Journal: Cell discovery

Article Title: The BRCA1/BARD1 complex recognizes pre-ribosomal RNA to facilitate homologous recombination.

doi: 10.1038/s41421-023-00590-8

Figure Lengend Snippet: Fig. 4 Pre-rRNA targets the BRCA1/BARD1 complex to DSBs. a Pre-rRNP exists at IRIF. Cells were treated with 10 Gy of IR. Pre-rRNA was examined by RNA probes. Ribosomal proteins were stained with indicated antibodies. The colocalization with BRCA1 was analyzed (bottom panel). b Foci number per cell and foci colocalization ratio are examined. c The IRIF of the BRCA1/BARD1 complex in the pol Ii-treated cells. HeLa cells were treated with RNA polymerase inhibitors prior to 10 Gy of IR. The IRIF of BRCA1 and BARD1 were stained with indicated antibodies. Foci number per cell is shown (right panel). The cells were also counterstained by DAPI. P values were calculated using Student’s t-test. Scale bars, 10 μm. d A schematic diagram showing that pre-incubation with pre-rRNA blocks the binding of the recombinant BRCA1 BRCT or BARD1 BRCT to IRIF (top left panel). Foci were examined with anti-GST antibody (bottom panel). DSB foci were marked with an anti-γH2AX antibody. Foci number per cell is shown (top right panel). P values were calculated using Student’s t test. n.s. nonsignificant, *P < 0.05, ***P < 0.001. Scale bars, 10 μm. e Enrichment of pre-rRNA at the unsynapsed axis of X and Y chromosomes. Analysis of pre-rRNA and SCP3 distribution is shown (right panel). f Pre-incubation of the BRCTs with pre- rRNA abolishes their localization onto the XY body. Recombinant BRCT proteins were pre-incubated with pre-rRNA. The circled area indicates the XY body. Scale bars, 10 μm.

Article Snippet: Anti-BARD1 antibody was purchased from Novus.

Techniques: Staining, Incubation, Binding Assay, Recombinant

Journal: bioRxiv

Article Title: LARP7 is a BRCA1 ubiquitinase substrate and regulates genome stability and tumorigenesis

doi: 10.1101/808535

Figure Lengend Snippet: a-b, IR did not suppress LARP7 mRNA transcription in HeLa cells (a) and MEFs (b), as detected by RT-qPCR. Data: mean±SD, Student’s t test, n=3. c-d, Reciprocal IP demonstrated that IR increased the interaction between LARP7 and wild-type BRCA1/BARD1 in 293T cells. e, A Co-IP experiment indicated that IR increased the interaction between LARP7 and BRCA1/BARD1. f-g, A BiFC assay showed that CDDP enhanced the interaction between LARP7 and BARD1. Green cells indicate cells with positive interactions. The JUN-FOS interaction was used as a positive control. Student’s t test, n=3, *: p<0.05, **: p<0.01. h, A GST pulldown assay showed that the BRCT domain of BARD1 had much lower affinity for LARP7 than the BRCT domain of BRCA1.

Article Snippet: To purify the BRCA1/BARD1 heterodimer RING domain, His-tagged N-terminals of BRCA1 (residues 1-304; Addgene, 12645) and BARD1 (residues 26-327; Addgene, 12646) were cotransfected into BL21/DE3 E. coli .

Techniques: Quantitative RT-PCR, Co-Immunoprecipitation Assay, Bimolecular Fluorescence Complementation Assay, Positive Control, GST Pulldown Assay

Journal: bioRxiv

Article Title: LARP7 is a BRCA1 ubiquitinase substrate and regulates genome stability and tumorigenesis

