Journal: Cell reports

Article Title: The Bromodomain Protein 4 Contributes to the Regulation of Alternative Splicing

doi: 10.1016/j.celrep.2019.10.066

Figure Lengend Snippet: (A) Schematic diagram depicting different types of alternatively spliced events (left). The bar graph (right) shows the distribution of alternatively spliced events among those that are differentially spliced in total thymus in BRD4 knock-out versus wild type (WT) (FDR < 0.05). Comparison of splicing events between WT and BRD4-deficient cells was based on transcripts expressed in both. (B) Developmental stages in thymocyte differentiation, DN (CD4, CD8 DN), ISP (CD8+ ISP), DP (CD4, CD8 DP), CD4, and CD8 single-positive thymocytes are shown. Arched arrows denote level of proliferative activity in DN and ISP thymocytes. (C) Bar graph showing the total number of differentially spliced events in the different thymocyte subpopulations in BRD4 knock-out versus wild-type thymus (FDR < 0.05), derived from RNA-seq analysis. BRD4 was conditionally deleted in DN thymocytes by LCK-Cre (Gegonne et al., 2018). Comparison of splicing events between WT and BRD4-deficient cells was based on transcripts expressed in both. See also Figure S1.

Article Snippet: The human Flag-tagged BRD4 ΔET in pCMV2 mammalian vector was obtained from Addgene (Addgene plasmid number 21938) ( Bisgrove et al., 2007 ).

Techniques: Knock-Out, Activity Assay, Derivative Assay, RNA Sequencing Assay

Journal: Cell reports

Article Title: The Bromodomain Protein 4 Contributes to the Regulation of Alternative Splicing

doi: 10.1016/j.celrep.2019.10.066

Figure Lengend Snippet: Summary of Immune Relevant Genes Whose Patterns of Splicing Are Altered by BRD4 Deficiency and the Thymocyte Subset in which the Alteration Occurs

Article Snippet: The human Flag-tagged BRD4 ΔET in pCMV2 mammalian vector was obtained from Addgene (Addgene plasmid number 21938) ( Bisgrove et al., 2007 ).

Techniques: Binding Assay

Journal: Cell reports

Article Title: The Bromodomain Protein 4 Contributes to the Regulation of Alternative Splicing

doi: 10.1016/j.celrep.2019.10.066

Figure Lengend Snippet: RNA from the different thymocyte subpopulations was subjected to RT-PCR for the indicated genes: CD45 (A and B), Arhgef1 (C and D), and Picalm (E and F). (A, C, and E) Upper panels: schematic diagrams depicting partial gene structure of the alternatively spliced genes CD45 (A), Arhgef1 (C), and Picalm (E). Rectangular boxes represent the exons, and the horizontal straight lines connecting the boxes represent the introns; the numbers below the boxes refer to the exon number of the gene, and numbers inside the boxes refer to the length of the exons; the numbers within the terminal exons do not refer to the actual exon length but the length amplifiable by the RT-PCR primers. The arrow heads show the approximate positions of the RT-PCR primers; boxes with hashed lines show the alternative exons; and curved lines connecting the boxes depict the splicing pattern. WT and KO refer to the splicing pattern prevalent in either the wild-type or knock-out thymocytes as determined by RNA-seq analysis. Lower panels: ethidium bromide stained agarose gels showing RT-PCR products derived from total RNA from BRD4 WT and KO thymocytes. (B, D, and F) Bar graphs of the RT-PCR results for CD45 (B), Arhgef1 (D), and Picalm (F). The ratios A/A+B (ratio of included exon transcript/total transcripts) were used as measure of alternative splicing and represent the average of three separate RT-PCR analyses. #, p < 0.05, significant difference between WT subpopulations, relative to WT DN; *p < 0.05, significant difference between WT and KO for the specific subpopulation. See also Figure S2.

Article Snippet: The human Flag-tagged BRD4 ΔET in pCMV2 mammalian vector was obtained from Addgene (Addgene plasmid number 21938) ( Bisgrove et al., 2007 ).

