human e2f4  (Addgene inc)

Journal: PLoS ONE

Article Title: Latent Transforming Growth Factor β-Binding Protein 4 Is Downregulated in Esophageal Cancer via Promoter Methylation

doi: 10.1371/journal.pone.0065614

Figure Lengend Snippet: In silico analysis of LTBP4 promoter sites identified a highly methylated binding site for GATA1 in the LTBP4L promoter of OE33 and KYSE180 cells. Highly methylated binding sites for SP1 and E2F4 are found in the LTBP4S promoter of both cell lines. A binding site for SMAD3 was identified nearby. The methylation status was analyzed by clonal bisulfite sequencing and at least ten clones were sequenced for each cell line. The percentage of methylation is visualized as pie chart. Exons are illustrated by black boxes and LTBP4S and LTBP4L promoters by red lines. The translation start sites are indicated (ATG). The dashed lines represent alternative splicing of LTBP4.

Article Snippet: The cells were cotransfected with expression vectors for murine Gata1 (Addgene plasmid 13626; , ), human SP1, human SMAD3 (Addgene plasmid 10920; ) or human E2F4 (Addgene plasmid 10914; ).

Techniques: In Silico, Methylation, Binding Assay, Methylation Sequencing, Clone Assay

Journal: PLoS ONE

Article Title: Latent Transforming Growth Factor β-Binding Protein 4 Is Downregulated in Esophageal Cancer via Promoter Methylation

doi: 10.1371/journal.pone.0065614

Figure Lengend Snippet: HEK293 cells were transiently transfected with pGL4.10-LTBP4L or pGL4.10-LTBP4S and a Renilla luciferase expression vector for normalization. Cells were also co-transfected with different concentrations of A) a Gata1 expression vector, B) a SP1 expression vector, C) a SMAD3 expression vector or D) an E2F4 expression vector. After 24 h cells were lysed and luciferase activities were determined. Experiments were repeated at least 3 times and the mean value was calculated. Data are presented ± standard deviation.

Article Snippet: The cells were cotransfected with expression vectors for murine Gata1 (Addgene plasmid 13626; , ), human SP1, human SMAD3 (Addgene plasmid 10920; ) or human E2F4 (Addgene plasmid 10914; ).

Techniques: Transfection, Luciferase, Expressing, Plasmid Preparation, Standard Deviation

Journal: PLoS ONE

Article Title: Latent Transforming Growth Factor β-Binding Protein 4 Is Downregulated in Esophageal Cancer via Promoter Methylation

doi: 10.1371/journal.pone.0065614

Figure Lengend Snippet: HEK293 cells were transiently transfected with pGL4.23-LTBP4L or pGL4.23-LTBP4S and a Renilla luciferase expression vector for normalization. Cells were also co-transfected with different concentrations of an E2F4 expression vector. After 24 h cells were lysed and luciferase activities were determined. Experiments were repeated at least 3 times and the mean value was calculated. Data are presented ± standard deviation.

Article Snippet: The cells were cotransfected with expression vectors for murine Gata1 (Addgene plasmid 13626; , ), human SP1, human SMAD3 (Addgene plasmid 10920; ) or human E2F4 (Addgene plasmid 10914; ).

Techniques: Transfection, Luciferase, Expressing, Plasmid Preparation, 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: Fig. 6 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/BWT) or mutations in the E2FA site (E2FΔA), the E2FB site (E2FΔB), or both E2F sites (E2FΔA/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 (E2FΔB). f EMSA with purified SFB-E2F4 and SFB-E2F1 using BRCA1 promoter probe with mutated E2FA site (E2FΔA). 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: Human ZC3H18 cDNA (Dharmacon, MHS6278202759301), E2F4 cDNA (Addgene plasmid #10914)43, and E2F1 cDNA (Addgene plasmid #24225)44 were subcloned into the pSFB vector that contains in-frame Nterminal S-peptide, FLAG, and streptavidin-binding peptide tags45.

Techniques: Binding Assay, Electrophoretic Mobility Shift Assay, Mutagenesis, 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: Fig. 7 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: Human ZC3H18 cDNA (Dharmacon, MHS6278202759301), E2F4 cDNA (Addgene plasmid #10914)43, and E2F1 cDNA (Addgene plasmid #24225)44 were subcloned into the pSFB vector that contains in-frame Nterminal S-peptide, FLAG, and streptavidin-binding peptide tags45.

Techniques: Expressing, Methylation, Disruption