Telomere homologous recombination (HR) is implicated in the ALT pathway1–4
. The increased telomere recombination is ALT-specific and extends the proliferative life of ALT cells5
. HR proteins and telomere associated proteins co-localize with telomeric DNA at ALT-associated promyelocytic leukemia (PML) nuclear bodies (APBs), which are present only in ALT cells6–9
. Additionally, APBs are enriched during the G2 phase of the cell cycle when HR is most active and ALT activity is likely occurring10,11
. MUS81 functions as an endonuclease by cleaving different DNA substrates12–17
and is required for the survival of cells undergoing aberrant replication and recombination12,14,17,18
, suggesting that MUS81 might be a candidate “ALT protein” involved in abnormal telomere recombination of ALT cells. Here, we report that MUS81 specifically associates with telomeres in ALT cells and is essential for ALT cell viability through regulation of telomere recombination.
APBs are usually found in <5% of ALT cells growing asynchronously and are seen as large TRF1 or TRF2 foci that appear much brighter than signals from individual telomeres6–9
. We confirmed the observation that TRF1, TRF2 and telomeric DNA co-localized with PML in APBs of GM847 cells (ALT positive)(Supplementary Information, Fig. S1A
). We next examined the nuclear localization of MUS81 in an asynchronous culture of ALT cells and found that MUS81 specifically formed discrete nuclear foci in APB-positive cells and no MUS81 foci were seen in APB-negative cells. These MUS81 foci co-localized with telomeric DNA and PML in APBs in < 5% of GM847 cells (). The presence of MUS81 in APBs was confirmed in another ALT cell line, U2OS cells (). The foci were specific for MUS81 since depletion of MUS81 led to disappearance of the foci (Supplementary Information, Fig. S1B
). These results indicate that MUS81 is an integral component of APBs in human ALT-positive cells.
Fig. 1 MUS81 localizes to APBs in ALT cells. A. MUS81 co-localizes with telomeres at APBs in GM847 cells. Cells were processed for immunofluoresence and telomeric DNA-FISH or for double immunofluoresence with MUS81 (anti-mouse) and PML antibodies. B. MUS81 co-localizes (more ...)
To determine whether the association of MUS81 with APBs is cell-cycle dependent, we examined MUS81 foci formation in the different phases of cell cycle. GM847 cells synchronized at the G1/S boundary by a double thymidine block were released into the cell cycle and then fixed at specific time points post-release. FACS analysis confirmed cell cycle distributions (Supplementary Information, Fig. S1C
). Consistent with previous reports10,11
, <5% of GM847 cells displayed APBs during G1/S and S phases, and MUS81 foci were only observed in APBs (). At G2 phase, ~40% of cells showed MUS81 foci that co-localized with APBs. We also observed less than 5% of cells with MUS81 foci when ALT cells were arrested at S phase with HU treatment. All together, our results demonstrate that the association of MUS81 with APBs is preferentially enriched at G2 phase.
GM847 cells arrested in G0/G1 phases by methionine restriction were accompanied by an induction of APBs in 50–60% of the population9
(). However, MUS81 only co-localized to the less than 5% of APBs (original APBs), not to the large population of APBs (induced APBs). Thus, we conclude that MUS81 foci formation was only enriched in APBs at G2 phase. The abundance of MUS81 was not increased in G2 phase of the ALT cells (, the lower panel), indicating that MUS81 may be recruited to APBs in ALT cells.
Gao and coworkers19
demonstrated that MUS81 localizes to nucleoli in human telomerase-positive (non-ALT) cells. We observed diffuse staining of MUS81 throughout the nucleoli in non-ALT HT1080 cells and also in ALT cells (Supplementary Information, Figs. S1D & E
), suggesting that MUS81 localizes to both APBs and nucleoli in ALT cells. Interestingly, co-localization of MUS81 with telomeric DNA was not observed in non-ALT cells (HT1080), suggesting that MUS81 may only contribute to telomere maintenance in ALT cells.
