Rev3, the catalytic subunit of Polζ, is required for maintaining fragile site stability in human cells

Abstract

It has been long speculated that mammalian Rev3 plays an important, yet unknown role(s) during mammalian development, as deletion of Rev3 causes embryonic lethality in mice, whereas no other translesion DNA synthesis polymerases studied to date are required for mouse embryo development. Here, we report that both subunits of Polζ (Rev3 and Rev7) show an unexpected increase in expression during G(2)/M phase, but they localize independently in mitotic cells. Experimental depletion of Rev3 results in a significant increase in anaphase bridges, chromosomal breaks/gaps and common fragile site (CFS) expression, whereas Rev7 depletion primarily causes lagging chromosome defect with no sign of CFS expression. The genomic instability induced by Rev3 depletion seems to be related to replication stress, as it is further enhanced on aphidicolin treatment and results in increased metaphase-specific Fanconi anemia complementation group D type 2 (FANCD2) foci formation, as well as FANCD2-positive anaphase bridges. Indeed, a long-term depletion of Rev3 in cultured human cells results in massive genomic instability and severe cell cycle arrest. The aforementioned observations collectively support a notion that Rev3 is required for the efficient replication of CFSs during G(2)/M phase, and that the resulting fragile site instability in Rev3 knockout mice may trigger cell death during embryonic development.

Figure 1.

Rev3 and Rev7 expression increases during G₂/M phase in HCT116 cells. (A) Cells stained with an anti-hRev3 antibody showing increased chromatin-associated fluorescence in metaphase cells (arrow) as compared with interphase cells (top row). Cells treated with siRNA against Rev3 (iRev3) for >48 h lost Rev3 staining (middle row). Cells arrested at G₂/M-phase by exposure to 100 ng/ml of nocodazole for 12 h showed increased fluorescence in chromatin areas (bottom row). (B) Western blot showing higher levels of Rev3 in nocodazole (Noco)-treated cells and abolition on treatment with iRev3. (C) Cells stained with an anti-hRev7 antibody either untreated or after treatment with nocodazole in a manner similar to that in (A) showing increased fluorescence in metaphase cells (arrow). (D) Western blot showing increased Rev7 levels in nocodazole-treated cells as compared with untreated cells. (E) Western blot showing Rev7 knockdown efficiency and specificity 72 h after siRNA transfection. A commercial mouse anti-Rev7 antibody was used for experiments shown in (C–E). (F) Cell cycle distribution as determined by flow cytometry after release from 48 h of serum starvation as indicated. (G) Relative REV3 and REV7 mRNA levels in samples collected from different times after serum starvation as shown in (F) as evaluated through qRT-PCR. Error bars, standard deviation from three samples versus an asynchronized sample (Asy) within the corresponding group. ***P < 0.0005, **P < 0.005 and *P < 0.05.

Figure 2.

Rev3 and Rev7 subcellular localization in HCT116 cells. Representative images from cells stained with the mouse anti-hRev3 (Red) and a rabbit anti-hRev7 (Green, received from Dr V. Maher, University of Michigan) antibodies (35) showing distinct subcellular distribution patterns in G₂/M cells. Cell cycle stages are indicated on the left panel. DAPI stain represents DNA.

Figure 3.

Rev3 and Rev7 suppression causes distinct types of genomic instability. (A) Representative anaphase HCT116 cells with DAPI staining showing bridges and lagging chromosomes. (B) Quantitative analysis of lagging chromosomes in cells treated for 48 h either with non-specific siRNA or siRNA against Rev1, Rev3 or Rev7. (C) Quantitative analysis of lagging chromosomes in HeLa cells transduced either with scrambled lentivirus or lentivirus expressing siRNA against Rev1, Rev3 or Rev7. (D) Quantitative analysis of anaphase bridges in same cells as in (B) but treated with siRNA for 72 h. (E) Quantitative analysis of anaphase bridges from same experiment as described in (C). (B–E) error bars represent standard deviations from three independent experiments with >100 anaphase cells for each experiment. *P < 0.05, **P < 0.005 and ***P < 0.0005 versus control.

Figure 4.

Loss of Rev3 causes DSBs in HCT116 cells. (A) Representative images of metaphase spreads showing breaks/gaps (arrow) 72 h after siRev3 treatment. (B) Percentage of metaphase cells containing breaks/gaps after the indicated siRNA treatment. (C) Quantitative analysis of average breaks/gaps per metaphase cell after siRNA treatment. Error bars represent standard deviations from three independent experiments with >50 metaphases for each experiment. **P < 0.005 versus control.

Figure 5.

Depletion of Rev3 in HCT116 cells causes fragile site expression and increased genomic instability. (A) Quantitative analysis of average breaks/gaps per metaphase after siRNA-treated cells were exposed to APH. Cells were incubated with the indicated siRNA for 45 h before a 24-h APH treatment followed by a 3-h colcemid treatment before being fixed and analysed. Shattered metaphases were not included in this analysis. P-value was calculated within each treatment group in comparison with the control siRNA treatment. **P < 0.005, *P < 0.05. (B) Representative metaphase spread from Rev3-depleted cells followed by 0.2 μM APH treatment showing the shattered chromosome phenotype. (C) Percentage of metaphases with shattered chromosome phenotype after siRNA and 0.2 μM APH treatment. (D) Representative chromosomes showing spontaneous breaks (arrow heads) at their respective fragile sites on Rev3 depletion, detected through FISH using indicated fluorescent fragile site probes. (E) Quantitative analysis of data as presented in (D). All error bars in this figure represent two independent experiments with >50 metaphases analysed for each experiment.

Figure 6.

Depletion of Rev3 leads to FANCD2 focus formation in HCT116 metaphase cells. (A) Representative images of cells depleted of Rev3 for 72 h and stained with an anti-FANCD2 (FD2) antibody showing the FANCD2 focus formation. A metaphase with no focus (top row), multiple foci on pro-metaphase (second row), cluster of foci on metaphase (third row) and two distinct foci on each side of the anaphase bridge (bottom row). (B) Quantitative measurement of metaphases/pro-metaphases with more than two foci per cell after 48, 72 and 85 h of treatment with siRNA. Error bars represent two experiments with >50 metaphase cells per experiment. *P < 0.05. (C) Representative images showing FANCD2 and γ-H2AX focus formation and their co-localization in metaphase cells. Cells were treated with gene-specific siRNA for 72 h and then stained with rabbit anti-FANCD2 and mouse anti-γH2AX antibodies simultaneously. Two distinct types of metaphase-specific FANCD2 foci were observed; one is co-localized with γH2AX foci (red arrow, enlarged box) and another does not correlate with γH2AX (white arrow, enlarged box). Boxed regions are enlarged at the bottom. (D) Proportion of metaphase FANCD2 foci with or without γH2AX focus co-localization.

Figure 7.

Prolonged depletion of Rev3 in HeLa cells causes multiple nuclear defects leading to cell death. (A) Relative REV3 mRNA levels of individual clones after 3 weeks of transduction by lentivirus expressing siRNA against REV3. (B) Nuclear defect frequency in selected clones. For each clone >100 cells were counted. (C) Representative images showing various forms of chromosomal abnormalities as indicated in (B).