REPORT Cell Cycle 14:24, 3929--3938; December 15, 2015; © 2015 Taylor & Francis Group, LLC
Rev7/Mad2B plays a critical role in the assembly of a functional mitotic spindle Audesh Bhat1, Zhaojia Wu1, Veronica M Maher2, J Justin McCormick2, and Wei Xiao1,3,* 1
Department of Microbiology and Immunology; University of Saskatchewan; Saskatchewan, Canada; 2Carcinogenesis Laboratory; Michigan State University; East Lansing, MI USA; 3 College of Life Sciences; Capital Normal University; Beijing, China
Keywords: aneuploidy, Mad2, mitosis, Rev7/Mad2B, RAN, spindle assembly checkpoint
The spindle assembly checkpoint (SAC) acts as a guardian against cellular threats that may lead to chromosomal missegregation and aneuploidy. Mad2, an anaphase-promoting complex/cyclosome-Cdc20 (APC/CCdc20) inhibitor, has an additional homolog in mammals known as Mad2B, Mad2L2 or Rev7. Apart from its role in Polz-mediated translesion DNA synthesis and double-strand break repair, Rev7 is also believed to inhibit APC/C by negatively regulating Cdh1. Here we report yet another function of Rev7 in cultured human cells. Rev7, as predicted earlier, is involved in the formation of a functional spindle and maintenance of chromosome segregation. In the absence of Rev7, cells tend to arrest in G2/M-phase and display increased monoastral and abnormal spindles with misaligned chromosomes. Furthermore, Rev7-depleted cells show Mad2 localization at the kinetochores of metaphase cells, an indicator of activated SAC, coupled with increased levels of Cyclin B1, an APCCdc20 substrate. Surprisingly unlike Mad2, depletion of Rev7 in several cultured human cell lines did not compromise SAC activity. Our data therefore suggest that besides its role in APC/CCdh1 inhibition, Rev7 is also required for mitotic spindle organization and faithful chromosome segregation most probably through its physical interaction with RAN.
Introduction A faithful distribution of chromosomes between the daughter cells is indispensable to prevent aneuploidy, a condition associated with many cancers.1 Following the assembly of a functional bipolar mitotic spindle with all the chromosomes properly attached and aligned at the metaphase plate, the spindle assembly checkpoint (SAC) becomes satisfied, allowing metaphase cells to enter anaphase.2 In the event of a misaligned chromosome(s) or defective mitotic spindle, SAC-mediated mitotic arrest is enforced through the sequestration of Cdc20, an anaphase-promoting complex/cyclosome (APC/C) activator. This in turn prevents Cyclin B1 degradation, thereby preventing the cells from entering anaphase.3 Through another pathway, the premature exit from metaphase is prevented by the regulatory effect of nucleocytoplasmic transport factors Rae1 and Nup98 on APC/CCdh1, thereby inhibiting the untimely degradation of securin.4,5 The SAC-mediated safeguard is considered vital for faithful segregation of chromosomes and hence has attracted tremendous attention in the past to understand the molecular mechanism especially in reference to cancer.1,6-8 It is now widely accepted that activated SAC inhibits APC/CCdc20 through a coordinated effort of several proteins including Mad1, Mad2, BubR1, Bub1, Bub3 and
Mps1, in which a closed form of Mad2 (c-Mad2) and BubR1 bind to Cdc20 thereby preventing its association with APC/C.7 However, some reports also suggest that BubR1 is the main player with Mad2 acting as a facilitator for the BubR1-Cdc20 association.9-11 The human REV7 gene was originally identified based on its sequence homology with the budding yeast REV7.12 Rev7 was subsequently found to interact with the Rev3 catalytic subunit of DNA polymerase z (Polz) as well as Y-family polymerase Rev1.12-14 Due to 48% sequence similarity to Mad2, Rev7 is also known as mitotic arrest deficient protein 2 B (Mad2B) or Mad2L2.15 This molecular similarity was further supported through in vitro functional studies more than a decade ago showing that Mad2B inhibits APC/C by binding to Cdc20 and Cdh116,17 and more recently in an in vivo study showing that Mad2B specifically blocks Cdh1.18 In addition to APC/C inhibition and the well-established translesion DNA synthesis (TLS) activity of Polz, several recent reports have shown that Rev7 interacts with an array of diverse proteins unrelated to TLS or APC/C, including Ets-like transcription factor (ELK-1), hepatocellular carcinoma-associated gene 2 (HCCA2), Ras-related nuclear protein (RAN), T cell factor 4 (TCF4), clathrin light chain A (CLTA) and single-minded 2 (Sim2),19-24 suggesting that Rev7 plays multiple roles either in response to DNA
*Correspondence to: Wei Xiao; Email: [email protected] Submitted: 07/10/2015; Revised: 10/24/2015; Accepted: 11/12/2015 http://dx.doi.org/10.1080/15384101.2015.1120922
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Results Subcellular localization of Rev7 We previously observed an increased expression and distinct subcellular localization of Rev7 during metaphase of the cell cycle.28 This led us to suspect that Rev7 plays a role in mitosis distinct from its TLS function. This is in agreement with a previous report that Rev7 interacts with a RAN GTPase,21 an essential component of a bipolar spindle formation,25,26 thereby having a possible role in the spindle formation. To first investigate the precise localization of Rev7 in mitotic cells, we performed double immunostaining with rabbit antib-tubulin and mouse anti-Rev7 antibodies in HeLa cells. As shown in Figure 1, Rev7 is highly concentrated around the metaphase plate and co-localized with mitotic spindles. Although this localization is in agreement with a previous report,21 our data indicate that Rev7 is actually around instead of being confined to the spindle structure. The Rev7 and Figure 1. Subcellular localization of Rev7. Co-immunostaining images of HeLa cells showing the subRAN interaction was confirmed in a cellular localization of Rev7 during metaphase and anaphase stages of the