Chromatin immunoprecipitation (ChIP) assays were performed to determine the association of MUS81 with telomeric DNA. We observed an enrichment of telomeric DNA coimmunoprecipitated with the MUS81 antibody in ALT cells (), suggesting that MUS81 binds to telomeres. MUS81 depletion decreased the telomeric DNA signal, indicating the specificity of the ChIP assay for MUS81. We did not detect telomeric DNA signal with the MUS81 antibody in non-ALT MCF7 cells, consistent with the immunostaining results that MUS81 associates with telomeres specifically in ALT cells. Immunostaining results point to localization of MUS81 to APBs specifically enriched during G2 phase. Telomere ChIP assays with the antibody to MUS81 in U2OS cells led to recovery of TTAGGG repeats in extracts from G2 phase cells (). We observed a significant enrichment of telomeric DNA co-immunoprecipitated with the MUS81 antibody from G2 phase cells. These results confirm that association of MUS81 with telomeres in APBs is enriched in G2 phase.
We next examined the role of MUS81 in ALT cell proliferation. Depletion of MUS81 caused growth arrest in the majority of ALT cells within 3–4 weeks. Colony formation assays showed that MUS81-shRNAs dramatically induced cell growth arrest in three ALT cell lines (GM847, U2OS and SAOS-2) ( and Supplementary Information, Fig. S2A
). Inhibition of MUS81 expression in non-ALT cells (HT1080 and MCF7) only decreased the cell viability to ~60%, suggesting that the effect of MUS81 on cell viability is specific for ALT cells. Accordingly, a significant decrease in bromodeoxyuridine incorporation was associated with reduction of MUS81 levels in ALT cells, but not in non-ALT cells (Supplementary Information, Fig. S2B
). Based on these results, we conclude that MUS81 plays a key role in the proliferation of ALT cells.
Fig. 2 Depletion of MUS81 induces the cell growth arrest, telomere loss and decreases telomere recombination in ALT cells. A. Knockdown of MUS81 induces the cell growth arrest in ALT cells. Colony formation assays in three ALT cells (U2OS, GM847 and SAOS-2) (more ...)
The effect of MUS81 depletion on cell cycle progression was analyzed by FACS (Supplementary Information, Fig. S3A
). Knockdown of MUS81 induced a diffused pattern of S and G2 phases, suggesting that depletion of MUS81 may induce a defective S phase. There was no significant change in HT1080 cells with expression of MUS81-shRNA. In addition, apoptotic cells did not increase in ALT and non-ALT cells upon depletion of MUS81 (Supplementary Information, Fig. S3B
). These data suggest that MUS81 may mediate ALT cell proliferation by regulation of the cell cycle.
To further investigate the role of MUS81 on telomere maintenance, we conducted telomere FISH assay in ALT GM847 cells. An elevated frequency of telomere signal loss was observed in the MUS81-shRNA transduced cells (). We detected 15% frequency of telomere signal loss per chromosome in the MUS81-shRNA transduced cells, which is approximately 3-fold higher than that in the control cells (5%, p < 0.001). Thus, depletion of MUS81 leads to increased loss of telomere signals.
Telomere sister chromatid exchange (T-SCE) is a hallmark of ALT cells associated with telomere recombination and ALT cell proliferation2,5,8
. To test whether depletion of MUS81 hinders telomere recombination we monitored the frequency of T-SCEs in GM847 cells by performing CO-FISH. Consistent with previous reports2,5
, we observed that a substantial proportion of metaphases had many chromosome extremities bearing “double signals (yellow)” in GM847 cells (). Quantitative analysis showed that GM847 cells presented ~15% frequency of these exchange events. Importantly, depletion of MUS81 significantly decreased the frequency of T-SCE to ~5%. Depletion of MUS81 also decreased the same frequency of T-SCE in other ALT cells (U2OS and SAOS-2), demonstrating that MUS81 participates in T-SCE of ALT cells.
To investigate the effect of MUS81 on global telomere maintenance, we analyzed telomere loss and T-SCE in non-ALT cells. Knockdown of MUS81 in HT1080 and MCF7 cells did not induce telomere loss or affect T-SCE rate (Supplementary Information, Fig. S4
). Moreover, Mus81
-deficient MEFs did not show telomere loss and T-SCE phenotypes, confirming that MUS81 may not be involved in global telomere maintenance, but have a more restricted role in maintenance of telomeres by recombination in ALT cells.
