The tumor suppressor FBXO31 preserves genomic integrity by regulating DNA replication and segregation through precise control of cyclin A levels

Abstract

F-box protein 31 (FBXO31) is a reported putative tumor suppressor, and its inactivation due to loss of heterozygosity is associated with cancers of different origins. An emerging body of literature has documented FBXO31's role in preserving genome integrity following DNA damage and in the cell cycle. However, knowledge regarding the role of FBXO31 during normal cell-cycle progression is restricted to its functions during the G₂/M phase. Interestingly, FBXO31 levels remain high even during the early G₁ phase, a crucial stage for preparing the cells for DNA replication. Therefore, we sought to investigate the functions of FBXO31 during the G₁ phase of the cell cycle. Here, using flow cytometric, biochemical, and immunofluorescence techniques, we show that FBXO31 is essential for maintaining optimum expression of the cell-cycle protein cyclin A for efficient cell-cycle progression. Stable FBXO31 knockdown led to atypical accumulation of cyclin A during the G₁ phase, driving premature DNA replication and compromised loading of the minichromosome maintenance complex, resulting in replication from fewer origins and DNA double-strand breaks. Because of these inherent defects in replication, FBXO31-knockdown cells were hypersensitive to replication stress-inducing agents and displayed pronounced genomic instability. Upon entering mitosis, the cells defective in DNA replication exhibited a delay in the prometaphase-to-metaphase transition and anaphase defects such as lagging and bridging chromosomes. In conclusion, our findings establish that FBXO31 plays a pivotal role in preserving genomic integrity by maintaining low cyclin A levels during the G₁ phase for faithful genome duplication and segregation.

Keywords: DNA replication; F-box protein 31 (FBXO31); LOH; MCM complex; SCF complex; anaphase-promoting complex; cell cycle; genomic instability; protein degradation; tumor suppressor gene.

Figure 1.

FBXO31 is essential for an error-free replication process and mitosis. A, graphical representation of percentage of S phase in MCF7 cells stably expressing NS and shFBXO31-1 and shFBXO31-2 at different time points following nocodazole release. Cells were grown in nocodazole-containing medium for 16 h; following that, the cells were allowed to re-enter the cell cycle in fresh medium and were collected at the indicated time points for flow cytometric analysis. Data points represent the mean of two independent experiments. B, cell-cycle profile of MCF7 cells stably expressing NS and shFBXO31-1 and shFBXO31-2 at different time points following nocodazole release. Cells were grown in nocodazole-containing medium for 16 h; following that, the cells were allowed to re-enter the cell cycle in fresh medium and were collected at the indicated time points for flow cytometric analysis. Cells were chased for BrdU incorporation for 2 h before harvesting, and then the cells were stained and analyzed for BrdU incorporation using flow cytometry. C, graphical representation of percentage of NS and shFBXO31 cells in different cell cycle phases at the indicated time points. Cells were grown in the presence of 0.25 mm HU for 22 h, then released by washing twice with media followed by culturing in fresh media, and then were collected at the indicated time points. Population of cells at different cell cycle phases was assessed using flow cytometry. Error bars represent S.E. from three independent experiments. D, graphical representation of percentage of NS and shFBXO31 cells in the G₂ and different mitotic phases after 12 h of hydroxyurea release. Cells were grown and collected as described in C and then stained with pH 3 Ser-10 antibody to score the number of cells at the G₂ and different phases of mitosis. Error bars represent S.E. from three independent experiments. E, graphical representation of percentage of NS and shFBXO31 cells having lagging and bridging chromosomes after 12 h of hydroxyurea release. Cells were grown and collected as described in C and were stained with pH 3 Ser-10 antibody to score the number of anaphase cells, and the percentage of cells was calculated with bridging and lagging chromosomes. Error bars represent S.E. from three independent experiments. F, graphical representation of percentage of sub-G₁ population of NS and shFBXO31 cells determined by flow cytometry following three times of repeated HU exposure. Cells expressing either NS or FBXO31 shRNA were exposed to replication stress using HU for 24 h, and then cells were released for 24 h; this cycle was repeated for three consecutive times. Error bars represent S.E. from three independent experiments. G, cells were grown as described in F and then the released cells were allowed to grow in fresh medium for the next 10 days. Colonies were then stained with crystal violet dye. Data are representative images of three independent experiments. H, graphical representation of percentage of γH2AX-positive NS and shFBXO31 cells as in G. n.s. represents nonsignificant; *, p ≤ 0.05; **, p < 0.01; ***, p < 0.001. Error bars represent S.E. from three independent experiments.