doi: 10.1101/808535

Figure Lengend Snippet: a, IR reduced the half-life of LARP7. b, In vivo ubiquitination assay results showing that IR induced the ubiquitination of LARP7. c, LARP7 interacted with BRCA1 and BARD1, which was independent of 7SK RNA and was increased by IR. d, IR enhanced the interaction of LARP7 with the BARD1 BRCT domain but not the BRCA1 BRCT domain. e, Ectopic expression of BRCA1/BARD1 promoted LARP7 degradation, an effect that was attenuated by ubiquitination inhibition (MG132). f, BRCA1/BARD1 coexpression induced LARP7 ubiquitination in 293T cells. g. Loss-of-function mutations in the BRCA1 RING domain (I26A and C61G) abolished LARP7 ubiquitination. h. An in vitro ubiquitination assay illustrated that LARP7 ubiquitination was mediated by the RING domain complex of BRCA1 (1-304) and BARD1 (26-327). i. An in vivo ubiquitination assay demonstrated that K48-linked polyubiquitin was the predominant form of BRCA1-elicited LARP7 ubiquitination. K48: ectopically expressed ubiquitin with all lysine residues mutated to arginine except K48. K63: ubiquitin with all lysine residues mutated to arginine except K63. WT: wild-type ubiquitin with all lysine residues intact.

Article Snippet: To purify the BRCA1/BARD1 heterodimer RING domain, His-tagged N-terminals of BRCA1 (residues 1-304; Addgene, 12645) and BARD1 (residues 26-327; Addgene, 12646) were cotransfected into BL21/DE3 E. coli .

Techniques: In Vivo, Ubiquitin Proteomics, Expressing, Inhibition, In Vitro

Journal: bioRxiv

Article Title: LARP7 is a BRCA1 ubiquitinase substrate and regulates genome stability and tumorigenesis

doi: 10.1101/808535

Figure Lengend Snippet: a, BRCA1 knockdown attenuated IR-induced LARP7 degradation. b, BARD1 knockdown attenuated IR-induced LARP7 degradation. c, LARP7 expression in variable breast cancer cell lines. w: wild-type, m: BRCA1 mutant, mut: P53 mutant. MDA-MB-436 and HCC1937 cells have a homozygous BRCA1 mutation; MCF7 cells have a heterozygous mutation. d, IR induced LARP7 downregulation in MDA-MB-231 cells but not in HCC1937 cells. e, Ectopic expression of BRCA1 and BARD1 augmented LARP7 ubiquitination in 293T cells.

Article Snippet: To purify the BRCA1/BARD1 heterodimer RING domain, His-tagged N-terminals of BRCA1 (residues 1-304; Addgene, 12645) and BARD1 (residues 26-327; Addgene, 12646) were cotransfected into BL21/DE3 E. coli .

Techniques: Knockdown, Expressing, Mutagenesis, Ubiquitin Proteomics

Journal: bioRxiv

Article Title: LARP7 is a BRCA1 ubiquitinase substrate and regulates genome stability and tumorigenesis

doi: 10.1101/808535

Figure Lengend Snippet: a, ATM phosphatase activity induced by DDR was required for LARP7 downregulation. KU55933: ATM inhibitor, 10 μM; VE-821: ATR inhibitor, 10 μM; NU7441: DNAPK inhibitor, 10 μM. b, IR induced phosphorylation at the ATM recognition motif of LARP7, as illustrated by a SQ/TQ phosphorylation antibody. c, T440 of LARP7 is evolutionarily conserved. d, T440 was phosphorylated after IR, as revealed by a phosphorylation-specific antibody. e, The ATM inhibitor attenuated IR-induced T440 phosphorylation. f, Immunoprecipitation assay showing that T440 phosphorylation enhanced the interaction of the LARP7 peptide with the BRCT domain of BARD1 but not with that of BRCA1. g, Co-IP results showing that T440A substitution attenuated the interaction of LARP7 with BARD1 but not with BRCA1. h, The T440A substitution attenuated IR-induced LARP7 polyubiquitination.

Article Snippet: To purify the BRCA1/BARD1 heterodimer RING domain, His-tagged N-terminals of BRCA1 (residues 1-304; Addgene, 12645) and BARD1 (residues 26-327; Addgene, 12646) were cotransfected into BL21/DE3 E. coli .

