Journal: Nature Communications

Article Title: ZC3H18 specifically binds and activates the BRCA1 promoter to facilitate homologous recombination in ovarian cancer

doi: 10.1038/s41467-019-12610-x

Figure Lengend Snippet: ZC3H18 promotes binding of E2F4 and activation of the BRCA1 promoter. a qRT-PCR analysis of BRCA1 mRNA expression. OVCAR-8 cells transfected with control luciferase (Luc), E2F2, E2F3, E2F4, E2F5, E2F6, E2F7, or E2F8 siRNAs with or without ZC3H18 siRNA were analyzed by qRT-PCR for BRCA1 mRNA levels, which were normalized to GAPDH mRNA. b Immunoblots of indicated proteins in OVCAR-8 and PEA1 cells transfected with control luciferase (Luc) and two independent E2F4 siRNAs. c BRCA1 mRNA expression, normalized to GAPDH mRNA, was determined by qRT-PCR in short-term ex vivo cultures of HGSOC tissues from PDX models electroporated with Luc or E2F4 siRNAs. d ZC3H18 promotes E2F4 occupancy on the BRCA1 promoter. OVCAR-8 cells transfected with control luciferase (Luc) or ZC3H18 siRNAs were analyzed by ChIP for E2F4 bound to the BRCA1 promoter. e , f Depletion of E2F4 or ZC3H18 promotes E2F1 and DNMT1 occupancy on the BRCA1 promoter. OVCAR-8 cells transfected with Luc, E2F4, and ZC3H18 siRNAs were analyzed by ChIP for E2F1 ( e ) and DNMT1 ( f ) accumulation on the BRCA1 promoter. g E2F4 depletion disrupts HR. OVCAR-8-DR-GFP cells transfected with pCβASceI plus indicated siRNAs were analyzed for GFP fluorescence by flow microfluorimitry 48 h after transfections. HR efficiencies were normalized to control (Luc) siRNA-transfected cells. h OVCAR-8 cells were transfected with control luciferase (Luc), E2F4, or BRCA1 siRNAs. Forty-eight hours later, the cells were trypsinized, re-plated, and allowed to adhere for 24 h. The indicated concentrations of olaparib were then added, and the cells were cultured for 10 days, stained with Coomassie Blue, and colonies were counted manually. Data are means ± SEM of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, unpaired Student’s t- test. Representative immunoblots in b are provided from three independent experiments. Unprocessed blots are provided in Source data file. The graph in h represents one of three independent experiments that gave similar results. Error bars are standard deviation of triplicate wells from a representative experiment

Article Snippet: Immunoblotting was done using the following primary antibodies: rabbit polyclonal ZC3H18 (1:1000, A304-682A, Bethyl Laboratories Inc.); mouse monoclonal BRCA1 (1:2000, sc-6954, Santa Cruz Biotechnology); mouse monoclonal E2F1 (1:500, ab4070, Abcam); rabbit polyclonal E2F4 (1:1000, NBP1-21374, Novus Biologicals); mouse monoclonal DNMT1 (1:5000, ab13537, Abcam); mouse monoclonal HSP90 (D. Toft, Mayo Clinic, H9010), and rabbit monoclonal HA-tag (1:1000, CST-3724S, Cell Signaling Technology).

Techniques: Binding Assay, Activation Assay, Quantitative RT-PCR, Expressing, Transfection, Control, Luciferase, Western Blot, Ex Vivo, Fluorescence, Cell Culture, Staining, Standard Deviation

