modified with K48- and K29-linked ubiquitin chains. Endogenous BRD4 was immunopurified from HCT116 cells treated with PDD (3 μM, 6 h) and MZ1 (100 nM, 1 h) together with MG132 (20 μM, 1 h) and subsequently subjected to PRM-based ubiquitin linkage quantification ( n = 2, biological replicates). d HCT116 cells were treated with PDD (3 μM, 6 h) and/or MZ1 (100 nM, 1 h), and BRD4-modified ubiquitin chains were analyzed using PRM. The data show abundance (normalized to the vehicle) of signature peptides for K29 ubiquitin linkages and total ubiquitin ( n = 2, biological replicates). e HT1080 cells transfected with the indicated siRNAs were treated as in ( a , b ) to pulldown K29-linked ubiquitin chains. f Knockdown of TRIP12 partially canceled PDD-dependent promotion of BRD4 degradation. HeLa cells were transfected with the indicated siRNAs and treated with PDD (3 μM, 6 h) and/or MZ1 (100 nM, 1 or 2 h). g BRD4–MZ1–CRL2 VHL ternary complex assembly. 293T cells were transfected with FLAG-BRD4 (lanes 2–5) and/or HA-VHL (lanes 1–5), and cell lysates were subjected to immunoprecipitation using anti-FLAG antibody. h The samples in ( g ) were subjected to LC-MS and label-free quantification ( n = 3, biological replicates, ANOVA). i Heatmaps of ChIP-seq signals of BRD4 in the cells treated either with control, PDD17273, or MZ1. BRD4-binding regions in control cells ( n = 3562) were subtracted for plotting, and peaks were divided into three clusters. j Percentage of BRD4-binding regions consisting of the three clusters. k Metaplots of ChIP-seq signals of BRD4 over the center of peaks. l Metaplots depicting H3K27ac ChIP-seq signals in HCT116 cells over BRD4-binding regions. m HCT116 cells were treated with PDD (5 μM, 4 h), and cell fractionation was performed. PARylation of chromatin fractions was analyzed.

Article Snippet: About 30 μg of sonicated chromatin was incubated with antibodies against BRD4 (Cell Signaling Technology, #13440, clone E2A7X, rabbit monoclonal, 10 μL) at 4 ̊C overnight.

Techniques: Concentration Assay, Purification, Western Blot, Modification, Ubiquitin Proteomics, Transfection, Knockdown, Immunoprecipitation, Liquid Chromatography with Mass Spectroscopy, Quantitative Proteomics, ChIP-sequencing, Control, Binding Assay, Cell Fractionation

Journal: Nature Communications

Article Title: Intrinsic signaling pathways modulate targeted protein degradation

doi: 10.1038/s41467-024-49519-z

Figure Lengend Snippet: a PERK inhibitors promote BRD4 degradation. HCT116 cells were treated with either GSK, GSK2656157, or AMG-PERK (10 μM, 6 h) and/or MZ1 (100 nM, 1 or 2 h). b HSP90 inhibitors promote BRD4 degradation. HCT116 cells were treated with either GSK, luminespib, or 17-AAG (10 μM, 6 h) and/or MZ1 (100 nM, 1 or 2 h). c BRD4 degradation in the presence of GSK is proteasome-dependent. HCT116 cells were treated with either GSK (10 μM, 6 h), MG132 (20 μM, 2 h), or MZ1 (100 nM, 1 or 2 h). d HCT116 cells were treated with either PDD, GSK, or luminespib (10 μM, 14 h) and/or 50 ng/mL CHX for the indicated number of hours. Total cell lysates were subjected to Western blotting. e , f HCT116 cells were transfected with the indicated siRNAs and treated with MZ1 (100 nM, 1 or 2 h). (Right) BRD4 band intensities were quantified ( n = 3 ( d ) or 2 ( e ), biological replicates). Asterisk: * P = 0.0014 or ** P = 0.0002 in ANOVA. g , h HCT116 cells were treated with either GSK, luminespib, or 17-AAG (10 μM, 6 h) and/or MZ1 (100 nM, 1 h). Cell lysates were subjected to pulldown using the indicated TUBEs. i 293T cells were transfected with FLAG-BRD4 (lanes 2–6) and/or HA-VHL (lanes 1–6) and then treated with the indicated chemicals; co-immunoprecipitation was subsequently performed.

Article Snippet: About 30 μg of sonicated chromatin was incubated with antibodies against BRD4 (Cell Signaling Technology, #13440, clone E2A7X, rabbit monoclonal, 10 μL) at 4 ̊C overnight.

Techniques: Western Blot, Transfection, Immunoprecipitation

Journal: Nature Communications

Article Title: Intrinsic signaling pathways modulate targeted protein degradation

