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Previous Article | Next Article 
Molecular and Cellular Biology, June 2003, p. 4247-4256, Vol. 23, No. 12
0270-7306/03/$08.00+0 DOI: 10.1128/MCB.23.12.4247-4256.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
Department of Molecular Pathology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030
Received 18 November 2002/ Returned for modification 26 January 2003/ Accepted 25 March 2003
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ABSTRACT |
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INTRODUCTION |
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B site (52). That study suggests that PML induces apoptosis by sensitizing the tumor necrosis factor death receptor pathway. PML is normally modified in vivo by the small ubiquitin-like modifier protein SUMO-1 at three different sites (K65, K160, and K490) (15, 60). This modification is essential for the organization of PML nuclear bodies (NBs) and for PML's role in transcription regulation and apoptosis (14, 42). Transfection of the PML SUMO-1 mutant into a normal human cell line forms normal PML NBs (23) but not in PML-deficient cells (10, 11), indicating that SUMO-1 modification of PML is essential for the formation of PML NBs (14, 60). SUMO-1 modification is essential for many of PML functions, including interaction with p53, Sp100, and Daxx (14, 22, 23, 60).
Substantial evidence demonstrates that PML plays a role in the regulation of gene expression (24, 59). PML interacts with transcription coactivator CREB-binding protein in vivo and activates the trancription of target genes (3, 6, 19). Documented evidence also shows that PML is associated with p53 in vivo and activates the transcription of p53 target genes (8, 9, 34). Activation of transcription of other genes by PML has also been reported (22, 46). Interestingly, evidence that PML plays a role in transcriptional repression has also been documented. When PML is fused downstream to the GAL4 DNA-binding domain, it represses GAL4-mediated transcription (45), possibly through a mechanism involving recruitment of the transcriptional corepressor histone deacetylases (51). PML interacts and associates in vivo with histone deacetylases and represses transactivation by deacetylation of the target promoter. The in vivo association of PML with other transcriptional corepressors has also been reported (16). Another mechanism by which PML represses transactivation is the interaction of PML with transcription factors, which are then prevented from binding to the target sites. PML sequesters and inhibits transcription mediated by Daxx (22, 23). PML interacts with Sp1 and inhibits Sp1-mediated transactivation of the epidermal growth factor receptor promoter (47). In addition, PML interacts with Nur77 (53) and NF-B (52) and represses transcription mediated by these transcription factors in a promoter-specific manner.
Recent studies suggest that PML may play a role in the maintenance of genome stability (58) and DNA repair (4, 58). PML colocalizes in vivo with BLM, a RecQ DNA helicase deficient in patients with Bloom syndrome (2, 58). Deficiency of BLM results in genome instability (7, 50). PML was also shown to colocalize with the alternative lengthening of telemeres (ALT), suggesting that PML plays a role in maintaining the stability of the telomere ends (10, 56). PML is associated in vivo with several DNA repair proteins, including Mre11, Rad51, and H2AX, and is localized to the single-stranded DNA (ssDNA) repair foci in response to ionizing radiation (IR) (4, 27, 36). These studies suggest that PML plays a role in the organization of the double-strand break (DSB) DNA repair complex.
TopBP1, a topoisomerase IIß-binding protein, was initially identified by yeast two-hybrid screening as the human homologue of the fission yeast Rad4/Cut5 protein, which involves in cellular responses to DNA damage and replication checkpoint controls (40, 41). TopBP1 is a DNA damage response gene, containing multiple copies of the Brca1 carboxyl-terminal motif, which has been shown to bind DNA (54). TopBP1 is a substrate of ATM kinase and is phosphorylated rapidly after exposure of the cells to IR (55). Specific antisense morpholino oligomers inhibit TopBP1 expression and induce cell death by apoptosis, indicating that TopBP1 function may be required for cell survival against IR (55). There is also evidence that TopBP1 functions in a manner similar to its fission yeast counterpart and likely involve a role in repair of DSB DNA damage and the replication checkpoint in mammalian cells (13, 25).
