bard1 plasmid  (Genecopoeia)

Journal: Cell Reports Medicine

Article Title: Epigenetic modulation of BARD1 to enhance anti-VEGF therapy

doi: 10.1016/j.xcrm.2025.102329

Figure Lengend Snippet: Aza induces transcriptomic and epigenetic changes (A) Heatmap of mRNA expression profiles in the SKOV3ip1-luc model ( n = 3 mice per group); includes thousands of probes. (B) Comparison of tumor-specific methylation probes and gene expression between B20 and Aza+B20 groups in the SKOV3ip1-luc mouse model. Some genes appear more than once because the array includes multiple probes per gene. (C) Relative mRNA expression of BARD1 in each treatment group in the SKOV3ip1-luc mouse model. (D) Protein expression of BARD1 in each treatment group in the SKOV3ip1-luc mouse model. (E) Petal diagram of BARD1-associated pathways identified using expression data shown in (B). (F) Relative mRNA expression of BARD1 in normal fallopian tube epithelium (FTE) cells, HIO180 cells, and OC cell lines. (G) Protein expression of BARD1 in normal FTE cells, non-transformed ovarian surface epithelial cells (HIO180), and OC cell lines. Data are presented as mean ± SD.

Article Snippet: Single guide RNA (sgRNA) expression vectors were generated by annealing BARD1 target-specific oligonucleotides and inserting them into the BsmBI restriction site of a modified gRNA backbone (Addgene plasmid #52963).

Techniques: Expressing, Comparison, Methylation, Gene Expression, Transformation Assay

Journal: Cell Reports Medicine

Article Title: Epigenetic modulation of BARD1 to enhance anti-VEGF therapy

doi: 10.1016/j.xcrm.2025.102329

Figure Lengend Snippet: BARD1 regulates tumor angiogenesis (A) Tube formation assay in RF24 endothelial cells transfected with BARD1 siRNA or control siRNA. Scale bars, 250 μm. (B) Tube formation in RF24 cells cultured with CM from SKOV3ip1-luc cells treated with BARD1 siRNA or control siRNA. Scale bars, 250 μm. (C) Gene Ontology analysis of gene and pathway enrichment in BARD1-silenced SKOV3ip1-luc cells. (D) Hemoglobin content in mice injected with CM from BARD1 siRNA or control siRNA-treated SKOV3ip1 cells. (E) Angiogenesis array results in SKOV3ip1-luc cells treated with BARD1 siRNA or control siRNA. (F) Schematic of the TetOn system: mice fed doxycycline (Dox) chow for BARD1 knockdown and treated with bevacizumab ± Aza. (G) Tumor weights in vivo with or without BARD1 knockdown ( n = 10 mice for untreated; n = 10 mice for untreated + Dox; n = 8 mice for Bev antibody; n = 10 mice for Bev antibody + Dox; n = 9 mice for combination; and n = 9 mice for combination + Dox). (H) Relative mRNA expression of BARD1 in vivo across treatment groups ± Dox. Data are presented as mean ± SD. Statistical comparisons were made using two-tailed Student’s t test (A, B, D, F, G, and H).

Article Snippet: Single guide RNA (sgRNA) expression vectors were generated by annealing BARD1 target-specific oligonucleotides and inserting them into the BsmBI restriction site of a modified gRNA backbone (Addgene plasmid #52963).

Techniques: Tube Formation Assay, Transfection, Control, Cell Culture, Injection, Knockdown, In Vivo, Expressing, Two Tailed Test

