Journal: Cell death & disease

Article Title: The RBPJ/DAPK3/UBE3A signaling axis induces PBRM1 degradation to modulate the sensitivity of renal cell carcinoma to CDK4/6 inhibitors.

doi: 10.1038/s41419-022-04760-6

Figure Lengend Snippet: Fig. 2 UBE3A promotes PBRM1 degradation in renal cancer cells. a 786-O cells were transfected with indicated plasmids for 24 h. Before harvested for western blotting analysis, cells were treated with or without 20 µM MG132 for 6 h. b 786-O cells were transfected with indicated plasmids. Twenty-four hours post transfection, cells were harvested for western blotting analysis. c 786-O cells were transfected with indicated plasmids. Twenty-four hours post transfection, cells were harvested for western blotting analysis. d 786-O cells were infected with indicated shRNAs. After 72 h, cells were treated with cycloheximide (CHX), and cells were collected for western blot analysis at different timepoints. e 786-O cells were transfected with indicated plasmids. After 24 h, cells were treated with CHX, and cells were collected for western blot analysis at different timepoints. f 786-O cells were infected with the indicated shRNAs. After 72 h, cells were collected for western blotting after treatment with MG132 for 8 h. g 786-O cells were transfected with the indicated plasmids. After 24 h, cells were collected for Western blotting after treatment with MG132 for 8 h. h 786-O cells were transfected with the indicated plasmids. After 24 h, cells were collected for western blotting after treatment with MG132 for 8 h.

Article Snippet: The antibodies used as follows: UBE3A (10344-1-AP, Proteintech; 1:1000 dilution), PBRM1 (12563-1-AP, Proteintech; 1:500 dilution), DAPK3 (2928, Cell signaling technology, 1:1000 dilution), RBPJ (14613-1-AP, Proteintech; 1:1000 dilution); P21 (10355-1-AP, Proteintech; 1:1000 dilution); GAPDH (10494-1-AP, Proteintech; 1:10000 dilution).

Techniques: Transfection, Western Blot, Infection

Journal: Cell death & disease

Article Title: The RBPJ/DAPK3/UBE3A signaling axis induces PBRM1 degradation to modulate the sensitivity of renal cell carcinoma to CDK4/6 inhibitors.

doi: 10.1038/s41419-022-04760-6

Figure Lengend Snippet: Fig. 7 A hypothesis model depicted that PKA phosphorylated UBE3A to prevent UBE3A degrading PBRM1. DAPK3 competed with PKA to bind with UBE3A and enhance the PBRM1 degradation in renal cancer cells. PBPJ transcriptionally regulated DAPK3 expression and then promoted UBE3A-mediated degradation of PBRM1. Then, PBRM1 increased the p21 expression and sensitized renal cancer cells to CDK4/6 inhibitors. In combination with RBPJ inhibitors, CDK4/6 inhibitors synergistically enhanced renal cancer cells.

Article Snippet: The antibodies used as follows: UBE3A (10344-1-AP, Proteintech; 1:1000 dilution), PBRM1 (12563-1-AP, Proteintech; 1:500 dilution), DAPK3 (2928, Cell signaling technology, 1:1000 dilution), RBPJ (14613-1-AP, Proteintech; 1:1000 dilution); P21 (10355-1-AP, Proteintech; 1:1000 dilution); GAPDH (10494-1-AP, Proteintech; 1:10000 dilution).

Techniques: Expressing

Journal: Cell Genomics

Article Title: Multi-omics integration identifies cell-state-specific repression by PBRM1-PIAS1 cooperation

doi: 10.1016/j.xgen.2023.100471

Figure Lengend Snippet:

Article Snippet: Primary antibodies used for western blotting include anti-PBRM1 (Bethyl, A700-019), anti-PIAS1 (D33A7) (Cell Signaling, 3550), anti-ARID2 (D8D8U) (Cell Signaling, 82342), anti-BRG1 (G-7) (Santa Cruz Biotechnology, sc-17796), anti-ARID1A (PSG3) (Santa Cruz Biotechnology, sc-32761), anti-BRD7 (Bethyl, A302-304A-T), anti-HA (C29F4) (Cell Signaling, 3724), and anti-Lamin A/C (E−1) (Santa Cruz Biotechnology, sc-376248).