In response to IR exposure TopBP1 forms IR-induced foci, a nuclear speckled staining pattern similar to that of the PML NBs in vivo (13, 55). Since TopBP1 plays a role in DNA damage response and replication checkpoint controls, we investigated whether PML NBs and TopBP1 foci are functionally associated in vivo. Our study demonstrated that PML colocalized with TopBP1 at the endogenous levels in response to IR. TopBP1 and PML colocalized with the IR-induced bromodeoxyuridine (BrdU) ssDNA foci and several proteins critical for DSB DNA repairs. PML and TopBP1 were coinduced by IR and interferon (IFN), and overexpression of PML significantly induced the expression of TopBP1 via stabilization of the protein. Our study demonstrated a PML-dependent induction of TopBP1 expression. The results presented here suggest that PML plays a role in regulating TopBP1 functions by association and stabilization of the protein in response to IR-induced DNA damage.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cell cultures and gene transfection. The human U2OS (osteosarcoma), SiHa (cervical), and NB4 (APL) cell lines were cultured in a humidified incubator containing 5% CO2 at 37°C. U2OS/PML and U2OS/pMEP4 stable cell lines were established, as described previously, by using PMLIV cDNA (8). PML-/- mouse embryo fibroblasts (MEFs) were obtained from P. P. Pandolfi (Department of Human Genetics and Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, New York, NY.). All of the adherent cells were grown as monolayers in Dulbecco modified Eagle medium (Gibco-BRL) containing 10% fetal bovine serum (FBS). The NB4 cells were cultured in RPMI 1640 medium supplemented with 10% FBS. NB4 cells were induced to differentiate with 10-6 M all-trans retinoic acid (ATRA) for 72 h (20). Gene transfection was performed with Fugene 6 transfection reagent (Roche Diagnostics Corp., Indianapolis, Ind.) in accordance with the manufacturer's instructions. Adenovirus-mediated gene transfer with the recombinant PML-adenovirus (Ad-PML) or the antisense PML-adenovirus was carried out as described in a previous study (21)
siRNA inhibition of PML expression. Two pairs of small interfering RNAs (siRNAs) were selected for the following target sequences of PML cDNA: 5'-AAGCACGAAGACAGACCTCTGG-3' and 5'-AACGACAGCCCAGAAGAGGAA-3' (Xeragon-Qiagen, Germantown, Md.). Proliferating U2OS cells were transfected with the two pairs of siRNA by using the Transmessager RNA transfection reagents (Qiagen). A total of 3.2 µg of siRNA was used per well in a six-well plate. Cells were then cultured for 48 h, and total proteins were isolated and analyzed for PML expression by immunofluorescence staining and Western blotting.
Antibodies. Mouse anti-TopBP1 monoclonal antibody was purchased from BD Biosciences-Transduction Laboratories (Lexington, Ky.). Anti-PML (H-238) and anti-BLM polyclonal antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, Calif.). Anti-ATM and anti-Brca1 polyclonal antibodies were obtained from Novus Biologicals, Inc. (Littleton, Colo.). BrdU monoclonal antibody was obtained from BD Biosciences-Pharmingen (San Diego, Calif.). BrdU polyclonal antibody was purchased from RDI Research Diagnostics, Inc. (Flanders, N.J.). Fluorescence- or horseradish peroxidase-conjugated secondary antibodies were obtained from Amersham Biosciences, Inc. (Piscataway, N.J.). The polyclonal PML antibody was raised in rabbits against the glutathione S-transferase-PML fusion protein, as described earlier (47).