Journal: Nature Communications

Article Title: ZC3H18 specifically binds and activates the BRCA1 promoter to facilitate homologous recombination in ovarian cancer

doi: 10.1038/s41467-019-12610-x

Figure Lengend Snippet: ZC3H18 binds the BRCA1 promoter and inhibits E2F1 binding. a Schematic of the BRCA1 proximal promoter with E2FA and E2FB sites indicated. Nucleotide sequences of the DNA probes used in the electrophoretic mobility shift assays (EMSA). E2FA and E2FB mutation sites are indicated in open rectangles. b EMSA with purified recombinant SFB-ZC3H18 using BRCA1 promoter probe with wild-type sequence (E2FA/B WT ) or mutations in the E2FA site (E2F ΔA ), the E2FB site (E2F ΔB ), or both E2F sites (E2F ΔA/B ). A probe with randomly shuffled sequences was used as negative control. For supershift assays, an anti-S-Tag monoclonal antibody, which binds the SFB tag in SFB-ZC3H18, was used. c , d ZC3H18 and E2F4 co-occupy the endogenous BRCA1 promoter. Sequential ChiP (ChIP-Re-ChIP) assays in OVCAR-8 cells using anti-ZC3H18 antibody for primary ChIP and anti-E2F4 antibody for secondary ChIP ( c ) and using anti-E2F4 antibody for primary ChIP and anti-ZC3H18 antibody for secondary ChIP ( d ). e EMSA with purified recombinant SFB-ZC3H18 and SFB-E2F1 using BRCA1 promoter probe with mutated E2FB site (E2F ΔB ). f EMSA with purified SFB-E2F4 and SFB-E2F1 using BRCA1 promoter probe with mutated E2FA site (E2F ΔA ). The images of EMSA in b , e , and f are representative of three independent experiments that gave similar results. Data in c and d are means ± SEM of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, unpaired Student’s t- test

Article Snippet: Immunoblotting was done using the following primary antibodies: rabbit polyclonal ZC3H18 (1:1000, A304-682A, Bethyl Laboratories Inc.); mouse monoclonal BRCA1 (1:2000, sc-6954, Santa Cruz Biotechnology); mouse monoclonal E2F1 (1:500, ab4070, Abcam); rabbit polyclonal E2F4 (1:1000, NBP1-21374, Novus Biologicals); mouse monoclonal DNMT1 (1:5000, ab13537, Abcam); mouse monoclonal HSP90 (D. Toft, Mayo Clinic, H9010), and rabbit monoclonal HA-tag (1:1000, CST-3724S, Cell Signaling Technology).

Techniques: Binding Assay, Electrophoretic Mobility Shift Assay, Mutagenesis, Purification, Recombinant, Sequencing, Negative Control, ChIP-chip

Journal: Nature Communications

Article Title: ZC3H18 specifically binds and activates the BRCA1 promoter to facilitate homologous recombination in ovarian cancer

doi: 10.1038/s41467-019-12610-x

Figure Lengend Snippet: ZC3H18 and E2F4 expression correlates with BRCA1 levels in HGSOC patient and PDX tumors. a Scatter plots of BRCA1 mRNA expression as a function of either ZC3H18 or E2F4 mRNA expression in HGSOC tumors from patients and PDX models. mRNA expression is in RPKM units. b Model for the role of ZC3H18 in BRCA1 transcription. Left panel: in ZC3H18-proficient cells, ZC3H18 directly binds to the E2FA site on the BRCA1 promoter, where it promotes E2F4 occupancy at the E2FB site, thereby preventing E2F1-dependent DNMT1 occupancy and promoter methylation and inducing BRCA1 transcription. Right panel: in ZC3H18-deficient cells, E2F1 occupies both E2FA and E2FB sites and causes DNMT1 loading onto the promoter, leading to methylation of the promoter, reduced expression of BRCA1, and disruption of HR. Spearman correlations are shown in the images

Article Snippet: Immunoblotting was done using the following primary antibodies: rabbit polyclonal ZC3H18 (1:1000, A304-682A, Bethyl Laboratories Inc.); mouse monoclonal BRCA1 (1:2000, sc-6954, Santa Cruz Biotechnology); mouse monoclonal E2F1 (1:500, ab4070, Abcam); rabbit polyclonal E2F4 (1:1000, NBP1-21374, Novus Biologicals); mouse monoclonal DNMT1 (1:5000, ab13537, Abcam); mouse monoclonal HSP90 (D. Toft, Mayo Clinic, H9010), and rabbit monoclonal HA-tag (1:1000, CST-3724S, Cell Signaling Technology).

