Journal: Nucleic Acids Research

Article Title: Single-molecule localization microscopy reveals molecular transactions during RAD51 filament assembly at cellular DNA damage sites

doi: 10.1093/nar/gkx1303

Figure Lengend Snippet: Topography of RAD51 and RPA at radiation-induced foci in EUFA423 and reconstituted EUFA423 + BRCA2 cells. Distribution of the RAD51 filament length measured by DT graph extension in ( A ) EUFA423 and ( D ) EUFA423 + BRCA2. Distribution of the number of RAD51 molecules inside RAD51 filament in EUFA423 ( B ) and EUFA423+BRCA2 ( E ). Fraction of RAD51 clusters co-localized with RPA in ( C ) EUFA423 and ( F ) EUFA423+BRCA2. Distribution of inter-point distance (P2P) from RAD51 filament to RPA cluster located at centroid-to-centroid (C2C) distances shorter than 200 nm in ( G ) EUFA423 and ( I ) EUFA423 + BRCA2 cells. Distribution of the number of RPA molecules inside the cluster in ( H ) EUFA423 and ( J ) EUFA423 + BRCA2 cells. Typical RAD51 filaments (violet) and RPA clusters (green) in EUFA423 + BRCA2 and EUFA423 at ( K, L ) 3 h and ( M, N ) 5 h after exposure to 5 Gy IR. Distributions were computed over data aggregated from five independent biological replicates. Abscissae indicate time after exposure to 5 Gy IR. The number of RAD51-RPA pairs that were analysed (EUFA423 + BRCA2/EUFA423) is 1352/1384, 382/559, 1119/1157 and 1158/920 at 30 min, 1 h 3h and 5 h, respectively. Exact P -values for multiple means max-t comparison test are given in . Stars indicate two sample two-sided Wilcoxon rank sum test between EUFA423 and EUFA423 + BRCA2 at a single time, *** P < 0.001, n.s. not significant. Scale bar—250 nm.

Article Snippet: BRCA2 knock-down was performed using short interfering (si)RNA reverse transfection according to manufacturer protocol simultaneously with plating cells in ibidi μ-slides with DharmaFECT1 (Dharmacon, UK) transfection reagent.

Techniques: Comparison

Journal: Nucleic Acids Research

Article Title: Single-molecule localization microscopy reveals molecular transactions during RAD51 filament assembly at cellular DNA damage sites

doi: 10.1093/nar/gkx1303

Figure Lengend Snippet: Topography of RAD51 and RPA at radiation-induced foci in Hela Kyoto cells after siRNA-induced depletion of BRCA2. ( A ) RAD51 (purple) and RPA (green) foci represented as DT. ( B ) Distribution of lengths of RAD51 assemblies measured by DT graph extension. ( C ) Distribution of the number of RAD51 molecules inside RAD51 filament. ( D ) Fraction of RAD51 filaments co-localized with RPA. Bivariate Ripley's function for RAD51 and RPA in ( E, H ) siRNA BRCA2 and ( F, I ) mock transfected control cells for 30 min (top) and 5 h (bottom) after exposure to 5 Gy IR. The purple line marks the experimental Ripley's function while the shaded blue areas and the blue lines show the 95% confidence intervals (CI) and mean obtained by repetitive randomization of cluster centroid positions. ( G ) Distribution of inter-point distance (P2P) for every RAD51 assembly to RPA cluster located at centroid-to-centroid (C2C) distance shorter than 200 nm. ( J ) Distribution of the number of molecules inside RPA cluster. Distributions were computed over data aggregated from three independent biological replicates. Abscissae indicate time after exposure to 5 Gy IR. The number of RAD51-RPA pairs that were analysed is 2099, 1283, 1721 and 2153 at 30 min, 1.5 h, and 3–h, respectively. Exact P -values for multiple means max-t comparison test are given in . Stars indicate two sample two-sided Wilcoxon rank sum test between siBRCA2 and matched control cells at a single time, *** P < 0.001, n.s. not significant. Scale bar—250 nm.

