Journal: Ophthalmology

Article Title: BAP1 Germline Mutations in Finnish Patients with Uveal Melanoma.

doi: 10.1016/j.ophtha.2016.01.008

Figure Lengend Snippet: Figure 2. Pedigrees showing the families of BAP1 pathogenic variant carriers. The cancer type, age at diagnosis, and the BAP1 pathogenic variant status when tested are shown. Core components of the BAP1 cancer predisposition syndrome are underlined. UMG# is an institutional patient identifier. Uveal melanoma patients with or without other cancers are shown in red and patients with other cancers only in black. BAPþ ¼ BAP1 pathogenic variant present; BAPe ¼ BAP1 pathogenic variant absent; ca ¼ carcinoma; CM ¼ cutaneous melanoma; UM ¼ uveal melanoma; y ¼ years of age.

Article Snippet: Author Contributions: Conception and design: Turunen, Lehesjoki, Kivelä Analysis and interpretation: Turunen, Markkinen, Saarinen, Lehesjoki, Kivelä Data collection: Turunen, Markkinen, Wilska, Saarinen, Raivio, Täll, Kivelä Obtained funding: Turunen, Markkinen, Kivelä Overall responsibility: Turunen, Markkinen, Wilska, Saarinen, Raivio, Täll, Lehesjoki, Kivelä Abbreviations and Acronyms: BAP1 1⁄4 BRCA1-associated protein-1; BRCA1 1⁄4 breast cancer 1; CI 1⁄4 confidence interval; ExAC 1⁄4 Exome Aggregation Consortium; LBD 1⁄4 largest basal diameter; TNM 1⁄4 Tumor, Node, Metastasis classification; UCSC 1⁄4 University of California Santa Cruz.

Techniques: Variant Assay, Biomarker Discovery

Journal: Frontiers in bioscience (Landmark edition)

Article Title: SMARCA2 and SMARCA4 Participate in DNA Damage Repair.

doi: 10.31083/j.fbl2907262

Figure Lengend Snippet: Fig. 3. Lacking the enzymatic activities of SMARCA2/4 extends the accumulation of γH2AX, Ring Finger Protein 8 (RNF8) and Breast cancer susceptibility gene 1 (BRCA1). HeLa cells were pre-treated with or without FHD286 (1 µM) or Compound 14 (1 µM)

Article Snippet: The following antibodies were purchased from their respective companies: anti-γH2AX (Novus, NB100-384), anti-RAD51 (Catalog Number: MA1-23271; Invitrogen, Carlsbad, CA, USA), anti-ring finger protein 8 (RNF8) (Catalog number: 14112-1-AP, Proteintech, Wuhan, Hubei, China), anti-breast cancer susceptibility gene 1 (BRCA1) (Catalog Number: NB100-404, Novus, Littleton, CO, USA), anti-SMARCA2 (Catalog number: HPA029981; Sigma, Louis, MO, USA).

Techniques:

Journal: Oncotarget

Article Title: Mitochondrial fission determines cisplatin sensitivity in tongue squamous cell carcinoma through the BRCA1–miR-593-5p–MFF axis

doi:

Figure Lengend Snippet: A , BRCA1 was analyzed using immunoblotting in Cal-27 cells under cisplatin treatment. B , BRCA1 attenuated the cisplatin-induced decrease of miR-593-5p. Cal-27 cells were transiently transfected with BRCA1 expressing plasmids or empty vector (Vec) and then treated with cisplatin for 24h. miR-593-5p were detected using qRT-PCR (upper panel), whereas BRCA1 was analyzed using immunoblotting (lower panel). *P< 0.01 versus cisplatin alone. C , ChIP-qPCR analysis of BRCA1 binding to the promoter of miR-593-5p in the BS3 region. **P< 0.001. D , ChIP-qPCR analysis of the association levels of BRCA1 with the miR-593-5p promoter in the BS3 region under cisplatin treatment. E , A luciferase assay indicated that cisplatin induced a reduction of miR-593-5p promoter activity in the BS3 region. Cal-27 cells were transfected with the wild-type promoter (wt) in the BS3 or empty vector (pGL3-Basic). F , A luciferase assay indicated that BRCA1 activated miR-593-5p promoter activity in the BS3 region. Cal-27 cells transiently transfected with BRCA1 expressing plasmids or empty vector (Vec) were treated with the wild-type promoter (wt) or a promoter with mutations in the BS3 (mut). **P< 0.001.