To test whether depletion of MUS81 impacts on telomere length homeostasis, we performed Quantitative Fluorescence In Situ
Hybridization (Q-FISH) on metaphase nuclei using a telomere specific PNA probe. We did not observe significant telomere length changes in the MUS81-shRNA transduced GM847 cells (Supplementary Information, Fig. S5A
), including average telomere length, the frequency of shorter telomeres (<5 kb) or longer telomeres (>50 kb). Telomere restricted fragment (TRF) analysis verified these results as there was no detectable change in telomere length upon depletion of MUS81 in ALT or non-ALT cells (Supplementary Information, Fig. S5B
). We did not observe obvious chromosome end-to-end fusion in ALT cells after depletion of MUS81 (data not shown). These results were consistent with the fact that knockdown of MUS81 did not induce the formation of telomere dysfunctional induced-foci (γH2
AX foci formation on telomeres, Supplementary Information, Fig. S6
), suggesting that MUS81 does not affect telomere end protection in ALT cells.
Expression of exogenous hTERT in ALT cells can induce telomerase activity, but does not abolish the ALT mechanism20,21
, suggesting coexistence of ALT and telomerase in hTERT-transfected ALT cells. To investigate the relationship between MUS81 mediated-ALT cell proliferation and telomere maintenance, we stably expressed hTERT in U2OS cells. Telomerase was activated in these cells, as detected by the telomerase TRAP assay (). The colony formation results showed that expression of hTERT partially rescued the arrested cell growth upon depletion of MUS81 (). We also confirmed the results in another ALT cell line (SAOS-2). Expression of hTERT in SAOS-2 rescued the cell growth arrest and about 70% of the cells survived upon depletion of MUS81. U2OS cells expressing telomerase still showed a high rate of T-SCE and knockdown of MUS81 decreased the T-SCE rate in these cells (), suggesting that rescue of MUS81-mediated ALT cell survival by telomerase is independent of the telomere recombination pathway. Thus, the results support that MUS81 regulates the growth of ALT cells mainly through maintenance of telomere by recombination.
Fig. 3 Expression of hTERT rescues the MUS81 depletion-mediated cell growth arrest in ALT cells. A. hTERT was stably expressed in U2OS cells and the TRAP assay was performed to evaluate the telomerase activity. Heated U2OS cell lysates and lysates from primary (more ...)
To investigate the role of endonuclease activity of MUS81 in the ALT pathway, we used a MUS81 construct in which the aspartic acid residues at 338 and 339 in VERK domain were substituted by alanine residues. Consistent with a previous report15
, no endonuclease activity was detected in HA immunoprecipitates when this construct was tested (Supplementary Information, Fig. S7A
). We observed that expression of wild-type MUS81 was sufficient to rescue the incidence of T-SCE in MUS81-depleted GM847 cells (). Importantly, expression of the mutant MUS81 did not significantly affect T-SCE frequency, indicating that the endonuclease activity is key for the function of MUS81 on telomere recombination in ALT cells. Furthermore, we hypothesized that endonuclease activity of MUS81 is required for the viability of ALT cells. U2OS cells transduced with shMUS81-B (targeting in 3’UTR region) were infected with lentiviral particles containing MUS81 wild-type or mutant protein. Expression of wild-type MUS81 entirely restored viable cell colonies to the levels seen in transduced control vector cultures ( and Supplementary Information, Fig. S7C
), demonstrating that the expression of wild-type MUS81 rescues the cell growth arrest induced by MUS81-shRNA. In contrast, the MUS81 endonuclease dead mutant did not significantly rescue the cell growth arrest upon MUS81 depletion. Thus, these results indicate that MUS81 enzymatic activity is required for the ALT cell survival and telomere recombination.
Fig. 4 MUS81 endonuclease activity is required for the recombination-based ALT cell survival. A. MUS81 endonuclease activity is required for the telomere recombination. GM847 cells were expressed shMUS81-B (in the 3’UTR region) for three days and then (more ...)
We used a proteomics approach to detect MUS81 interacting partners in the ALT pathway. A stable U2OS cell line expressing His- and Flag-tagged MUS81 was grown. After purification by using the His- and Flag-tag with chromatography columns, the MUS81 immunoprecipitates were analyzed by LC–MS/MS. MUS81 binding partner Eme1 and TRF2 were found in the MUS81 complex. Furthermore, we found that endogenous MUS81 immunoprecipitated with endogenous TRF2 (). Treatment with DNase I did not affect interaction of TRF2-MUS81, excluding bridging effects of nucleic acids. Moreover, the MUS81 immunocomplex contained TRF2, but not TRF2ΔB, a dominant negative mutant of TRF2 lacking the N-terminal domain (), confirming the specificity of the association of MUS81 with TRF2.