Journal: Cell Reports Medicine

Article Title: Epigenetic modulation of BARD1 to enhance anti-VEGF therapy

doi: 10.1016/j.xcrm.2025.102329

Figure Lengend Snippet: BARD1 is epigenetically regulated under hypoxic conditions (A and B) Relative mRNA expression of HIF1α and BARD1 in SKOV3ip1 cells grown under normoxic (UT) and hypoxic conditions (1% O 2 ): (A) HIF1α and (B) BARD1. Data are presented as mean ± SD ( n = 3 samples per group). (C) Protein expression levels of HIF1α in normoxic (N) and hypoxic (H) conditions. (D) Protein expression of BARD1 under normoxic (N) and hypoxic (H) conditions. (E) Relative BARD1 mRNA levels in SKOV3ip1-luc cells treated with HIFα versus control siRNA. (F) Relative DNMT3A mRNA levels in SKOV3ip1 cells grown under normoxic (UT) or hypoxic conditions (1% O 2 ). (G) Relative DNMT3A mRNA expression levels in SKOV3ip1-luc cells treated with HIF1α siRNA versus control siRNA. (H) Chromatin immunoprecipitation followed by qPCR (ChIP-qPCR) results showing HIF1α enrichment under hypoxic and normoxic conditions ( n = 3 samples per group). ∗ p < 0.05. (I) HIF1α and DNMT3A enrichment at BARD1 promoter at site 4 under hypoxic and normoxic conditions. Data are presented as mean ± SD ( n = 3 samples per group). ∗ p < 0.05. (J) Relative TET1 mRNA expression levels under hypoxic and normoxic conditions. (K) Tumor weight in mice treated with DOPC-dCas9-TET1-sgBARD1-3 (sgBARD1-3) versus sgControl. ( n = 12 mice per group). (L) Relative BARD1 mRNA expression in mice treated with sgBARD1-3 or sgControl ( n = 4 mice per group). (M) Tumor weight in mice treated with sgBARD1-3, alone or in combination with bevacizumab (Bev) ( n = 13 mice for untreated, n = 12 mice for DOPC-TET1-sgControl, n = 12 mice for DOPC-TET1-sgBARD1-3, n = 12 mice for DOPC-TET1-sgControl + Bev antibody treatment, and n = 12 mice for DOPC-TET1-sgBARD1-3 + Bev antibody treatment group). (N) Number of nodules in mice treated with sgBARD1-3, alone or in combination with Bev (same group sizes as in M). (O) Tumor weight in mice treated with sgBARD1-1 or control ( n = 8 mice per group). Data are presented as mean ± SD (A, B, H, I, and K) or SEM (L–O). Statistical comparisons were made using two-tailed Student’s t test.

Article Snippet: Single guide RNA (sgRNA) expression vectors were generated by annealing BARD1 target-specific oligonucleotides and inserting them into the BsmBI restriction site of a modified gRNA backbone (Addgene plasmid #52963).

Techniques: Expressing, Control, Chromatin Immunoprecipitation, ChIP-qPCR, Two Tailed Test

Journal: Cell Reports Medicine

Article Title: Epigenetic modulation of BARD1 to enhance anti-VEGF therapy

doi: 10.1016/j.xcrm.2025.102329

Figure Lengend Snippet: Proposed mechanism of BARD1 epigenetic regulation in response to AVA therapy Prolonged AVA treatment induces adaptive resistance characterized by increased tumor hypoxia. Hypoxia stabilizes HIF1α, which enhances DNMT3A expression. DNMT3 then methylates the BARD1 promoter, repressing its transcription. Reduced BARD1 expression in cancer cells is associated with upregulation of proangiogenic factors, promoting tumor angiogenesis and progression. Targeted demethylation of BARD1 restores its expression and suppresses angiogenesis (created in BioRender).

Article Snippet: Single guide RNA (sgRNA) expression vectors were generated by annealing BARD1 target-specific oligonucleotides and inserting them into the BsmBI restriction site of a modified gRNA backbone (Addgene plasmid #52963).

Techniques: Expressing

Journal: bioRxiv

Article Title: Distinct Mechanisms of Recognition of Phosphorylated RNAPII C- Terminal Domain by BRCT Repeats of the BRCA1–BARD1 Complex: Insights from Structural and Functional Analyses

