Journal: Journal of Ovarian Research

Article Title: The transcriptome of corona radiata cells from individual MІІ oocytes that after ICSI developed to embryos selected for transfer: PCOS women compared to healthy women

doi: 10.1186/s13048-014-0110-6

Figure Lengend Snippet: Comparison between microarray and qRT-PCR results for 5 selected genes in PCOS and control CRC samples. Fold change (PCOS/controls) ± Standard Error of the Mean (SEM). Red bars represented microarray results and blue bars represented qRT-PCR results. Microarray experiment: 6 PCOS CRC arrays vs. 6 control CRC arrays. qRT-PCR experiment: 10 PCOS CRC samples vs. 10 control CRC samples. The up-regulation of UBE2C, HIST1H4C and E2F4 in PCOS CRCs found in the microarray experiment was in line with the qRT-PCR results, whereas the 1.4 fold up-regulation of CCND2 and CCNT2 in PCOS CRCs found in the microarray experiment could not be confirmed by qRT-PCR.

Article Snippet: The following TaqMan® Gene Expression Assays (pre-designed) (Applied Biosystems, Life Technologies Europe, Nærum, Denmark) were used (Assay ID-No: Hs00168719_m1 (Cyclophillin B/PPIB), Hs00171034_m1 (Cyclin T2, CCNT2), Hs00543883_s1 (histone cluster 1, H4c/HIST1H4C), Hs00608098_m1 (E2F transcription factor 4, p107/p130-binding/E2F4), and Hs00964100_g1 (ubiquitin-conjugating enzyme E2C/UBE2C)).

Techniques: Comparison, Microarray, Quantitative RT-PCR, Control

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