BRE/BRCC45 regulates CDC25A stability by recruiting USP7 in response to DNA damage

BRCA2 is essential for maintaining genomic integrity. BRCA2-deficient primary cells are either not viable or exhibit severe proliferation defects. Yet, BRCA2 deficiency contributes to tumorigenesis. It is believed that mutations in genes such as TRP53 allow BRCA2 heterozygous cells to overcome growth arrest when they undergo loss of heterozygosity. Here, we report the use of an insertional mutagenesis screen to identify a role for BRE (Brain and Reproductive organ Expressed, also known as BRCC45), known to be a part of the BRCA1-DNA damage sensing complex, in the survival of BRCA2-deficient mouse ES cells. Cell viability by BRE overexpression is mediated by deregulation of CDC25A phosphatase, a key cell cycle regulator and an oncogene. We show that BRE facilitates deubiquitylation of CDC25A by recruiting ubiquitin-specific-processing protease 7 (USP7) in the presence of DNA damage. Additionally, we uncovered the role of CDC25A in BRCA-mediated tumorigenesis, which can have implications in cancer treatment.


Expression analysis
Total RNA was prepared using RNA-BEE (Tel-Test, Inc.) according to the manufacturer's protocol and cDNA samples were prepared from total RNA using SuperScript III first strand synthesis kit (Invitrogen). To quantitate expression levels a Brilliant II SYBR Green QPCR kit (Stratagene) was performed according to the manufacturer's instructions. GAPDH was used as internal control. Gene-specific primer sequences and thermo cycling conditions are available on request.

Cell cycle analysis
For BrdU (5-bromo-2'-deoxy-uridine) staining, cells were irradiated with 6 Gy irradiation, incubated for 1hr and then pulse-labeled with 10 µM BrdU (Boehringer Mannheim, Mannheim, Germany) for 15 minutes. Cells were harvested and fixed in 70 % ethanol. Cells were then stained for both DNA content and BrdU incorporation by the acid denaturation-protease method by using fluorescein isothiocyanate (FITC)-conjugated anti-BrdU (Becton Dickinson) antibody.
Cells were incubated with RNase-propidium iodide solution (BD biosciences) for 30 minutes before analyzing by flow cytometry.
For the transient G2/M transition checkpoint assay, asynchronous cells were exposed to 2 Gy IR and then incubated at 37°C for 8 h. Cells were fixed with 70% ethanol and stained with FITCconjugated rabbit anti-phospho-histone H3 antibody (#9701S, Cell signaling). The stained cells were treated with RNase-propidium iodide solution (BD biosciences) for 30 min at room temperature and then analyzed by flow cytometry.
For radio-resistant DNA synthesis analysis log phase cells were treated with media containing 10 nCi/ml 14 C-thymidine overnight, cells were then washed and further incubated for 24 hrs. Cells were exposed to various dose of irradiation, incubated for 30 minutes and then pulsed 2.5 µCi /ml 3 H-thymidine for 15 minutes. Cells were harvested and fixed with 70% methanol. Fixed cells were adsorbed onto Whatman filter disks using vacuum manifold, washed, dried and counted using a liquid scintillation counter. The tritium counts were normalized against 14 C counts. DNA synthesis is expressed as percent of counts per minute in DNA of irradiated cells compared to unirradiated controls.

Drug and radiation sensitivity assay
For drug sensitivity assay to different drugs and ionizing radiation 10,000 ES cells per well were plated (20,000 cells per well for mutants to compensate for lower seeding and growth efficiency) in 96-well plates. The next day, cells were treated with M15 medium containing appropriate concentrations of drugs. For gamma-irradiation, plates were exposed to a 137 Cs source at 146.3 RAD min-1 without media change. After 48 hours of drug treatment, cell proliferation was measured by XTT (2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2Htetrazolium hydroxide) assay as described previously 2 .
For clonogenic survival assay 100,000 cells were seeded in 3.5 cm dish. The next day plates were irradiated with media and incubated to form colonies. Colonies were stained with methylene blue [2% methylene blue (wt/vol) in 70% ethanol for 15 min followed by washing with 70% ethanol]. Colonies were quantified using ImageJ software.
All samples were analyzed in triplicate. For each cell lines two independent clones were checked and they behaved identically. The percentages of surviving cells were expressed as compared to untreated cells.

RAD51 foci formation assay
Cells were grown on lysine-coated coverslips and 5 hrs after 6 Gy IR cells were fixed with 4% paraformaldehyde for 5 min and permeabilized in PBS buffered 0.1% Triton X-100 for 10 min.
Antibody staining was performed as described previously 3 . Nuclei were imaged using a Zeiss LSM 510 inverted confocal laser-scanning microscope equipped with a ZEISS Axiovert 100 microscope with a 63X immersion oil objective. Two independent clones were used for each genotype and they behaved identically.

Karyotyping
ES cells were treated with colcemid (Invitrogen) for 1.5 hr and karyotyping was performed as described previously 2 . For each genotype randomly selected 50 metaphase spreads with 40 chromosomes were counted for aberrations blindly. For each genotype two independent clones were analyzed.    Values represent mean ± s.d.