doi: 10.1038/s41467-024-49519-z

Figure Lengend Snippet: a , b PDD or GSK promotes BRD4 degradation induced by SIM1. HeLa ( a ) or HCT116 ( b ) cells were treated with PDD, GSK, and/or MZ1 as indicated, and cell lysates were subjected to western blotting. c HiBiT-BRD4 cells were treated as indicated, and luminescence was measured ( n = 3, biological replicates). d , e PDD or GSK promotes cell death induced by SIM1. HeLa cells were treated with PDD, GSK, and/or MZ1 for 3 days, as indicated. Asterisk: ( d ) * P = 0.0006 or ** P < 0.0001 in ANOVA ( n = 5, biological replicates). e ** P < 0.0001 in ANOVA ( n = 5, biological replicates). f , h – j HeLa ( f ), HCT116 ( h ), MCF7 ( i ), or MDA-MB231 ( j ) cells were treated with the indicated chemicals for 6 h or for the indicated number of hours (100 nM ThalSNS, 30 nM ARV471, 0.5 μM SJF8240). Total cell lysates were subjected to Western blotting. The lower panel shows the band intensities of the blots from two biological replicates. g HeLa cells were treated with the indicated chemicals for 3 days, and cell viability was quantified. Asterisk: * P = 0.026 or ** P < 0.0001 in ANOVA ( n = 5, biological replicates). k Schematic model. Various cell-intrinsic pathways spontaneously counteract target degradation at multiple steps. Inhibitors to PARG, PERK, or HSP90 robustly enhance the targeted degradation of BRD4 as well as BRD2/3 and sensitize cells to PROTAC-induced apoptosis. PARG inhibition promotes TRIP12-mediated K29/K48-branched ubiquitylation of BRD4 by facilitating the BRD4-PROTAC-CRL2 VHL ternary complex, while HSP90 inhibition promotes BRD4 degradation after the ubiquitylation step.

Article Snippet: About 30 μg of sonicated chromatin was incubated with antibodies against BRD4 (Cell Signaling Technology, #13440, clone E2A7X, rabbit monoclonal, 10 μL) at 4 ̊C overnight.

Techniques: Western Blot, Inhibition

Journal: Cancer research

Article Title: BET inhibitors potentiate chemotherapy and killing of SPOP -mutant colon cancer cells via induction of DR5

doi: 10.1158/0008-5472.CAN-18-3223

Figure Lengend Snippet: (A) HCT116 cells transfected with control scrambled or BRD4 siRNA were analyzed by RNA-Seq at 24 hr after transfection. Left, verification of BRD4 knockdown and DR5 induction by western blotting; right, heat map for comparing the expression of genes related to the death receptor and mitochondrial apoptotic pathways with an indication of the ratio of DR5 FPKM (fragments per kilobase of transcript per million mapped reads). (B) Western blotting of DR5 in HCT116 cells treated with the BETi JQ1, I-BET151, OTX015 or I-BET762 at indicated concentrations for 36 hr. (C) Western blot (upper) and RT-PCR (lower) analyses of DR5 expression at indicated time points in HCT116 cells treated with indicated BETi (1 μM). (D) Western blotting of indicated proteins in CRC cell lines with indicated p53, KRAS, BRAF and PIK3CA status at 24 hr after transfection with control scrambled or BRD4 siRNA. (E) Western blotting of indicated proteins (upper) and RT-PCR of DR5 (lower) in indicated CRC cell lines treated with JQ1 or OTX015 (1 μM) for 36 hr. In (A)-(E), relative expression of BRD4 and DR5 was quantified using the Image J program, normalized to that of β-actin, and expressed as a ratio relative to that in untreated cells or those transfected with control siRNA. In (C) and (E), results were expressed as means ± SD of three independent experiments. *, P <0.05; **, P <0.01.

Article Snippet: SiRNA transfection was performed 24 hr before drug treatment using 200 pmole of control scrambled, CHOP (5’-GCACAGCUAGCUGAAGAGAdTdT-3’), DR5 (5’-AAGACCCUUGUGCUCGUUGUCdTdT-3’) (Dharmacon), BRD4 (sc-43639), or SPOP (sc-63056) siRNA (Santa Cruz Biotechnology). mRNA sequencing (RNA-Seq) Total RNA was prepared from HCT116 cells transfected with either control scrambled or BRD4 siRNA for 24 hr using the Quick-RNA Kit (Zymo Research) according to manufacturer’s instructions.

Techniques: Transfection, RNA Sequencing Assay, Western Blot, Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: Cancer research

Article Title: BET inhibitors potentiate chemotherapy and killing of SPOP -mutant colon cancer cells via induction of DR5

doi: 10.1158/0008-5472.CAN-18-3223

Figure Lengend Snippet: (A) Western blot (left) and RT-PCR (right) analyses of DR5 and CHOP in HCT116 cells at 24 hr after transfection with control scrambled or BRD4 siRNA. (B) Western blotting of CHOP and indicated ER stress markers (upper) and RT-PCR (lower) of CHOP in indicated CRC cell lines treated with indicated BETi (1 μM) for 36 hr. p-eIF2α: phospho-eIF2α, Ser51; p-PERK: phospho-PERK, Thr980. (C) Western blotting of CHOP and DR5 (upper) and RT-PCR (lower) of DR5 in HCT116 and RKO cells transfected with control scrambled or CHOP siRNA and treated with JQ1 (1 μM) for 36 hr. (D) Chromatin immunoprecipitation (ChIP) analysis of the binding of CHOP to the DR5 promoter in HCT116 cells treated with JQ1 (1 μM) for 36 hr with IgG as a negative control. PCR was performed using primers surrounding the CHOP-binding site in the DR5 promoter, followed by analysis of PCR products by agarose gel electrophoresis. (E) HCT116 cells transfected with a luciferase reporter of the DR5 promoter containing either wild-type (WT) or mutant CHOP-binding site, along with the transfection control β-galactosidase reporter pCMVβ. After 24 hr, cells were treated with JQ1 (1 μM) for 36 hr. Upper, a schematic representation of the CHOP-binding site in the DR5 promoter with “xxxx” indicating mutated nucleotides in the mutant reporter; lower, luciferase activities of the WT and mutant DR5 promoter reporters normalized to that of pCMVβ. In (A) and (C), relative expression of BRD4 and CHOP was quantified using Image J, normalized to that of β-actin, and expressed as a ratio relative to that in cells transfected with control siRNA. In (A), (B), (C) and (E), results were expressed as means ± SD of three independent experiments. *, P <0.05; **, P <0.01.