cell cycle with respect to pull-down assay (See Fig. S3A), consisthe spindle. During metaphase Rev7 looks to be concentrated around the metaphase plate as tent with an early report.21 The specificdetected with a mouse anti-Rev7 antibody, and co-localizes with the spindle as detected with rabbit anti-b-tubulin (top panel). During early anaphase there is an increased Rev7 staining between the ity of the Rev7 antibody used in this anaphase plates (middle panel) and in late anaphase Rev7 staining is reduced and more uniform study was validated by various means as (bottom panel). described previously.28 The subcellular distribution of Rev7 was also confirmed in HCT116 cells and by using a previdamage19 or during cell cycle progression that are not necessarily ously-validated rabbit anti-Rev7 antibody 29 (data not shown). related to APC/C inhibition. Its interaction with RAN throughout the cell cycle, especially during metaphase 19 is of particular interest since GTP-bound RAN plays a key role in spindle assemDepletion of Rev7 causes G2/M arrest whereas Mad2 or bly.25-27 RAN is a member of the Ras superfamily, which when RAN depletion causes cell death active (RAN-GTP), forms a concentration gradient with higher In order to understand the cellular functions of Rev7 that are levels of RAN-GTP in the nucleus during interphase and around independent of TLS, we established a highly-efficient Rev7 the metaphase plate during metaphase to assist in nucleocytoplas- depletion protocol (Fig. S1A). Following siRev7 treatment for mic transport and spindle assembly, respectively.25,27 However 48 hrs, there was an apparent increase in the number of round, the biological relevance of the Rev7-RAN interaction with presumably metaphase cells, which became more obvious after respect to the mitotic functions remains to be illustrated. 72 hrs of treatment. In contrast, the percentage of round cells in Here we report that Rev7 plays a role in spindle assembly, the mock- and control-treated cells remained constant (Fig. 2Amostly probably through its association with RAN. Loss of B). To determine the biological nature of the round cells, we perRev7 induces Mad2-mediated mitotic arrest with increased formed flow cytometeric analysis, which confirmed that the frequency of abnormal spindles, misaligned chromosomes and round cells are in fact metaphase cells but not dead cells. The peraccumulation of Cyclin B1. Furthermore, unlike Mad2, Rev7 centage of G2/M-phase cells was significantly higher in the depletion does not affect SAC activity and its long-term siRev7-treated compared to the control-treated cells (Fig. 2C-D). depletion results in aneuploidy. Experimental depletion of The G2/M arrest after siRev7 treatment was further confirmed in RAN shows mitotic phenotypes comparable to Rev7 deple- NF1604 and HCT116 cells albeit at a different frequency tion, and Rev7 depletion appears to affect RAN distribution (Fig. S2). In sharp contrast, cells treated with siRev3 did not during mitosis. These data indicate that Rev7 has a mitotic show such a G2/M arrest (Fig. 2C-D), indicating that Rev7function distinct from APC/C inhibition. mediated G2/M activity is distinct from its TLS function.
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Figure 2. Rev7 depletion causes mitotic arrest in HeLa cells. (A) Representative images showing increased number of round cells (red arrowheads) in siRev7-treated cells after 72 hrs of treatment. (B) Quantitative analysis of the percentage of round cells in mock-, siCTRL- and siRev7-treated cells 72 hrs post-transfection. Results are the average of 3 experiments with approximately 500 cells in each experiment. Round cells were considered to be mitotic cells and the rest to be other phases of the cell cycle. (C) Cell-cycle distribution as determined by flow cytometry after 72 hrs of treatment with indicated siRNAs. siMad2 cells were analyzed after 48 hrs of treatment. (D) Percentage distribution of cells in different phases of the cell cycle averaged from 3 experiments as analyzed from the flow cytometry data in (C). (E) Representative images of DAPI-stained cells with Rev7, RAN or Mad2 depletion showing different types of nuclear abnormalities such as nuclear constrictions (siRAN), nuclear fragmentation (siMad2), interphase bridge and micronuclei formation (siMad2). siRAN images were captured at 36 hrs post-transfection, siMad2 at 48 hrs post-transfection and siRev7 at 72 hrs post-transfection. (F) Quantitative analysis of various nuclear abnormalities observed after the siRNA treatment. I-bridges (Interphase bridges), N-fragmentation (Nuclear fragmentation). Error bars represent standard deviation. *p < 0 .05, **p < 0 .005, ***p < 0 .0005 vs. siCTRL.
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Figure 3. Rev7 and RAN depletion causes abnormal mitotic spindle formation and chromosome misalignment in HeLa cells. (A) Representative images of b-tubulin and pericentrin co-stained cells showing monoastral and multiastral spindles. (B) Quantitative analysis showing the percentage of metaphase cells carrying monoastral and multiastral spindles in 72-h siCTRL- and siRev7-treated samples. (C) Quantitative analysis of the percentage of metaphase cells carrying monoastral and multiastral spindles after 36-h treatment of siRev7 or siRAN. (D) Representative images of b-tubulin-stained cells showing normally-aligned (top panel), mildly-misaligned (middle panel) and severely-misaligned (bottom panel) chromosomes at the metaphase plate. (E) Quantitative analysis of the percentage of metaphase cells carrying misaligned chromosomes after 36 hrs of siRNA treatment. (F) Quantitative analysis of the percentage of metaphase cells carrying misaligned chromosomes after 72 hrs of siRev7 treatment. Error bars represent standard deviation from 3 independent experiments for all figures except B, which were calculated from 6 experiments. *p < 0 .05, **p < 0 .005, ***p < 0 .0005 vs. siCTRL.