Figure 2.

FBXO31 regulates cyclin A expression at the proteasomal level. A, immunoblot monitoring the level of cyclin A and FBXO31 in MCF7 cells expressing either NS or FBXO31 shRNAs. Whole-cell protein extracts were immunoblotted to probe for the indicated proteins. B, cytoplasmic and nuclear fractions of cells expressing either NS or FBXO31 shRNA were immunoblotted with the indicated antibodies. Tubulin and Lamin B1 were used as cytoplasmic and nuclear loading controls, respectively. C, real time RT-PCR was performed to monitor cyclin A mRNA expression in NS and shFBXO31 cells. Cyclin A mRNA level was normalized to the GAPDH mRNA level. Error bars represent S.E. from three independent experiments. D, immunoblots monitoring the turnover profile of cyclin A in NS and shFBXO31 cells following cycloheximide (CHX) (40 μg/ml) chase for the indicated time periods. E, quantification of levels of cyclin A in D. Expression of cyclin A was normalized with tubulin, and then cyclin A expression at 0 h was considered as 100% relative to the values of other time points that were plotted. F, immunoblot monitoring the turnover of cyclin A in cells expressing either empty vector or myc-FBXO31 following cycloheximide (40 μg/ml) chase for the indicated periods. Cells were transfected with the indicated constructs for 36 h and following that whole-cell protein extracts were immunoblotted to probe for the indicated proteins. G, quantitation of half-life of cyclin A from F. Expression of cyclin A was normalized with tubulin, and the degradation profile was plotted considering the expression of cyclin A at 0 h as 100%. H, cells were transfected either with empty vector or increasing concentrations of myc-FBXO31 for 48 h. Cells were then harvested, and whole-cell protein lysates were immunoblotted to probe for indicated proteins. I, cells were transfected with either empty vector or myc-FBXO31 for 36 h and were grown in the presence or absence of 10 μm proteasome inhibitor MG132 for 6 h and were immunoblotted and probed with the indicated antibodies. Error bars where shown represent S.E. from three independent experiments, and n.s. represents nonsignificant; *, p ≤ 0.05; **, p < 0.01; ***, p < 0.001.

Figure 3.

FBXO31 interacts with cyclin A to promote its polyubiquitylation through SCF complex. A, immunoblots monitoring the expression of cyclin A in MCF7 cells expressing either vector or myc-FBXO31 or F-box–deleted mutant of FBXO31 (myc-ΔF-FBXO31). Cells were transfected with the indicated plasmids for 48 h, and then whole-cell protein extracts were analyzed by immunoblotting. B, MCF7 cells were co-transfected with the indicated plasmids for 36 h, and transfected cells were then grown in the presence of 10 μm MG132 for 6 h. Whole-cell protein extracts were co-immunoprecipitated using anti-myc antibody. Immunoprecipitates and input protein extracts were immunoblotted for the indicated proteins. C, immunoblot monitoring the interaction between FBXO31 and cyclin A at the endogenous level in MCF7 cells treated with MG132 (10 μm for 6 h) in a co-immunoprecipitation assay. Whole-cell protein extracts were immunoprecipitated with the indicated antibodies as described under “Materials and methods.” Immunoprecipitates and input protein extracts were immunoblotted for the indicated proteins. D, MCF7 cells were transfected with FLAG–cyclin A in combination with either HA–ubiquitin or myc-FBXO31, or both, for 36 h. Transfected cells were then incubated with 10 μm MG132 for 6 h and harvested, and the whole-cell lysates were immunoprecipitated with anti-FLAG antibody. Immunoprecipitates and input protein extracts were immunoblotted for the indicated proteins. E, immunoblots monitoring the lysine 48 (Lys-48) (IB) linkage-specific ubiquitylation of cyclin A in MCF7 cells expressing NS or FBXO31 shRNA. Whole-protein extracts were immunoprecipitated using the anti-cyclin A antibody. Immunoprecipitates and input protein extracts were immunoblotted to probe for the indicated proteins.