Article Snippet: SiRNA transfection was performed 24 hr before drug treatment using 200 pmole of control scrambled, CHOP (5’-GCACAGCUAGCUGAAGAGAdTdT-3’), DR5 (5’-AAGACCCUUGUGCUCGUUGUCdTdT-3’) (Dharmacon), BRD4 (sc-43639), or SPOP (sc-63056) siRNA (Santa Cruz Biotechnology). mRNA sequencing (RNA-Seq) Total RNA was prepared from HCT116 cells transfected with either control scrambled or BRD4 siRNA for 24 hr using the Quick-RNA Kit (Zymo Research) according to manufacturer’s instructions.

Techniques: Western Blot, Reverse Transcription Polymerase Chain Reaction, Transfection, Chromatin Immunoprecipitation, Binding Assay, Negative Control, Agarose Gel Electrophoresis, Luciferase, Mutagenesis, Expressing

Journal: Cancer research

Article Title: BET inhibitors potentiate chemotherapy and killing of SPOP -mutant colon cancer cells via induction of DR5

doi: 10.1158/0008-5472.CAN-18-3223

Figure Lengend Snippet: (A) MTS analysis of HCT116 cells transfected with control scrambled or BRD4 siRNA and treated with 5-FU or oxaliplatin (OXA) at indicated concentrations for 72 hr. (B) MTS analysis of HCT116 cells treated with 5-FU or OXA at indicated concentrations with or without JQ1 (1 μM) or OTX015 (1 μM) for 72 hr. (C) MTS analysis of WT and DR5-KO HCT116 cells transfected as in (A) and treated with 5-FU and OXA as in (A) and (B). (D) WT and DR5-KO HCT116 cells treated with 5-FU (15 μg/mL) or OXA (15 μM) for 36 hr with or without BRD4 knockdown as in (A) or BETi treatment as in (B). Apoptosis was analyzed by counting cells containing condensed and fragmented nuclei after nuclear staining with Hoechst 33258. (E) Western blotting of cleaved (C) caspases 3, 8 and 9 in cells treated as in (D). Relative BRD4 expression was quantified using the Image J program, normalized to that of β-actin, and expressed as a ratio relative to that in cells transfected with control siRNA. (F) Colony formation of cells treated as in (D) as analyzed by crystal violet staining. Upper, representative images of colonies; lower, enumeration of colony numbers. In (D) and (F), results were expressed as means ± SD of three independent experiments. *, P <0.05; **, P <0.01; ***, P <0.001.

Article Snippet: SiRNA transfection was performed 24 hr before drug treatment using 200 pmole of control scrambled, CHOP (5’-GCACAGCUAGCUGAAGAGAdTdT-3’), DR5 (5’-AAGACCCUUGUGCUCGUUGUCdTdT-3’) (Dharmacon), BRD4 (sc-43639), or SPOP (sc-63056) siRNA (Santa Cruz Biotechnology). mRNA sequencing (RNA-Seq) Total RNA was prepared from HCT116 cells transfected with either control scrambled or BRD4 siRNA for 24 hr using the Quick-RNA Kit (Zymo Research) according to manufacturer’s instructions.

Techniques: Transfection, Staining, Western Blot, Expressing

Journal: Cancer research

Article Title: BET inhibitors potentiate chemotherapy and killing of SPOP -mutant colon cancer cells via induction of DR5

doi: 10.1158/0008-5472.CAN-18-3223

Figure Lengend Snippet: (A) MTS analysis of SPOP-WT (HCT116, RKO, SW48, Lim1215 and HT29) and SPOP-mutant (NCI-H508 and SNU-407) CRC cells treated with JQ1 or OTX015 at indicated concentrations for 72 hr. (B) Colony formation of the indicated CRC cells treated with JQ1 (0.5 μM) for 36 hr was analyzed by crystal violet staining. Upper, representative images of colonies; lower, enumeration of colony numbers. (C) Western blotting of BRD4 and SPOP in indicated SPOP-WT and -mutant CRC cell lines. (D) Upper, western blotting of DR5 in indicated SPOP-WT and -mutant CRC cell lines treated with JQ1 at indicated concentrations for 36 hr; lower, quantification of DR5 signals from both isoforms using the NIH Image J program, normalized to that of β-actin, and expressed as a ratio relative to untreated controls. (E) Western blotting of indicated proteins in NCI-H508 and SNU-407 cells transfected with control scrambled or SPOP siRNA and treated with JQ1 (0.5 μM) or OTX015 (0.5 μM) for 36 hr. Relative SPOP expression was quantified using Image J, normalized to that of β-actin, and expressed as a ratio relative to that in untreated cells that were transfected with control siRNA. (F) NCI-H508 and SNU-407 cells transfected with control scrambled or SPOP siRNA were treated with JQ1 (0.5 µM) or OTX015 (0.5 µM) for 36 hr. Apoptosis was analyzed by counting cells containing condensed and fragmented nuclei after nuclear staining. (G) MTS analysis of NCI-H508 and SNU-407 cells transfected with control scrambled or SPOP siRNA and treated with JQ1 or OTX015 at indicated concentrations for 72 hr. In (B) and (F), results were expressed as means ± SD of three independent experiments. *, P <0.05; **, P <0.01; ***, P <0.001.

