Journal: iScience

Article Title: Creatine kinase brain-type regulates BCAR1 phosphorylation to facilitate DNA damage repair

doi: 10.1016/j.isci.2023.106684

Figure Lengend Snippet: BCAR1 associates with RBBP4 to modulate H4K16Ac level (A) Workflow to carry out IP-MS of BCAR1-Flag WT and Y327F from 293T cells. (B) Statistical analysis of IP-MS results from (A) (see also ). Proteins with “ratio BCAR1 WT/Y327F > 5, p value <0.01” or “ratio BCAR1 WT/Y327F < 0.20, p value <0.01” are categorized in “UP” or “Down” group, respectively. (C) Co-IP experiments of RBBP4-Myc with BCAR1-Flag WT, Y327F, or Y327E in 293T cells. (D) Mapping of BCAR1 interacting domain by co-IP of various constructs of Flag-BCAR1 with RBBP4-Myc in 293T cells. (E-G) Examination of H4K16Ac levels in: (E) MCF-7 shGFP, shBCAR1#1 or shBCAR1#2 cells; (F) MCF-7 BCAR1 KD cells rescued with rBCAR1-Flag WT/Y327F/Y327E or empty vector (EV); and (G) MCF-7 shGFP, shCKB#1, or shCKB#2 cells. See also Figure S3 .

Article Snippet: RBBP4 antibody , Novus , Cat#NBP1-41201; RRID: AB_2238239.

Techniques: Protein-Protein interactions, Co-Immunoprecipitation Assay, Construct, Plasmid Preparation

Journal: iScience

Article Title: Creatine kinase brain-type regulates BCAR1 phosphorylation to facilitate DNA damage repair

doi: 10.1016/j.isci.2023.106684

Figure Lengend Snippet: BCAR1 binds to the promoter region of RAD51 to regulate its transcription together with RBBP4 (A) ChIP re-ChIP assay. The assay was carried out to evaluate the coexistence of BCAR1 and RBBP4 in the promoter regions of RAD51 and MGMT in 293T cells co-transfected with RBBP4-Myc and BCAR1-Flag WT/Y327F. (B and C) ChIP assays were performed: (B) to examine H4K16Ac modification and the existence of BCAR1 or RBBP4 in the promoter region of RAD51 in MCF-7 BCAR1 KD cells rescued with rBCAR1-Flag WT/Y327F or GFP-Flag; and (C) to examine H4K16Ac modification and the existence of RBBP4 in the promoter region of RAD51 in MCF-7 shGFP, shCKB#1 or shCKB#2 cells. (D and E) mRNA levels of RAD51 in: (D) MCF-7 BCAR1 KD cells rescued with rBCAR1-Flag WT/Y327F or GFP-Flag; and (E) MCF-7 shGFP, shCKB#1 or shCKB#2 cells. (F) ChIP assays were performed to examine H4K16Ac modification and the existence of BCAR1-Flag WT/Y327F(YF) in the promoter region of RAD51 in MCF-7 shGFP, shRBBP4#1 or shRBBP4#2 cells overexpressing BCAR1-Flag WT/Y327F(YF) or GFP-Flag Cells were cultured with 5 ng/mL TGF-β for 24 h before examination. The data in all figures are presented as mean ± SD from three independent experiments (n = 3) and analyzed by Student’s t tests.

Article Snippet: RBBP4 antibody , Novus , Cat#NBP1-41201; RRID: AB_2238239.

Techniques: Transfection, Modification, Cell Culture

Journal: iScience

Article Title: Creatine kinase brain-type regulates BCAR1 phosphorylation to facilitate DNA damage repair

doi: 10.1016/j.isci.2023.106684

Figure Lengend Snippet: BCAR1 Y327 phosphorylation elevates RAD51 expression and H4K16Ac level mediated by RBBP4 (A-F).WB analysis of RAD51 and H4K16Ac levels in: (A) MCF-7 shGFP, shBCAR1#1 or shBCAR1#2 cells; (B) MCF-7 BCAR1 KD cells rescued with rBCAR1-Flag WT/Y327F/Y327E or empty vector (EV); (C) MCF-7 shGFP, shCKB#1 or shCKB#2 cells; (D) MCF-7 shGFP, shCKB#1 or shCKB#2 cells overexpressing BCAR1-Flag WT/Y327F/Y327E or empty vector (EV); (E) MCF-7 shGFP, shRBBP4#1 or shRBBP4#2 cells; and (F) MCF-7 shGFP, shRBBP4#1 or shRBBP4#2 cells overexpressing BCAR1-Flag WT/Y327F/Y327E or empty vector (EV).

