High levels of BRC4 induced by a Tet-On 3G system suppress DNA repair and impair cell proliferation in vertebrate cells

Abstract

Transient induction or suppression of target genes is useful to study the function of toxic or essential genes in cells. Here we apply a Tet-On 3G system to DT40 lymphoma B cell lines, validating it for three different genes. Using this tool, we then show that overexpression of the chicken BRC4 repeat of the tumor suppressor BRCA2 impairs cell proliferation and induces chromosomal breaks. Mechanistically, high levels of BRC4 suppress double strand break-induced homologous recombination, inhibit the formation of RAD51 recombination repair foci, reduce cellular resistance to DNA damaging agents and induce a G2 damage checkpoint-mediated cell-cycle arrest. The above phenotypes are mediated by BRC4 capability to bind and inhibit RAD51. The toxicity associated with BRC4 overexpression is exacerbated by chemotherapeutic agents and reversed by RAD51 overexpression, but it is neither aggravated nor suppressed by a deficit in the non-homologous end-joining pathway of double strand break repair. We further find that the endogenous BRCA2 mediates the cytotoxicity associated with BRC4 induction, thus underscoring the possibility that BRC4 or other domains of BRCA2 cooperate with ectopic BRC4 in regulating repair activities or mitotic cell division. In all, the results demonstrate the utility of the Tet-On 3G system in DT40 research and underpin a model in which BRC4 role on cell proliferation and chromosome repair arises primarily from its suppressive role on RAD51 functions.

Keywords: BRC4; BRCA2; DT40 cells; Homologous recombination; RAD51; Tet-On system.

Fig. 1

Cellular effects associated with inducible p53 in chicken DT40 cells. (A) Growth curves. WT or WT + p53 (Tet-On) cells (1 × 10⁵) were inoculated in 1 ml of medium and passaged every 12 h. Dox was added at time 0. (B) DNA fragmentation assay. Genomic DNA was prepared and subjected to agarose gel electrophoresis. DNA ladders were detected by staining with 0.5 μg/ml ethidium bromide. M indicates the 100 bp DNA Ladder marker. (C) Monitoring of p53-FLAG. Whole cell lysates were prepared from cells cultured in the absence or the presence of Dox for 12 h. p53-FLAG and α-tubulin (loading control) were detected by Western blotting.

Fig. 2

Measurement of homologous recombination-dependent DSB repair. (A) WT + I-SceI (Tet-On) cells containing the DR-GFP construct were incubated with Dox. mCherry and GFP expressing fractions were measured at the indicated time points. The vertical axis represents the expression level of mCherry and horizontal axis represents the expression level of GFP. R2, R3 and R4 indicate cells that underwent recombination, I-SceI -induced cells and I-SceI negative cells, respectively. (B) The percentage of the cell population expressing mCherry and GFP are plotted.

Fig. 3

High levels of BRC4 cause cytotoxicity. (A) Relative BRC4 expression level measured by quantitative polymerase chain reaction (qPCR). WT, BRCA2^−/− (negative control), and individually obtained 2 clones of BRC4 cells (WT + BRC4 (Tet-On) cells) were incubated in the presence or absence of Dox for 12 h when RNA was isolated and converted to cDNA. The cDNA was amplified with GoTaq^® qPCR Master Mix. (B) Expression of the BRC4–FLAG peptide. Whole cell lysates were prepared from BRC4 #1 cells cultured in the presence of Dox for the indicated times. BRC4–FLAG and α-tubulin (loading control) were detected by Western blotting. (C) Growth curves. Individually obtained 2 clones of BRC4 cells (1 × 10⁵) were inoculated in 1 ml of medium and passaged every 12 h. Dox was added at time 0. (D) Growth curves of mCherry positive cells. The number of cells expressing mCherry was counted every 12 h. (E) Number of chromosomal aberrations in RAD51^−/− cells expressing hsRAD51 from a Tet-inducible promoter and in BRC4 cells after treatment with Dox for 18 h (RAD51^−/− + hsRAD51 cells) or for 24 h (BRC4 cells).