In this study, we also monitored cellular phenotypes after Mad2 or RAN depletion. While depletion of Mad2 was rather effective (Fig. S1B), we were unable to achieve a highly-efficient RAN depletion despite using 3 different siRNAs at varying concentrations and in different cells lines. The specificity of Mad2 antibodies used in this study was verified by overexpressing
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pCDNA-Mad2 vector in HCT116 cells (Fig. S1C). All three RAN siRNAs resulted in approximately 50% depletion (Fig. S1D). Nevertheless, treatment of HeLa cells with siMad2 or siRAN for 48 hrs resulted in extensive cell death. The apparent difference between Rev7 and RAN depletion was that cells died 48 hrs after siRAN treatment but no immediate cell death was
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when there was no apparent cell death and the knockdown efficiency was approximately 50%. Interestingly, treatment with all 3 siRANs resulted in some increase in monoastral phenotype but no change in multiastral spindles (Fig. 3C). Since an abnormal spindle can cause chromosomal misalignment at the metaphase plate, we next wished to quantify chromosome misalignment in siRev7-treated cells and compare it with siRAN-treated cells. As shown in Figure 3D-F, there was a significant increase in the number of misaligned metaphases ranging from minor to severely misaligned in both siRev7- and siRAN-treated cells, suggesting that Rev7 and RAN may function in the same pathway during mitosis. Although Rev7 indeed physically interacts with RAN in vivo (Fig. S3A), depletion of Rev7 did not alter total levels of cellular RAN Figure 4. HeLa cells depleted of Rev7 or Mad2 respond differently to nocodazole-induced G2/M cell (Fig. S3B). However, subcellular discycle arrest. (A) Western blots showing partial and complete depletion of Mad2 using different concentrations of siMad2. b-tubulin was used as an internal control. (B) Representative images taken from cells tribution of RAN in metaphase cells treated with siRev7 or 6.5 nM siMad2 for 48 hrs followed by treatment with 200 ng/ml nocodazole for an appeared to be affected. Interestingly, additional 24 hrs before taking photographs. (C) Quantitative analysis of the percentage of interphase almost all metaphase cells with mis(flat) and metaphase (round) cells in different treatment groups averaged from 2 experiments with aligned chromosomes showed abnorapproximately 300 cells in each experiment. mal RAN distribution around the chromatin in comparison to normal seen in siRev7-treated cells even after 72-hr treatment (Fig. 2C, metaphases (Fig. S3C-D). Unfortunately, due to technical diffiE). The siMad2-associated cell death was distinct from siRAN, as culties we were unable to observe the reported RAN-GTP conloss of Mad2 caused multiple cellular defects including micronu- centration gradient in the metaphase cells 25 and hence cannot clei formation, nuclear fragmentation and interphase bridges conclude whether and how Rev7 functions to maintain such a (Fig. 2F) but siRAN resulted in a deformed nucleus.(Fig. 2E) gradient. Both Rev7 and RAN are essential for proper spindle formation To further look into possible causes for the observed G2/M arrest, we examined the mitotic spindle for possible defects based on our observations and previous reports.21,24 As shown in Figure. 3A-C, depletion of Rev7 results in a substantial increase in the number of monoastral spindles with duplicate centrosomes and some increase in the number of multiastral and abnormal spindles. The most significant increase in monoastral (threefold) and multiastral (twofold) spindles was observed 72 hrs after siRev7 transfection (Fig. 3B). Other than the reported physical interaction between Rev7 and RAN,21 the functional importance of this interaction remains unknown. To address functional relevance between Rev7 and RAN, we depleted RAN from HeLa cells using 3 different siRANs and compared the phenotypes with Rev7-depleted cells. Since all our RAN siRNAs caused cell death at 48 hrs post-transfection, we were unable to look for a similar phenotype at the 72-hr time point. Instead, we observed some comparable phenotypes 36 hrs after siRNA transfection
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Rev7 depletion leads to Mad2 activation A recent study using U2OS cells indicates that Rev7 specifically sequesters Cdh1, thereby preventing early activation of APC/CCdh1.18 In this case, the Rev7-deficient cells exit mitosis faster than control cells after release from nocodazole arrest. In contrast we observed an apparent G2/M arrest in HeLa cells after Rev7 depletion (Fig. 2A-D), a phenomenon that was also observed in HCT116 and lung fibroblast NF1604 cells (Fig. S2). Our choice of HeLa cells was based on their extensive use in SAC-related studies and a report showing that these cells have a robust SAC.30 We have previously shown that the Rev7 siRNA used in this study did not cause any change in the Mad2 protein levels.28 To further rule out any SAC-related activity like its Mad2 homolog, we examined the effect of siRev7 on nocodazole-induced cell cycle arrest. Since complete depletion of Mad2 results in cell death, we managed a partial and transient depletion of Mad2 by using siRNA at a low concentration (6.5 nM) (Fig. 4) to mimic haplo-insufficient Mad2 cells known to fail to arrest after nocodazole treatment.31 Indeed the partial depletion