Article Snippet: RBBP4 antibody , Novus , Cat#NBP1-41201; RRID: AB_2238239.

Techniques: Phospho-proteomics, Expressing, Plasmid Preparation

Journal: iScience

Article Title: Creatine kinase brain-type regulates BCAR1 phosphorylation to facilitate DNA damage repair

doi: 10.1016/j.isci.2023.106684

Figure Lengend Snippet: RBBP4 mediates the promotion of DNA damage repair by BCAR1 Y327 phosphorylation (A and C) Comparison of tail moments in comet assays for: (A) MCF-7 shGFP, shRBBP4#1 or shRBBP4#2 cells (n = 32, 37 and 32 for shGFP, shRBBP4#1 and shRBBP4#2, respectively); and (C) MCF-7 shGFP, shRBBP4#1 or shRBBP4#2 cells overexpressing BCAR1-Flag WT/Y327F/Y327E or empty vector (EV) (n = 39, 35, 39 and 32 for EV, BCAR1 WT, Y327F, and Y327E in shGFP cells, respectively; n = 37, 39, 42 and 39 for EV, BCAR1 WT, Y327F, and Y327E in shRBBP4#1 cells, respectively; n = 40, 43, 41 and 40 for EV, BCAR1 WT, Y327F, and Y327E in shRBBP4#2 cells, respectively). The data are presented as mean ± SD and analyzed by Student’s t tests. (B and D) WB analysis of RAD51 and γH2AX in: (B) MCF-7 shGFP, shRBBP4#1 or shRBBP4#2 cells; and (D) MCF-7 shGFP, shRBBP4#1 or shRBBP4#2 cells overexpressing BCAR1-Flag WT/Y327F/Y327E or empty vector (EV). (E) A proposed model that BCAR1 Y327 phosphorylation facilitates the association with RBBP4 and its recruitment to RAD51 promoter to enhance H4K16 acetylation leading to transcriptional upregulation. (F) WB analysis of RAD51 and γH2AX in MCF-7 BCAR1 KD cells rescued with rBCAR1-Flag WT/Y327F/Y327E or empty vector (EV) Cells in (A-D) were treated with 20 μM etoposide or DMSO for 4 h before examination. Cells in (F) were treated with 20 μM etoposide for 4 h and then released into the fresh medium without etoposide for indicated times. See also Figure S4 .

Article Snippet: RBBP4 antibody , Novus , Cat#NBP1-41201; RRID: AB_2238239.

Techniques: Phospho-proteomics, Comparison, Plasmid Preparation

Journal: iScience

Article Title: Creatine kinase brain-type regulates BCAR1 phosphorylation to facilitate DNA damage repair

doi: 10.1016/j.isci.2023.106684

Figure Lengend Snippet:

Article Snippet: RBBP4 antibody , Novus , Cat#NBP1-41201; RRID: AB_2238239.

Techniques: Virus, Recombinant, Magnetic Beads, Membrane, Protease Inhibitor, Cloning, Purification, Sonication, Chromatin Immunoprecipitation, Molecular Cloning, Expressing, ChIP-qPCR, shRNA, Plasmid Preparation, Software, Imaging

Journal: Nucleic Acids Research

Article Title: Direct interaction between the PRDM3 and PRDM16 tumor suppressors and the NuRD chromatin remodeling complex

doi: 10.1093/nar/gky1192

Figure Lengend Snippet: The first 12 residues of PRDM3/16 interact with RBBP4. ( A–C ) In vitro measurement of dissociation constant ( K D ) and molar stoichiometry ( N ) of (A) PRDM3, (B) PRDM16 and (C) Histone H3 peptides with RBBP4 measured by ITC (experiment performed in triplicate, standard deviation is shown). ( D ) Cellular interaction between PRDM3 and RBBP4 measured by LacO/LacR chromatin immobilization assay. PRDM3-mCherry-LacR-NLS with WT PRDM3 or PRDM3 lacking the 12 N-terminal residues (ΔN12) were assessed for co-localization with GFP-RBBP4. ( E ) Quantification of the PRDM3 (WT or ΔN12) co-localization with RBBP4 shown in (D). The violin plots represent the GFP intensity increase over background in the mCherry foci, calculated from n = 100 cells.