Techniques: Reverse Transcription Polymerase Chain Reaction, Knock-Out, RNA Sequencing Assay, Staining, Derivative Assay

Journal: Cell reports

Article Title: The Bromodomain Protein 4 Contributes to the Regulation of Alternative Splicing

doi: 10.1016/j.celrep.2019.10.066

Figure Lengend Snippet: (A) Effect of JQ1 or dBET6 treatment on the binding of BRD4 across the gene. ALL, TSS+gene body+TTS+intergenic; TSS, transcription start site; Gene body, between TSS and TTS; TTS, transcription termination site; intergenic, all remaining sequences. The peak distribution, in the absence of treatment is as follows: TSS, 1123; gene body, 4065; TTS, 226; intergenic, 1827. (B) Bar graph showing the distribution of alternative splice events among the differentially spliced events in response to JQ1 treatment or dBET6 treatment in T-ALL cells. (C) Bar graph showing the fraction of alternative splice (AS) genes that also have BRD4 associated with them at the TSS (pkAS). The total number of BRD4 peaks detected at the TSS across the genome was 1123. (D) Bar graph showing the fraction of AS genes that are also differentially expressed (DE) in response to JQ1 or dBET6 treatment. p values for (C) and (D) were obtained using a hypergeometric test, which tests the probability that the frequency of AS genes derived from either DE genes (overlap) or genes with BRD4-bound TSS peaks is larger than expected from the population; a low p value suggests the enrichment of AS genes in either DE genes or genes with BRD4 TSS peaks. See also Figures S3 and S4.

Article Snippet: The human Flag-tagged BRD4 ΔET in pCMV2 mammalian vector was obtained from Addgene (Addgene plasmid number 21938) ( Bisgrove et al., 2007 ).

Techniques: Binding Assay, Derivative Assay

Journal: Cell reports

Article Title: The Bromodomain Protein 4 Contributes to the Regulation of Alternative Splicing

doi: 10.1016/j.celrep.2019.10.066

Figure Lengend Snippet: (A) Immunoblot of BRD4 immunoprecipitates from thymocyte nuclear extracts (with and without benzonase treatment) with indicated antibodies to splicing factors FUS, HnRNPL, and U1–70. The immunoprecipitates from a single extract were run on either a 6% gel to visualize BRD4 and Fus or on a 10% gel to visualize HnRNPL and U1–70. The values under the IP lanes indicate the enrichment of anti-BRD4 co-IP, relative to the IgG control. (B, left) Immunoblot of BRD4 immunoprecipitates from HeLa nuclear extracts with indicated antibodies to splicing factors FUS, HnRNPM, U1–70, and U1-A. (B, right) Immunoblot of FUS immunoprecipitates from HeLa nuclear extracts with indicated antibodies to BRD4 and splicing factors HnRNPM, U1–70, and U1-A. (C) Schematic representation of BRD4 and BRD4-deletion mutants. The coordinates of the mouse BRD4 mutations are as follows. WT BRD4, 1402 aa; DN, 722–1402 aa; ΔC, 1–699aa; ΔBD1, 146–1402 aa; ΔBD2+B, 1–349/599–1402 aa; ΔB, 1–502/549–1402 aa; ΔET, 1–600/684–1402 aa; ΔHAT, 1–1156/1198–1402 aa. (D) Immunoblots showing pull-down analysis of recombinant BRD4 with recombinant HnRNPM. rHnRNPM (0.25 μg) was pulled down with rflag-BRD4 (0.5 μg) immobilized on Flag beads. Immunoblots were with anti-HnRNPM (upper) and anti-BRD4 (lower). (E) Immunoblots showing pull-down analysis of recombinant BRD4 with recombinant FUS. rFUS (0.25 μg) was pulled down with rflag-BRD4 (0.5 μg) WT or equimolar amounts of N-terminal or C-terminal BRD4 truncation mutants immobilized on Flag beads. Immunoblots were with anti-FUS (upper) and anti-BRD4 (lower). (F) Binding of HnRNPM (left panel) and FUS (right panel) to BRD4 mutants was assessed in pull-down assays with rBRD4 immobilized on Flag beads and immunoblotting with appropriate antibodies. The results represent the average of two experiments. (G) Retention of FUS and HnRNPM to BRD4 mutants, relative to the WT, was quantified as the fraction of input and normalized to the extent of binding to BRD4 WT. All results are representative of at least two independent experiments. See also Figure S5.