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Cyclin B1 accumulation at selected time points in mitotic shake-off cells collected after release from RO-0303 (Cdk1 inhibitor) block (Fig. 5C). The above observations collectively indicate that the mitotic arrest caused by Rev7 depletion is at least partially due to the activation of Mad2 checkpoint. Loss of Rev7 causes aneuploidy but not premature segregation In order to further delineate Mad2 and Rev7 functions, we looked at the premature sister chromatid segregation defect in Rev7-depleted cells, a well-recognized phenotype of Mad2-depletion.31 As shown in Figure 6A-B, a significantly higher percentage of metaphases had premature segregation and DNA double-strand breaks in the Mad2-depleted HCT116 cells than expected; however, no such an increase was observed in the Rev7-depleted Figure 5. Rev7 depletion induces Mad2 focus formation and Cyclin B1 accumulation in HeLa cells. (A) Representative images of Mad2- and b-tubulin-immunostained cells showing a normal metaphase cell with no cells when compared to the Mad2 foci (left panel) and a misaligned metaphase cell with Mad2 foci and an abnormal spindle (right panel). siCTRL cells. Since Rev7(B) Quantitative analysis of metaphase cells positive for Mad2 foci 72 hrs after siRev7 treatment. (C) Western depleted cells display an apparent blots showing increased levels of Cyclin B1 after Rev7 depletion at 0 hr post-release from RO-3306 block and misalignment (Fig. 3F) and lagdelayed degradation at 45 and 96 minutes post-release when compared to siCTRL treated cells. Mad2 levels ging chromosome defects,28 an remained unchanged. Actin was used as an internal control. Error bars represent standard deviation from 3 experiments. **p < 0.005 vs. siCTRL. obvious prediction is that loss of Rev7 could cause aneuploidy. To address this possibility, we first of Mad2 was sufficient to interfere with its SAC activity in examined transiently-transfected cells using siRNAs and did not response to the nocodazole treatment (Fig. 4B-C). In comparifind an apparent difference in aneuploidy between the treatment son, HeLa cells depleted of Rev7 and treated with nocodazole for groups (data not shown). Next, we counted metaphase chromo24 and 48 hrs arrested normally like siCTRL-treated cells somes in 2 stable Rev7-depleted MSU1.1 cell lines (Clone 2.2 (Fig. 4B-C). We also examined nocodazole-induced G2/M arrest and 2.6) (Fig. 6C) 29 to see if a long-term depletion causes aneuin HCT116 cells and did not notice alteration of mitotic index ploidy. Surprisingly, both clones showed a significant increase in in Rev7-depleted cells (Fig. S4). the number of metaphases with 42 chromosomes as compared Although Rev7 depletion causes increased mitotic cells with to vector control cells (Fig. 6D-E). Consistently, in a freshly-genmonoastral spindle that may lead to mitotic arrest, the defective erated stable Rev7-depleted HCT116 cell line (shRev7-C4), an bipolar spindles with misaligned chromosomes may cause SAC increase in the number of metaphases with fewer than normal activation that may also lead to mitotic arrest. During SAC actinumber of chromosomes was observed in comparison to the vecvation, Mad2 localizes at the unattached kinetochores during tor-transfected cells after 16 passages post-selection (Fig. S5). metaphase.32 We performed immunostaining using a rabbit antiMad2 antibody to look for Mad2 focus formation. Metaphases with misaligned chromosomes showed Mad2 foci with signifiDiscussion cantly higher frequency in siRev7-treated cells than in siCTRL cells (Fig. 5A-B), indicating that loss of Rev7 in turn results in The functional complexity of Rev7/Mad2B in human cells has the activation of SAC. It has been previously reported that acti- just stared to unravel. Besides its role in TLS, Rev7 was initially vated SAC causes an increase in the Cyclin B1 levels through believed to be a SAC protein like Mad215; however, it was later APC/CCdc20 inhibition.3 As expected, Rev7 depletion resulted in described as an APC/C inhibitor but surprisingly not a SAC
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around the metaphase spindle. It is unlikely that the discrepancy was due to our experimental variations as we used 2 independent anti-Rev7 antibodies and 3 different cell lines. Third, experimental depletion of either Rev7 or RAN results in increased monoastral cells and cells with abnormal spindles and misaligned chromosomes, indicating a defect in the spindle assembly. Assembly of a bipolar spindle can happen in the absence of a centrosome but requires the RAN-GTP gradient.35 The unique Rev7 localization pattern in mitotic cells and its physical interaction with RAN led us to hypothesize that Rev7 is involved in the maintenance of the RAN-GTP gradient. Although experimental limitations did not allow us to test the RAN-GTP gradient around the metaphase plate directly, depletion of Rev7 appears to cause redistribution of RAN around the chromatin throughout metaphase without affecting the total cellular level of RAN. Based on these data we envisage that when there is no nuclear membrane to maintain the RAN-GTP gradient in metaphase cells, Rev7 play a critical role in the recruitment and/or retention of RAN-GTP toward the metaphase plate. In this aspect, Rev7 can be considered as Figure 6. Mad2 depletion causes premature segregation and DNA damage whereas long-term deplean accessory protein of RAN-GTP. tion of Rev7 causes aneuploidy. (A) Representative images of HCT116 metaphase spread showing a A role of Rev7 in mediating RAN normal metaphase in siCTRL- and siRev7-treated cells but premature sister chromatid segregation and spindle assembly does not rule out (red arrow heads) and chromosome breaks (yellow arrow heads) in siMad2-treated cells 48 hrs postits possible involvement in the mitotic transfection. (B) Quantitative analysis of premature segregation (Pre-segregation) and chromosomal spindle checkpoint, particularly given its breaks in images taken from (A). Error bars represent standard deviations from 3 experiments. **p < 0 .005, ***p
Rev7/Mad2B plays a critical role in the assembly of a functional mitotic spindle Audesh Bhat1, Zhaojia Wu1, Veronica M Maher2, J Justin McCormick2, and Wei Xiao1,3,* 1
