DNA Damage Response-Independent Role for MDC1 in Maintaining Genomic Stability

Abstract

MDC1 is a central player in checkpoint activation and subsequent DNA repair following DNA damage. Although MDC1 has been studied extensively, many of its known functions, to date, pertain to the DNA damage response (DDR) pathway. Herein we report a novel function of phosphorylated MDC1 that is independent of ATM and DNA damage and is required for proper mitotic progression and maintenance of genomic stability. We demonstrate that MDC1 is an in vivo target of Plk1 and that phosphorylated MDC1 is dynamically localized to nuclear envelopes, centrosomes, kinetochores, and midbodies. Knockdown of MDC1 or abrogation of Plk1 phosphorylation of MDC1 causes a delay of the prometaphase-metaphase transition. It is significant that mice with reduced levels of MDC1 showed an elevated level of spontaneous tumors in aged animals. Our results demonstrate that MDC1 also plays a fundamentally significant role in maintenance of genomic stability through a DDR-independent pathway.

Keywords: MDC1; Plk1; genomic instability; mitosis; phosphorylation.

FIG 1

MDC1 heterozygous mice are cancer prone by a DDR-independent pathway. (A) Representative images of cancers from MDC1^+/− mice. Arrows indicate tumors in various organs. (B) Spontaneous tumor incidence and spectrum in WT (n = 26) and MDC1^+/− (n = 43) mice for up to 60 weeks. (C and D) Survival curves for WT, MDC1^+/−, and MDC1^−/− mice that received a single dose of whole-body gamma radiation with 15 Gy (C) or 5 Gy (D) at the age of 5 to 6 weeks. (E) MDC1^+/+ and MDC1^+/− MEFs were irradiated with 2 Gy gamma radiation, incubated for 6 h, and harvested for immunoblotting (IB) with an antibody against phosphorylated H2AX.

FIG 2

MDC1 reduction causes aneuploidy both in vitro and in vivo. (A) Whole-cell extracts from MDC1^+/+ and MDC1^+/− MEFs were blotted with MDC1 antibody. MDC1 protein levels were reduced by 50% in MDC1^+/− MEFs compared to MDC1^+/+ MEFs. (B) MDC1 insufficiency causes various chromosome segregation errors. (Top) The left image shows a metaphase cell with misaligned chromosomes, while the middle image is an example of an anaphase cell with lagging chromosomes. The right image shows cells with a chromosome bridge. Arrows indicate the various chromosome segregation defects described above. (Bottom) Histograms showing the percentages of cells with various chromosome segregation errors in MDC1-defective cells. (C) Representative chromosome spreading of MDC1^+/+ and MDC1^+/− MEFs, with WT MEFs showing 40 chromosomes. Bars, 10 μm. (D) Increased aneuploidy in MDC1^+/− cells. The percentages of aneuploid cells were calculated from metaphase spreads of MDC1^+/− and MDC1^+/+ MEFs at different passages cultured according to the 3T3 protocol (n = 3 experiments with 50 spreads each). (E) Histogram of chromosome spreads at passage 7. (F and G) MDC1 is required to maintain chromosome stability in vivo. (F) Average aneuploidy in 6-month-old or 10-month-old mouse splenocytes (n = 3 independent experiments with 50 spreads each). (G) Average aneuploidy in 10-month-old mouse peripheral blood cells (n = 3 independent experiments with 50 spreads each). *, P < 0.05.