Article Snippet: The whole 30 μl were processed for western blot using antibodies for GFP (Living Colors JL-8, Clontech), RBBP4 (R&D systems, MAB7416-SP), MTA1 (CST, 5647) and HDAC2 (CST, 5113).

Techniques: In Vitro, Standard Deviation

Journal: Nucleic Acids Research

Article Title: Direct interaction between the PRDM3 and PRDM16 tumor suppressors and the NuRD chromatin remodeling complex

doi: 10.1093/nar/gky1192

Figure Lengend Snippet: Crystal structure of RBBP4 in complex with the PRDM3 (1–12 amino acid) peptide. ( A ) Stick representation of PRDM3 peptide [Oxygen atoms (red) and nitrogen (blue)] bound to the ribbon representation of RBBP4. ( B ) Electrostatic surface potential representation of the binding pocket with aligned PRDM3 peptide. RBBP4 surface color indicates electrostatic potential ranging from −7kT/e (red) to +7kT/e (blue). Electrostatic surface potentials were calculated using the APBS.

Article Snippet: The whole 30 μl were processed for western blot using antibodies for GFP (Living Colors JL-8, Clontech), RBBP4 (R&D systems, MAB7416-SP), MTA1 (CST, 5647) and HDAC2 (CST, 5113).

Techniques: Binding Assay

Journal: Nucleic Acids Research

Article Title: Direct interaction between the PRDM3 and PRDM16 tumor suppressors and the NuRD chromatin remodeling complex

doi: 10.1093/nar/gky1192

Figure Lengend Snippet: Data for X-ray Crystal Structure of PRDM3/16 (1–12aa) with RBBP4.

Article Snippet: The whole 30 μl were processed for western blot using antibodies for GFP (Living Colors JL-8, Clontech), RBBP4 (R&D systems, MAB7416-SP), MTA1 (CST, 5647) and HDAC2 (CST, 5113).

Techniques:

Journal: Nucleic Acids Research

Article Title: Direct interaction between the PRDM3 and PRDM16 tumor suppressors and the NuRD chromatin remodeling complex

doi: 10.1093/nar/gky1192

Figure Lengend Snippet: Amino acid interactions at the PRDM3 peptide-RBBP4 interface. ( A ) The interface between PRDM3 residues (tan) and RBBP4 residues (green). Interactions between PRDM3 centered at ( B ) arginine 2, ( C ) lysine 4, ( D ) arginine 6 and ( E ) lysine 9 are indicated with dashed lines. Interactions within 4 Å (purple) and 3 Å (yellow) are detailed in .

Article Snippet: The whole 30 μl were processed for western blot using antibodies for GFP (Living Colors JL-8, Clontech), RBBP4 (R&D systems, MAB7416-SP), MTA1 (CST, 5647) and HDAC2 (CST, 5113).

Techniques:

Journal: Nucleic Acids Research

Article Title: Direct interaction between the PRDM3 and PRDM16 tumor suppressors and the NuRD chromatin remodeling complex

doi: 10.1093/nar/gky1192

Figure Lengend Snippet: PRDM3 and PRDM16 mimic the RBBP4-histone H3 interaction. ( A ) Structure alignment of all reported peptides that bind perpendicular to the RBBP4 β-propeller axis. ( B ) Structure alignment of arginine, threonine/serine and lysine from H3, PRDM3 and PRDM16 (top) and arginine and lysine from FOG1, PHF6, BCL11a and AEBP2 (bottom). ( C ) Sequence alignment of all reported peptides that bind to the RBBP4 top hole. Colors correspond to A and B panels. ( D ) A model illustrating a potential mechanism for how full-length PRDM3 and PRDM16 could tethering the NuRD complex to chromatin and subsequently regulate transcription. Dashed line and arrows indicate potential secondary interactions between MBD3 of NuRD and the first zinc finger motifs of PRDM3 and PRDM16.