Department of Microbiology and Immunology; University of Saskatchewan; Saskatchewan, Canada; 2Carcinogenesis Laboratory; Michigan State University; East Lansing, MI USA; 3 College of Life Sciences; Capital Normal University; Beijing, China
Keywords: aneuploidy, Mad2, mitosis, Rev7/Mad2B, RAN, spindle assembly checkpoint
The spindle assembly checkpoint (SAC) acts as a guardian against cellular threats that may lead to chromosomal missegregation and aneuploidy. Mad2, an anaphase-promoting complex/cyclosome-Cdc20 (APC/CCdc20) inhibitor, has an additional homolog in mammals known as Mad2B, Mad2L2 or Rev7. Apart from its role in Polz-mediated translesion DNA synthesis and double-strand break repair, Rev7 is also believed to inhibit APC/C by negatively regulating Cdh1. Here we report yet another function of Rev7 in cultured human cells. Rev7, as predicted earlier, is involved in the formation of a functional spindle and maintenance of chromosome segregation. In the absence of Rev7, cells tend to arrest in G2/M-phase and display increased monoastral and abnormal spindles with misaligned chromosomes. Furthermore, Rev7-depleted cells show Mad2 localization at the kinetochores of metaphase cells, an indicator of activated SAC, coupled with increased levels of Cyclin B1, an APCCdc20 substrate. Surprisingly unlike Mad2, depletion of Rev7 in several cultured human cell lines did not compromise SAC activity. Our data therefore suggest that besides its role in APC/CCdh1 inhibition, Rev7 is also required for mitotic spindle organization and faithful chromosome segregation most probably through its physical interaction with RAN.
Introduction A faithful distribution of chromosomes between the daughter cells is indispensable to prevent aneuploidy, a condition associated with many cancers.1 Following the assembly of a functional bipolar mitotic spindle with all the chromosomes properly attached and aligned at the metaphase plate, the spindle assembly checkpoint (SAC) becomes satisfied, allowing metaphase cells to enter anaphase.2 In the event of a misaligned chromosome(s) or defective mitotic spindle, SAC-mediated mitotic arrest is enforced through the sequestration of Cdc20, an anaphase-promoting complex/cyclosome (APC/C) activator. This in turn prevents Cyclin B1 degradation, thereby preventing the cells from entering anaphase.3 Through another pathway, the premature exit from metaphase is prevented by the regulatory effect of nucleocytoplasmic transport factors Rae1 and Nup98 on APC/CCdh1, thereby inhibiting the untimely degradation of securin.4,5 The SAC-mediated safeguard is considered vital for faithful segregation of chromosomes and hence has attracted tremendous attention in the past to understand the molecular mechanism especially in reference to cancer.1,6-8 It is now widely accepted that activated SAC inhibits APC/CCdc20 through a coordinated effort of several proteins including Mad1, Mad2, BubR1, Bub1, Bub3 and
Mps1, in which a closed form of Mad2 (c-Mad2) and BubR1 bind to Cdc20 thereby preventing its association with APC/C.7 However, some reports also suggest that BubR1 is the main player with Mad2 acting as a facilitator for the BubR1-Cdc20 association.9-11 The human REV7 gene was originally identified based on its sequence homology with the budding yeast REV7.12 Rev7 was subsequently found to interact with the Rev3 catalytic subunit of DNA polymerase z (Polz) as well as Y-family polymerase Rev1.12-14 Due to 48% sequence similarity to Mad2, Rev7 is also known as mitotic arrest deficient protein 2 B (Mad2B) or Mad2L2.15 This molecular similarity was further supported through in vitro functional studies more than a decade ago showing that Mad2B inhibits APC/C by binding to Cdc20 and Cdh116,17 and more recently in an in vivo study showing that Mad2B specifically blocks Cdh1.18 In addition to APC/C inhibition and the well-established translesion DNA synthesis (TLS) activity of Polz, several recent reports have shown that Rev7 interacts with an array of diverse proteins unrelated to TLS or APC/C, including Ets-like transcription factor (ELK-1), hepatocellular carcinoma-associated gene 2 (HCCA2), Ras-related nuclear protein (RAN), T cell factor 4 (TCF4), clathrin light chain A (CLTA) and single-minded 2 (Sim2),19-24 suggesting that Rev7 plays multiple roles either in response to DNA
*Correspondence to: Wei Xiao; Email: [email protected] Submitted: 07/10/2015; Revised: 10/24/2015; Accepted: 11/12/2015 http://dx.doi.org/10.1080/15384101.2015.1120922
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Results Subcellular localization of Rev7 We previously observed an increased expression and distinct subcellular localization of Rev7 during metaphase of the cell cycle.28 This led us to suspect that Rev7 plays a role in mitosis distinct from its TLS function. This is in agreement with a previous report that Rev7 interacts with a RAN GTPase,21 an essential component of a bipolar spindle formation,25,26 thereby having a possible role in the spindle formation. To first investigate the precise localization of Rev7 in mitotic cells, we performed double immunostaining with rabbit antib-tubulin and mouse anti-Rev7 antibodies in HeLa cells. As shown in Figure 1, Rev7 is highly concentrated around the metaphase plate and co-localized with mitotic spindles. Although this localization is in agreement with a previous report,21 our data indicate that Rev7 is actually around instead of being confined to the spindle structure. The Rev7 and Figure 1. Subcellular localization of Rev7. Co-immunostaining images of HeLa cells showing the subRAN interaction was confirmed in a cellular localization of Rev7 during metaphase and anaphase stages of the cell cycle with respect to pull-down assay (See Fig. S3A), consisthe spindle. During metaphase Rev7 looks to be