Article Snippet: The human Flag-tagged BRD4 ΔET in pCMV2 mammalian vector was obtained from Addgene (Addgene plasmid number 21938) ( Bisgrove et al., 2007 ).

Techniques: Western Blot, Co-Immunoprecipitation Assay, Recombinant, Binding Assay

Journal: Cell reports

Article Title: The Bromodomain Protein 4 Contributes to the Regulation of Alternative Splicing

doi: 10.1016/j.celrep.2019.10.066

Figure Lengend Snippet: (A) Proximity ligation assays (PLAs) were performed on primary thymocytes with anti-BRD4 and the antibodies for the indicated splicing factors. The PLAs are all significantly above the single antibody controls (Figure S6C). (B) PLA was performed using anti-BRD4 and the antibodies for the indicated splicing factors on fixed HeLa cells that had been treated with JQ1 (500 nM)/ DMSO for 6 hr. There is no significant difference (p > 0.05) between the treated and control PLA samples for either HnRNPM or Fus; both PLAs are significantly above single antibody alone controls (Figure S6C). PLA interaction is shown in red; DAPI staining in blue. See also Figures S6C and S6D.

Article Snippet: The human Flag-tagged BRD4 ΔET in pCMV2 mammalian vector was obtained from Addgene (Addgene plasmid number 21938) ( Bisgrove et al., 2007 ).

Techniques: Ligation, Staining

Journal: Cell reports

Article Title: The Bromodomain Protein 4 Contributes to the Regulation of Alternative Splicing

doi: 10.1016/j.celrep.2019.10.066

Figure Lengend Snippet: (A) Metagene profile of BRD4 and FUS CHIP datasets showing colocalization of BRD4 and FUS at the TSS. (B) Log2 enrichment of reads in genomic features along the metagene body. (C) Enrichment heatmap showing co-localization of BRD4 with FUS across the genome. (D) Genome browser views of DNAAF3, ROBO3, and MAN1A1, showing BRD4 and FUS co-localization around the TSS and gene body. See also Figures S6A and S6B.

Article Snippet: The human Flag-tagged BRD4 ΔET in pCMV2 mammalian vector was obtained from Addgene (Addgene plasmid number 21938) ( Bisgrove et al., 2007 ).

Techniques:

Journal: Cell reports

Article Title: The Bromodomain Protein 4 Contributes to the Regulation of Alternative Splicing

doi: 10.1016/j.celrep.2019.10.066

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: The human Flag-tagged BRD4 ΔET in pCMV2 mammalian vector was obtained from Addgene (Addgene plasmid number 21938) ( Bisgrove et al., 2007 ).

Techniques: Recombinant, In Situ, Plasmid Preparation, Mutagenesis, Clone Assay, Software, Sequencing

Journal: Oncotarget

Article Title: PATZ1 induces PP4R2 to form a negative feedback loop on IKK/NF-κB signaling in lung cancer

doi: 10.18632/oncotarget.10427

Figure Lengend Snippet: Lung cancer cells (A549 and CL1-5) were transfected either with control empty vector (EV) or pCMV6- PATZ1 ( pPATZ1 ) for 24 h and then treated for 24 h with 20 μg/ml of PGE2 or 100 ng/ml growth factors as indicated. A. The cell lysates were examined by immunoblotting for protein expression. PATZ1, phospho-IKKα/β S176/180 , IKKα/β, phospho-NF-κB p65 S536 and NF-κB were measured at 1 h, all the others were determined at 24 h after PGE2 and growth factor treatment. B and C. The migration and invasion abilities of cells were examined by transwell assays after 20 h incubation. The activities of MMP-2 in cell-conditioned media were analyzed by gelatin zymography after culturing cells in serum-free medium for 24 h. Blots are representative of three independent experiments. Data represent means ± s.d. of three independent experiments; * P < 0.05 by t-test.