Article Snippet: The whole 30 μl were processed for western blot using antibodies for GFP (Living Colors JL-8, Clontech), RBBP4 (R&D systems, MAB7416-SP), MTA1 (CST, 5647) and HDAC2 (CST, 5113).

Techniques: Sequencing

Journal: Cell reports

Article Title: Retinoblastoma Binding Protein 4 Modulates Temozolomide Sensitivity in Glioblastoma by Regulating DNA Repair Proteins

doi: 10.1016/j.celrep.2016.02.045

Figure Lengend Snippet: (A) Absolute levels of RBBP4 shRNA in GBM22 cells following treatment with either DMSO or TMZ. (B) Effect of RBBP4 siRNA, with and without TMZ treatment, on the primary neurosphere (NS) formation in GBM12 and GBM22 cells. (C) RBBP4 expression in T98G cells stably expressing shNT and 4 different RBBP4 shRNA lentiviral constructs (upper panel) and in vitro TMZ cytotoxicity CyQuant assay in the same cell constructs (lower panel). (D) Effect of control shNT and 4 different RBBP4 shRNA on RBBP4 levels in U138 GBM cells (upper panel) and in vitro cytotoxicity. Shown is the data from 3 independent experiments conducted in triplicate (error bar =S.E.M; * = p <0.05).

Article Snippet: The shRNA-resistant Myc3-tagged RBBP4 pCDNA3 construct (Plasmid #20715) was purchased from Addgene (Cambridge, MA).

Techniques: shRNA, Expressing, Stable Transfection, Construct, In Vitro, CyQUANT Assay

Journal: Cell reports

Article Title: Retinoblastoma Binding Protein 4 Modulates Temozolomide Sensitivity in Glioblastoma by Regulating DNA Repair Proteins

doi: 10.1016/j.celrep.2016.02.045

Figure Lengend Snippet: (A) Real time PCR evaluating RBBP4 and MGMT expression using total RNA extracted from T98G cells expressing control shNT and 4 different constructs of RBBP4 shRNA (B) A representative western blot depicting RBBP4 and MGMT protein levels in T98G cells expressing NT-shRNA and two RBBP4 shRNA constructs (C) Effects of MGMT inhibitor O6-BG on TMZ sensitivity in T98G cells expressing control shNT and two RBBP4 shRNA (RBBP4-1 and RBBP4-3) constructs as measured in a CyQuant assay. (D) Western blot for RBBP4 and MGMT in T98G shNT compared with T98G-shRBBP4 cells stably expressing pCDNA3 vector or an shRNA resistant pCDNA3-RBBP4 expression construct (clones R1-R5). (E) TMZ sensitivity in T98G-shNT compared with T98G-shRBBP4 reconstituted with either pCDNA3 or shRNA resistant RBBP4 (clones R1 and R4). Shown in each bar graph is the data from 3 independent experiments conducted in triplicate (error bar = S.E.M; * = p <0.05).

Article Snippet: The shRNA-resistant Myc3-tagged RBBP4 pCDNA3 construct (Plasmid #20715) was purchased from Addgene (Cambridge, MA).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Construct, shRNA, Western Blot, CyQUANT Assay, Stable Transfection, Plasmid Preparation, Clone Assay

Journal: Cell reports

Article Title: Retinoblastoma Binding Protein 4 Modulates Temozolomide Sensitivity in Glioblastoma by Regulating DNA Repair Proteins

doi: 10.1016/j.celrep.2016.02.045

Figure Lengend Snippet: (A) Western blot depicting the time-course activation of DNA damage signaling following TMZ comparing T98G shNT and shRBBP4-3 clone (B) Nuclear p-H2AX foci in response to 72 hour exposure of T98G cells expressing shNT compared with shRBBP4-3 clone (upper panel), while the lower panel shows the fraction of cells with mean γ-H2AX foci of 3 experiments conducted in triplicate (C) Effect of RBBP4 disruption by shRNA clone shRBBP4 on PARP inhibitor ABT-888 sensitization in T98G GBM cells measured in a CyQuant assay (D) Real time PCR showing RAD51 and BRCA1 expression in T98G-shNT cells compared to T98G-shRBBP4. (E) Real time PCR evaluating RAD51 expression comparing T98G-shRBBP4_pCDNA3 with the RBBP4 reconstituted T98G-shRBBP4_R1 and _R4 cells (F) CyQuant evaluation of ABT-888 sensitivity after RBBP4 reconstitution in T98G-shRBBP4 clone compared with the T98G-shRBBP4_pcDNA3 and T98G-shNT cells Error bar = S.E.M.; * = p<0.05).