Article Snippet: SiRNA transfection was performed 24 hr before drug treatment using 200 pmole of control scrambled, CHOP (5’-GCACAGCUAGCUGAAGAGAdTdT-3’), DR5 (5’-AAGACCCUUGUGCUCGUUGUCdTdT-3’) (Dharmacon), BRD4 (sc-43639), or SPOP (sc-63056) siRNA (Santa Cruz Biotechnology). mRNA sequencing (RNA-Seq) Total RNA was prepared from HCT116 cells transfected with either control scrambled or BRD4 siRNA for 24 hr using the Quick-RNA Kit (Zymo Research) according to manufacturer’s instructions.

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

Journal: PLOS Genetics

Article Title: Resf1 is a compound G4 quadruplex-associated tumor suppressor for triple negative breast cancer

doi: 10.1371/journal.pgen.1011236

Figure Lengend Snippet: (A) From the N 2 backcross cohort in , 131 mice were categorized into Resf1 over and under-expressed groups and plotted as Kaplan-Meier survival curves showing worse DMFS survival in animals under-expressing Resf1 . (B) RNA-seq of paired spontaneous mammary tumors and lung metastases from FVB/NJ and MOLF/EiJ mice that were crossed with MMTV-PyMT were analyzed for Resf1 expression levels and shows higher expression levels in the less metastatic MOLF/EiJ strain. (C) Query of the METABRIC human breast cancer tumor database shows significant reduction in survival at lower expression levels of RESF1 . High (red), intermediate (gray), low (blue). (D) Query of the UCSC BLAT Genome Browser for the 5’ UTR and upstream region of Resf1 displays DHS peaks (green) in the highlighted yellow area. Included are locations of primers used for PCR and cloning of the promoter enhancer region. (E) Upstream regions of Resf1 in FVB/NJ and MOLF/EiJ were cloned into the luciferase pGL4.23 reporter plasmid, and dually transfected with the Renilla hRluc plasmid into HEK293T cells, with empty vector as a negative control. Values show ratio of Firefly luciferase to Renilla in empty vector control, FVB/NJ upstream region, and MOLF/EiJ upstream region. P-value based on unpaired t-test.

Article Snippet: To generate the donor vector containing dTAG-2xHA sequences, pCRIS-PITChv2-dTAG-Puro (RESF1) (Addgene, #91796) vector was utilized, serving as a template for PCR amplification of the dTAG-2xHA sequence.

Techniques: Expressing, RNA Sequencing, Cloning, Clone Assay, Luciferase, Plasmid Preparation, Transfection, Negative Control, Control

Journal: PLOS Genetics

Article Title: Resf1 is a compound G4 quadruplex-associated tumor suppressor for triple negative breast cancer

doi: 10.1371/journal.pgen.1011236

Figure Lengend Snippet: (A) qRT-PCR analysis of Resf1 in primary tumors from WT and genetrap hypomorph crossed with MMTV/PyMT mice (n = 5 per group). Control and hypomorph mice were crossed with MMTV-PyMT mice to induce spontaneous mammary tumors and pulmonary metastases (B-E). (B) Primary mammary fatpad tumors were collected and weighed for WT (n = 27) and hypomorph (n = 38) and resulted in significantly larger tumors in hypomorph mice, p-value calculated by Mann-Whitney test. (C) Surface lung metastases were counted, resulting in more metastases in hypomorph mice, p-value calculated by Mann-Whitney test. (D) Normalization of lung metastases per gram tumor to account for larger tumor size remained significantly higher in hypomorph mice, p-value calculated by Mann-Whitney test. (E) Metastatic incidence was higher in hypomorph mice compared to control.

Article Snippet: To generate the donor vector containing dTAG-2xHA sequences, pCRIS-PITChv2-dTAG-Puro (RESF1) (Addgene, #91796) vector was utilized, serving as a template for PCR amplification of the dTAG-2xHA sequence.

Techniques: Quantitative RT-PCR, Control, MANN-WHITNEY

Journal: PLOS Genetics

Article Title: Resf1 is a compound G4 quadruplex-associated tumor suppressor for triple negative breast cancer

doi: 10.1371/journal.pgen.1011236

Figure Lengend Snippet: (A) qRT-PCR analysis of 6DT1 shRNA-mediated Resf1 stable KD cells. (B) Weight of primary tumors from 6DT1 Control (scramble), H4, and H6 cells orthotopically injected into the 4 th mammary fatpad of syngeneic FVB/NJ mice, n = 15 mice per group. (C) Surface pulmonary metastasis in mice from (B). (D) Pulmonary metastases normalized per gram tumor from (B). (E) qRT-PCR analysis of Mvt1 shRNA-mediated Resf1 stable KD cells. (F) Weight of primary tumors from Mvt1 Control (scramble), H4, and H6 cells orthotopically injected into the 4 th mammary fatpad of syngeneic FVB/NJ mice, n = 10 mice per group. (G) Surface pulmonary metastasis in mice from (F). (H) Pulmonary metastases normalized per gram tumor from (F). (I) qRT-PCR analysis of MDA-MB-231 shRNA-mediated RESF1 stable KD cells. (J) Weight of primary tumors from MDA-MB-231 Control (scramble) and G5 KD cells orthotopically injected into the 4th mammary fatpad of nu/nu mice, n = 10 mice per group. (K) Surface pulmonary metastasis in mice from (J). (L) Pulmonary metastases normalized per gram tumor from (J). P-values in B-D and F-H calculated by ordinary one-way ANOVA with Dunnett’s multiple comparison test. P-values in J-L calculated by Mann-Whitney test.