Techniques: Expressing, Methylation, Disruption

Journal: Cells

Article Title: Weighted Correlation Network Analysis Reveals CDK2 as a Regulator of a Ubiquitous Environmental Toxin-Induced Cell-Cycle Arrest

doi: 10.3390/cells9010143

Figure Lengend Snippet: E2F1 and E2F4 were downregulated by OTA exposure. ( A ) E2F1 and E2F4 were significantly downregulated following 100 nM OTA exposure according to RNA-sequencing data in both cell lines, while E2F7 was found to be upregulated only in HEK293-T cells. Due to the consistent results between the two cell lines, the expression of E2F1 and E2F4 only was verified by RT-qPCR ( B ) and Western blot ( C ) (mean ± SD, * p < 0.05, N = 3).

Article Snippet: CDKN1A/p21 (Cell Signaling Technology, cat. no. 2946, Frankfurt, Germany), CDK2 (Cell Signaling Technology, cat. no. 2546), and E2F4 (R&D, cat. no. AF5139) were detected using IRDye-coupled fluorescent secondary antibodies (LI-COR Biosciences, Bad Homburg, Germany) and an Odyssey imaging system from LI-COR Biosciences.

Techniques: RNA Sequencing Assay, Expressing, Quantitative RT-PCR, Western Blot

Journal:

Article Title: Anchorage-Independent Transcription of the Cyclin A Gene Induced by the E7 Oncoprotein of Human Papillomavirus Type 16

doi:

Figure Lengend Snippet: Constitutive expression of the cyclin A gene in HPV-16 E7-expressing cells. (A) Western blot showing cyclin A protein levels in E7-expressing and control cells under normal growth conditions (lanes A) and after 20 h of culture in suspension (lanes S). (B) Northern blot analysis of RNA prepared in parallel and probed with a labelled fragment of the murine cDNAs for cyclin A (upper blot) and GAPDH (lower blot). (C) Cyclin A mRNA levels were quantitated and normalized relative to the GAPDH signal. Relative levels of expression (in percentages) are given by the bars. (D) Analysis of cyclin A promoter activity. NIH 3T3 cells were transfected with luciferase reporter gene constructs containing the promoter region of the human cyclin A gene. Twenty-four hours posttransfection cultures were trypsinized and plated in aliquots on uncoated (open bars) and agarose-coated (shaded bars) dishes for 20 h. The left part shows the relative luciferase activity of the construct containing the wild-type promoter (cycA-89/+11), whereas the right part shows the activity of a construct in which the E2F binding site was altered by clustered point mutations (cycA-89/+11mut). The values are the means of results of four independent experiments and are normalized to the activity of a cotransfected CMV-based β-galactosidase expression vector.

Article Snippet: E2F-associated proteins were analyzed by addition of specific antibodies to the bandshift reaction followed by incubation on ice for 50 min, prior to electrophoresis. p107 was detected by the monoclonal antibody SD15 (a gift from N. Dyson, Charlestown, S.C.), cdk2 was detected by a polyclonal antiserum (M-2; Santa Cruz Inc.), DP-1 was detected by the monoclonal antibody TFD10 (Neomarkers), E2F-4 was detected by a monoclonal antibody (WUF-11; a gift from N. Dyson), and p130 was detected by a polyclonal antiserum (C-20; Santa Cruz Inc.).