FIG 3

MDC1 heterozygosity sensitizes transformation of immortalized MEFs. (A) Immortalized MDC1^+/− MEFs promote focus formation. After cells were plated onto 6-cm dishes in triplicate for 14 days, plates were stained with dye and photographed. Data are representative of at least 3 independent experiments using 2 spontaneously immortalized clones. (B) Histogram depicting data from at least 3 independent experiments conducted for panel A. Mean colony numbers ± standard deviations are shown. (C) Immortalized MDC1^+/− MEFs facilitate growth in soft agar. After 3 × 10³ cells per well were grown for 4 weeks, colonies were stained and images were acquired. Arrows indicate tumors. (D) Histogram depicting data from at least 3 independent experiments conducted for panel C. Mean colony numbers ± standard deviations are shown. (E and F) Immortalized MDC1^+/− cells promote tumor-forming potential. Immortalized MDC1^+/+ or MDC1^+/− MEFs (2 × 10⁶) in 100 μl of phosphate-buffered saline (PBS) were injected subcutaneously into immunocompromised NSG mice. Arrows indicate tumors. *, P < 0.05.

FIG 4

MDC1 is required for timely progression through mitosis. (A) HeLa cells were infected with a lentivirus encoding shRNA to deplete MDC1, selected with puromycin, synchronized by use of a double thymidine block protocol (16 h of thymidine treatment, 8 h of release, and a second thymidine treatment for 16 h) to arrest cells at the G₁/S boundary, released for the indicated times, and harvested for fluorescence-activated cell sorting (FACS). MDC1 depletion led to an apparent mitotic progression delay, as indicated by the arrowhead. Numbers at two late time points (10 h and 11 h) show percentages of cell populations with 4n content. As shown in panel C, partial MDC1 depletion was achieved under these conditions. (B) Depletion of MDC1 causes a transient prometaphase delay. The distribution of mitotic stages in control or shMDC1 cells was determined by microscopy. Different submitotic stages were differentiated by DAPI (4′,6-diamidino-2-phenylindole) and α-tubulin staining. A minimum of 100 mitotic cells were counted in three independent experiments. (C) HeLa cells (WT or MDC1 depleted) were treated with nocodazole (Noc) overnight and subjected to a mitotic shake-off protocol to collect prometaphase cells. Cells were then incubated in drug-free medium to allow mitotic exit and harvested at different times for IB. (D) HeLa cells stably expressing GFP-H2B were subjected to MDC1 depletion as described above, followed by time-lapse microscopic analysis to examine mitotic progression of live cells. Bars, 10 μm. (E) Quantification of the time needed from nuclear envelope breakdown to metaphase plate formation of HeLa cells stably expressing GFP-H2B. (F) Knockdown of MDC1 causes the formation of disorganized mitotic spindles. Mitotic spindles (α-tubulin) and chromosomes (DAPI) were detected 11 h after release from the double thymidine block, and cells with disorganized spindles were quantified (n ≥ 1,000). (G) The progression through mitosis of cells stably expressing GFP–α-tubulin was monitored by live-cell microscopy, and typical examples of image sequences are given. Bars, 10 μm. *, P < 0.05.