doi: 10.1101/2025.01.22.634233

Figure Lengend Snippet: The interaction between the BRCA1-BARD1 complex and RNAPII is direct and mediated by the phosphorylated CTD of RNAPII and the BRCT domains of BRCA1-BARD1. A) Schematic representation of BRCA1 and BARD1 domains. Positions of investigated binding variants, the tags, and cleavage sites are indicated. B) BRCA1-BARD1 interacts with RNAPII via the CTD phosphorylated on Ser2 and Ser5. Western blot analysis of pull-downs from HEK293 lysates. HEK293 cells were lysed, and the lysate was cleared by centrifugation. To the supernatant, FLAG-BRCA1-BARD1 was added, and the samples were incubated with α-FLAG beads. As a control, the HEK293 lysate with no added BRCA1-BARD1 was used. The proteins were eluted using 3xFLAG peptide and the samples were analysed using western blots. C) BRCA1-BARD1 interacts directly with pS2pS5 GST-(CTD) 26 and pS5pS7 GST-(CTD) 26 in vitro . SDS-PAGE analysis of in vitro pull-down assay between GST-(CTD) 26 and BRCA1-BARD1. Purified BRCA1-BARD1 was incubated with phosphorylated and non-phosphorylated GST-(CTD) 26 bound to glutathione beads. The samples were centrifuged and the input, unbound (supernatant) and bound (pellet) fractions were analysed using the SDS PAGE. D) Substitutions in the phosphoserine binding site (BRCA1 S1655F, K1702M , BARD1 S575F, K619A ) abolish the binding. SDS-PAGE analysis of in vitro pull-down assay between GST-(CTD) 26 and BRCA1 BRCT and BARD1 BRCT, respectively. Purified BRCA1 BRCT and BARD1 BRCT, respectively, were incubated with phosphorylated and non-phosphorylated GST-(CTD) 26 bound to glutathione beads. The samples were centrifuged and the input, unbound (supernatant) and bound (pellet) fractions were analysed using the SDS PAGE.

Article Snippet: To generate a vector for co-expression of the full-length human BRCA1-BARD1 complex in insect cells, fragment of DNA, containing FLAG-tagged BRCA1 was first cloned into 2BcT plasmid (pET His6 LIC cloning vector; addgene #37236) to add C-terminal (His) 6 tag to the construct.

Techniques: Binding Assay, Western Blot, Centrifugation, Incubation, Control, In Vitro, SDS Page, Pull Down Assay, Purification

Journal: bioRxiv

Article Title: Distinct Mechanisms of Recognition of Phosphorylated RNAPII C- Terminal Domain by BRCT Repeats of the BRCA1–BARD1 Complex: Insights from Structural and Functional Analyses

doi: 10.1101/2025.01.22.634233

Figure Lengend Snippet: The BRCT domains of the BRCA1–BARD1 complex exhibit differences in their binding kinetics to the phosphorylated CTD of RNAPII. A) Substitutions in the phosphoserine binding site of the BRCT repeats (BRCA1 S1655F, K1702M , BARD1 S575F, K619A ) abolish the binding to phosphorylated CTD. Sensograms obtained by biolayer-interferometry (BLI). The sensograms represent the mean of 3 measurements for each concentration. The data were analysed in Octet® Analysis Studio Software using 1:2 Bivalent analyte model. The data were plotted using Prism GraphPad 9 software. B) BARD1 BRCT associates with GST-p(CTD) 26 more dynamically than BRCA1 BRCT. Sensograms obtained by biolayer-interferometry (BLI) and their respective fits (left). Comparison of association ( K as ), dissociation ( K dis ), and dissociation ( K D ) constants for pS5pS7 GST-(CTD) 26 and BRCA1 and BARD1 BRCT, respectively, obtained by biolayer interferometry (right). The sensograms represent the mean of 3 measurements for each concentration. The association and dissociation constants and the coefficient of determination (R2) indicating the appropriateness of the fit were calculated in Octet® Analysis Studio Software using 1:2 Bivalent analyte model. The data were plotted using Prism GraphPad 9 software. C) Structural alignment of BRCA1 BRCT (PDB: 1JNX, teal) and BARD1 BRCT (PDB: 2NTE, purple) obtained in UCSF Chimera. D) Comparison of the amino-acid composition of the hydrophobic pocket of BRCA1 BRCT (1JNX, teal) and the residues present on the homologous positions in BARD1 BRCT (2NTE, purple). Close up from (C). E) Comparison of sensograms obtained by biolayer-interferometry (BLI) and their respective fits (top) of pS5pS7 GST-(CTD) 26 binding to BRCA1 BRCT M1775H and BARD1 BRCT H686H . Kinetic parameters (association ( K as ), dissociation ( K dis ), and dissociation ( K D ) constants) (bottom). The sensograms represent the mean of 3 measurements for each concentration. The data were analysed in Octet® Analysis Studio Software using 1:2 Bivalent analyte model. The data were plotted using Prism GraphPad 9 software.