Techniques: Recombinant, SYBR Green Assay, Protease Inhibitor, In Situ, Blocking Assay, Isolation, Sequencing, shRNA, Software

Journal: Oncotarget

Article Title: Functional role of SETD2, BAP1, PARP-3 and PBRM1 candidate genes on the regulation of hTERT gene expression

doi: 10.18632/oncotarget.18712

Figure Lengend Snippet: (A) qRT-PCR analysis of pre-spliced hTERT expression levels (mean ± S.E n=3) within PB1-human chromosome 3 (PB1-#3fragment) hybrid clones, relative to wild-type PB1 cells. (B) FISH analysis of a single mouse A9-clone 8#3fragment hybrid generated by retro-transfer of the #3 fragment from clone 8 into mouse A9 cells. Representative DAPI-stained metaphase spreads of mouse A9-clone 8#3fragment clones, hybridised with, (i) TexasRed-labelled total human genomic paint and (ii) FITC-labelled chromosome 3-specific painting probes. (C) Summary of chromosome 3 microsatellite sequence-tagged site (STS) analysis of two mouse A9-clone 8#3 fragment hybrids (clones 4 and 9). A list of genes located within the 490kb region of deletion observed in clone 9 is indicated. (NRQ-Normalised relative quantity) Open circle represents loss of the marker while filled circles retained the marker.

Article Snippet: The plasmid vectors pCMV6AC- BAP1 (OriGene Technologies Inc., Rockville, MD, USA), pBABEpuro- BAF180/PBRM1 [ ], pCMVNeo-PARP-3 and associated empty vector controls were used to generate stable BAP1, PBRM1 and PARP-3, 21NT transfection clones, respectively.

Techniques: Quantitative RT-PCR, Expressing, Clone Assay, Generated, Staining, Sequencing, Marker

Journal: Oncotarget

Article Title: Functional role of SETD2, BAP1, PARP-3 and PBRM1 candidate genes on the regulation of hTERT gene expression

doi: 10.18632/oncotarget.18712

Figure Lengend Snippet: (A) Graphical representation of the approximate positions of candidate genes within the 3p21.1-p21.3 region of human chromosome 3. Genomic coordinates of all candidate genes were obtained from the National Centre of Biotechnology Information (NCBI) database. (B) Candidate gene copy number (CN) variation analysis of HMEC strains and a panel of nine breast cancer cell lines. (C) Real-time qPCR mRNA expression analysis for SETD2, PBRM1, BAP1 and PARP-3 in breast cancer cell lines and normal breast cells (HMEC’s).

Article Snippet: The plasmid vectors pCMV6AC- BAP1 (OriGene Technologies Inc., Rockville, MD, USA), pBABEpuro- BAF180/PBRM1 [ ], pCMVNeo-PARP-3 and associated empty vector controls were used to generate stable BAP1, PBRM1 and PARP-3, 21NT transfection clones, respectively.

Techniques: Expressing

Journal: Oncotarget

Article Title: Functional role of SETD2, BAP1, PARP-3 and PBRM1 candidate genes on the regulation of hTERT gene expression

doi: 10.18632/oncotarget.18712

Figure Lengend Snippet: qRT-PCR analysis of average (Ai) PARP-3 , (Bi) BAP1 , (Ci) SETD2 and (Di) PBRM1 expression levels (mean ± S.E n=3) and (Aii, Bii, Cii, and Dii) pre-spliced hTERT expression levels (mean ± S.E n=3) across five independent stable 21NT-candidate gene transfection clones and five independent stable 21NT-empty vector (EV) clones relative to parental 21NT cells (*p<0.05). Figure 3C shows (iii) SETD2 and (iv) hTERT expression levels (mean ± SD) within 21NT cells 48-144 hours following transient transfection of 21NT cells with pCMVNeo (EV) and pCMVNeo- SETD2 vector constructs, expressed relative to untreated 21NT cells. (NRQ-Normalised Relative Quantity).