concentrated around the metaphase plate as tent with an early report.21 The specificdetected with a mouse anti-Rev7 antibody, and co-localizes with the spindle as detected with rabbit anti-b-tubulin (top panel). During early anaphase there is an increased Rev7 staining between the ity of the Rev7 antibody used in this anaphase plates (middle panel) and in late anaphase Rev7 staining is reduced and more uniform study was validated by various means as (bottom panel). described previously.28 The subcellular distribution of Rev7 was also confirmed in HCT116 cells and by using a previdamage19 or during cell cycle progression that are not necessarily ously-validated rabbit anti-Rev7 antibody 29 (data not shown). related to APC/C inhibition. Its interaction with RAN throughout the cell cycle, especially during metaphase 19 is of particular interest since GTP-bound RAN plays a key role in spindle assemDepletion of Rev7 causes G2/M arrest whereas Mad2 or bly.25-27 RAN is a member of the Ras superfamily, which when RAN depletion causes cell death active (RAN-GTP), forms a concentration gradient with higher In order to understand the cellular functions of Rev7 that are levels of RAN-GTP in the nucleus during interphase and around independent of TLS, we established a highly-efficient Rev7 the metaphase plate during metaphase to assist in nucleocytoplas- depletion protocol (Fig. S1A). Following siRev7 treatment for mic transport and spindle assembly, respectively.25,27 However 48 hrs, there was an apparent increase in the number of round, the biological relevance of the Rev7-RAN interaction with presumably metaphase cells, which became more obvious after respect to the mitotic functions remains to be illustrated. 72 hrs of treatment. In contrast, the percentage of round cells in Here we report that Rev7 plays a role in spindle assembly, the mock- and control-treated cells remained constant (Fig. 2Amostly probably through its association with RAN. Loss of B). To determine the biological nature of the round cells, we perRev7 induces Mad2-mediated mitotic arrest with increased formed flow cytometeric analysis, which confirmed that the frequency of abnormal spindles, misaligned chromosomes and round cells are in fact metaphase cells but not dead cells. The peraccumulation of Cyclin B1. Furthermore, unlike Mad2, Rev7 centage of G2/M-phase cells was significantly higher in the depletion does not affect SAC activity and its long-term siRev7-treated compared to the control-treated cells (Fig. 2C-D). depletion results in aneuploidy. Experimental depletion of The G2/M arrest after siRev7 treatment was further confirmed in RAN shows mitotic phenotypes comparable to Rev7 deple- NF1604 and HCT116 cells albeit at a different frequency tion, and Rev7 depletion appears to affect RAN distribution (Fig. S2). In sharp contrast, cells treated with siRev3 did not during mitosis. These data indicate that Rev7 has a mitotic show such a G2/M arrest (Fig. 2C-D), indicating that Rev7function distinct from APC/C inhibition. mediated G2/M activity is distinct from its TLS function.
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Figure 2. Rev7 depletion causes mitotic arrest in HeLa cells. (A) Representative images showing increased number of round cells (red arrowheads) in siRev7-treated cells after 72 hrs of treatment. (B) Quantitative analysis of the percentage of round cells in mock-, siCTRL- and siRev7-treated cells 72 hrs post-transfection. Results are the average of 3 experiments with approximately 500 cells in each experiment. Round cells were considered to be mitotic cells and the rest to be other phases of the cell cycle. (C) Cell-cycle distribution as determined by flow cytometry after 72 hrs of treatment with indicated siRNAs. siMad2 cells were analyzed after 48 hrs of treatment. (D) Percentage distribution of cells in different phases of the cell cycle averaged from 3 experiments as analyzed from the flow cytometry data in (C). (E) Representative images of DAPI-stained cells with Rev7, RAN or Mad2 depletion showing different types of nuclear abnormalities such as nuclear constrictions (siRAN), nuclear fragmentation (siMad2), interphase bridge and micronuclei formation (siMad2). siRAN images were captured at 36 hrs post-transfection, siMad2 at 48 hrs post-transfection and siRev7 at 72 hrs post-transfection. (F) Quantitative analysis of various nuclear abnormalities observed after the siRNA treatment. I-bridges (Interphase bridges), N-fragmentation (Nuclear fragmentation). Error bars represent standard deviation. *p < 0 .05, **p < 0 .005, ***p < 0 .0005 vs. siCTRL.
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Figure 3. Rev7 and RAN depletion causes abnormal mitotic spindle formation and chromosome misalignment in HeLa cells. (A) Representative images of b-tubulin and pericentrin co-stained cells showing monoastral and multiastral spindles. (B) Quantitative analysis showing the percentage of metaphase cells carrying monoastral and multiastral spindles in 72-h siCTRL- and siRev7-treated samples. (C) Quantitative analysis of the percentage of metaphase cells carrying monoastral and multiastral spindles after 36-h treatment of siRev7 or siRAN. (D) Representative images of b-tubulin-stained cells showing normally-aligned (top panel), mildly-misaligned (middle panel) and severely-misaligned (bottom panel) chromosomes at the metaphase plate. (E) Quantitative analysis of the percentage of metaphase cells carrying misaligned chromosomes after 36 hrs of siRNA treatment. (F) Quantitative analysis of the percentage of metaphase cells carrying misaligned chromosomes after 72 hrs of siRev7 treatment. Error bars represent standard deviation from 3 independent experiments for all figures except B, which were calculated from 6 experiments. *p < 0 .05, **p < 0 .005, ***p < 0 .0005 vs. siCTRL.