Article Snippet: To generate a vector for co-expression of the full-length human BRCA1-BARD1 complex in insect cells, fragment of DNA, containing FLAG-tagged BRCA1 was first cloned into 2BcT plasmid (pET His6 LIC cloning vector; addgene #37236) to add C-terminal (His) 6 tag to the construct.

Techniques: Binding Assay, Concentration Assay, Software, Comparison

Journal: bioRxiv

Article Title: Distinct Mechanisms of Recognition of Phosphorylated RNAPII C- Terminal Domain by BRCT Repeats of the BRCA1–BARD1 Complex: Insights from Structural and Functional Analyses

doi: 10.1101/2025.01.22.634233

Figure Lengend Snippet: Structural characterisation of the BRCA1 BRCT domain bound to pS5 CTD peptide. A) Crystal structure of BRCA1 BRCT (gray) with bound pS5 CTD peptide (yellow). B) Detail of the BRCA1 BRCT phospho-peptide binding site (gray) with bound pS5 CTD peptide (yellow). C) Detail of the phosphoserine binding site of BRCA1 BRCT (gray) with bound pS5 CTD peptide (yellow). The pS5 of the CTD peptide is depicted in purple, the amino-acid residues interacting with pS5 are depicted in light violet. The hydrogen bonds were displayed as pseudo bonds using the structural analysis tool Hydrogen bonds in UCSF ChimeraX and are depicted in turquoise. The relax distance tolerance was 1.0 Å and the relax angle tolerance was 20.0°. D) Detail of the aromatic amino-acid binding pocket of BRCA1 BRCT (gray) with bound pS5 CTD peptide (yellow). The Y1 of the CTD is depicted in navy, the interacting amino-acid residues in light blue. The hydrogen bonds (left) were displayed as pseudobonds using the structural analysis tool Hydrogen bonds in UCSF ChimeraX and are depicted in turquoise. The relax distance tolerance was 1.0 Å and the relax angle tolerance was 20.0°. Van der Waals, hydrophobic and stacking interactions (right) were displayed as pseudobonds using the structural analysis tool in UCSF ChimeraX and are depicted in black. The interacting atoms were indentified based on VDW overlap ≥-0.4 Å. E) Validation of the obtained structural model by fluorescence anisotropy measurement. The assays were performed between BRCA1 BRCT domain and the indicated ligands (at 25nM). Anisotropy data were plotted as a function of protein concentration and fitted to a single-site saturation with non-specific binding model using XMGrace. F) Comparison of binding of different ligands to BRCA1 BRCT. Alignment of the structures was created using UCSF ChimeraX. BRCA1 BRCT (from the structure with pCTD) is depicted in gray, pCTD peptide in yellow, BACH1 phosphopeptide (PDB: 1T29) in red, CtIP phosphopeptide (PDB: 1Y98) in magenta, Abraxas singly phosphorylated peptide (PDB: 4Y2G) in turquoise, ATRIP phosphopeptide (PDB: 4IGH) in blue.

Article Snippet: To generate a vector for co-expression of the full-length human BRCA1-BARD1 complex in insect cells, fragment of DNA, containing FLAG-tagged BRCA1 was first cloned into 2BcT plasmid (pET His6 LIC cloning vector; addgene #37236) to add C-terminal (His) 6 tag to the construct.

Techniques: Binding Assay, Fluorescence, Protein Concentration, Comparison

Journal: bioRxiv

Article Title: Distinct Mechanisms of Recognition of Phosphorylated RNAPII C- Terminal Domain by BRCT Repeats of the BRCA1–BARD1 Complex: Insights from Structural and Functional Analyses