Article Snippet: BRCA2 knock-down was performed using short interfering (si)RNA reverse transfection according to manufacturer protocol simultaneously with plating cells in ibidi μ-slides with DharmaFECT1 (Dharmacon, UK) transfection reagent.

Techniques: Transfection, Control, Comparison

Journal: Nucleic Acids Research

Article Title: Single-molecule localization microscopy reveals molecular transactions during RAD51 filament assembly at cellular DNA damage sites

doi: 10.1093/nar/gkx1303

Figure Lengend Snippet: Schematic model of steps in DNA end-resection and RAD51 filament assembly during HR initiation at cellular DSBs. (0–30 min) Initiation of end-resection, RPA assembles on ssDNA. RAD51 assemblies abut RPA/ssDNA clusters, and requires the BRC repeats of BRCA2. (30–150 min) Continuing end-resection enlarges ssDNA and RPA clusters abutting RAD51. Elongation of RAD51 clusters into filaments is dependent on the C-terminal domain of BRCA2 (150–300 min). RPA cluster size levels off. RAD51 filaments assume a more compact and coiled structure, which could mark the homology search, leading eventually to strand exchange.

Article Snippet: BRCA2 knock-down was performed using short interfering (si)RNA reverse transfection according to manufacturer protocol simultaneously with plating cells in ibidi μ-slides with DharmaFECT1 (Dharmacon, UK) transfection reagent.

Techniques:

Journal: Nucleic Acids Research

Article Title: Genome-wide CRISPR screens reveal cyclin C as synthetic survival target of BRCA2

doi: 10.1093/nar/gkab540

Figure Lengend Snippet: Genome-wide CRISPR/Cas9 screens identify genetic vulnerabilities in BRCA2-deficient cells. ( A ) Strategy for CRISPR-based mAID-EGFP tagging at the C-terminal BRCA2 genomic loci in HEK293A OsTIR1 pInducer20 cells. TAA: stop coden; BSD: blasticidin; HR: homologous recombination. ( B ) Genomic PCR was performed to validate the homozygous clone with correct insertion of mAID-EGFP tag. MW, molecular weight; WT: wild-type; KI: knock-in. ( C ) Western blot showing that after cells were treated with Dox and IAA (indole-3-aceticacid), BRCA2 was degraded within 24 h. ( D ) Colony survival assay was performed to determine cell viability upon BRCA2 depletion in HEK293A OsTIR1 pInducer20 BRCA2 mAID-EGFP KI cells. ( E ) Schematic of the workflow for the CRISPR screen performed in HEK293A OsTIR1 pInducer20 BRCA2 mAID EGFP KI cells with TKOv3 whole-genome gRNA library. MOI, multiplicity of infection; DMSO, dimethyl sulfoxide; NGS, next-generation sequencing. ( F ) Ranking of BRCA2 –/– co-essential genes based on drugZ analysis of the results of CRISPR/Cas9 screen. The z -score was used to define possible vulnerability of cell lethality with BRCA2 ablation. The higher the score the more viable upon co-depletion of BRCA2. Likewise, the lower the score the greater vulnerability with BRCA2 co-depletion. All genes targeted in the TKOv3 library were scored according to the fold change in the abundance of their sgRNAs (IAA-based versus DMSO-based treatment). ( G ) The top 10 significantly enriched Gene Ontology (GO) Biological Process terms ( P < 0.001) with the high-confidence candidate genes whose loss of function led to survival of BRCA2 –/– cells.

Article Snippet: Then control and CCNC knocking-down cells were transfected with 50 pmol control siRNA or BRCA2 siRNA (SR300471, OriGene Technologies) with Lipofectamine RNAiMAX (ThermoFisher Scientific).