Detection of ssDNA after IR-induced DNA damage. Cells were grown on coverslips in 10% fetal calf serum containing 10 µg of BrdU/ml for 30 h and then exposed to IR in BrdU-free medium, as described previously, with modifications (2, 38). Briefly, cells were fixed with 4% paraformaldehyde for 20 min and then washed three times with ice-cold phosphate-buffered saline (PBS). Cells were then permeabilized in 1% Triton X-100 and 0.5% Nonidet P-40 (NP-40) for 20 min, washed three times in PBS for 5 min each, and blocked in 1% bovine serum albumin containing 20 µg of DNase-free RNase A/ml. Double-color immunofluorescence staining was performed with anti-BrdU and anti-TopBP1 or anti-PML antibodies. Fluorescein isothiocyanate- or Cy3-conjugated secondary antibodies were used. Cells were counterstained with DAPI (4',6'-diamidino-2-phenylindole) and viewed under a Leica (DM LB) fluorescence microscope. Images were captured by the Kodak digital imaging system.
Northern blot analysis. Cells were washed once with PBS buffer, and total RNA was isolated by using an RNeasy minikit from Qiagen (Valencia, Calif.). For Northern blot analysis, 30 µg of total RNA was loaded onto a formaldehyde-agarose gel. RNA was transferred onto a positively charged membrane by using the Northern Max-Plus kit from Ambion (Austin, Tex.). Hybridization was performed with a 32P-labeled TopBP1 probe.
IR. Cells were exposed to IR (3.5 Gy/min) in the presence of 10% fetal calf serum by using a Nasatron generator and then immediately transferred to a humidified incubator at 37°C in 5% CO2. After incubation for the indicated times, cells were harvested for Western blot analysis or immunofluorescence staining as described previously (53).
Immunofluorescence staining. Double-color immunofluorescence staining was performed in accordance with the procedure described by Mirzoeva and Petrini (27), with some modifications. Cells were cultured on coverslips in six-well plates and fixed with 4% paraformaldehyde at 4°C for 20 min. After fixation, the cells were permeabilized in 1% Triton X-100 and 0.5% NP-40 for 30 min. Samples were blocked with 1% bovine serum albumin and then incubated with primary antibody for 2 h and secondary (fluorescein isothiocyanate-, rhodamine-, or Cy3-labeled) antibody for 1 h. Cells were then stained with DAPI to visualize nuclear DNA. To reduce the background signals in the cytoplasm and nucleoplasm, cells were pretreated with cytoskeletal stripping buffer before fixation. Cells on coverslips were washed twice in PBS, incubated in cytoskeleton buffer {PIPES [piperazine-N,N'-bis(2-ethanesulfonic acid)]; pH 6.8], 100 mM NaCl, 300 mM sucrose, 3 mM MgCl2, 1 mM EGTA, 0.5% Triton X-100} for 5 min on ice, and then incubated in stripping buffer (10 mM Tris-HCl [pH 7.4], 10 mM NaCl, 3 mM MgCl2, 1% Tween 20, 0.25% sodium deoxycholate) for 3 min on ice. The cells were washed three times in ice-cold PBS and then were fixed and processed as described above. The coverslips were mounted onto glass slides with VectaShield antifade (Vector Laboratories, Burlingame, Calif.) and visualized by using a Leica DM LB fluorescence microscope or a Zeiss LSM 5 confocal microscope. Images were captured with a Kodak digital imaging system.
Pulse-chase labeling of cells with [35S]methionine and [35S]cysteine. U2OS/PML and U2OS/pMEP4 cells were grown to 60% confluency in 100-mm culture dishes and treated with 100 µM ZnSO4 overnight to induce PML expression. Cells were washed twice with 10 ml of prewarmed pulse-labeling medium (methionine- and cysteine-free Dulbecco modified Eagle medium, dialyzed overnight against saline solution and 25 mM HEPES [pH 7.4; Gibco-BRL]) containing 10% FBS. Next, 5 ml of prewarmed pulse-labeling medium was added to the cells, which were then incubated for 15 min in a humidified incubator at 37°C containing 5% CO2. The medium was removed and replaced with 2 ml of labeling solution containing 0.15 mCi of [35S]methionine and [35S]cysteine (Perkin-Elmer Life Sciences, Boston, Mass.)/ml in prewarmed pulse-labeling medium, and the cells were incubated for 45 min in a CO2 incubator. The labeling solution was removed by two washes with 10 ml of 37°C chase medium (pulse-labeling medium containing 15 mg of unlabeled methionine and 31.5 mg of unlabeled cysteine/liter), and then 10 ml of 37°C chase medium was added. The cells were incubated for 0, 2, 4, 8, and 12 h in a CO2 incubator. Cells were harvested, and total proteins were isolated. Immunoprecipitation was performed with specific antibodies, and the precipitated proteins were resolved by sodium dodecyl sulfate-10% polyacrylamide gel electrophoresis (SDS-10% PAGE). Gels were fixed, dried, and autoradiographed. Signals were quantified by densitometry by using the ImageQuant software (Molecular Dynamics).