Article Snippet: An MFF shRNA retrovirus vector (pSR-puro-MFF1 shRNA, 37247) [ ] and a BRCA1 (pBABE-puro HA BRCA1, 14999) [ ] retrovirus vector were obtained from Addgene (MA, USA).

Techniques: Western Blot, Transfection, Expressing, Plasmid Preparation, Quantitative RT-PCR, Binding Assay, Luciferase, Activity Assay

Journal: Oncotarget

Article Title: Mitochondrial fission determines cisplatin sensitivity in tongue squamous cell carcinoma through the BRCA1–miR-593-5p–MFF axis

doi:

Figure Lengend Snippet: A , BRCA1 attenuated mitochondrial fission in Cal-27 cells under cisplatin treatment. Cal-27 cells transiently transfected with BRCA1 expressing plasmids or vector control (Vec) were treated with cisplatin for 24h. Scale bar equals 3 μm. **P < 0.001 versus cisplatin alone. B , C and D , Apoptosis was detected using TUNEL, flow cytometry, and caspase-3/7 activity assays. *P < 0.01 versus cisplatin alone; **P < 0.001 versus cisplatin alone. E , The knockdown of miR-593-5p leads to the attenuation of the BRCA1 inhibitory effect on MFF protein levels under cisplatin treatment. Cal-27 cells stably expressing BRCA1 or vector control (Vec) were transfected with miR-593-5p inhibitors or inhibitor-negative control (inhibitor-NC). MFF levels were analyzed using immunoblotting. F , Mitochondrial fission and apoptosis were detected via staining with MitoTracker Red and TUNEL.*P < 0.01.

Article Snippet: An MFF shRNA retrovirus vector (pSR-puro-MFF1 shRNA, 37247) [ ] and a BRCA1 (pBABE-puro HA BRCA1, 14999) [ ] retrovirus vector were obtained from Addgene (MA, USA).

Techniques: Transfection, Expressing, Plasmid Preparation, TUNEL Assay, Flow Cytometry, Activity Assay, Stable Transfection, Negative Control, Western Blot, Staining

Journal: Oncotarget

Article Title: Mitochondrial fission determines cisplatin sensitivity in tongue squamous cell carcinoma through the BRCA1–miR-593-5p–MFF axis

doi:

Figure Lengend Snippet: A, B, C, BALB/c-nu mice bearing Cal-27 cells with the stable expression of MFF shRNA or its scramble form (sc) were treated with saline or cisplatin. ( A ) Tumor growth curves for Cal-27 tumors treated with saline or cisplatin. ( B ) Representative photomicrographs of tumors from each group at day 35. ( C ) Apoptosis was detected via TUNEL assay. n=6 for each group. For TUNEL assay, n=24 slices from 6 xenograft tumors were sampled per group. Bar=20 μm; # P < 0.05 versus cisplatin alone. D, E, F, BALB/c-nu mice bearing Cal-27 cells with the stable expression of miR-593-5p or its control (con) were treated with saline or cisplatin. ( D ) Tumor growth curves for Cal-27 tumors treated with saline or cisplatin. ( E ) Representative photomicrographs of tumors from each group at day 35. ( F ) Apoptosis was detected via TUNEL assay. n=6 for each group. For TUNEL assay, n=24 slices from 6 xenograft tumors were sampled per group. Bar=20 μm; # P < 0.05 versus cisplatin alone. G, H, I, BALB/c-nu mice bearing Cal-27 cells with the stable expression of BRCA1 or empty vector (Vec) were treated with saline or cisplatin. ( G ) Tumor growth curves for Cal-27 tumors treated with saline or cisplatin. ( H ) Representative photomicrographs of tumors from each group at day 35. ( I ) Apoptosis was detected via TUNEL assay. n=6 for each group. For TUNEL assay, n=24 slices from 6 xenograft tumors were sampled per group. Bar=20 μm; # P < 0.05 versus cisplatin alone; *P< 0.01 versus cisplatin alone.