Article Snippet: To generate the donor vector containing dTAG-2xHA sequences, pCRIS-PITChv2-dTAG-Puro (RESF1) (Addgene, #91796) vector was utilized, serving as a template for PCR amplification of the dTAG-2xHA sequence.

Techniques: Quantitative RT-PCR, shRNA, Control, Injection, Comparison, MANN-WHITNEY

Journal: PLOS Genetics

Article Title: Resf1 is a compound G4 quadruplex-associated tumor suppressor for triple negative breast cancer

doi: 10.1371/journal.pgen.1011236

Figure Lengend Snippet: BioTAP XL data was mapped against the T2Tv2 human genome and showed significant associations between Resf1 and (A) 7S RNAs, (B) exons of protein-coding genes, and (C) ribosomal RNA repeats. (D) Histogram graph showing the majority of the BioTAP XL sites within 0–5 kb downstream of the TSS. (E) Comparisons of the top 1000 RESF1 BioTAP XL binding sites versus the bottom 1000 sites revealed a significant association between RESF1 and GC-enriched sequences. (F) Identification of the de novo motif with the potential to form G4s within the BioTAP XL sites.

Article Snippet: To generate the donor vector containing dTAG-2xHA sequences, pCRIS-PITChv2-dTAG-Puro (RESF1) (Addgene, #91796) vector was utilized, serving as a template for PCR amplification of the dTAG-2xHA sequence.

Techniques: Binding Assay

Journal: PLOS Genetics

Article Title: Resf1 is a compound G4 quadruplex-associated tumor suppressor for triple negative breast cancer

doi: 10.1371/journal.pgen.1011236

Figure Lengend Snippet: (A) BioTAP XL analysis showing a significant association between RESF1 and RPL27 exon 3 containing G4s on both strands. (B) Gel mobility shift assays with RPL27 compound G4s (G4.1 and G4.2) on the template strand. (C-D) Circular dichroism analysis of RPL27 compound G4s on the template strand with the presence of lithium, sodium, or potassium ions.

Article Snippet: To generate the donor vector containing dTAG-2xHA sequences, pCRIS-PITChv2-dTAG-Puro (RESF1) (Addgene, #91796) vector was utilized, serving as a template for PCR amplification of the dTAG-2xHA sequence.

Techniques: Mobility Shift, Circular Dichroism

Journal: PLOS Genetics

Article Title: Resf1 is a compound G4 quadruplex-associated tumor suppressor for triple negative breast cancer

doi: 10.1371/journal.pgen.1011236

Figure Lengend Snippet: Confocal fluorescence microscopy images of (A) a 30-minute OPP (o-propargyl-puromycin) pulse, (B) a 30-minute EU (ethinyl uridine) pulse, and (C) PolyA mRNA FISH, (D) 5.8S, 18S, and 28S rRNA FISH of 4T1 CRISPR KO cell lines. (E) Western blot analysis showing shRNA-mediated KDs of Resf1 in 6DT1, Met1, and MDA-MB-231 cell lines. (F) Tumorsphere assays with 4T1 and 6DT1 CRISPR KO cells, 6DT1, Met1, and MDA-MB-231 KD cells. P-values were calculated by ordinary one-way ANOVA with Dunnett’s multiple comparison test.

Article Snippet: To generate the donor vector containing dTAG-2xHA sequences, pCRIS-PITChv2-dTAG-Puro (RESF1) (Addgene, #91796) vector was utilized, serving as a template for PCR amplification of the dTAG-2xHA sequence.

Techniques: Fluorescence, Microscopy, CRISPR, Western Blot, shRNA, Comparison

Journal: PLOS Genetics

Article Title: Resf1 is a compound G4 quadruplex-associated tumor suppressor for triple negative breast cancer

doi: 10.1371/journal.pgen.1011236

Figure Lengend Snippet: (A-D) GSEA analysis snapshots of MYC-target pathways that are upregulated in 6DT1 shRNA KD, 4T1 CRISPR KO, HEK293T siRNA, and MDA-MB-231 shRNA KD. (E) BioTAP XL analysis of the association between RESF1 and MYC exons.

Article Snippet: To generate the donor vector containing dTAG-2xHA sequences, pCRIS-PITChv2-dTAG-Puro (RESF1) (Addgene, #91796) vector was utilized, serving as a template for PCR amplification of the dTAG-2xHA sequence.

Techniques: shRNA, CRISPR

Journal: Molecular cell

Article Title: Phosphorylation by JNK switches BRD4 functions.

doi: 10.1016/j.molcel.2024.09.030

Figure Lengend Snippet: Figure 1. JNK directly interacts with and phosphorylates BRD4 (A) BRD4 co-localizes with kinase active JNK. Proximity ligation assays (PLAs) with anti-BRD4 and anti-pJNK on fixed HCT116 cells. Negative control; anti- nucleolin, and anti-BRD4 (scale bars, 20 mM). (B) JNK co-immunoprecipitates with BRD4. BRD4 was immunoprecipitated from HeLa nuclear extract using anti-BRD4 and immunoblotted with anti-JNK. (C) BRD4 binds JNK directly. Recombinant JNK1 (0.1 and 0.2 mg) was pulled down with 0.5 mg rBRD4 immobilized on FLAG beads. (D) JNK phosphorylates BRD4. Upper: map of BRD4 and deletion mutants. Lower: autoradiograph of kinase assays with GST-JNK1 and WT-BRD4 or deletion mutants. (E) JNK phosphorylation sites on BRD4. Upper: JNK consensus phosphorylation sites located on human/mouse BRD4. Lower: autoradiograph of kinase assays with His-JNK1 and BRD4 WT or the point mutants. (F) BRD4 is phosphorylated at Thr1186 and Thr1212 JNK activation. HCT116 cells were treated with anisomycin, heat shock, LPS treatment, or UV stress. BRD4 phosphorylation was assessed by immunoblotting (upper) and densitometric quantification (lower). (G) BRD4’s interaction with JNK is abrogated by phosphorylation. CoIP of JNK with BRD4 following anisomycin treatment of WT- and 3A-BRD4-expressing HCT116 cells. See also Figures S1 and S2.