Techniques: Genome Wide, CRISPR, Homologous Recombination, Molecular Weight, Knock-In, Western Blot, Clonogenic Cell Survival Assay, Infection, Next-Generation Sequencing

Journal: Nucleic Acids Research

Article Title: Genome-wide CRISPR screens reveal cyclin C as synthetic survival target of BRCA2

doi: 10.1093/nar/gkab540

Figure Lengend Snippet: The RNA Pol II transcription Mediator complex is important for survival and PARPi sensitivity in BRCA2-depleted cells. ( A ) Western blots performed to determine the efficacy of sgRNAs targeting CCNC, CDK8, MED24 and MED25. HEK293A BRCA2 mAID-EGFP cells were infected with lentiviruses expressing the indicated sgRNAs and selected with puromycin. After treatment with Dox and IAA for 24 hrs, cells were collected and lysates were blotted with the indicated antibodies. ( B ) Results of long-term clonogenic assays performed using cells transduced with the indicated sgRNA constructs and given the indicated treatments. 5×10 3 cells were seeded in each well and incubated with the indicated treatments for 7 or 14 days. Colonies were fixed and stained with 0.5% crystal violet. Ola, olaparib. Hereafter BRCA2+/+ cells represent HEK293A BRCA2 mAID-EGFP cells without IAA and Dox treated cells; BRCA2-/- cells represent HEK293A BRCA2 mAID-EGFP cells with IAA and Dox treated cells. ( C ) Quantification of relative cell survival in the clonogenic assays in (B). All treated groups were normalized to the indicated untreated group. And the relative cell growth was compared with LentiV2 (V2) which was set as 1. The data are presented as means (±SD). ** P < 0.01; *** P < 0.001; ns, not significant (Student t-test). ( D ) Western blots performed to determine the efficacy of sgRNAs targeting MED12 and MED13. ( E ) Results of long-term clonogenic assays performed to determine the viability of the cells in D. 5 × 10 3 cells were seeded in each well and incubated with the indicated agents for 14 days. Colonies were fixed and stained with 0.5% crystal violet. ( F ) Quantification of relative cell survival in the clonogenic assays in (E). The data are presented as means ± SD. * P < 0.05; ** P < 0.01; *** P < 0.001 (Student's t -test).

Article Snippet: Then control and CCNC knocking-down cells were transfected with 50 pmol control siRNA or BRCA2 siRNA (SR300471, OriGene Technologies) with Lipofectamine RNAiMAX (ThermoFisher Scientific).

Techniques: Western Blot, Infection, Expressing, Transduction, Construct, Incubation, Staining

Journal: Nucleic Acids Research

Article Title: Genome-wide CRISPR screens reveal cyclin C as synthetic survival target of BRCA2

doi: 10.1093/nar/gkab540

Figure Lengend Snippet: Loss of CCNC is important for survival and PARPi sensitivity in BRCA2-deficient Cells. ( A ) Validation of CCNC KO in HEK293A BRCA2 mAID-EGFP KI cells by western blot using the indicated antibodies. ( B ) Results of long-term clonogenic assays conducted using HEK293A BRCA2 mAID-EGFP CCNC-WT and -KO cells with the indicated treatments. Ola: Olaparib. ( C ) Quantification of relative cell survival in the clonogenic assays in (B). All treated groups were normalized to the indicated untreated group. The data are presented as means ± SD. *** P < 0.001 (Student's t -test). ( D ) Results of long-term clonogenic assays conducted using HEK293A BRCA2 mAID-EGFP CCNC-WT and -KO cells with the indicated treatments. Tal: Talazoparib. ( E ) Quantification of relative cell survival in the clonogenic assays in (D). All treated groups were normalized to the indicated untreated group. The data are presented as means ± SD. *** P < 0.001 (Student's t -test). ( F ) Capan-1 cells were infected with control pLentiCRISPRv2 (Lenti V2), or CCNC gRNA-1 (g1) virus and selected with puromycin. Western blot was conducted to validate CCNC knock down efficiency with the indicated antibodies. ( G ) Results of long-term clonogenic assays conducted using Capan-1 LentiV2 and CCNC g1 cells with different concentration of Olaparib treatments. Ola: Olaparib. ( H ) Quantification of relative cell survival in clonogenic assays in (G). All treated groups were normalized to the indicated untreated group. The data are presented as means ± SD. *** P < 0.001 (Student's t -test). ( I ) Results of long-term clonogenic assays conducted using Capan-1 LentiV2 and CCNC g1 cells with different concentration of Talazoparib treatments. Tal: Talazoparib. ( J ) Quantification of relative cell survival in the clonogenic assays in (I). All treated groups were normalized to the indicated untreated group. The data are presented as means ± SD. ** P < 0.01, *** P < 0.001 (Student's t -test).