Article Snippet: An MFF shRNA retrovirus vector (pSR-puro-MFF1 shRNA, 37247) [ ] and a BRCA1 (pBABE-puro HA BRCA1, 14999) [ ] retrovirus vector were obtained from Addgene (MA, USA).

Techniques: Expressing, shRNA, TUNEL Assay, Plasmid Preparation

Journal: Oncotarget

Article Title: Mitochondrial fission determines cisplatin sensitivity in tongue squamous cell carcinoma through the BRCA1–miR-593-5p–MFF axis

doi:

Figure Lengend Snippet: A , MFF, miR-593-5p and BRCA1 expression and apoptosis were demonstrated in cisplatin-sensitive versus non-sensitive TSCCs. Left panel: MFF and BRCA1 expression were analyzed via immunohistochemistry; miR-593-5p expression was analyzed using in situ hybridization (×200). Apoptosis was detected using a TUNEL assay. Bar=20 μm. Right panel: Quantification of MFF, miR-593-5p and BRCA1 expression in cisplatin-sensitive versus non-sensitive TSCCs. # P < 0.05; *P < 0.01. B , Associations between MFF, miR-593-5p and BRCA1 expression in TSCCs were analyzed via Spearman order correlation. C , Kaplan-Meier survival curves for TSCCs are plotted for MFF, miR-593-5p and BRCA1 expression, and survival differences were analyzed using a log-rank test. D , Model of the BRCA1–miR-593-5p–MFF axis in regulating mitochondrial fission and cisplatin sensitivity. The dotted line indicates the commonly accepted mechanism of BRCA1 regulation of cisplatin sensitivity, whereas the solid line represents the novel mechanism of BRCA1-mediated cisplatin sensitivity identified in the present study.

Article Snippet: An MFF shRNA retrovirus vector (pSR-puro-MFF1 shRNA, 37247) [ ] and a BRCA1 (pBABE-puro HA BRCA1, 14999) [ ] retrovirus vector were obtained from Addgene (MA, USA).

Techniques: Expressing, Immunohistochemistry, In Situ Hybridization, TUNEL Assay

Journal: Oncotarget

Article Title: Mitochondrial fission determines cisplatin sensitivity in tongue squamous cell carcinoma through the BRCA1–miR-593-5p–MFF axis

doi:

Figure Lengend Snippet: Correlation among clinicopathological status and the expression of MFF, miR-593-5p or BRCA1 in TSCC patients

Article Snippet: An MFF shRNA retrovirus vector (pSR-puro-MFF1 shRNA, 37247) [ ] and a BRCA1 (pBABE-puro HA BRCA1, 14999) [ ] retrovirus vector were obtained from Addgene (MA, USA).

Techniques: Expressing

Journal: iScience

Article Title: Histone H2A ubiquitination resulting from Brap loss of function connects multiple aging hallmarks and accelerates neurodegeneration.

doi: 10.1016/j.isci.2022.104519

Figure Lengend Snippet: Figure 6. Histone H2A ubiquitination accompanied by BRCA1 activation is the hallmark phenotype of BRAP LOF (A) Immunoblotting of histone extracts from NPCs as well as from embryonic, neonatal, adult cerebral cortical tissues, and quantification (Mean G SD) of increases in histone H2Aub (total H2Aub and H2AubK119, respectively) resulted from Brap LOF. n = 3–6 biological replicates. p-values calculated by Student’s t test are indicated. (B) Immunoblotting of Brca1 in various cells and tissues, showing that Brap LOF results in increased Brca1 abundance. (C) Immunoblotting of nuclear vs cytoplasmic fractions of MEFs at P1, showing increased nuclear localization of Brca1 in Brap/ cells. (D) Brca1 (red) and NeuN (green) double immunohistology images of cerebral cortical sections of BrapcKONPC and control mice at four months of age. Representative images are shown. Note the increased intensity and density of Brca1 puncta in the nuclei of BrapcKONPC cortical neurons (NeuN+). (E and F) Immunoblotting analyses of histone extracts from cerebral cortical tissues of three-month-old mice, showing increased ubiquitination of H2A variants targeted by Brca1 (E) along with total histone H2A ubiquitination (F). (G) Double immunohistology staining of cortical sections of 4-month old WT or BrapcKONPC mice with antibodies against Gfap (green) and histone H3 (red), showing reduced nuclear histones in cells surrounded by reactive astrocytes (circles) in BrapcKONPC cortical tissues. Representative images are shown. Nuclear DNA was stained with Hoechst 33342. Bars: 50 um or as indicated. See also Figure S3.