Article Snippet: The primary antibodies used were as follows: anti-BRD4 rabbit monoclonal antibody (Bethyl; [BL-149-2H5]) (1:100 dilution), anti-phospho JNK mouse monoclonal antibody (G-7, Santa cruz biotechnology) (1:100 dilution), and anti-Nucleolin (sc-8031, Santa cruz biotechnology) (1:100 dilution).

Techniques: Ligation, Negative Control, Immunoprecipitation, Recombinant, Autoradiography, Phospho-proteomics, Activation Assay, Western Blot, Expressing

Journal: Molecular cell

Article Title: Phosphorylation by JNK switches BRD4 functions.

doi: 10.1016/j.molcel.2024.09.030

Figure Lengend Snippet: Figure 2. JNK phosphorylation of BRD4 releases it from chromatin (A) BRD4 is released from the chromatin upon JNK activation by anisomycin. Immunoblots of chromatin-free (CF) and chromatin-bound (CB) BRD4 in HCT116 cells following treatment with anisomycin. (B) Inhibition of JNK kinase blocks BRD4’s release from chromatin. Immunoblots of CF and CB BRD4 in HCT116 cells transfected with WT JNK1 or JNK1 and dominant-negative kinase mutants JNK1/JNK2 (APF) individually or in combination, followed by anisomycin treatment. (C) BRD4 is released from the chromatin upon JNK activation by heat shock. Left: immunoblots of CF and CB BRD4 in HCT116 cells grown at 37C or heat shocked at 42C for 15 min in the presence or absence of JNK inhibitor SP600125. Right: immunoblots showing pJNK levels under the above conditions. (D) JNK preferentially phosphorylates BRD4 bound to mononucleosomes. Anti-BRD4 pT1212 and anti-BRD4 immunoblots of kinase assays with recombinant JNK1 and BRD4 after pre-incubating BRD4 with or without assembled mononucleosomes (MN) for 10 or 20 min. (E) Mutation of BRD4 phosphorylation sites prevents BRD4 release from chromatin. Left: immunoblots of CF and CB BRD4 in HCT116 cells transfected with WT or 3A-BRD4 and subjected to heat shock treatment. Right: immunoblots showing pJNK levels following heat shock in WT- and 3A-BRD4-expressing cells. (F) JNK activation results in global loss of CB BRD4. Total BRD4 peaks detected in BRD4 ChIP-seq of control-untreated and anisomycin-treated DLD1 BRD4- IAA7 cells expressing endogenous BRD4 or exogenous WT or 3A-BRD4 following auxin treatment. (G) Loss of JNK-phosphorylated BRD4 from chromatin is widespread. Distribution of BRD4 ChIP-seq peaks across the genomes of cells described in (F).

Article Snippet: The primary antibodies used were as follows: anti-BRD4 rabbit monoclonal antibody (Bethyl; [BL-149-2H5]) (1:100 dilution), anti-phospho JNK mouse monoclonal antibody (G-7, Santa cruz biotechnology) (1:100 dilution), and anti-Nucleolin (sc-8031, Santa cruz biotechnology) (1:100 dilution).

Techniques: Phospho-proteomics, Activation Assay, Western Blot, Inhibition, Transfection, Dominant Negative Mutation, Recombinant, Mutagenesis, Expressing, ChIP-sequencing, Control

Journal: Molecular cell

Article Title: Phosphorylation by JNK switches BRD4 functions.

doi: 10.1016/j.molcel.2024.09.030

Figure Lengend Snippet: Figure 3. JNK-mediated BRD4 release from chromatin disrupts its nucleosome clearance function (A) BRD4 binding to mononucleosomes is abrogated upon phosphorylation by JNK. Anti-histone H3 immunoblot of assembled mononucleosomes pulled down by recombinant WT or 3A-BRD4 that was either unphosphorylated or pre-phosphorylated (*) by JNK and immobilized on FLAG beads. (B) JNK phosphorylation of BRD4 inhibits H3K122 acetylation. Anti-histone H3K122ac immunoblot of assembled mononucleosomes subjected to an in vitro HAT assay with recombinant WT or 3A-BRD4 that was either unphosphorylated or pre-phosphorylated (*) by JNK. (C) H3K122 acetylation is reduced in vivo upon JNK activation. Immunoblots of whole-cell extracts (WCEs) of HCT116 cells that were untreated (control) or treated with either DMSO (mock) or anisomycin. (D) In vivo H3K122 acetylation by BRD4 is regulated by JNK. Immunoblots of WCEs of HCT116 cells that were transfected with WT or 3A-BRD4 and treated with or without anisomycin. (E) In vivo H3K122 acetylation is regulated by JNK kinase activity. Immunoblots of WCEs of HCT116 cells that were transfected with WT JNK1, JNK2, or their respective dominant-negative mutants (JNK APF), either individually or together, and treated with or without anisomycin. Densitometric quantification of H3K122ac levels is shown below. (F) Nucleosome clearance activity by BRD4 is controlled by JNK phosphorylation. Autoradiograph of an in vitro nucleosome clearance assay showing disso- ciation of assembled mononucleosomes by unphosphorylated or JNK pre-phosphorylated (*) WT or 3A-BRD4 upon being subjected to a HAT assay in the presence or absence of AcCoA. See also Figure S4.