Article Snippet: Then control and CCNC knocking-down cells were transfected with 50 pmol control siRNA or BRCA2 siRNA (SR300471, OriGene Technologies) with Lipofectamine RNAiMAX (ThermoFisher Scientific).

Techniques: Western Blot, Infection, Concentration Assay

Journal: Nucleic Acids Research

Article Title: Genome-wide CRISPR screens reveal cyclin C as synthetic survival target of BRCA2

doi: 10.1093/nar/gkab540

Figure Lengend Snippet: Determination of the viability of CCNC-KO cells treated with different DNA-damaging agents. ( A–G ) Cell viability assays performed in HEK293A BRCA2 mAID-EGFP KI CCNC-WT and -KO cells. Cells were treated with the indicated doses of ATRi (ATR inhibitor AZD6738), CPT(Camptothecin), MMC (Mitomycin C), Ola (Olaparib), Irradiation (IR), HU (Hydroxyurea), ETO (Etoposide). The data are presented the mean ± SD. n = 2 independent experiments. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: Then control and CCNC knocking-down cells were transfected with 50 pmol control siRNA or BRCA2 siRNA (SR300471, OriGene Technologies) with Lipofectamine RNAiMAX (ThermoFisher Scientific).

Techniques: Irradiation

Journal: Nucleic Acids Research

Article Title: Genome-wide CRISPR screens reveal cyclin C as synthetic survival target of BRCA2

doi: 10.1093/nar/gkab540

Figure Lengend Snippet: Depletion of CCNC decrease DNA damage signal in BRCA2 deficient cells. ( A ) CCNC KO and WT HEK293A BRCA2 mAID cells with or without depletion of BRCA2 were untreated (NT) or treated with Olaparib (2 μM) for 24 h and collected for immunostaining with γH2AX antibody. ( B ) Statistical quantification of γH2AX foci formation from (A). Mean number of γH2AX foci were analyzed in >100 cells. ** P < 0.01. ( C ) Immunostaining of γH2AX in Capan-1 LentiV2 and CCNC g1 cells with or without Olaparib (2 μM) treatment for 24 h. ( D ) Statistical quantification of γH2AX foci formation from (C). Percentage of cells with > 10 γH2AX foci were counted. Data are represented as mean ± SD ( n = 3), ** P < 0.01, ns, not significant (Student's t -test).

Article Snippet: Then control and CCNC knocking-down cells were transfected with 50 pmol control siRNA or BRCA2 siRNA (SR300471, OriGene Technologies) with Lipofectamine RNAiMAX (ThermoFisher Scientific).

Techniques: Immunostaining

Journal: Nucleic Acids Research

Article Title: Genome-wide CRISPR screens reveal cyclin C as synthetic survival target of BRCA2

doi: 10.1093/nar/gkab540

Figure Lengend Snippet: Depletion of CCNC does not restore HR repair. ( A ) Depletion of CCNC does not affect RAD51 foci formation upon DNA damage. HEK293A BRCA2 mAID CCNC-KO and WT cells with BRCA2 depletion (+IAA) or without BRCA2 depletion were treated with Olaparib (2 μM) for 24 h and collected for immunostaining with RAD51 antibody. ( B ) Statistical quantification of percentage of cells with >10 RAD51 foci from (A). ( C ) Validation of CCNC and BRCA2 knock down efficiency in U2OS DR-GFP cells by western blot with indicated antibodies. ( D ) Statistical quantification of HR repair efficiency in cells from (C). Data are represented as mean ± SD ( n = 3). ns, not significant (Student t-test).

Article Snippet: Then control and CCNC knocking-down cells were transfected with 50 pmol control siRNA or BRCA2 siRNA (SR300471, OriGene Technologies) with Lipofectamine RNAiMAX (ThermoFisher Scientific).