In this study, we also monitored cellular phenotypes after Mad2 or RAN depletion. While depletion of Mad2 was rather effective (Fig. S1B), we were unable to achieve a highly-efficient RAN depletion despite using 3 different siRNAs at varying concentrations and in different cells lines. The specificity of Mad2 antibodies used in this study was verified by overexpressing
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pCDNA-Mad2 vector in HCT116 cells (Fig. S1C). All three RAN siRNAs resulted in approximately 50% depletion (Fig. S1D). Nevertheless, treatment of HeLa cells with siMad2 or siRAN for 48 hrs resulted in extensive cell death. The apparent difference between Rev7 and RAN depletion was that cells died 48 hrs after siRAN treatment but no immediate cell death was
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when there was no apparent cell death and the knockdown efficiency was approximately 50%. Interestingly, treatment with all 3 siRANs resulted in some increase in monoastral phenotype but no change in multiastral spindles (Fig. 3C). Since an abnormal spindle can cause chromosomal misalignment at the metaphase plate, we next wished to quantify chromosome misalignment in siRev7-treated cells and compare it with siRAN-treated cells. As shown in Figure 3D-F, there was a significant increase in the number of misaligned metaphases ranging from minor to severely misaligned in both siRev7- and siRAN-treated cells, suggesting that Rev7 and RAN may function in the same pathway during mitosis. Although Rev7 indeed physically interacts with RAN in vivo (Fig. S3A), depletion of Rev7 did not alter total levels of cellular RAN Figure 4. HeLa cells depleted of Rev7 or Mad2 respond differently to nocodazole-induced G2/M cell (Fig. S3B). However, subcellular discycle arrest. (A) Western blots showing partial and complete depletion of Mad2 using different concentrations of siMad2. b-tubulin was used as an internal control. (B) Representative images taken from cells tribution of RAN in metaphase cells treated with siRev7 or 6.5 nM siMad2 for 48 hrs followed by treatment with 200 ng/ml nocodazole for an appeared to be affected. Interestingly, additional 24 hrs before taking photographs. (C) Quantitative analysis of the percentage of interphase almost all metaphase cells with mis(flat) and metaphase (round) cells in different treatment groups averaged from 2 experiments with aligned chromosomes showed abnorapproximately 300 cells in each experiment. mal RAN distribution around the chromatin in comparison to normal seen in siRev7-treated cells even after 72-hr treatment (Fig. 2C, metaphases (Fig. S3C-D). Unfortunately, due to technical diffiE). The siMad2-associated cell death was distinct from siRAN, as culties we were unable to observe the reported RAN-GTP conloss of Mad2 caused multiple cellular defects including micronu- centration gradient in the metaphase cells 25 and hence cannot clei formation, nuclear fragmentation and interphase bridges conclude whether and how Rev7 functions to maintain such a (Fig. 2F) but siRAN resulted in a deformed nucleus.(Fig. 2E) gradient. Both Rev7 and RAN are essential for proper spindle formation To further look into possible causes for the observed G2/M arrest, we examined the mitotic spindle for possible defects based on our observations and previous reports.21,24 As shown in Figure. 3A-C, depletion of Rev7 results in a substantial increase in the number of monoastral spindles with duplicate centrosomes and some increase in the number of multiastral and abnormal spindles. The most significant increase in monoastral (threefold) and multiastral (twofold) spindles was observed 72 hrs after siRev7 transfection (Fig. 3B). Other than the reported physical interaction between Rev7 and RAN,21 the functional importance of this interaction remains unknown. To address functional relevance between Rev7 and RAN, we depleted RAN from HeLa cells using 3 different siRANs and compared the phenotypes with Rev7-depleted cells. Since all our RAN siRNAs caused cell death at 48 hrs post-transfection, we were unable to look for a similar phenotype at the 72-hr time point. Instead, we observed some comparable phenotypes 36 hrs after siRNA transfection
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Rev7 depletion leads to Mad2 activation A recent study using U2OS cells indicates that Rev7 specifically sequesters Cdh1, thereby preventing early activation of APC/CCdh1.18 In this case, the Rev7-deficient cells exit mitosis faster than control cells after release from nocodazole arrest. In contrast we observed an apparent G2/M arrest in HeLa cells after Rev7 depletion (Fig. 2A-D), a phenomenon that was also observed in HCT116 and lung fibroblast NF1604 cells (Fig. S2). Our choice of HeLa cells was based on their extensive use in SAC-related studies and a report showing that these cells have a robust SAC.30 We have previously shown that the Rev7 siRNA used in this study did not cause any change in the Mad2 protein levels.28 To further rule out any SAC-related activity like its Mad2 homolog, we examined the effect of siRev7 on nocodazole-induced cell cycle arrest. Since complete depletion of Mad2 results in cell death, we managed a partial and transient depletion of Mad2 by using siRNA at a low concentration (6.5 nM) (Fig. 4) to mimic haplo-insufficient Mad2 cells known to fail to arrest after nocodazole treatment.31 Indeed the partial depletion