Article Snippet: The primary antibodies used were as follows: anti-BRD4 rabbit monoclonal antibody (Bethyl; [BL-149-2H5]) (1:100 dilution), anti-phospho JNK mouse monoclonal antibody (G-7, Santa cruz biotechnology) (1:100 dilution), and anti-Nucleolin (sc-8031, Santa cruz biotechnology) (1:100 dilution).

Techniques: Binding Assay, Phospho-proteomics, Western Blot, Recombinant, In Vitro, HAT Assay, In Vivo, Activation Assay, Control, Transfection, Activity Assay, Dominant Negative Mutation, Autoradiography

Journal: Molecular cell

Article Title: Phosphorylation by JNK switches BRD4 functions.

doi: 10.1016/j.molcel.2024.09.030

Figure Lengend Snippet: Figure 4. JNK-mediated BRD4 release from chromatin activates BRD4 kinase (A) JNK activation induces phosphorylation of BRD4 kinase substrates. Immunoblots of WCEs of HCT116 cells that were treated with DMSO (mock), anisomycin, or anisomycin with JNK peptide inhibitor D-JNK1. (B) Blocking JNK activity inhibits induction of BRD4 kinase. Immunoblots of WCEs of HCT116 cells that were transfected with FLAG-tagged WT JNK1, JNK2, or respective dominant-negative mutants (JNK APF), either individually or together, and treated with or without anisomycin. (C) JNK phosphorylation of BRD4 is necessary for induction of BRD4 kinase. Immunoblots of WCEs of HCT116 cells transfected with WT or 3A-BRD4 and subjected to heat shock. (D) BRD4 phosphorylation regulates Myc stability. Immunofluorescence images showing Myc levels in HCT116 cells transfected with either WT or 3A-BRD4 or empty vector (control) and subjected to heat shock (scale bars, 25 mM). See also Figure S5.

Article Snippet: The primary antibodies used were as follows: anti-BRD4 rabbit monoclonal antibody (Bethyl; [BL-149-2H5]) (1:100 dilution), anti-phospho JNK mouse monoclonal antibody (G-7, Santa cruz biotechnology) (1:100 dilution), and anti-Nucleolin (sc-8031, Santa cruz biotechnology) (1:100 dilution).

Techniques: Activation Assay, Phospho-proteomics, Western Blot, Blocking Assay, Activity Assay, Transfection, Dominant Negative Mutation, Plasmid Preparation, Control

Journal: Molecular cell

Article Title: Phosphorylation by JNK switches BRD4 functions.

doi: 10.1016/j.molcel.2024.09.030

Figure Lengend Snippet: Figure 5. JNK phosphorylation toggles BRD4 enzymatic activities and is reversed by PP4 phosphatase (A) BRD4 kinase and HAT activities are cross-regulated by its substrates. Top: autoradiograph of an in vitro kinase assay with BRD4 and RNA Pol II CTD in the presence or absence of assembled mononucleosomes. Bottom: immunoblots of an in vitro HAT assay with BRD4 and histone H3 in the presence or absence of RNA Pol II CTD. (B) JNK activation enhances the interaction between BRD4 and its kinase substrates. Immunoblots showing co-immunoprecipitated total and T1212-phos- phorylated BRD4 from HCT116 cells treated with or without anisomycin. Top: BRD4 co-immunoprecipitated with RNA Pol II CTD. Bottom: BRD4 co-immu- noprecipitated with CDK9. (C) JNK-mediated phosphorylation of BRD4 is transient. Immunoblots of WCEs of HCT116 cells grown under normal conditions, subjected to heat shock or heat shocked and then rescued for 20 min. (D) Inhibition of phosphatases enhances phosphorylated BRD4 levels. Immunoblots of WCEs of HCT116 cells that were treated with or without anisomycin alone or anisomycin with phosphatase inhibitor, nodularin. (E) Phosphatase PP4 dephosphorylates JNK-phosphorylated BRD4. Immunoblots of WCEs of HCT116 cells that were transfected with either control, PP2Ac, or PP4c siRNA and treated with or without anisomycin. (F) BRD4’s interaction with RNA Pol II CTD is modulated by PP4. Immunoblots showing total and pT1212 BRD4 co-immunoprecipitated with RNA Pol II CTD from HCT116 cells transfected with either control or PP4c siRNA and treated with anisomycin.

Article Snippet: The primary antibodies used were as follows: anti-BRD4 rabbit monoclonal antibody (Bethyl; [BL-149-2H5]) (1:100 dilution), anti-phospho JNK mouse monoclonal antibody (G-7, Santa cruz biotechnology) (1:100 dilution), and anti-Nucleolin (sc-8031, Santa cruz biotechnology) (1:100 dilution).