Techniques: Immunostaining, Western Blot

Journal: Nucleic Acids Research

Article Title: Genome-wide CRISPR screens reveal cyclin C as synthetic survival target of BRCA2

doi: 10.1093/nar/gkab540

Figure Lengend Snippet: Depletion of CCNC restore replication fork stability in BRCA2 deficient cells upon replication stress. ( A ) Schematic of ldU/CIdU pulse-labeling followed by a 4 hrs hydroxyurea (HU; 2 mM) treatment (top). Representative images of ldU and CIdU replication tracks in HU-treated HEK293A BRCA2 mAID CCNC-KO and WT cells with (+IAA) or without BRCA2 depletion are shown (bottom). ( B ) Dot plots of CIdU to ldU tract length ratios for individual replication forks in cells from (A). The median value of 100 or more IdU and CldU tracts per experimental condition is indicated. Statistical analysis was conducted using Mann-Whitney test (n.s. not significant, *** P < 0.001). ( C ) Schematic of ldU/CIdU pulse-labeling followed by a 4 h hydroxyurea (HU; 2 mM) treatment (top). Representative images of ldU and CIdU replication tracks in DMSO or HU-treated Capan-1 LentiV2 and CCNC g1 cells are shown (bottom). ( D ) Dot plots of CIdU to ldU tract length ratios for individual replication forks in cells from (C). The median value of 100 or more IdU and CldU tracts per experimental condition is indicated. Statistical analysis was conducted using Mann–Whitney test (n.s. not significant, *** P < 0.001).

Article Snippet: Then control and CCNC knocking-down cells were transfected with 50 pmol control siRNA or BRCA2 siRNA (SR300471, OriGene Technologies) with Lipofectamine RNAiMAX (ThermoFisher Scientific).

Techniques: Labeling, MANN-WHITNEY

Journal: Molecular Cancer

Article Title: Cell cycle-dependent regulation of the bi-directional overlapping promoter of human BRCA2/ZAR2 genes in breast cancer cells

doi: 10.1186/1476-4598-9-50

Figure Lengend Snippet: Sequences of the oligonucleotides used in this study.

Article Snippet: We cloned the 497 bp promoter DNA sequence (-187 to +310) of human BRCA2 gene in front of Renilla luciferase ( Rluc ) gene in pRL-Null plasmid (Promega) to obtain clones with the insert either in the forward or in the reverse orientation with respect to the luciferase gene (Fig. ).

Techniques: Sequencing, Amplification, Control

Journal: Molecular Cancer

Article Title: Cell cycle-dependent regulation of the bi-directional overlapping promoter of human BRCA2/ZAR2 genes in breast cancer cells

doi: 10.1186/1476-4598-9-50

Figure Lengend Snippet: Cell cycle dependent bi-directional activities of human BRCA2 gene promoter . (A) Genomic context of human BRCA2 gene bi-directional (BD) promoter studied. The numbers shown are with respect to the transcription start site of human BRCA2 gene. (B) Maps of the reporter constructs in pRL-Null vector used in the study. (i) The single reporter constructs: pRL-FP (forward construct) and pRL-RP (reverse construct); (ii) the dual reporter construct. URS: upstream regulatory sequence; Ex-1: exon 1; Int-1: intron 1; Rluc: Renilla luciferase; Fluc: firefly luciferase; ORF: open reading frame. (C) Activities of the BRCA2 (forward) and the ZAR2 (reverse) promoters in different lines of human breast cancer cells at G0/G1 and S/G2 phases of their cell cycles. Results are mean ± SE (n = 6). The differences between the G0/G1 and S/G2 phase cells were statistically significant (shown by '*'; p < 0.001).

Article Snippet: We cloned the 497 bp promoter DNA sequence (-187 to +310) of human BRCA2 gene in front of Renilla luciferase ( Rluc ) gene in pRL-Null plasmid (Promega) to obtain clones with the insert either in the forward or in the reverse orientation with respect to the luciferase gene (Fig. ).