Immunoprecipitation and Western blot analysis. U2OS/PML cells were lyzed with cell lysis buffer (50 mM PIPES, pH 7.5; 400 mM NaCl; 1 mM EGTA; 1 mM EDTA; 1% Triton X-100; 0.5% NP-40; 10% glycerol; protease inhibitors). The lysates were precleared by treatment with protein A/G-Sepharose beads (Santa Cruz Biotechnology), antibody-conjugated beads were added, and the lysates were incubated for 4 h. After five washes with buffer (50 mM PIPES, pH 7.5; 100 mM NaCl; 0.25 mM EGTA; 0.25 mM EDTA; 0.25% Triton X-100; 0.125% NP-40; 2.5% glycerol; protease inhibitors) and one wash with ice-cold PBS, precipitated proteins were dissolved in an equal volume of 2x SDS loading buffer and then resolved by SDS-10% PAGE. Western blot analysis was carried out according to standard procedures.
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RESULTS |
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PML and TopBP1 are colocalized with the IR-induced BrdU ssDNA foci. PML NBs colocalized with ssDNA repair foci in response to IR (30, 31). These ssDNA foci formed primarily in response to DNA DSBs, presumably the sites of late-stage DNA repair (2, 38). Only a low number (1%) of proliferating BrdU-labeled cells were found to contain the ssDNA foci, suggesting that the foci do not represent the sites of active DNA replication. The formation of ssDNA foci may require several kilobases of ssDNA as described by Raderschall et al. (38); such long stretches of ssDNA are rare in normal cultured cells. The ssDNA foci detected after IR exposure were not a result of apoptosis because no PARP degradation or FISEL staining was detected (2, 38). In addition, UV did not induce any significant degree of ssDNA foci (2). Our results show that no significant degree of BrdU foci was found at 0 h after cells were exposed to IR (Fig. 2). This result is in agreement with the previous reports (2, 38).
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The results shown in Fig. 1A and 3A and B show that cells with higher numbers of PML and TopBP1 foci increased progressively over the 12-h period after IR exposure. However, cells with high numbers of ssDNA foci (i.e., >40) increased dramatically in 1 h, and cells with lower numbers of ssDNA foci predominated after 2 h of IR exposure (Fig. 3C). This result shows that the increase in the number of PML and TopBP1 foci coincided with the decrease in the number of ssDNA foci, suggesting that an increased number of PML and TopBP1 foci promotes repair of DNA DSBs induced by IR.
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significantly increased PML expression, a finding similar to the result reported previously (43). Interestingly, TopBP1 was also coinduced by IFN-. We next sought to determine whether the increased TopBP1 expression was directly related to the PML induction by examining the effects of inducible overexpression of PML on TopBP1 expression. Zn- or IR-induced PML expression significantly increased the TopBP1 expression levels (Fig. 4D). This conclusion was further supported in an experiment with Ad-PML. U2OS cells infected with various concentrations of Ad-PML expressed increasing amounts of the PML protein, and the TopBP1 expression levels in these cells increased dramatically in response to PML (Fig. 4E). TopBP1 expression levels did not increase in cells infected with the negative controls.
PML-dependent expression of TopBP1 protein.