Techniques: Activity Assay, Phospho-proteomics, Immunoprecipitation, Co-Immunoprecipitation Assay

Journal: bioRxiv

Article Title: LARP7 is a BRCA1 ubiquitinase substrate and regulates genome stability and tumorigenesis

doi: 10.1101/808535

Figure Lengend Snippet: a. Breast cancer patients receiving chemotherapy were stratified by LARP7 expression (LARP7 High= top 25%; LARP7 Low=bottom 25%), and RFS was analyzed by the Kaplan-Meier method. HR: hazard ratio. The numbers of patients that died at different times are labeled at the bottom of the plots. Statistical significance was calculated with logrank tests, and p<0.05 indicated significance. b. IHC showing the LARP7 protein levels in CDDP- and IR-treated tumors. CDDP and IR induced significant downregulation and extranuclear shuttling of LARP7 in MDA-MB-231 xenograft tissue but not in HCC1937 xenograft tissue. c. Tumor sizes of wild-type or LARP7 -/- HCC1937 xenografts treated with CDDP or X-rays. Wild-type tumors with or without treatment: n=7, LARP7 -/- tumors with or without treatment: n=8, mean±SD. The intersection of each curve with the dotted line indicates the time (from treatment initiation) at which the tumor size reached 1000 mm 3 . d. Tumor sizes of wild-type or LARP7 OE MDA-MB-231 xenografts treated with CDDP or X-rays. Wild-type tumors with or without treatment: n=7, LARP7 OE tumors with or without treatment: n=7, mean±SD. The intersection of each curve with the dotted line indicates the time (from treatment initiation) at which the tumor size reached 1000 mm 3 . e. Schematic of the mechanism by which LARP7 attenuated HDR. DNA DSBs activated ATM, which phosphorylated LARP7 at T440, increased the interaction of LARP7 with BARD1 and triggering the ubiquitination and degradation of LARP7 through the 26S proteasome. Under ordinary conditions, LARP7 promotes the expression of CCC. Depletion of LARP7 suppressed CCC expression, which arrested the cell cycle at the G2/M checkpoint. In addition, LARP7 depletion decreased BRCA2 phosphorylation, which enhanced RAD51 recruitment to DNA damage sites and thus increased RAD51-mediated homologous recombination repair.

Article Snippet: To purify the BRCA1/BARD1 heterodimer RING domain, His-tagged N-terminals of BRCA1 (residues 1-304; Addgene, 12645) and BARD1 (residues 26-327; Addgene, 12646) were cotransfected into BL21/DE3 E. coli .

Techniques: Expressing, Labeling, Ubiquitin Proteomics, Phospho-proteomics, Homologous Recombination

Journal: Journal of Translational Medicine

Article Title: Survival advantage of native and engineered T cells is acquired by mitochondrial transfer from mesenchymal stem cells

doi: 10.1186/s12967-024-05627-4

Figure Lengend Snippet: MitoT increases the expression of anti-apoptotic Bcl-2/BARD1 pathways. A Fold change of differentially expressed (DE) genes contained within the GO terms related to apoptosis, cell death, and/or responses to different stimuli. B qRT-PCR analysis of BARD1 mRNA expression levels in FACS-sorted CD3+ T cells after 24-h post-mitoception with MSC derived-MT (n = 4). C Representative western blots of BARD1 in FACS-sorted CD3+ MitoT pos cells and MitoT neg cells after 24 or 48 h post-mitoception. D Fold change quantification of proteins showed in C (n = 3). E Ingenuity Pathway analysis for Bcl-2 gene network for the DE genes denoted in A . F qRT-PCR of Bcl-2 family gene expression in FACS-sorted CD3+ MitoT pos cells and MitoT neg cells after 24 h post-mitoception (n = 4). For figures B and D : graphs show mean ± SEM and statistical analysis by unpaired t-test. For figure F : graph shows mean ± SEM and statistical analysis by unpaired Mann–Whitney test (*p < 0.05, relative to MitoT neg control). All replicates are biological

Article Snippet: Then membranes were incubated with 1:1000 primary antibodies, rabbit anti-Caspase 3, or mouse anti-β-actin (Cell Signaling Technology #9662 and #3700, respectively), and rabbit anti-BRCA1-associated RING domain protein 1 (BARD1) (Novus Biological #NB100-319) and incubated overnight at 4 °C with shaking.

Techniques: Expressing, Quantitative RT-PCR, Derivative Assay, Western Blot, Gene Expression, MANN-WHITNEY, Control

Journal: PLoS ONE

Article Title: Preparation and purification of mono-ubiquitinated proteins using Avi-tagged ubiquitin

doi: 10.1371/journal.pone.0229000

Figure Lengend Snippet: Plasmids used in this study.