Techniques: Construct, Plasmid Preparation, Sequencing, Luciferase

Journal: Molecular Cancer

Article Title: Cell cycle-dependent regulation of the bi-directional overlapping promoter of human BRCA2/ZAR2 genes in breast cancer cells

doi: 10.1186/1476-4598-9-50

Figure Lengend Snippet: Relative activities of the forward and the reverse promoters of BRCA2 gene in different unsynchronized human breast cancer cells at 95% confluency.

Article Snippet: We cloned the 497 bp promoter DNA sequence (-187 to +310) of human BRCA2 gene in front of Renilla luciferase ( Rluc ) gene in pRL-Null plasmid (Promega) to obtain clones with the insert either in the forward or in the reverse orientation with respect to the luciferase gene (Fig. ).

Techniques: Luciferase, Activity Assay

Journal: Molecular Cancer

Article Title: Cell cycle-dependent regulation of the bi-directional overlapping promoter of human BRCA2/ZAR2 genes in breast cancer cells

doi: 10.1186/1476-4598-9-50

Figure Lengend Snippet: Ratio of the forward (BRCA2) and the reverse (ZAR2) activity in the G0/G1 and S/G2 growth phases of different breast cancer cells using transient transfection with the dual reporter/promoter construct (see Fig. 1B).

Article Snippet: We cloned the 497 bp promoter DNA sequence (-187 to +310) of human BRCA2 gene in front of Renilla luciferase ( Rluc ) gene in pRL-Null plasmid (Promega) to obtain clones with the insert either in the forward or in the reverse orientation with respect to the luciferase gene (Fig. ).

Techniques: Activity Assay, Transfection, Construct

Journal: Molecular Cancer

Article Title: Cell cycle-dependent regulation of the bi-directional overlapping promoter of human BRCA2/ZAR2 genes in breast cancer cells

doi: 10.1186/1476-4598-9-50

Figure Lengend Snippet: The transcription start sites of the reverse transcript from BRCA2 gene bi-directional promoter . (A) GeneRacer amplification product for ZAR2. See Materials and methods for details. (B) Nucleotide sequence of human ZAR2/BRCA2 bi-directional promoter. The transcriptional start sites (TSSs), as determined by GeneRacer technique, are shown. The segment in green color is the sequence complementary to part of the intron 1 sequence of human BRCA2 gene, the segment in red color is from exon 1 and the blue part is from the upstream sequence of BRCA2 gene. The E-box sequence essential for BRCA2 gene expression [ , ] is underlined. The splice donor site at the ZAR2 gene exon 1/intron 1 junction is indicated by a downward arrow. The 'G' residue at the SNP site at -26 from BRCA2 gene transcription start site is shown by a red *. (C) Cartoon showing the human BRCA2 (upper panel) and ZAR2 (lower panel) gene promoter studied. The identities of ZAR2 exon1 (Ex-1) and intron 1 (Int-1) were experimentally determined in this study. TSS: transcription start site (designated as +1); URS: upstream regulatory sequence.

Article Snippet: We cloned the 497 bp promoter DNA sequence (-187 to +310) of human BRCA2 gene in front of Renilla luciferase ( Rluc ) gene in pRL-Null plasmid (Promega) to obtain clones with the insert either in the forward or in the reverse orientation with respect to the luciferase gene (Fig. ).

Techniques: Amplification, Sequencing, Gene Expression, Residue

Journal: Molecular Cancer

Article Title: Cell cycle-dependent regulation of the bi-directional overlapping promoter of human BRCA2/ZAR2 genes in breast cancer cells

doi: 10.1186/1476-4598-9-50

Figure Lengend Snippet: Conservation of the BRCA2/ZAR2 genetic arrangements in vertebrates . (A) Relative chromosomal locations of BRCA2 and ZAR2 genes in different vertebrates. The maps were obtained from NCBI site for Entrez genes http://www.ncbi.nlm.nih.gov/sites/entrez?db=gene&term=BRCA2+ Not drawn to the scale. (B) Dendrogram with branch lengths for the ZAR2 proteins from different vertebrates. The putative ZAR2 protein amino acid sequences were mined from the NCBI Entrez database and dendrogram with branch length was analyzed by CLUSTALW program http://align.genome.jp/ .