Article Snippet: The plasmid vectors pCMV6AC- BAP1 (OriGene Technologies Inc., Rockville, MD, USA), pBABEpuro- BAF180/PBRM1 [ ], pCMVNeo-PARP-3 and associated empty vector controls were used to generate stable BAP1, PBRM1 and PARP-3, 21NT transfection clones, respectively.

Techniques: Quantitative RT-PCR, Expressing, Transfection, Clone Assay, Plasmid Preparation, Construct

Journal: Oncotarget

Article Title: Functional role of SETD2, BAP1, PARP-3 and PBRM1 candidate genes on the regulation of hTERT gene expression

doi: 10.18632/oncotarget.18712

Figure Lengend Snippet: Sequences of the synthetic oligonucleotides used as primers for qRT-PCR and thermal cycling parameters

Article Snippet: The plasmid vectors pCMV6AC- BAP1 (OriGene Technologies Inc., Rockville, MD, USA), pBABEpuro- BAF180/PBRM1 [ ], pCMVNeo-PARP-3 and associated empty vector controls were used to generate stable BAP1, PBRM1 and PARP-3, 21NT transfection clones, respectively.

Techniques: Sequencing

Journal: bioRxiv

Article Title: PBRM1 BD2 and BD4 associate with RNA to facilitate chromatin association

doi: 10.1101/2022.02.07.479474

Figure Lengend Snippet: (a) The domain architecture of the PBRM1 subunit of PBAF complex. BD2 and BD4 are colored dark grey. (b) Structures of double stranded (top) and single stranded (bottom) DNA (green) and RNA (purple) used for NMR titrations shown in c. (c) Overlay of 1 H- 15 N HSQC spectra of 15 N-BD2 (top row) or 15 N-BD4 (bottom row) upon titration with dsDNA, ssDNA, dsRNAI and ssRNAI. The spectra are color coded according to protein:nucleic acid molar ratio as shown in the legend. For clarity, only 4 titration points are displayed. For BD2 dsDNA titration was collected at 1:0, 1:1, 1:2, 1:4, 1:6, 1:10, 1:12, 1:15, ssDNA was collected at 1:0, 1:0.5, 1:1, 1:2, 1:4, 1:7, 1:10, dsRNAI was collected at 1:0, 1:0.1, 1:0.25, 1:0.5, 1:1, 1:2, 1:4, 1:6 and ssRNAI was collected at 1:0, 1:1, 1:2, 1:4, 1:6, 1:8. For BD4 sDNA titration was collected at 1:0, 1:0.5, 1:1, 1:2, 1:2.5, 1:5, 1:7.5, 1:10, 1:13.5, ssDNA was collected at 1:0, 1:0.5, 1:1, 1:4, 1:8, 1:12, 1:16, 1:24, dsRNAI was collected at 1:0, 1:0.5, 1:1, 1:2, 1:3, 1:4, 1:6, 1:10 and ssRNAI was collected at 1:0, 1:1, 1:2, 1:4, 1:6, 1:8. (d , e) Binding curves calculated from the normalized chemical shifts for BD2 (d) or BD4 (e) upon binding to dsRNAI. Titration points are fit to a single-site binding model under ligand-depleted conditions.

Article Snippet: Codon optimized plasmids encoding the individual His-tagged PBRM1 bromodomain constructs were received from Nicola Burgess-Brown (Addgene plasmid numbers 38999, 39013, 39027, 39028, 39030, 39103).