doi: 10.1101/2025.01.22.634233

Figure Lengend Snippet: The BRCA1-BARD1 complex forms liquid-like condensates in vitro , which accommodate phosphorylated CTD domain of RNAPII and RNA. A) Schematic representation of BRCA1 and BARD1 domains. Positions of investigated binding mutants are indicated. B) Liquid-liquid phase separation (LLPS) assays with purified, Alexa488-labelled BRCA1- BARD1. BRCA1-BARD1 (at 1.25 µM, 2.5 µM, and 5 µM) was mixed with the crowding agent (10% dextran). Bar chart (top) representing quantification (n = 3) of the number of droplets per frame from the LLPS experiments with BRCA1-BARD1. Statistical significance was determined by unpaired t-test. A nested scatterplot (middle) represents quantification (n = 3) of an area of individual droplets from three independent experiments with BRCA1-BARD1, with median area determined per dataset. Statistical significance was determined by nested t- test. Representative images from three experiments (bottom) are depicted as an overlay of differential interference contrast (DIC) and GFP. Where indicated, hexane-1,6-diol (hex; at 10%) was added to inhibit hydrophobic interactions or ATP (at 5mM) to inhibit electrostatic interactions. Scale bars, 10 µm. C) LLPS assays with purified, Alexa488-labelled BRCA1-BARD1, pS5pS7 mCherry-hCTD and Cy5-RNA. BRCA1-BARD1 (at 5 µM) was mixed with phosphorylated CTD (2.5 µM) or Cy5-ITS1 RNA (at 15 nM) in the presence of a crowding agent (10% dextran). Representative images from three experiments are depicted as an overlay of differential interference contrast (DIC), Alexa488, and Cy5. Scale bars, 10 µm. D) Bar chart (top) representing quantification (n = 3) of the number of droplets per frame from the LLPS experiments with the BRCA1-BARD1 complex shown in (C). Statistical significance was determined by unpaired t-test. A nested scatterplot (bottom) represents quantification (n = 3) of an area of individual droplets from three independent experiments with the BRCA1- BARD1 complex shown in (C), with median area determined per dataset. Statistical significance was determined by nested t-test. E) LLPS assays with purified, BRCA1-BARD1, pS5pS7 GFP-(CTD) 26 and Cy5-RNA. BRCA1-BARD1 (at 5 µM) was mixed with phosphorylated CTD (2.5 µM) and Cy5-ITS1 RNA (at 15 nM) in the presence of a crowding agent (10% dextran). Representative images from three experiments are depicted as an overlay of differential interference contrast (DIC), GFP, and Cy5. Scale bars, 10 µm.

Article Snippet: To generate a vector for co-expression of the full-length human BRCA1-BARD1 complex in insect cells, fragment of DNA, containing FLAG-tagged BRCA1 was first cloned into 2BcT plasmid (pET His6 LIC cloning vector; addgene #37236) to add C-terminal (His) 6 tag to the construct.

Techniques: In Vitro, Binding Assay, Purification

Journal: bioRxiv

Article Title: Distinct Mechanisms of Recognition of Phosphorylated RNAPII C- Terminal Domain by BRCT Repeats of the BRCA1–BARD1 Complex: Insights from Structural and Functional Analyses

doi: 10.1101/2025.01.22.634233

Figure Lengend Snippet: The BRCT repeats of the BRCA1-BARD1 form liquid-like condensates in vitro , which accommodate phosphorylated CTD domain of RNAPII and RNA. A) LLPS assays with pS5pS7 mCherry-hCTD and Alexa488-labelled BRCA1 BRCT (top) and BARD1 BRCT (bottom). The BRCT (at 160 µM) in absence or presence of phosphorylated CTD (2.5 µM) was mixed with the crowding agent (10% dextran). Representative images from three experiments are depicted as an overlay of differential interference contrast (DIC) with the signal for Alexa488 or mCherry. Scale bars 10µm. B) Bar chart (top) representing quantification (n = 3) of the number of droplets per frame from the LLPS experiments with BRCA1 and BARD1 BRCT shown in (A). Statistical significance was determined by unpaired t test. A nested scatterplot (middle) represents quantification (n = 3) of an area of individual droplets from three independent experiments with BRCA1-BARD1 shown in (A), with median area determined per dataset. Statistical significance was determined by nested t test. Bar chart (bottom) depicts the proportion of droplets containing signals for Alexa488-labelled BRCT (green) and pS5pS7 mCherry-hCTD (red). C) LLPS assays with purified BRCA1 BRCT and pS5pS7 mGFP-hCTD. BRCA1 BRCT (at 160 µM, w. t. and S1655F,K1702M, respectively) was mixed with phosphorylated CTD (2.5 µM) in the absence or presence of a crowding agent (10% dextran). Representative images (top) from three experiments are depicted as an overlay of differential interference contrast (DIC) and GFP. Hexane-1,6-diol (hex; at 10%) was added to inhibit hydrophobic interactions. Scale bars, 10 µm. Bar chart (middle) representing quantification (n = 3) of the number of droplets per frame from the LLPS experiments. Statistical significance was determined by unpaired t-test. A nested scatterplot (bottom) represents quantification (n = 3) of an area of individual droplets from three independent experiments, with median area determined per dataset. Statistical significance was determined by nested t-test. D) LLPS assays with purified BARD1 BRCT and pS5pS7 mGFP-hCTD. BARD1 BRCT (at 160 µM, w. t. and S575F,K619A) was mixed with phosphorylated CTD (2.5 µM) in the absence or presence of a crowding agent (10% dextran). Representative images (top) from three experiments are depicted as an overlay of differential interference contrast (DIC) and GFP. Hexane-1,6-diol (hex; at 10%) was added to inhibit hydrophobic interactions. Scale bars, 10 µm. Bar chart (middle) representing quantification (n = 3) of the number of droplets per frame from the LLPS experiments. Statistical significance was determined by unpaired t-test. A nested scatterplot (bottom) represents quantification (n = 3) of an area of individual droplets from three independent experiments, with median area determined per dataset. Statistical significance was determined by nested t-test.