Article Snippet: We cloned the 497 bp promoter DNA sequence (-187 to +310) of human BRCA2 gene in front of Renilla luciferase ( Rluc ) gene in pRL-Null plasmid (Promega) to obtain clones with the insert either in the forward or in the reverse orientation with respect to the luciferase gene (Fig. ).

Techniques:

Journal: Molecular Cancer

Article Title: Cell cycle-dependent regulation of the bi-directional overlapping promoter of human BRCA2/ZAR2 genes in breast cancer cells

doi: 10.1186/1476-4598-9-50

Figure Lengend Snippet: The exon-intron structure and mRNA sequence of human ZAR2 gene . (A) Cartoon showing the exon-intron structure of human ZAR2 gene. The first exon of ZAR2 overlaps with the exon 1 of the BRCA2 gene (not drawn to the scale). (B) Nucleotide sequence of human ZAR2 mature mRNA. The 5'-UTR sequence was experimentally determined (see text for details). The putative protein coding sequence (ORF) is shown in blue and highlighted in gray. The upstream AUG (uAUG) codons at the 5'-UTR are highlighted: out-of-frame uAUGs are in yellow shades; in-frame uAUGs are in green shades. The 5'-UTR sequence overlapping with BRCA2 mRNA sequences are shaded yellow. Rest of the 5'-UTR sequence of ZAR2 mRNA is derived from the newly identified exon 1 and is shown in red.

Article Snippet: We cloned the 497 bp promoter DNA sequence (-187 to +310) of human BRCA2 gene in front of Renilla luciferase ( Rluc ) gene in pRL-Null plasmid (Promega) to obtain clones with the insert either in the forward or in the reverse orientation with respect to the luciferase gene (Fig. ).

Techniques: Sequencing, Derivative Assay

Journal: Molecular Cancer

Article Title: Cell cycle-dependent regulation of the bi-directional overlapping promoter of human BRCA2/ZAR2 genes in breast cancer cells

doi: 10.1186/1476-4598-9-50

Figure Lengend Snippet: Relative expressions of BRCA2 and ZAR2 mRNAs at different cell cycle stages of human breast cells . (A) RT-PCR analysis showing the expressions of BRCA2 and ZAR2 mRNAs in the unsynchronized (mostly dividing) cells. β-Actin mRNA was used as a loading control. (B) Real-time RT-PCR evaluation of the relative levels of BRCA2 and ZAR2 mRNAs in different human breast cancer cells at G0/G1 and S/G2 phases. The differences between the G0/G1 and S/G2 phase cells were statistically significant (shown by '*'; p < 0.001). (C) Immunofluorescence confocal microscopy showing growth phase-dependent localization of N-terminal FLAG-tagged ZAR2 protein in the synchronized MCF7 cells. Anti-FLAG M2 antibody was used for the detection of FLAG-tagged ZAR2 protein in the cells. The cells were transiently transfected with the expression plasmid constructs and thus not all cells are expressing the recombinant protein.

Article Snippet: We cloned the 497 bp promoter DNA sequence (-187 to +310) of human BRCA2 gene in front of Renilla luciferase ( Rluc ) gene in pRL-Null plasmid (Promega) to obtain clones with the insert either in the forward or in the reverse orientation with respect to the luciferase gene (Fig. ).

Techniques: Reverse Transcription Polymerase Chain Reaction, Control, Quantitative RT-PCR, Immunofluorescence, Confocal Microscopy, Transfection, Expressing, Plasmid Preparation, Construct, Recombinant

Journal: Molecular Cancer

Article Title: Cell cycle-dependent regulation of the bi-directional overlapping promoter of human BRCA2/ZAR2 genes in breast cancer cells