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Cyclin B1 accumulation at selected time points in mitotic shake-off cells collected after release from RO-0303 (Cdk1 inhibitor) block (Fig. 5C). The above observations collectively indicate that the mitotic arrest caused by Rev7 depletion is at least partially due to the activation of Mad2 checkpoint. Loss of Rev7 causes aneuploidy but not premature segregation In order to further delineate Mad2 and Rev7 functions, we looked at the premature sister chromatid segregation defect in Rev7-depleted cells, a well-recognized phenotype of Mad2-depletion.31 As shown in Figure 6A-B, a significantly higher percentage of metaphases had premature segregation and DNA double-strand breaks in the Mad2-depleted HCT116 cells than expected; however, no such an increase was observed in the Rev7-depleted Figure 5. Rev7 depletion induces Mad2 focus formation and Cyclin B1 accumulation in HeLa cells. (A) Representative images of Mad2- and b-tubulin-immunostained cells showing a normal metaphase cell with no cells when compared to the Mad2 foci (left panel) and a misaligned metaphase cell with Mad2 foci and an abnormal spindle (right panel). siCTRL cells. Since Rev7(B) Quantitative analysis of metaphase cells positive for Mad2 foci 72 hrs after siRev7 treatment. (C) Western depleted cells display an apparent blots showing increased levels of Cyclin B1 after Rev7 depletion at 0 hr post-release from RO-3306 block and misalignment (Fig. 3F) and lagdelayed degradation at 45 and 96 minutes post-release when compared to siCTRL treated cells. Mad2 levels ging chromosome defects,28 an remained unchanged. Actin was used as an internal control. Error bars represent standard deviation from 3 experiments. **p < 0.005 vs. siCTRL. obvious prediction is that loss of Rev7 could cause aneuploidy. To address this possibility, we first of Mad2 was sufficient to interfere with its SAC activity in examined transiently-transfected cells using siRNAs and did not response to the nocodazole treatment (Fig. 4B-C). In comparifind an apparent difference in aneuploidy between the treatment son, HeLa cells depleted of Rev7 and treated with nocodazole for groups (data not shown). Next, we counted metaphase chromo24 and 48 hrs arrested normally like siCTRL-treated cells somes in 2 stable Rev7-depleted MSU1.1 cell lines (Clone 2.2 (Fig. 4B-C). We also examined nocodazole-induced G2/M arrest and 2.6) (Fig. 6C) 29 to see if a long-term depletion causes aneuin HCT116 cells and did not notice alteration of mitotic index ploidy. Surprisingly, both clones showed a significant increase in in Rev7-depleted cells (Fig. S4). the number of metaphases with 42 chromosomes as compared Although Rev7 depletion causes increased mitotic cells with to vector control cells (Fig. 6D-E). Consistently, in a freshly-genmonoastral spindle that may lead to mitotic arrest, the defective erated stable Rev7-depleted HCT116 cell line (shRev7-C4), an bipolar spindles with misaligned chromosomes may cause SAC increase in the number of metaphases with fewer than normal activation that may also lead to mitotic arrest. During SAC actinumber of chromosomes was observed in comparison to the vecvation, Mad2 localizes at the unattached kinetochores during tor-transfected cells after 16 passages post-selection (Fig. S5). metaphase.32 We performed immunostaining using a rabbit antiMad2 antibody to look for Mad2 focus formation. Metaphases with misaligned chromosomes showed Mad2 foci with signifiDiscussion cantly higher frequency in siRev7-treated cells than in siCTRL cells (Fig. 5A-B), indicating that loss of Rev7 in turn results in The functional complexity of Rev7/Mad2B in human cells has the activation of SAC. It has been previously reported that acti- just stared to unravel. Besides its role in TLS, Rev7 was initially vated SAC causes an increase in the Cyclin B1 levels through believed to be a SAC protein like Mad215; however, it was later APC/CCdc20 inhibition.3 As expected, Rev7 depletion resulted in described as an APC/C inhibitor but surprisingly not a SAC
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around the metaphase spindle. It is unlikely that the discrepancy was due to our experimental variations as we used 2 independent anti-Rev7 antibodies and 3 different cell lines. Third, experimental depletion of either Rev7 or RAN results in increased monoastral cells and cells with abnormal spindles and misaligned chromosomes, indicating a defect in the spindle assembly. Assembly of a bipolar spindle can happen in the absence of a centrosome but requires the RAN-GTP gradient.35 The unique Rev7 localization pattern in mitotic cells and its physical interaction with RAN led us to hypothesize that Rev7 is involved in the maintenance of the RAN-GTP gradient. Although experimental limitations did not allow us to test the RAN-GTP gradient around the metaphase plate directly, depletion of Rev7 appears to cause redistribution of RAN around the chromatin throughout metaphase without affecting the total cellular level of RAN. Based on these data we envisage that when there is no nuclear membrane to maintain the RAN-GTP gradient in metaphase cells, Rev7 play a critical role in the recruitment and/or retention of RAN-GTP toward the metaphase plate. In this aspect, Rev7 can be considered as Figure 6. Mad2 depletion causes premature segregation and DNA damage whereas long-term deplean accessory protein of RAN-GTP. tion of Rev7 causes aneuploidy. (A) Representative images of HCT116 metaphase spread showing a A role of Rev7 in mediating RAN normal metaphase in siCTRL- and siRev7-treated cells but premature sister chromatid segregation and spindle assembly does not rule out (red arrow heads) and chromosome breaks (yellow arrow heads) in siMad2-treated cells 48 hrs postits possible involvement in the mitotic transfection. (B) Quantitative analysis of premature segregation (Pre-segregation) and chromosomal spindle checkpoint, particularly given its breaks in images taken from (A). Error bars represent standard deviations from 3 experiments. **p < 0 .005, ***p