Fig. 4

BRC4 suppresses homologous recombination and activates the G2/M checkpoint, but not the ATR replication checkpoint. (A) Homologous recombination (HR) repair assay using the I-SceI induced DSB formation at the DR-GFP reporter (see Fig. 2). Percentage of cells that underwent HR (measured as cells expressing GFP) is plotted. (B) Rad51 focus formation. BRC4 cells were incubated in the presence or absence of Dox for 18 h. Cells were exposed to 500 ng/ml of MMC for 1 h, then washed, and sampled after 1 h. At least 100 cells were scored for each preparation. Identical trends were observed in two independent experiments. (C) Cell cycle distribution of BRC4 cells. Cells were cultured in the presence of Dox for the indicated times, pulse-labeled with BrdU for 15 min, and harvested. The cells were stained with FITC anti-BrdU to detect BrdU uptake and with propidium iodide (PI) to detect DNA. The vertical axis represents BrdU uptake and horizontal axis represents total DNA. The gates represent SubG1 (apoptotic cells), G1, S and G2/M phase from left to right, in this order. Numbers show the percentage of cells falling in each gate. (D) BRC4 cells were cultured in the presence of Dox for 12 h, further cultured in the presence or absence of Caffeine for 1 h and used for FACS analysis. (E) BRC4 cells were cultured in the presence of Dox for indicated times and whole cell lysates were prepared from each time points. RAD51, γ-H2AX, pCHK1 S345, BRC4–FLAG and MCM7 (loading control) were detected by Western blotting.

Fig. 5

BRC4 cytotoxicity depends on endogenous BRCA2. (A) Relative BRC4 expression level measured by qPCR. BRC4 cells and individually obtained 2 clones of BRCA2^−/+ BRC4 cells were incubated in the presence or absence of Dox for 12 h and RNA was isolated. Isolated cDNA was used for qPCR. (B) Expression of BRC4–FLAG peptide. Whole cell lysates were prepared from each cell line cultured in the presence of Dox for the indicated times. BRC4–FLAG and MCM7 (loading control) were detected by Western blotting. (C) Growth curves. BRC4 or individually obtained 2 clones of BRCA2^−/+ BRC4 cells (1 × 10⁵) were inoculated in 1 ml of medium and passaged every 12 h. Dox was added at time 0. (D) Growth curves of mCherry positive cells. The number of cells expressing mCherry was counted every 12 h. (E) Number of chromosomal aberrations in BRC4 and BRCA2^−/+ BRC4 cells after treatment with Dox for 24 h.

Fig. 6

BRC4-induced cytotoxicity is not dependent on non-homologous end joining. (A) Growth curves. BRC4 and individually obtained 2 clones of XRCC4/⁻ BRC4 cells (1 × 10⁵) were inoculated in 1 ml of medium and passaged every 12 h. Dox was added at time labeled 0. (B) Number of chromosomal aberrations in BRC4 and XRCC4/⁻ BRC4 cells after treatment with Dox for 24 h.

Fig. 7

RAD51 overexpression rescues BRC4-induced cytotoxicity. (A) Expression of RAD51 and BRC4–FLAG peptide. Whole cell lysates were prepared from each cell lines cultured in the presence of Dox for the indicated times. RAD51, BRC4–FLAG and MCM7 (loading control) were detected by Western blotting. The upper and lower bands of RAD51 show chicken and human RAD51, respectively. (B) Growth curves. BRC4 and individually obtained 2 clones of BRC4 + hsRAD51 cells (1 × 10⁵) were inoculated in 1 ml of medium and passaged every 12 h. Dox was added at the time point labeled 0. (C) Growth curves of BRC4 and BRC4-A1504S cells in the presence or absence of Dox. 1 × 10⁵ cells of the indicated genotype were inoculated in 1 ml of medium and passaged every 12 h. Dox was added at time 0. (D) Growth curves of mCherry positive BRC4 and BRC4-A1504S cells. The number of cells expressing mCherry was counted every 12 h.

Fig. 8

Rad51 overexpression rescues BRC4-induced homologous recombination repair defects. (A) and (B) growth curves. BRC4 cells (1 × 10⁵) were inoculated in 1 ml of medium and passaged every 12 h. Dox and olaparib (A) or CPT (B) were added at time 0. (C) Sensitivity of indicated cells to olaparib. Dox was added 24 h before the experiment. Cell survival percentage is displayed as the ratio of the number of surviving cells following olaparib treatment relative to the untreated control. Each line and error bar represents the mean value and SD from three independent experiments, respectively. (D) and (E) Rad51 focus formation. BRC4 cells and BRC4 + hsRAD51 cells were incubated in the presence or absence of Dox for 20 h. Then cells were exposed to 500 ng/ml of MMC for 6 h. Cytoplasmic RAD51 was washed out by pre-triton treatment prior to fixation to reduce the background. At least 100 cells were scored for each preparation. Identical trends were observed in independently conducted experiments.