doi: 10.1186/1476-4598-9-50

Figure Lengend Snippet: In vivo binding of ZAR2 protein to the BRCA2/ZAR2 gene promoter . (A) PCR amplification of the immunoprecipitated chromatin DNA fragment pulled down with FLAG antibody from synchronized MCF7 cells over-expressing C-terminal FLAG-tagged ZAR2 protein at the G0/G1 and S/G2 phases. Input DNA (5% was used as control. Chromatin DNA fragments mock precipitated with mouse IgG did not significantly amplified any detectable DNA. BRCA2 gene promoter specific primers were used for PCR amplifications. (B) Quantitative ChIP analysis of ZAR2 recruitment to BRCA2/ZAR2 bi-directional promoter in MCF7 cells at G0/G1 and S/G2 phases. qChIP-PCR analyses were performed with chromatin extracts harvested from cells over expressing C-terminal FLAG-tagged ZAR2. The mean values from triplicate data points are plotted and error bars indicate ± SE. The amplification values are normalized by subtraction with IgG control antibody and then division with 1% input DNA. Data shown were representative of three independent experiments (mean + SE) and the difference between the G0/G1 phase and the S/G2 phase cells was statistically significant (shown by '*'; p < 0.001).

Article Snippet: We cloned the 497 bp promoter DNA sequence (-187 to +310) of human BRCA2 gene in front of Renilla luciferase ( Rluc ) gene in pRL-Null plasmid (Promega) to obtain clones with the insert either in the forward or in the reverse orientation with respect to the luciferase gene (Fig. ).

Techniques: In Vivo, Binding Assay, Amplification, Immunoprecipitation, Expressing, Control

Journal: Molecular Cancer

Article Title: Cell cycle-dependent regulation of the bi-directional overlapping promoter of human BRCA2/ZAR2 genes in breast cancer cells

doi: 10.1186/1476-4598-9-50

Figure Lengend Snippet: Effects of over-expression (OVEX) of the C-terminal FLAG-tagged ZAR2 in synchronized MCF7 cells on the BRCA2 and ZAR2 mRNA levels (A) and on the activities of BRCA2 and ZAR2 gene promoters (B) at the S/G2 phase . MCF7 cells were stably transfected with C-terminally FLAG-tagged ZAR2 and evaluated for their ZAR2 over expression. Levels of the mRNAs were determined by real-time RT-PCR . Promoter activities were measured in MCF7 cells transiently transfected with the single-reporter constructs (Fig. 1B) following the dual luciferase assay protocols (Promega). pGL3-Control was used as normalization control as described in the 'Methods' section. Results are mean ± SE (n = 6). '*' indicates the difference between the corresponding control and the experimental sets is statistically significant (p < 0.001).

Article Snippet: We cloned the 497 bp promoter DNA sequence (-187 to +310) of human BRCA2 gene in front of Renilla luciferase ( Rluc ) gene in pRL-Null plasmid (Promega) to obtain clones with the insert either in the forward or in the reverse orientation with respect to the luciferase gene (Fig. ).

Techniques: Over Expression, Stable Transfection, Transfection, Quantitative RT-PCR, Construct, Luciferase, Control

Journal: Molecular Cancer

Article Title: Cell cycle-dependent regulation of the bi-directional overlapping promoter of human BRCA2/ZAR2 genes in breast cancer cells

doi: 10.1186/1476-4598-9-50

Figure Lengend Snippet: Effect of knockdown of ZAR2 in synchronized MCF7 cells on the BRCA2 and ZAR2 mRNA levels (A) and on the activities of BRCA2 and ZAR2 gene promoters (B) at the G0/G1 phase . ZAR2 was knocked down in MCF7 cells with two different double-stranded stealth siRNAs (Invitrogen). Levels of the mRNAs were determined by real-time RT-PCR. Promoter activities were measured in MCF7 cells transiently transfected with the single-reporter constructs (Fig. 1B) following the dual luciferase assay protocols (Promega). pGL3-Control was used as normalization control as described in the 'Methods' section. Results are mean ± SE (n = 6). '*' indicates the difference between the corresponding control and the experimental sets is statistically significant (p < 0.001).

Article Snippet: We cloned the 497 bp promoter DNA sequence (-187 to +310) of human BRCA2 gene in front of Renilla luciferase ( Rluc ) gene in pRL-Null plasmid (Promega) to obtain clones with the insert either in the forward or in the reverse orientation with respect to the luciferase gene (Fig. ).

Techniques: Knockdown, Quantitative RT-PCR, Transfection, Construct, Luciferase, Control