Fig. 5 MUS81 physically and functionally interacts with TRF2. A. Endogenous MUS81 binds to TRF2 in U2OS cells by coimmunoprecipitation. Nuclear extracts (0.5 mg) were treated with DNase I and then subjected to immunoprecipitation with the anti-TRF2 antibody (more ...)
We then determined whether TRF2 regulates MUS81 enzymatic activity. Immunoprecipitates of MUS81 from U2OS cells alone could cleave approximately 17% of the total D-loop substrates (). However, depletion of TRF2 by shRNA significantly enhanced MUS81-mediated cleavage in ALT cells, but not in non-ALT cells. More than 43% of the total substrates were cleaved upon transduction of TRF2-shRNA in U2OS cells. To exclude that the putative endonuclease activity detected in the nuclease assays derives from another protein co-immunoprecipitating with endogenous MUS81, HA-tagged MUS81 wild-type or nuclease dead construct was expressed and immunoprecipitations and nuclease assays were performed by using a HA antibody. We observed that knockdown of TRF2 also increased the nuclease activity of HA-MUS81 (Supplementary Information, Fig. S7A
). Thus, our results suggest that TRF2 may inhibit the MUS81 enzymatic activity. In addition, depletion of TRF2 increased MUS81 mediated-cleavage of 3’ flap substrate made by telomere sequences or non-telomere sequences (Supplementary Information, Fig. S7B
), indicating that inhibition of the MUS81 enzymatic activity by TRF2 is not sequence specific. Quantitative Western analysis showed equivalent amounts of immunoprecipitated MUS81 from cells with or without TRF2-shRNA (), suggesting that TRF2 does not affect the expression levels of MUS81.
Next, we tested whether recombinant TRF2 regulated the binding of HA-MUS81 to DNA structures. Immunoblot analysis with anti-HA antibody showed that in the absence of TRF2, HA-MUS81 was associated with biotinylated-DNA substrates, as retrieved by streptavidin beads (; Supplementary Information, Fig. S7D
). The addition of TRF2 inhibited the binding of HA-MUS81 to the DNA in a dose-dependent manner. These data indicate that TRF2 may suppress MUS81 endonuclease activity through inhibition of MUS81 binding to the DNA substrates. To confirm the results in cells, ChIP assays were performed to determine whether TRF2 regulates the ability of MUS81 to bind to telomeric DNA. Knockdown of TRF2 increased the telomere-bound levels of MUS81 (). Thus, our results suggest that TRF2 inhibits the MUS81 enzymatic activity by regulation of MUS81 loading onto telomeres during telomere recombination process.
To investigate the functional relationship between TRF2 and MUS81, we examined their effects on telomere recombination. T-SCE rate increased with 20% frequency in GM847 cells upon depletion of TRF2 (), suggesting that TRF2 suppresses telomere recombination in ALT cells, consistent with a previous model22,23
. Knockdown of MUS81 decreased the frequency of T-SCE in cells expressing TRF2-shRNA (), demonstrating that TRF2-mediated telomere recombination requires MUS81 in ALT cells.
We observed that there was no significant change of telomere circle (t-circle formation) after depletion of MUS81 (Supplementary Information, Fig. S8
), suggesting that t-circles may be not involved in MUS81-mediated ALT cell survival. However, because a lower level of t-circles may be insufficient to detect, we can not rule out a role for MUS81 on t-circle formation. TRF2 mediates t-loop formation and disruption of t-loop by TRF2ΔB
mutant generates t-circles3,24,25
. MUS81 does not interact with TRF2ΔB
mutant, implicating that the MUS81 endonuclease may be involved in the disruption of t-loop, which is mediated by TRF2.
In summary, our results demonstrate that MUS81 endonuclease is specifically involved in telomere recombination and in the maintenance of ALT cell survival. We propose a model where telomeres move into APBs for processing recombination based telomere maintenance during G2 phase. MUS81 is recruited into recombination sites to cleave the recombination structure. TRF2 negatively regulates MUS81 enzymatic activity to balance this process. In this manner, ALT cells maintain their telomeres and extend the proliferative lifespan.