Fig 2: ZC3H18 occupies the BRCA1 promoter and regulates E2F1. a OVCAR-8, OVCAR-5, and PEA1 cells were processed for ChIP assays using anti-ZC3H18 or IgG control antibodies and primers specific for the BRCA1 promoter. b OVCAR-8 cells transiently expressing SFB-ZC3H18 were processed for ChIP assays using FLAG or IgG control antibodies. c, d ChIP assays showing increased occupancy of E2F1 on the BRCA1 promoter when ZC3H18 is depleted. OVCAR-8 (c) and PEA1 (d) cells transfected with control luciferase (Luc) or ZC3H18 siRNAs (siZC3) were processed for ChIP using anti-E2F1 or IgG control antibodies 48 h after transfection. e E2F1 depletion does not affect BRCA1 levels. OVCAR-8 cells were transfected with control luciferase (Luc) or two independent E2F1 siRNAs and analyzed for BRCA1, E2F1, and HSP90 levels by immunoblotting 48 h later. f DNMT1 occupancy on the BRCA1 promoter requires E2F1. OVCAR-8 cells transfected with Luc, E2F1, or ZC3H18 siRNA with our without E2F1 siRNAs were processed for ChIP with DNMT1 or control IgG antibodies 48 h after transfection. g Depletion of E2F1 restores BRCA1 expression when ZC3H18 is depleted. OVCAR-8 cells transfected with Luc, E2F1, or ZC3H18 siRNAs with our without E2F1 siRNAs were analyzed for BRCA1 mRNA levels by qRT-PCR. BRCA1 mRNA levels were normalized to GAPDH mRNA levels. Data in a–g are means ± SEM from three independent experiments. **p < 0.01, ***p < 0.001, unpaired Student’s t-test. Representative immunoblots in e are provided from three independent experiments. Unprocessed blots are provided in Source data file

Fig 3: ZC3H18 depletion reduces BRCA1 mRNA and protein levels. a and b Indicated cell lines were transfected with control luciferase (Luc), ZC3H18 (ZC3), or BRCA1 siRNAs. Forty-eight hours after transfection, the cells were immunoblotted for the indicated antigens (a) and analyzed by qRT-PCR for ZC3H18 and BRCA1 mRNA levels, which are expressed relative to GAPDH mRNA levels as an internal control (b). c Rescue of BRCA1 mRNA and protein in ZC3H18-depleted OVCAR-8 cells with siRNA-resistant SFB-ZC3H18. OVCAR-8 cells were transfected with Luc or ZC3H18 siRNA plus empty vector (EV) or siRNA-resistant ZC3H18 plasmid (SFB-ZC3H18). BRCA1 mRNA and proteins levels were assessed by qRT-PCR (c) and immunoblotting (Supplementary Fig. 4b), respectively. d Ectopic BRCA1 expression rescues the HR defect caused by ZC3H18 depletion. OCVAR-8 DR-GFP cells were transfected with empty vector (EV) or HA-BRCA1-expressing plasmid with either control luciferase (Luc) or ZC3H18 (ZC3) siRNAs. Seventy-two hours later, the cells were analyzed for GFP by flow cytometry. Representative immunoblots in a are provided from three independent experiments. Unprocessed blots are provided in Source data file. Shown are means ± SEM from three independent experiments in b–d. *p < 0.05, **p < 0.01, ***p < 0.001, unpaired Student’s t-test

Fig 4: ZC3H18 depletion causes hypermethylation of the BRCA1 promoter. a Map of the divergent BRCA1/NBR2 promoter with CpG islands indicated by circles below the map. Methylation patterns obtained by bisulfite sequencing of 10 individual clones of PCR products from genomic DNA of control luciferase (Luc) and ZC3H18 siRNA (siZC3)-transfected OVACR-8 cells. Methylated (filled circles) and unmethylated (open circles) CpG positions are shown. b ChIP assays showing increased DNMT1 occupancy on the BRCA1 promoter in ZC3H18-depleted cells. OVCAR-8 (top panel) and PEA1 (bottom panel) cells transfected with control luciferase (Luc) or ZC3H18 siRNAs were harvested 48 h after transfection and processed for ChIP to detect DNMT1 on the BRCA1 promoter. c and d OVCAR-8 (c) and PEA1 (d) cells were transfected with control (Luc) or ZC3H18 siRNAs and treated with vehicle or 5-aza-2'-deoxycytidine (5 µM) for 3 days. BRCA1 mRNA (top panel) and ZC3H18 mRNA (bottom panel) levels were analyzed by qRT-PCR. The mRNA levels are normalized to GAPDH mRNA levels as the internal control. Data are means ± SEM from three independent experiments in b–d. *p < 0.05, **p < 0.01, ***p < 0.001, unpaired Student’s t-test

Fig 5: 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 (E2F?A), the E2FB site (E2F?B), or both E2F sites (E2F?A/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?B). f EMSA with purified SFB-E2F4 and SFB-E2F1 using BRCA1 promoter probe with mutated E2FA site (E2F?A). 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