FIG 5

Plk1 phosphorylates MDC1 T4 in vitro and in vivo. (A) HeLa cells were treated with mimosine, hydroxyurea (HU), and nocodazole (Noc) to arrest cells at the G₁, S, and M phases, respectively, and then harvested for both regular and Phos-tag SDS-PAGE followed by IB. (B) Extracts from randomly growing cells or cells arrested in mitosis by use of nocodazole or paclitaxel (Taxol) were resolved by Phos-tag SDS-PAGE followed by IB. Where indicated, the extract was incubated with λ-phosphatase (λ-PPase) for 30 min at 30°C before electrophoresis. Arrowheads indicate lanes 2 and 3. (C) HeLa cells were transfected with HA-MDC1, treated with nocodazole or paclitaxel for 12 h, and harvested. Where indicated, extracts were incubated with λ-phosphatase before electrophoresis. (D) HeLa cells were transfected with HA-MDC1, treated with nocodazole in the presence of RO-3306, VX-680, SB202190, or BI6727, and harvested. (E) Physical interaction between Plk1 and MDC1. HeLa cells were transfected with Flag-MDC1 for 3 days, treated with mimosine (Mimo.), hydroxyurea, or nocodazole, and harvested for anti-Plk1 immunoprecipitation (IP) followed by IB. (F) Plk1 phosphorylates MDC1 in vitro. After purified Plk1 was incubated with recombinant GST-MDC1 regions in the presence of [γ-³²P]ATP, the reaction mixtures were resolved by SDS-PAGE, stained with Coomassie brilliant blue (Coom.), and detected by autoradiography. F-1 to F-10 are 10 different regions of MDC1. (G) Plk1 targets MDC1 T4. Plk1 was incubated with various GST–MDC1-F-1 mutants as described for panel F. (H) Alignment of MDC1 protein sequences containing the equivalent of T4 in different species. (I) The phospho-T4-MDC1 antibody is specific. Plk1 (WT or kinase-dead K82M mutant) was incubated with GST–MDC1-F-1 (WT or T4A) in the presence of unlabeled ATP, followed by IB with pT4-MDC1 antibody. (J) MDC1 T4 is phosphorylated in vivo. HEK 293T cells were transfected with HA-MDC1 constructs (WT, T4A, and T4D constructs), treated with nocodazole, and harvested for IB. (K) Plk1 but not ATM is responsible for MDC1 T4 phosphorylation during mitosis. HEK 293T cells were depleted of Plk1 with RNAi, treated with nocodazole for 12 h in the absence or presence of caffeine, and harvested for IB. (L) HeLa cells expressing different HA-MDC1 constructs (WT, T4A, and T4E constructs) were treated with nocodazole and harvested for IB. (M) Extracts from asynchronous or nocodazole-arrested HeLa cells expressing different HA-MDC1 constructs were treated with λ-phosphatase, followed by IB.

FIG 6

Subcellular localization of phosphorylated MDC1 during mitosis. (A to C) Exponentially growing HeLa cells were extracted with 1% Triton X-100 at room temperature for 5 min, fixed in 4% paraformaldehyde for 15 min, and stained with the indicated antibodies. While lamin A/C is a marker of the nuclear envelope, γ-tubulin and CENP-A stain centrosomes/spindle poles and centromeres, respectively. Bars, 10 μm. (D) Randomly growing U2OS cells were analyzed as described above. (E) U2OS cells were treated with 50 nM BI2536 for 1 h and then subjected to IF staining.

FIG 7

Plk1-dependent phosphorylation of MDC1 T4 is required for mitotic progression. (A) MDC1 T4 phosphorylation is required for normal mitotic progression. MDC1-depleted HeLa cells were transfected with RNAi-resistant MDC1-WT or MDC1-T4A, synchronized with a double thymidine block, released for different times, and harvested for FACS. Arrows indicate 4N. (B) HeLa cells expressing different forms of RNAi-resistant MDC1 (WT or T4A mutant) were depleted of endogenous MDC1, treated with nocodazole, and subjected to the mitotic shake-off protocol, followed by release into fresh medium for the indicated time. Cells expressing MDC1-T4A showed a much slower mitotic exit than cells expressing MDC1-WT. (C) MDC1 T4 phosphorylation is critical for normal mitotic spindle assembly. HeLa cells treated as described for panel A were released from the double thymidine block for 11 h and harvested for anti-α-tubulin staining to monitor spindle formation. Cells with disorganized spindles were quantified (n ≥ 1,000). Bars, 10 μm. Arrows indicate the difference of α-tubulin. (D) Phosphorylation of MDC1 T4 is required for timely progression of mitosis. HeLa cells treated as described for panel A were analyzed by time-lapse microscopy to monitor the mitotic progression of live cells, and the box-and-whisker plot shows the duration from nuclear envelope breakdown to the onset of anaphase. (E) Plk1 phosphorylation of MDC1 is involved in chromosome stability. HeLa cells treated as described for panel A were cultured for 30 generations, and the proportions of aneuploid cells were quantified (n = 150). The dashed line indicates the aneuploidy percentage of MDC1-WT. *, P < 0.05.