Article Snippet: The shRNA-resistant Myc3-tagged RBBP4 pCDNA3 construct (Plasmid #20715) was purchased from Addgene (Cambridge, MA).

Techniques: Western Blot, Activation Assay, Expressing, shRNA, CyQUANT Assay, Real-time Polymerase Chain Reaction

Journal: Cell reports

Article Title: Retinoblastoma Binding Protein 4 Modulates Temozolomide Sensitivity in Glioblastoma by Regulating DNA Repair Proteins

doi: 10.1016/j.celrep.2016.02.045

Figure Lengend Snippet: (A) ChIP evaluating the H3K9Ac and H3K9me3 levels within MGMT promoter region comparing T98G-shNT versus T98G-shRBBP4 cells. (B) ChIP evaluating recruitment of SP1, C-JUN, NF-kB (p65) and H3K9Ac to bind MGMT promoter region comparing T98G-shNT versus T98G-shRBBP4 cells. (C) ChIP assessing the recruitment of E2F1 to the RAD51 promoter in T98G-shNT compared with the T98G-shRBBP4. (D) ChIP evaluating RBBP4 and p300 recruitment to the MGMT promoter in T98G-shNT comparing with T98G-RBBP4 cells. (E) RBBP4 and p300 ChIP re-ChIP depicting co-occupancy of RBBP4 within the MGMT promoter region and (F) the RAD51 promoter.

Article Snippet: The shRNA-resistant Myc3-tagged RBBP4 pCDNA3 construct (Plasmid #20715) was purchased from Addgene (Cambridge, MA).

Techniques:

Journal: Cell reports

Article Title: Retinoblastoma Binding Protein 4 Modulates Temozolomide Sensitivity in Glioblastoma by Regulating DNA Repair Proteins

doi: 10.1016/j.celrep.2016.02.045

Figure Lengend Snippet: Representative line graphs displaying the H3K9Ac binding tags within 8-kb region surrounding the transcriptional start site (TSS) of (A) under-expressed and (B) over-expressed genes associated with shRBBP4 (C) Top 20 genes suppressed by RBBP4 shRNA (positively regulated by nativeRBBP4) (D) Top 20 genes elevated by RBBP4 shRNA (negatively regulated by native RBBP4). Representative H3K9Ac ChIP-seq display of a gene that was (E) suppressed and (F) elevated by RBBP4 shRNA.

Article Snippet: The shRNA-resistant Myc3-tagged RBBP4 pCDNA3 construct (Plasmid #20715) was purchased from Addgene (Cambridge, MA).

Techniques: Binding Assay, shRNA, ChIP-sequencing

Journal: Cell reports

Article Title: Retinoblastoma Binding Protein 4 Modulates Temozolomide Sensitivity in Glioblastoma by Regulating DNA Repair Proteins

doi: 10.1016/j.celrep.2016.02.045

Figure Lengend Snippet: (A) RNAseq was performed using RNA extracted from T98G-shNT and T98G-shRBBP4 constructs and shown are the gene counts for FIGNL1 and EYA1. (B) ChIPseq display of H3K9Ac marks within FIGNL1 (upper panel) and EYA1 (lower panel) promoter regions. (C) Upper panel shows the effect of shRBBP4 on TMZ induced p-H2AX (Y142) and p-H2AX (S139), whereas the lower panel depicts the effect on cleaved-PARP. (D) RBBP4, FIGNL1 and EYA1 expression in U138 cells expressing shNT compared with shRBBP4 (E) A proposed model of RBBP4 associated with CBP/p300, which leads to histone acetylation and an open chromatin structure that facilitates transcription factor binding and expression of target genes.

Article Snippet: The shRNA-resistant Myc3-tagged RBBP4 pCDNA3 construct (Plasmid #20715) was purchased from Addgene (Cambridge, MA).

Techniques: Construct, Expressing, Binding Assay