Figure 4.

Accumulation of cyclin A during G₁ phase leads to compromised chromatin loading of MCM4 in FBXO31-depleted cells. A, NS or shFBXO31-expressing MCF7 cells were synchronized using nocodazole for 16 h. Synchronized cells were then released and collected at the indicated time points. Whole-cell protein extracts were immunoblotted to probe for the indicated proteins. B, quantitation of cyclin A expression from A. Cyclin A expression was normalized with respective loading control tubulin at the corresponding time points and then normalized to 100% at each time point for NS cells. Then, cyclin A levels in shFBXO31-expressing cells at the indicated time points were quantified with respect to corresponding NS cells. Error bars represent S.E. from three independent experiments. C, immunoblot monitoring the interaction between FBXO31–cyclin A and CDH1–cyclin A at the endogenous levels at different phases of the cell cycle. MCF7 cells were synchronized using nocodazole for 16 h. Synchronized cells were then released and allowed to grow in fresh medium until harvested. MG132 (10 μm) was added to the medium for 2 h before harvesting the cells at the indicated time points. Whole-cell protein extracts were immunoprecipitated with the indicated antibodies. The immunoprecipitates and input protein extracts were immunoblotted and probed for the indicated proteins. D, MCF7 cells expressing either NS or FBXO31 shRNA were transfected with FLAG–cyclin A in combination with either His–Lys-11–only ubiquitin or His–Lys-48–only ubiquitin for 24 h, and transfected cells were then incubated with 10 μm MG132 for 2 h. Cells were then harvested, and whole-cell lysates were incubated with Ni-NTA beads. The pulled down fractions and input protein lysates were immunoblotted for the indicated proteins. E, immunoblot (IB) analysis monitoring MCM4 level in chromatin and nonchromatin fractions during mitosis and G₁ phase of NS, FBXO31KD, and FBXO31–cyclin A knockdown cells. Error bars where shown represent S.E. from three independent experiments, and n.s. represents nonsignificant; *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Figure 5.

Depletion of FBXO31 leads to premature S-phase entry and reduced DNA replication accompanied by increased DNA damage. A, immunofluorescence analysis of number of cells showing S-phase–specific pattern of PCNA in NS and shFBXO31 cells. Both NS and shFBXO31-expressing cells were synchronized using nocodazole, and then cells were allowed to grow in the absence of nocodazole for 12 h. Cells were fixed and stained for PCNA (red) and Hoechst (blue). The experiment was repeated three times, and 10 random fields were observed in each time. Image represents one of the fields. B, percentage of S-phase cells was quantified from A. C, flow cytometric representation of cell-cycle profile of NS and shFBXO31-expressing cells at 12 h post-nocodazole release. D, immunofluorescence analysis of DNA damage foci represented by γ-H2AX (cyan) in S-phase cells. Both NS and shFBXO31-expressing cells were synchronized by nocodazole, and then cells were allowed to grow in the absence of nocodazole for 12 h. Cells were fixed and stained for γ-H2AX (cyan), PCNA (red), and Hoechst (blue). E, graphical representation of mean fluorescence intensity of PCNA of S phase in NS and shFBXO31-expressing cells. Region of interest was drawn at the periphery of nucleus displaying S-phase–specific PCNA distribution. Experiment was repeated three times, and five random fields were observed each time. F, quantitation of NS and shFBXO31 cells having γ-H2AX foci in S-phase cells (n = 100). Error bars represent S.E. from three independent experiments, and n.s. represents nonsignificant; *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Figure 6.

Elevated level of cyclin A in FBXO31-depleted cells is responsible for premature S-phase entry, replication stress, and mitotic delay and defects. A, percentage of S-phase cells in NS, shFBXO31, and co-expressing shFBXO31 and shcyclin A cells at 8 h post-nocodazole release. B, percentage of NS, shFBXO31, and shFBXO31 and shcyclin A co-depleted cells in different phases of the cell cycle at 0, 9, and 12 h post-HU release. C, graphical representation of cells in G₂ phase and different mitotic phases following 12 h of hydroxyurea release. Cells were fixed and stained with Hoechst and pH 3 Ser-10 antibody to score G₂ and different phases of mitotic cells. D, percentage of NS, shFBXO31, and co-expressing shFBXO31 and shcyclin A cells, harboring anaphase bridges following 12 h of HU release. Cells were stained as described in Fig. 1D, and anaphase cells were scored to calculate the percentage of cells with bridging chromosomes. E, percentage of NS, shFBXO31, and co-expressing shFBXO31 and shcyclin A cells harboring lagging chromosomes following 12 h of HU release. Cells were stained as in Fig. 1D, and anaphase cells were scored to calculate the percentage of cells with lagging chromosomes. Error bars represents S.E. from three independent experiments, and n.s. represents nonsignificant; *, p ≤ 0.05; **, p < 0.01; ***, p < 0.001.