To further investigate whether TopBP1 expression is PML-dependent, siRNA specific for PML mRNA was designed as described in Materials and Methods. The results presented in Fig. 5A and D show that inhibition of PML expression by siRNA was associated with a significant reduction in IR-induced TopBP1 expression. We next investigated the cellular localization of TopBP1 in the PML-deficient APL-derived NB4 cells. In these cells, the normal PML staining pattern has been altered to an abnormal microspeckled pattern as a result of forming a heterodimer with the PML-RAR fusion protein (26). ATRA induced a rapid degradation of the fusion protein and reorganization of the normal PML NBs. The staining pattern and the in vivo localization of TopBP1 was altered in NB4 cells (Fig. 5F). The signal intensity of TopBP1 appeared to be weak, and the staining pattern was similar to the typical microspeckled pattern of PML. In NB4 cells, TopBP1 failed to form IR-induced foci. Interestingly, after treatment of NB4 cells with ATRA, both PML NBs and TopBP1 foci were reorganized, and the two proteins colocalized in vivo 8 h after IR exposure. These results strongly support the concept that a functional in vivo interaction exists between PML and TopBP1. The results of Western blot analysis showed that TopBP1 protein was expressed at low levels even in cells exposed to 10 Gy of IR (Fig. 5B). After the NB4 cells were treated with ATRA, TopBP1 expression levels increased significantly, with or without IR treatment. Finally, we investigated the expression of the TopBP1 protein in PML-/- MEFs and normal MEFs. We found that PML-/- MEFs express low levels of TopBP1 and that its expression was not inducible by treatment with IFN- (Fig. 5C). When PML expression was reintroduced into the PML-/- MEFs by adenovirus-mediated gene transfer, a significant increase in TopBP1 expression was found, which colocalized with the PML NBs (Fig. 5G). Together, our findings support the conclusion that IR-induced upregulation of TopBP1 is PML dependent. PML either activates TopBP1 expression or, alternatively, recruits TopBP1 to the PML NBs and stabilizes the protein.
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Association of PML and TopBP1 in vivo. To further support the concept that PML associated with TopBP1 in vivo, we performed a coimmunoprecipitation assay using PML and TopBP1 antibodies. The results of the present study showed that TopBP1 antibody coimmunoprecipitated the PML protein, and that PML antibody coimmunoprecipitated the TopBP1 protein (Fig. 7A and B), supporting the idea that the two proteins physically associate in vivo. To elucidate the domain of PML, which is associated with TopBP1 in vivo, His-tagged PML mutant plasmids were created and cotransfected with the Myc-tagged TopBP1 expression plasmid (55). Total proteins were isolated, and coimmunoprecipitation was performed with Myc-tagged specific antibody (against the Myc-tagged TopBP1 fusion protein). The precipitated proteins were analyzed by Western blot analysis with His-tagged specific antibody (for detecting His-tagged PML protein). The present study showed that the N-terminal domain of PML is required for its in vivo association with TopBP1 (Fig. 7C and D).
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Functional association of PML with TopBP1 in response to IR. TopBP1 consists of eight Brca1 carboxyl-terminal motifs, a human homologue of the Rad4/Cut5 protein of fission yeast, which is required for DNA damage repair, the regulation of DNA replication checkpoint, or both (13, 40, 41). A recent study demonstrated that a human ubiquitin ligase, hHYD, interacts with TopBP1 and that ubiquitination leads to degradation of the protein through the proteosome pathway (13). IR stabilizes TopBP1 by reducing ubiquitination, probably through phosphorylation by the ATM kinase (55) and increased colocalization with H2AX in the DNA breaks. This study suggests that hHYD regulates TopBP1 in response to IR-induced DNA damage. Our results in the present study showed that PML and TopBP1 (together with other proteins involved in DSB DNA repair) are colocalized at the endogenous level in response to IR. Furthermore, both PML NBs and the IR-induced TopBP1 foci are colocalized with the ssDNA foci, presumably the sites of DSB DNA repair (2, 38). An increase in the number of PML NBs and TopBP1 foci coincided with the rapid decrease in the number of the ssDNA foci, suggesting that these two proteins are probably involved in DSB DNA repair (Fig. 3).