Techniques: Expressing, Western Blot, Northern Blot, Activity Assay, Transfection, Luciferase, Construct, Binding Assay, Plasmid Preparation

Journal:

Article Title: Anchorage-Independent Transcription of the Cyclin A Gene Induced by the E7 Oncoprotein of Human Papillomavirus Type 16

doi:

Figure Lengend Snippet: E7-expressing cells retain free E2F in suspension. (A) Bandshift analysis of whole-cell extracts from E7-expressing (E7/4 and E7/2) and control (pMo) cells after 20 h of culture under normal (lanes A) or suspension (lanes S) conditions with an oligonucleotide encompassing the E2F binding site of the human cyclin A promoter as the radioactive probe. The positions of specific complexes are marked. (B) Complexes obtained with whole-cell extracts as described for panel A were analyzed by addition of specific antibodies to p107 (lanes 2, 5, 9, 11, and 15) and cdk2 (lanes 3, 6, 9, 12, and 16). The positions of complexes 1 and 2 and of bands representing free E2F are marked. susp., suspension. (C) Bandshift analysis of nuclear extracts from asynchronously growing NIH 3T3 cells with an oligonucleotide encompassing the E2F binding site of the human cyclin A promoter as the radioactive probe. Prior to electrophoresis, extracts either remained untreated (lane 1) or were incubated with the recombinant GST-E7 wild type (wt) (lane 2), GST-E7 PRO2 (lane 3), and GST-E7 GLY24 (lane 4).

Article Snippet: E2F-associated proteins were analyzed by addition of specific antibodies to the bandshift reaction followed by incubation on ice for 50 min, prior to electrophoresis. p107 was detected by the monoclonal antibody SD15 (a gift from N. Dyson, Charlestown, S.C.), cdk2 was detected by a polyclonal antiserum (M-2; Santa Cruz Inc.), DP-1 was detected by the monoclonal antibody TFD10 (Neomarkers), E2F-4 was detected by a monoclonal antibody (WUF-11; a gift from N. Dyson), and p130 was detected by a polyclonal antiserum (C-20; Santa Cruz Inc.).

Techniques: Expressing, Binding Assay, Electrophoresis, Incubation, Recombinant

Journal:

Article Title: Anchorage-Independent Transcription of the Cyclin A Gene Induced by the E7 Oncoprotein of Human Papillomavirus Type 16

doi:

Figure Lengend Snippet: Free E2F is nuclear in E7-expressing but not in control cells. Nuclear extracts prepared from E7-expressing (E7/2) and control (pMo) cells after 20 h of culture under normal (lanes A) or suspension (lanes S) conditions were analyzed for complexes binding to the E2F site of the cyclin A promoter by bandshift experiments. (A) Western blot controlling the separation of cytoplasmic (left part) and nuclear (right part) compartments in the fractionation procedure. As markers for the nuclear fractions, antibodies to SP1 were used. Purity of the cytoplasmic fraction was controlled by a antibodies to M2 pyruvate kinase (PKM2). (B) Bandshift experiment with the cytoplasmic fractions and nuclear extracts shown in panel A with an oligonucleotide encompassing the E2F site of the human cyclin A promoter as the labelled probe. Positions of specific complexes are marked. (C) Analysis of complexes obtained with nuclear extracts of control cells (pMo) by addition of antibodies specific for p107 (lanes 2 and 7), p130 (lanes 3 and 8), E2F-4 (lanes 4 and 9), and DP-1 (lanes 5 and 10). The positions of complexes I and II are marked. (D) Analysis of complexes obtained with E7/2 cells by addition of antibodies specific for p107 (lanes 2 and 6), p130 (lanes 3 and 7), E2F-4 (lanes 9 and 13), and DP-1 (lanes 10 and 14). Free E2F was detected by its ability to bind to a recombinant GST fusion protein containing the pocket domain of pRb [GST–Rb(379–928)], which resulted in a retarded band (lanes 4 and 8).

Article Snippet: E2F-associated proteins were analyzed by addition of specific antibodies to the bandshift reaction followed by incubation on ice for 50 min, prior to electrophoresis. p107 was detected by the monoclonal antibody SD15 (a gift from N. Dyson, Charlestown, S.C.), cdk2 was detected by a polyclonal antiserum (M-2; Santa Cruz Inc.), DP-1 was detected by the monoclonal antibody TFD10 (Neomarkers), E2F-4 was detected by a monoclonal antibody (WUF-11; a gift from N. Dyson), and p130 was detected by a polyclonal antiserum (C-20; Santa Cruz Inc.).