Figure 7.

FBXO31 controls the progression of mitosis and G₁ phase through maintaining the optimum levels of cyclin A. A, image representing flow cytometric analysis of BrdU-pulsed MCF7 cells ectopically expressing cyclin A alone or along with either WT FBXO31 or mutant FBXO31, released from nocodazole block at the indicated time periods. Cells were pulsed with BrdU for 2 h before harvesting and following that the cells were collected by trypsinization, stained, and analyzed by flow cytometry. B, image representing flow cytometric analysis of BrdU-pulsed MCF7 cells, ectopically expressing cyclin A alone or along with either WT FBXO31 or mutant FBXO31. Transfected cells were released from HU block. Released cells were pulsed with BrdU for 2 h before harvesting. Pulsed cells were collected by trypsinization for FACS analysis. C, immunoblot analysis monitoring MCM4 level in chromatin and nonchromatin fractions during mitosis and G₁ phase of MCF7 cells expressing either vector or cyclin A, or co-expressing cyclin A and WT FBXO31, or co-expressing cyclin A and mutant FBXO31. Transfected cells were synchronized with nocodazole and then released for the indicated time periods. D, graphical representation of percentage of G₂ and different mitotic phases of NS and FBXO31KD cells expressing either vector or WT FBXO31 or cyclin A-binding defective FBXO31 mutant or F-box motif-deleted FBXO31 mutant following 12 h of hydroxyurea release. Cells were grown and collected as described in Fig. 1C. Cells were then stained with pH 3 Ser-10 antibody to score the number of cells at the G₂ and different phases of mitosis. Error bars represent S.E. from three independent experiments. E, representative image of anaphase defects as observed in NS and FBXO31KD cells expressing either vector or WT FBXO31 or cyclin A-binding defective FBXO31 mutant or F-box motif–deleted FBXO31 mutant following 12 h of hydroxyurea release. Arrow (white) indicates cells are either depleted for FBXO31 or expressing cyclin A degradation-defective FBXO31 mutant or F-box motif–deleted FBXO31 mutant, and arrow (yellow) indicates cells are having WT FBXO31 expression. F, graphical representation of percentage of lagging and bridging chromosomes in NS and FBXO31KD cells expressing either vector or WT FBXO31 or cyclin A-binding defective FBXO31 mutant or F-box motif–deleted FBXO31 mutant following 12 h of hydroxyurea release. Cells were grown and collected as described in Fig. 1C and were stained with pH 3 Ser-10 antibody to score the number of anaphase cells and calculated the percentage of cells with bridging and lagging chromosomes. Error bars represent S.E. from three independent experiments. Error bars represents S.E. from three independent experiments, and n.s. represents nonsignificant; *, p ≤ 0.05; **, p < 0.01; ***, p < 0.001.

Figure 8.

Proposed model depicts the role of FBXO31 in coordinated cell-cycle progression. Polyubiquitination of cyclin A by FBXO31 facilitates its degradation during mitosis and G₁ phase thus maintaining low cyclin A–CDK activity during G₁. This environment is favorable for MCM complex loading and licensing of origins and efficient DNA replication. Cells lacking FBXO31 accumulate cyclin A during mitosis and throughout G₁ phase resulting in high cyclin A–CDK activity, which promotes dissociation of MCM complex from origins and hence hampers origin licensing. High level of cyclin A being a rate-limiting factor for S-phase progression stimulates S-phase progression with reduced number of licensed origins resulting in inefficient DNA replication and DNA damage. Segregation of damaged DNA during mitosis results in genomic instability in the form of lagging and bridging chromosomes.