Article Snippet: pFastbac1-StrepII-BARD1 , Codon optimized human BARD1 , Amp, Gent , gene synthesis by Gene Universal. Deposited Addgene: 137166 , a.

Techniques: Selection, Ubiquitin Proteomics

Journal: PLoS ONE

Article Title: Preparation and purification of mono-ubiquitinated proteins using Avi-tagged ubiquitin

doi: 10.1371/journal.pone.0229000

Figure Lengend Snippet: (A) Coomassie stained 4–12% Bis-Tris gel run in 1X MES buffer showing purified human FANCI:FANCD2, BRCA1 Δexon11 :BARD1, reconstituted nucleosome, PCNA, UBCH5C (S22R), Avi-ubiquitin, FANCB-FANCL-FAAP100 (BL100), FANCC-FANCE-FANCF (CEF), UBE1 and UBE2T (lanes 2–11). (B) Western blot of the time course ubiquitination reaction of human FANCI:FANCD2 at 25 °C, 30 °C and 37 °C. The percentage of mono-ubiquitinated FANCD2 or FANCI were calculated and showed under each western blot panel. (C) PCNA mono-ubiquitination time course experiments using UBCH5C as an E2 enzyme. (D) Western blots of time course experiments revealing that BRCA1 Δexon11 and BARD1 were auto-ubiquitinated at multiple lysine residues in the presence of UBCH5C. (E) Mono-ubiquitination timecourse of nucleosomes as substrates in the presence of BRCA1 Δexon11 :BARD1 and UBCH5C. Experiments in (B-E) were performed in triplicate with similar results.

Article Snippet: pFastbac1-StrepII-BARD1 , Codon optimized human BARD1 , Amp, Gent , gene synthesis by Gene Universal. Deposited Addgene: 137166 , a.

Techniques: Staining, Purification, Ubiquitin Proteomics, Western Blot

Journal: Journal of Biological Chemistry

Article Title: The BRCA1 RING and BRCT Domains Cooperate in Targeting BRCA1 to Ionizing Radiation-induced Nuclear Foci

doi: 10.1074/jbc.m408879200

Figure Lengend Snippet: FIG. 1. Endogenous BRCA1 and ec- topic YFP-BRCA1 form nuclear foci with BARD1 and MDC1. A, endogenous BRCA1 nuclear foci (stained with Ab-4 and Texas Red) co-localize with BARD1 and MDC1 (stained with fluorescein iso- thiocyanate) in T47D cells before and af- ter 15-Gy IR treatment and 4 h of recov- ery. B, YFP-tagged wild-type BRCA1 was transfected into MCF-7 cells and assessed 48 h later for nuclear foci, which also co- localized with BARD1 and MDC1 (stained with Texas Red). Note the redistribution of BRCA1 from few and larger spots in untreated cells to small and more dis- persed foci in IR-treated cells.

Article Snippet: For BARD1 and MDC1 staining, rabbit BARD1 antibody 59L (gift from Professor Richard Baer) and rabbit MDC1 BL578 antibody (Bethyl Laboratories) were used, respectively.

Techniques: Staining, Transfection

Journal: Journal of Biological Chemistry

Article Title: The BRCA1 RING and BRCT Domains Cooperate in Targeting BRCA1 to Ionizing Radiation-induced Nuclear Foci

doi: 10.1074/jbc.m408879200

Figure Lengend Snippet: FIG. 2. Effect of BRCA1 cancer mutations on nuclear focus formation. A, diagram of BRCA1 showing the location of RING and BRCT domains and of published nuclear localization (NLS) and nuclear export (NES) transport signals. YFP-tagged BRCA1 wild-type and mutant proteins were co-transfected with pFLAG-BARD1 into MCF-7 cells and either left untreated or irradiated with 15 Gy of IR with 4 h of recovery. Representative images of nuclei and foci are shown. B, nuclear foci scoring results for the wild-type and mutant BRCA1. Scores were categorized into no focus, 1–10 foci, and 10 foci, and for each category an average percentage of transfected cells (S.E.) was obtained from two separate experiments (100 cells per experiment). Data for wild-type BRCA1 was from four experiments (mean S.D.). White bars represent untreated samples, and black bars represent irradiated samples.