Article Snippet: The materials were purchased and obtained as follows: Recombinant human EGF, HGF and IGF-1 were from R&D Systems (Abingdon, UK); pCMV6- PP4R1 (NM_005134), pCMV6- PP4R2 (NM_174907), pCMV6 -PP4C (NM_002720), pCMV6 -PATZ1 variant 3 (NM_032052), and pCMV6 -PATZ1 variant 4 (NM_032051) plasmids were from ORIGENE (Rockville, MD); PATZ1 variant 4 and PP4R2 genes from pCMV6- PATZ1 and pCMV6- PP4R2 plasmids subcloned into pCMV6-AC-mCFP plasmids (ORIGENE) were applied for the establishment of PATZ1- and PP4R2-transduced stable cells. pCMV2-IKKβ WT (wild type; WT), pCMV2-IKKβ S177ES181E (dominant active; DA) and pCMV2-IKKβ K44M (dominant negative; DN) were obtained from Addgene (Cambridge, MA; plasmid numbers 11103 and 11104) and were originally created by Dr. Anjana Rao laboratory. control siRNAs, PP4R1-siRNA, PP4R2-siRNA, PP4C-siRNA and PATZ1-siRNA were all from Santa Cruz Biotechnology (Santa Cruz, CA); two shRNA HuSH 29mer shRNA (TF302311A and TF302311C) constructs against PP4R2 in pRFP-C-RS vector and the non-effective 29-mer scrambled shRNA (TR30015) were from ORIGENE; mouse anti-Vimentin (V9) antibodies and recombinant PGE2 were from Sigma-Aldrich (St Louis, MO); phosphatidylinositol (3,4,5)-trisphosphate diC16 (PI(3,4,5)P3 diC16) were from Echelon Biosciences (Salt Lake City, UT); Anti-PP4R1 (ab70624), anti-PP4R2 (ab70631), anti-PP4C (ab16475), anti-PP4R3α (ab70635), anti-PP4R3β (ab70622), anti-PP4R4 (ab111419), and anti-PATZ1 (ab154025) rabbit polyclonal antibodies were from Abcam (Cambridge, UK); mouse anti-PP1 (E-9), mouse anti-PP5 (H-7), rabbit anti-PHB (H-80), rabbit anti-IKKα/β (H-470), mouse anti-β-actin (C-4), goat anti-COX-2 (M-19), rabbit anti-E-cadherin (H-108), rabbit anti-ZEB1 (H-102), rabbit anti-Snail (H-130), rabbit anti-Twist (H-81), and rabbit anti-PATZ1 (H-300) antibodies as well as horseradish peroxidase-conjugated anti-mouse IgG, horseradish peroxidase-conjugated anti-goat IgG and horseradish peroxidase-conjugated anti-rabbit IgG antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA); rabbit anti-PI3Kp85, rabbit anti-PI3Kp85 Y458 , rabbit anti-Akt, rabbit anti-phospho-Akt Ser473 , rabbit anti-phospho-IKKα/β S176/S180 and rabbit anti-phospho-NF-κBp65 S536 , horseradish peroxidase-conjugated mouse-anti-rabbit IgG (Conformation Specific; L27A9) antibodies were from Cell Signaling Technology (Beverly, MA); and rabbit anti-IκB, rabbit anti-NF-κBp65 and rabbit anti-Raf-1 antibodies were from Millipore (Temecula, CA).

Techniques: Transfection, Plasmid Preparation, Western Blot, Expressing, Migration, Incubation, Zymography