Article Snippet: Phospho-p53 (Ser15) Abcam Cat# ab1431; RRID:AB_301090 Phospho-p53 (Ser15) (D4S1H) Cell Signaling Technology Cat# 12571; RRID:AB_2714036 Phospho-p53 (Ser15) (16G8) Cell Signaling Technology Cat# 9286; RRID:AB_331741 Phospho-ATM(Ser1981) Novus Biologicals Cat# AF1655 Phospho-ATR (Ser 428) Cell Signaling Technology Cat# 2853; RRID:AB_2290281 53 BP1 Novus Biologicals Cat# NB100-304 Brca1 Santa Cruz Biotechnology Cat# sc-642; RRID:AB_630944 Brca1 Novus Biologicals Cat# MAB22101 Brca1 (clone 8F7) Novus Biologicals Cat# NBP1-41186 LC3B (G-9) Santa Cruz Biotechnology Cat# sc-376404; RRID:AB_11150489 LC3B Cell Signaling Technology Cat# 2775; RRID:AB_915950 LC3B Novus Biologicals Cat# NB100-2220 p62SQSTM1 Novus Biologicals Cat# MAB8028 p62SQSTM1 GeneTex Cat# GTX100685; RRID:AB_2038029

Techniques: Ubiquitin Proteomics, Activation Assay, Western Blot, Control, Staining

Journal: Open biology

Article Title: Kaiso mediates transcription and RNA splicing in colorectal carcinoma: role of BRCA1 in the Kaiso enhanceosome.

doi: 10.1098/rsob.240329

Figure Lengend Snippet: Figure 1. Visual inspection of VDA3 and GRB2 promoters (ENCODE ChIP-seq profile) (lower panels) and bioinformatic profiles (upper panels right) of top 15 ranked eKBS co-occupants for BRCA1 and ZBTB33/Kaiso across ENCODE data across multiple cancer cell lines. (A) Diagram of the Kaiso domains. (B) Co-occupants of BRCA1 and Kaiso/ZBTB33 are rank ordered by Z score. Purple fill mark top core hits. Light grey mark hits common to both. (C) The location of the eKBS in the VDA3 and GRB2 promoters is indicated by the ZBTB33/Kaiso peak; the BRCA1 and CHD2 peaks are highlighted with red arrows. eKBS, encode-derived Kaiso-binding site.

Article Snippet: Immunoprecipitations of crosslinked DNA/proteins were performed with BRCA1 antibody (Cell Signaling, cat. no. 9010) or IgG control antibody of the same class.

Techniques: ChIP-sequencing, Derivative Assay, Binding Assay

Journal: Open biology

Article Title: Kaiso mediates transcription and RNA splicing in colorectal carcinoma: role of BRCA1 in the Kaiso enhanceosome.

doi: 10.1098/rsob.240329

Figure Lengend Snippet: Figure 2. Kaiso and BRCA1 interact directly. Left three panels, BRCA1 and Kaiso interact specifically by PLA—the interaction takes place primarily but not exclusively in the nucleus. M, mouse, R, rabbit. Right panels: DNA damage causes striking translocation of Kaiso into the nucleus. Detection is by a single antibody variation of PLA. Each dot represents one molecule of Kaiso.

Article Snippet: Immunoprecipitations of crosslinked DNA/proteins were performed with BRCA1 antibody (Cell Signaling, cat. no. 9010) or IgG control antibody of the same class.

Techniques: Translocation Assay

Journal: Open biology

Article Title: Kaiso mediates transcription and RNA splicing in colorectal carcinoma: role of BRCA1 in the Kaiso enhanceosome.

doi: 10.1098/rsob.240329

Figure Lengend Snippet: Figure 5. ChIP-seq analysis of Kaiso and BRCA1 DNA-binding sites. (A) Number of gene promoters, exons, introns or intergenic regions bound by Kaiso or BRCA1 antibody in HCT116 cells. (B) Distribution of Kaiso and BRCA1 antibody binding to various regions of the genes. (C) sVenn diagram visualizing the gene promoters with or without overlap of BRCA1 and Kaiso antibody binding.