Article Snippet: For BARD1 and MDC1 staining, rabbit BARD1 antibody 59L (gift from Professor Richard Baer) and rabbit MDC1 BL578 antibody (Bethyl Laboratories) were used, respectively.

Techniques: Mutagenesis, Transfection, Irradiation

Journal: Journal of Biological Chemistry

Article Title: The BRCA1 RING and BRCT Domains Cooperate in Targeting BRCA1 to Ionizing Radiation-induced Nuclear Foci

doi: 10.1074/jbc.m408879200

Figure Lengend Snippet: FIG. 7. RING/BARD1-binding fragment inhibits endogenous BRCA1 nuclear focus formation. A, five short YFP-BRCA1 constructs were transfected into T47D cells to assess their effect on endogenous BRCA1 (stained with Ab-4 and Texas Red). Cells were left untreated or treated with 15 Gy of IR and 4 h of recovery. Cell images show representative transfected cells and co-staining for endogenous BRCA1 and cell nuclei (stained with Hoechst). The arrows point to transfected cell nuclei. B, endogenous BRCA1 nuclear foci were counted in cells transfected with the YFP-BRCA1 constructs (cells with no visible BRCA1 not shown in graphs). Cells were scored for 0, 1–10, and 10 foci; white bars and black bars represent untreated and irradiated samples, respectively.

Article Snippet: For BARD1 and MDC1 staining, rabbit BARD1 antibody 59L (gift from Professor Richard Baer) and rabbit MDC1 BL578 antibody (Bethyl Laboratories) were used, respectively.

Techniques: Binding Assay, Construct, Transfection, Staining, Irradiation

Journal: iScience

Article Title: BRCA1 mediates protein homeostasis through the ubiquitination of PERK and IRE1

doi: 10.1016/j.isci.2022.105626

Figure Lengend Snippet: BRCA1 protein interacts with and ubiquitinates PERK and IRE1 (A–D) Total protein extracts were isolated from (A) control or BRCA1-depleted MCF7, (B) control or BRCA1-depleted MDA-MB-231cells, (C) MDA-MB-436 cells (+ or def), and (D) control, BRCA1 or BARD1 over-expressing MDA-MB-436 BRCA1-def cells. (E and F) Immunoprecipitation (IP) of BRCA1 protein pull-downs of PERK and IRE1 from MCF7 (E) or MDA-MB-231 (F) cytoplasmic protein extract. BRCA1 IB: ∗ = hyperphosphorylated BRCA1, ∗∗ = truncated BRCA1. ∗∗∗ = delta11q isoform. IRE1 IB: ∗ = possible ubiquitinated IRE1. (G and H) Ubiquitination analysis of PERK and IRE1 in (G) MCF7 cells or (H) MDA-MB-231 cells. Cells were transfected with control or BRCA1 siRNA. BRCA1 siRNA transfected cells were either untreated or treated with DMSO or Bortezomib overnight before harvesting for protein analysis. (I and J) In vitro ubiquitination analysis of (I) PERK or (J) IRE1 with E1, UBE2J1, BRCA1, BARD1, and/or ubiquitin. Ubiquitinated PERK or IRE1 was detected with an anti-Ub antibody. Ub = Ubiquitin. Bortz = Bortezomib. Data are represented as mean ± SD. P value was calculated by Student’s two-tailed, unpaired t -test. ∗ <0.05. See also Figures S4–S6 .

Article Snippet: BARD1 , OriGene , Cat# HP200444.

Techniques: Isolation, Expressing, Immunoprecipitation, Transfection, In Vitro, Two Tailed Test

Journal: iScience

Article Title: BRCA1 mediates protein homeostasis through the ubiquitination of PERK and IRE1

doi: 10.1016/j.isci.2022.105626

Figure Lengend Snippet:

Article Snippet: BARD1 , OriGene , Cat# HP200444.

Techniques: Recombinant, SYBR Green Assay, Isolation, Plasmid Preparation, Software