Article Snippet: To generate a vector for co-expression of the full-length human BRCA1-BARD1 complex in insect cells, fragment of DNA, containing FLAG-tagged BRCA1 was first cloned into 2BcT plasmid (pET His6 LIC cloning vector; addgene #37236) to add C-terminal (His) 6 tag to the construct.

Techniques: In Vitro, Purification

Journal: bioRxiv

Article Title: Distinct Mechanisms of Recognition of Phosphorylated RNAPII C- Terminal Domain by BRCT Repeats of the BRCA1–BARD1 Complex: Insights from Structural and Functional Analyses

doi: 10.1101/2025.01.22.634233

Figure Lengend Snippet: Characterisation of disease-associated variants within the BRCT repeats of the BRCA1-BARD1 complex on their ability to promote condensation in vitro . B) Positions of the investigated mutations of BRCT domains. The crystal structure of BRCA1 BRCT with the pS5 CTD ligand (left) and the AlphaFold 3-generated model of the BARD1 BRCT with the pS5 CTD ligand (right) were used. Investigated mutations are not located near the phospho-peptide binding sites and therefore should not interfere with the binding. C) LLPS assays with purified BRCA1 BRCT and pS5pS7 mGFP-hCTD. BRCA1 BRCT (at20 µM, 40 µM, 80 µM, and 160 µM), w. t., E1682K, and E1754K, was mixed with phosphorylated CTD (2.5 µM) in the presence of a crowding agent (10% dextran). Representative images from three experiments are depicted as an overlay of differential interference contrast (DIC) and GFP. Scale bars, 10 µm. D) Bar chart (top) representing quantification (n = 3) of the number of droplets per frame from the LLPS experiments with BRCA1 BRCT (w.t. and mutated variants, at 160 µM) and pS5pS7 mGFP-hCTD, shown in (B), using the green fluorescent signal. Statistical significance was determined by unpaired t test. A nested scatterplot (bottom) representing quantification (n = 3) of an area of individual droplets from three independent experiments with BRCA1 BRCT, pS5pS7 mGFP-hCTD, shown in (B), with median area determined per dataset. Statistical significance was determined by nested t test. E) LLPS assays with purified BARD1 BRCT and pS5pS7 mGFP-hCTD. BARD1 BRCT (at 40 µM, 80 µM, 160 µM), w.t., E587K, E665K, S711R, and K754N, respectively, was mixed with phosphorylated CTD (2.5 µM) in the presence of a crowding agent (10% dextran). Representative images from three experiments are depicted as an overlay of differential interference contrast (DIC) and GFP. Scale bars, 10 µm. F) Bar chart (top) representing quantification (n = 3) of the number of droplets per frame from the LLPS experiments with BARD1 BRCT (w.t. and mutated variants, at 160 µM), and pS5pS7 mGFP-hCTD, shown in (D), using the green fluorescent signal. The analysis and visualisation were performed as in (C).

Article Snippet: To generate a vector for co-expression of the full-length human BRCA1-BARD1 complex in insect cells, fragment of DNA, containing FLAG-tagged BRCA1 was first cloned into 2BcT plasmid (pET His6 LIC cloning vector; addgene #37236) to add C-terminal (His) 6 tag to the construct.

Techniques: In Vitro, Generated, Binding Assay, Purification