Article Snippet: Immunoprecipitations of crosslinked DNA/proteins were performed with BRCA1 antibody (Cell Signaling, cat. no. 9010) or IgG control antibody of the same class.

Techniques: ChIP-sequencing, Binding Assay

Journal: Open biology

Article Title: Kaiso mediates transcription and RNA splicing in colorectal carcinoma: role of BRCA1 in the Kaiso enhanceosome.

doi: 10.1098/rsob.240329

Figure Lengend Snippet: Figure 4. (A) Encode data showing how CHD2, BRCA1, ZBT33 and ETS binding sites overlap on the CCNC promoter. (B) BRCA1 transactivates Kaiso as eKBS sites. The CCNC promoter was subcloned into the ‘empty’ vector PGL3basic and assayed for in vitro transcriptional activity after transient transfection of the empty vector (lanes 1−4) or the same vector expressing the constructs indicated across the bottom. (C) As in (B), but the experimental constructs compare the effects of endogenous Kaiso (lane 4), to transiently overexpressed Kaiso (lane 5) and a dominant negative (DN) Kaiso (lacking N-terminal BTB domain; lane 6). BRCA1 elevates transcription compared with Kaiso overexpression alone by 30%. DN-Kaiso suppresses transcription relative to endogenous Kaiso alone.

Article Snippet: Immunoprecipitations of crosslinked DNA/proteins were performed with BRCA1 antibody (Cell Signaling, cat. no. 9010) or IgG control antibody of the same class.

Techniques: Binding Assay, Plasmid Preparation, In Vitro, Activity Assay, Transfection, Expressing, Construct, Dominant Negative Mutation, Over Expression

Journal: Open biology

Article Title: Kaiso mediates transcription and RNA splicing in colorectal carcinoma: role of BRCA1 in the Kaiso enhanceosome.

doi: 10.1098/rsob.240329

Figure Lengend Snippet: Figure 6. Schematic showing hypothetical interactions of eKBS co-occupants in proximal promoter transcriptional elongation. Components of the complex are anchored to DNA via Kaiso, which binds directly to the TCTCGCGAGA motif (aka the eKBS) near the transcriptional start site (TSS). BRCA1 is recruited to the CHD4/Kaiso complex, RNA Polymerase II+GTFs bind to BRCA1 at the TATA box of a promoter, and transcription is activated with the help of INRs.

Article Snippet: Immunoprecipitations of crosslinked DNA/proteins were performed with BRCA1 antibody (Cell Signaling, cat. no. 9010) or IgG control antibody of the same class.

Techniques:

Journal: Open biology

Article Title: Kaiso mediates transcription and RNA splicing in colorectal carcinoma: role of BRCA1 in the Kaiso enhanceosome.

doi: 10.1098/rsob.240329

Figure Lengend Snippet: Figure 9. (A) Bar graph of enriched terms across input gene lists that bind both BRCA1 and Kaiso in HCT116 cells, coloured by p-value. (B) Summary of enrichment analysis in transcription factor targets from analysis of genes binding both BRCA1 and Kaiso in HCT116 cells. (C) Summary of enrichment analysis in transcriptional regulatory relationships unraveled by sentence-based text mining (TRRUST) analysis of genes binding both BRCA1 And Kaiso in HCT116 cells.

Article Snippet: Immunoprecipitations of crosslinked DNA/proteins were performed with BRCA1 antibody (Cell Signaling, cat. no. 9010) or IgG control antibody of the same class.