Techniques: Expressing, Binding Assay, Western Blot, Fractionation, Recombinant

Journal:

Article Title: Anchorage-Independent Transcription of the Cyclin A Gene Induced by the E7 Oncoprotein of Human Papillomavirus Type 16

doi:

Figure Lengend Snippet: Nuclear localization of E2F-4 after transient expression of HPV-16 E7. U2-OS cells were transfected with expression vectors for E2F-4, DP-1, p107, and HPV-16 E7, as indicated. (A) Immunofluorescence staining of transfected cells with a monoclonal antibody against E2F-4 (left side). Seven to 10% of cells show a bright staining, indicating expression of the transfected protein. Nuclei were counterstained with DAPI (right side). (B) Whole-cell extracts prepared from transfected U2-OS cells were analyzed by bandshift experiments with the E2F binding site of the human cyclin A promoter as the labelled probe. The complex formed after transfection of expression vectors for E2F-4 and DP-1 (free E2F; lane 2) is marked. This complex is absent in extracts from cells transfected with expression vectors carrying genes encoding E2F-4, DP-1, and p107 but reappears when an expression vector for HPV-16 E7 is included. The complex obtained after coexpression of E7 (lane 4) was analyzed by addition of antibodies to E2F-4 (lane 5) and addition of recombinant GST fusion protein containing the pocket domain of pRb [GST–Rb(379–928); lane 6], which resulted in a retarded band.

Article Snippet: E2F-associated proteins were analyzed by addition of specific antibodies to the bandshift reaction followed by incubation on ice for 50 min, prior to electrophoresis. p107 was detected by the monoclonal antibody SD15 (a gift from N. Dyson, Charlestown, S.C.), cdk2 was detected by a polyclonal antiserum (M-2; Santa Cruz Inc.), DP-1 was detected by the monoclonal antibody TFD10 (Neomarkers), E2F-4 was detected by a monoclonal antibody (WUF-11; a gift from N. Dyson), and p130 was detected by a polyclonal antiserum (C-20; Santa Cruz Inc.).

Techniques: Expressing, Transfection, Immunofluorescence, Staining, Binding Assay, Plasmid Preparation, Recombinant

Journal: Molecular Neurobiology

Article Title: A Mutant Variant of E2F4 Triggers Multifactorial Therapeutic Effects in 5xFAD Mice

doi: 10.1007/s12035-022-02764-z

Figure Lengend Snippet: Expression of tau, E2F4 and E2F4DN-myc in the hippocampus and cerebral cortex of transgenic mice. a Western blot analysis of hippocampal extracts from 3-month-old mice from the indicated genotypes ( n = 3 females/genotype, except WT/E2F4DN: 1 male and 2 females) using antibodies against tau protein, E2F4, myc tag and Actin (as a loading control). Original western blots from this and the rest of figures can be seen in Supplementary Fig. . b Quantification of the ratios of Tau and E2F4 vs Actin in the hippocampus from the indicated genotypes. * p < 0.05 (two-way ANOVA, followed by post hoc Student’s t test). c Western blot analysis of cortical extracts from 3-month-old mice from the indicated genotypes ( n = 3 females/genotype, except WT/E2F4DN: 1 male and 2 females) using antibodies against tau protein, E2F4, myc tag, and Actin (as a loading control). d Quantification of the ratios of Tau and E2F4 vs Actin in the cerebral cortex from the indicated genotypes. ** p < 0.01 (two-way ANOVA, followed by post hoc Student’s t test)

Article Snippet: The rabbit anti-E2F4 polyclonal antibody (LS-B1532; LSBio) was used at 1:400 for proximity ligation assay (PLA) and immunohistochemistry.

Techniques: Expressing, Transgenic Assay, Western Blot, Control