Techniques: Phospho-proteomics, Autoradiography, In Vitro, Kinase Assay, Western Blot, HAT Assay, Activation Assay, Immunoprecipitation, Inhibition, Transfection, Control

Journal: Molecular cell

Article Title: Phosphorylation by JNK switches BRD4 functions.

doi: 10.1016/j.molcel.2024.09.030

Figure Lengend Snippet: Figure 6. JNK-mediated BRD4 release from chromatin activates transcription (A) JNK activation enhances expression of BRD4-regulated genes. Volcano plots showing differential gene expression observed in RNA-seq analysis of WT- and 3A-BRD4-expressing HCT116 cells after anisomycin treatment. (B) Inflammatory and immune response pathways are enriched among the BRD4-regulated genes induced by JNK activation. GO analysis of genes induced in anisomycin-treated WT-BRD4-expressing cells relative to control HCT116 cells. (C) Induction of key inflammatory and immune response genes depends on JNK phosphorylation of BRD4. RT-qPCR of cDNA from anisomycin-treated WT- and 3A-BRD4-expressing cells relative to control cells. Error bars, SEM (n = 3 independent experiments; *p < 0.001 by two-tailed Student’s t tests). (D) JNK activation leads to increased BRD4-RNA Pol II interaction at BRD4-regulated inflammatory and immune response genes. Sequential-ChIP assays showing RNA Pol II and RNA Pol II-bound BRD4 at the promoter and gene body regions of CCL20, CXCL1, BIRC3, and control Myc genes. Error bars, SEM (n = 4 technical replicates from 2 independent experiments; *p < 0.05 by two-tailed Student’s t tests). See also Figure S6.

Article Snippet: The primary antibodies used were as follows: anti-BRD4 rabbit monoclonal antibody (Bethyl; [BL-149-2H5]) (1:100 dilution), anti-phospho JNK mouse monoclonal antibody (G-7, Santa cruz biotechnology) (1:100 dilution), and anti-Nucleolin (sc-8031, Santa cruz biotechnology) (1:100 dilution).

Techniques: Activation Assay, Expressing, Gene Expression, RNA Sequencing, Control, Phospho-proteomics, Quantitative RT-PCR, Two Tailed Test

Journal: Molecular cell

Article Title: Phosphorylation by JNK switches BRD4 functions.

doi: 10.1016/j.molcel.2024.09.030

Figure Lengend Snippet: Figure 7. BRD4 phosphorylation and chromatin release are correlated with thymocyte activation and EMT (A) Thymocyte activation correlates with JNK phosphorylation of BRD4. Left: flow cytometry profiles of thymocytes activated by 0.3 ng PMA/0.3 mg ionomycin or by 10 ng PMA/3.75 mg ionomycin. FACS analysis of CD4/CD8 (upper) and CD69 expression (lower). Right: immunoblots of WCEs from unstimulated and stimulated thymocytes. Densitometric quantification of relative BRD4 phosphorylation levels is shown below. (B) Thymocyte activation is correlated with JNK-mediated release of BRD4 from chromatin. Immunoblot of chromatin-free (CF) and chromatin-bound (CB) BRD4 in thymocytes unstimulated or stimulated as described above. Densitometric quantification of CF:CB BRD4 ratio is shown below. Anti-histone H3 immunoblot monitors purity of separation. (C) Immunoblots of WCEs from PC3 cells at day 0 and day 5 of treatment with or without EMT-inducing media supplement. (D) EMT induction correlates with JNK-mediated release of BRD4 from chromatin. Immunoblot of CF and CB BRD4 in PC3 cells after 5 days of treatment with or without (control) EMT-inducing media. (E) EMT induction and BRD4 phosphorylation are both dependent on JNK activity. Immunoblots of WCEs from PC3 cells that were treated, or not, with EMT- inducing media alone or in combination with JNK peptide inhibitor D-JNK-1. (F) EMT induction and expression of EMT regulators are dependent on BRD4 phosphorylation. Immunoblots of WCEs from PC3 cells that were treated, or not, with EMT-inducing media and transfected with WT-BRD4, 3A-BRD4, or empty vector control on day 3 of treatment.

Article Snippet: The primary antibodies used were as follows: anti-BRD4 rabbit monoclonal antibody (Bethyl; [BL-149-2H5]) (1:100 dilution), anti-phospho JNK mouse monoclonal antibody (G-7, Santa cruz biotechnology) (1:100 dilution), and anti-Nucleolin (sc-8031, Santa cruz biotechnology) (1:100 dilution).

Techniques: Phospho-proteomics, Activation Assay, Cytometry, Expressing, Western Blot, Control, Activity Assay, Transfection, Plasmid Preparation

Journal: Molecular cell

Article Title: Phosphorylation by JNK switches BRD4 functions.

doi: 10.1016/j.molcel.2024.09.030

Figure Lengend Snippet: Figure 8. Model of BRD4-JNK interaction and the switching of BRD4 functions BRD4 primarily functions as a chromatin regulator by acetylating H3K122 and dissociating nucleosomes. Upon activation, JNK phosphorylates BRD4, releasing it from chromatin and activating its kinase. Chromatin-free BRD4 is then dephosphorylated by PP4, enhancing its interaction with and phosphorylation of RNA Pol II CTD, PTEFb, and Myc, thereby activating transcription at specific genes. A portion of dephosphorylated BRD4 returns to chromatin to renew its chromatin regulatory function.

Article Snippet: The primary antibodies used were as follows: anti-BRD4 rabbit monoclonal antibody (Bethyl; [BL-149-2H5]) (1:100 dilution), anti-phospho JNK mouse monoclonal antibody (G-7, Santa cruz biotechnology) (1:100 dilution), and anti-Nucleolin (sc-8031, Santa cruz biotechnology) (1:100 dilution).

Techniques: Activation Assay, Phospho-proteomics