Techniques: Titration, Binding Assay

Journal: bioRxiv

Article Title: PBRM1 BD2 and BD4 associate with RNA to facilitate chromatin association

doi: 10.1101/2022.02.07.479474

Figure Lengend Snippet: (a , b) Overlay of 1 H- 15 N HSQC spectra of 15 N-BD2 (a) or 15 N-BD4 (b) upon titration with different dsRNA substrates (see c). The spectra are color coded according to protein:RNA molar ratio as shown in the legend. For clarity, only 4 points are displayed. For BD2, all dsRNA titrations were collected at 1:0, 1:0.1, 1:0.25, 1:0.5, 1:1, 1:2, 1:4, 1:6. For BD4, all titrations were collected at 1:0, 1:0.5, 1:1, 1:2, 1:3, 1:4, 1:6, 1:10. (c) Schematics of dsRNA substrates used for NMR titrations in the study (d) Dissociation constants (K d ) determined from NMR titrations with 15 N-PBRM1 BD2 and BD4 with double stranded RNA substrates. K d values are fit to a single-site binding model under ligand-depleted conditions using 7-9 titration points. Shown are the averages over individual residues with significant CSPs and associated standard deviation. For dsRNAIV association with BD2 the calculated K d was less than 1/10 the protein concentration and thus stated as the upper limit.

Article Snippet: Codon optimized plasmids encoding the individual His-tagged PBRM1 bromodomain constructs were received from Nicola Burgess-Brown (Addgene plasmid numbers 38999, 39013, 39027, 39028, 39030, 39103).

Techniques: Titration, Binding Assay, Standard Deviation, Protein Concentration

Journal: bioRxiv

Article Title: PBRM1 BD2 and BD4 associate with RNA to facilitate chromatin association

doi: 10.1101/2022.02.07.479474

Figure Lengend Snippet: (a) Dissociation constants (K d ) determined by NMR for BD2 and BD4 wild-type and mutant. (b) Immunoblot analysis of lysates from Caki2 renal clear cell carcinoma cells expressing full length WT PBRM1 or PBRM1 containing triple mutants in BD2 and BD4. hhnRNPA1 is included as a loading control. ( c) Crosslinking immunoprecipitation (CLIP) performed in Caki2 cells. Phosphorimage of 32P-labeled immunoprecipitations of exogenous V5 tagged PBRM1, as well as positive control hnRNPA1 from UV crosslinked Caki2 cells. ( d) (left) Representative immunoblot of PBRM1 (PBAF) and ARID1A (cBAF) elution by sequential salt extraction (SSE) in Caki2 cells with WT PBRM1 and PBRM1 mutants. (right) Analysis of binding affinity to chromatin by SSE of PBRM1 in the BD mutants indicated by the percentage of PBRM1 eluted at increasing NaCl concentrations. n = 4 independent biological replicates. A designation of * = p <0.05 (paired Student t -test). Error bars represent s.d. ( e) . CellTiter-Glo® measurement of viable cells for seven days of culture of 2,000 cells plated in 96-well plates. A designation of * = p <0.05, ** = p <0.01, *** = p<0.001, **** = p<0.0001 (paired Student t -test). Error bars represent s.d. n = 8. ( f) The change in the proportion of GFP negative cells compared to GFP positive cells as measured by flow cytometry. Equal numbers of GFP-labeled Caki2 cells and Caki2 cells expressing inducible PBRM1 were plated on day 0 and cells were harvested on designated time points for analysis. A designation of **** = p<0.0001 (paired Student t -test). Error bars represent s.d. n = 4.

Article Snippet: Codon optimized plasmids encoding the individual His-tagged PBRM1 bromodomain constructs were received from Nicola Burgess-Brown (Addgene plasmid numbers 38999, 39013, 39027, 39028, 39030, 39103).

Techniques: Mutagenesis, Western Blot, Expressing, Cross-linking Immunoprecipitation, Labeling, Positive Control, Binding Assay, Flow Cytometry

Journal: Cell Genomics

Article Title: Multi-omics integration identifies cell-state-specific repression by PBRM1-PIAS1 cooperation

doi: 10.1016/j.xgen.2023.100471

Figure Lengend Snippet:

Article Snippet: PBRM1 cDNA sequence was amplified from the TetO-FUW-PBRM1-pgk-puro construct (Addgene #85746).

Techniques: Recombinant, SYBR Green Assay, Protease Inhibitor, In Situ, Blocking Assay, Isolation, Sequencing, shRNA, Software