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: 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