Our study showed that PML and TopBP1 proteins are coinduced by IR. IFN, which induces PML expression at the transcriptional level (43), also induced TopBP1 expression. Overexpression of PML in U2OS cells induced by Zn2+ or by infection with Ad-PML significantly increased TopBP1 expression. Furthermore, inhibition of PML expression by specific siRNA reduced TopBP1 expression in cells exposed to IR and that reintroduction of PML expression in PML-/- MEFs is associated with a dramatic increase in the TopBP1 protein that colocalized with the PML NBs. The results of the present study demonstrated that expression of the TopBP1 protein in response to DNA damage induced by IR is PML dependent. These results strongly support a functional link between the two proteins. Results obtained from the pulse-chase labeling experiment showed that overexpression of PML substantially increased the stability of TopBP1 but not Mre11, which was also found to colocalize with PML (27). PML plays a role in regulating TopBP1 function by recruiting and stabilizing the protein in response to IR.
Our results showed that PML colocalizes with the IR-induced ssDNA foci within 1 h after the cells were exposed to IR; however, it took more than 2 h to detect any significant levels of TopBP1 colocalized with the ssDNA. Our results also showed that inducible overexpression of PML stabilized the Rad51 protein (unpublished result). The results of the present study suggest that PML recruits TopBP1 and other proteins involved in DSB DNA repair to the PML NBs in response to IR-induced DNA damage and stabilized the protein complex.
PML NBs as organizer of the DSB-DNA repair enzyme complex. The proteins found to colocalize with PML and TopBP1 play important roles in DSB-DNA repair by homologous recombination (5, 44). For example, ATM is the master controller of IR-induced responses (33, 39); Rad51 is a DNA recombinase, the human homologue of the RecA protein in bacteria that plays a critical role in DSB DNA repair (17); the Mre11/Rad50/NBS complex plays a critical role in DSB DNA repair (18); H2AX is the earliest protein found to associate with the site of DSB DNA repair (28); BLM is a RecQ-like DNA helicase, possibly functioning in DNA replication, recombination, and DNA repair (2, 58); and Brca1 function is required in both homologous recombination and nonhomologous end joining DNA repair (29, 48, 57). Although the functions of both TopBP1 and Rad9 are not well understood, there is evidence that these two proteins are involved in the cellular responses following DNA damage (13, 55). The important question to be addressed is how is PML involved in cellular responses to DNA DSBs after IR exposure? It is intriguing that proteins that colocalize with PML, including the upstream regulator ATM, are involved in different steps of the DSB DNA repair mechanism. This argues against PML having a role in a specific step of the DNA repair mechanism. Our pulse-chase analysis showed that overexpression of PML substantially stabilized TopBP1 and that PML-deficient cells were unable to form IR-induced foci of TopBP1, Brca1, BLM, and Rad51 (Fig. 5 and our unpublished results), strongly suggesting that PML plays an essential role in the organization and stabilization of the IR-induced DNA-repair enzyme complex. In addition, recent findings demonstrated a metabolic-energy-dependent movement of the PML NBs in living cells, suggesting that PML NBs act as a nuclear sensor for detecting foreign materials (32). Together, we speculate that PML acts as a nuclear sensor and an organizer, detecting the sites of DNA DSBs during the late phase of DNA repair in response to DNA damage and organizing the DNA repair enzymes complex. PML NBs are macronuclear structures that are up to 0.5 µm in size. Their ability to move rapidly within the nucleus may provide an efficient mechanism for sensing and an anchorage point for assembling and organizing the enzyme complex.
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ACKNOWLEDGMENTS |
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This study was supported by grant CA55577 from the National Institutes of Health to K.-S.C.
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FOOTNOTES |
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