Techniques: Binding Assay

Journal: Journal of cell science

Article Title: Poly(ADP-ribosyl)ation links the chromatin remodeler SMARCA5/SNF2H to RNF168-dependent DNA damage signaling.

doi: 10.1242/jcs.109413

Figure Lengend Snippet: Fig. 7. SMARCA5 promotes cH2AX ubiquitylation and BRCA1 accumulation at DSBs. (A) Cells were transfected with the indicated siRNAs, exposed to 1 Gy IR and 1 h later immunostained for conjugated ubiquitin (FK2) to visualize IRIF. Scale bar: 10 mm. (B) Quantitative representation of FK2 IRIF formation in A. The average number of foci/nucleus 6 s.e.m. is presented. More than 150 nuclei were scored per treatment in independent experiments. *P,0.05, compared with siLuc (control). (C) Cells transfected with the indicated siRNAs were exposed to 10 Gy IR. 1 h later chromatin-enriched extracts (CEE) were prepared. CEE were subjected to IP with cH2AX antibody. Western blot analysis of IP with cH2AX antibody, and CEE with SMARCA5 antibody are shown. Ponceau S staining was used as a loading control. (D) Graphical representation of relative cH2AX ubiquitylation levels determined using results from C. Ratios of unmodified cH2AX and either mono-ubiquitylated or di-ubiquitylated cH2AX were normalized to that of control cells, which was set to 1. The means 6 s.e.m. of two independent experiments is shown. (E) As in A, except that cells were immunostained for BRCA1. (F) As in B, except that cells from E were analyzed.

Article Snippet: Antibodies Immunofluorescence and western blot analysis were performed using antibodies to cH2AX, phospho-histone H3 S10p (Millipore), a-Tubulin (Sigma), Streptavidin (IBA BioTAGnology), GFP (Roche), ubiquitin and PARP2 (FK2, Enzo Life Sciences), PAR (Trevigen), BRCA1 (Calbiochem and Santa Cruz), 53BP1 (Novus), SMARCA5/SNF2h, histone H3 and MDC1 (Abcam) and PARP1 (Cell Signaling).

Techniques: Transfection, Ubiquitin Proteomics, Control, Western Blot, Staining

Journal: Journal of cell science

Article Title: Poly(ADP-ribosyl)ation links the chromatin remodeler SMARCA5/SNF2H to RNF168-dependent DNA damage signaling.

doi: 10.1242/jcs.109413

Figure Lengend Snippet: Fig. 8. SMARCA5 chromatin remodeling activity is required for BRCA1 accumulation at DSBs. (A) U2OS cells were transfected with the indicated siRNAs and either GFP, SMARCA5-WT-GFP or SMARCA5-K211R-GFP plasmids, exposed to 2 Gy IR and stained for BRCA1 and DAPI. Scale bar: 5 mm (B) Quantitative representation of BRCA1 IRIF formation in A. The percentage of cells with more than 10 foci per nucleus is presented. Error bars indicate the s.d. More than 100 cells from at least two independent experiments were scored. (C) SMARCA5 and SMARCA5-GFP levels were monitored by western blot analysis using whole cell extracts of cells in A. Tubulin was used as a loading control. (D) Model for the role of SMARCA5 in the DSB response. A DSB in chromatin triggers PARP1 recruitment and cH2AX formation. PARP1 elicits local chromatin expansion, whereas cH2AX promotes the accumulation of MDC1 and RNF8, which ubiquitylates H2A-type histones. This leads to RNF168 recruitment, which amplifies RNF8-induced ubiquitylation, promoting the accumulation of ubiquitin-binding factors, including the RAP80–BRCA1 complex. PARP1 promotes the spatial distribution of SMARCA5 and factors of the RNF168 signaling cascade (RNF168, UBC13, RAD18, RAP80, BRCA1; only RNF168 and BRCA1 are shown for simplicity), and stimulates SMARCA5 and RNF168 interaction throughout DSB-flanking chromatin, most likely through PARylation of RNF168, thereby regulating RNF168-dependent DSB signaling. SMARCA5 also promotes efficient repair of DSB by NHEJ and HR (see text for details).

Article Snippet: Antibodies Immunofluorescence and western blot analysis were performed using antibodies to cH2AX, phospho-histone H3 S10p (Millipore), a-Tubulin (Sigma), Streptavidin (IBA BioTAGnology), GFP (Roche), ubiquitin and PARP2 (FK2, Enzo Life Sciences), PAR (Trevigen), BRCA1 (Calbiochem and Santa Cruz), 53BP1 (Novus), SMARCA5/SNF2h, histone H3 and MDC1 (Abcam) and PARP1 (Cell Signaling).

Techniques: Activity Assay, Transfection, Staining, Western Blot, Control, Ubiquitin Proteomics, Binding Assay