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1 Virology Division, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuohku, Tokyo 104-0045, Japan
2 Nara Institute of Science and Technology, Takayama, Ikoma, Nara 630-0101, Japan
3 Faculty of Advanced Life Science, Hokkaido University, Kita-21, Nishi-11, Sapporo 001-0021, Japan
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Abstract |
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Introduction |
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TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
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ORC, consisting of six subunits ORC1-6, was initially identified in budding yeast as a protein complex binding sequence specifically to autonomously replicating sequences (Bell & Stillman 1992). However, the interaction of mammalian ORC with chromosomal DNA is not simply determined by the primary DNA sequence (Vashee et al. 2003). Certain transcription factors appear to recruit ORC through physical interaction so that the binding sites of the transcription factors promote DNA replication (Danis et al. 2004; Minami et al. 2006). In human cells, ORC2-5 constitute a tight complex throughout the cell cycle and ORC1 interacts with this complex to form a complete complex during the period to assemble pre-RCs (Vashee et al. 2001; Ohta et al. 2003). In pre-RC assembly, ORC functions as the landing pad for other factors. In addition, many ORC subunits possess essential ATPase activities (Bell & Dutta 2002).
In addition to its role in pre-RC formation, ORC has also been implicated in other chromatin transactions. In budding yeast, ORC, together with Rap1, ABF1 and Sir proteins, functions in transcriptional silencing at silent mating-type loci (reviewed by Fox & McConnell 2005). Also in higher eukaryotes, ORC has been implicated in chromatin silencing. This was first suggested in Drosophila, where ORC physically interacts with HP1, a major structural component of heterochromatin, and mutations in ORC2 disrupt HP1 association with heterochromatin and suppress heterochromatin formation (Pak et al. 1997; Huang et al. 1998). Drosophila ORC1 has a strong binding site for HP1. Also in mammalian cells, ORC2 co-localizes to heterochromatic foci containing HP1 proteins and their physical interactions have been showed (Prasanth et al. 2004). The association of ORC2 with prominent heterochromatic foci is cell cycle regulated, disappearing as cells pass through S phase except for co-localization with centromeric heterochromatin. Depletion of ORC2 by siRNA disrupts HP1 localization to the heterochromatin. In addition, it has been suggested that ORC2 localizes to centrosomes to participate in its duplication (Prasanth et al. 2004). Mammalian ORC6 might furthermore be involved in chromosome segregation and cytokinesis in addition to DNA replication (Prasanth et al. 2002).
In the present report, we describe a novel link between pre-RC assembly regulation and telomere homeostasis in human cells. Biochemical analyses showed that human ORC binds to TRF2, a telomere sequence-binding protein (reviewed by Smogorzewska & de Lange 2004), and that the binding is dependent on the ORC1 subunit. Chromatin immunoprecipitation analyses (ChIP) revealed that pre-RCs are assembled around telomeres in a cell cycle-dependent manner. Further analyses suggested that ORC and TRF2 are involved in pre-RC formation at telomeres and telomere homeostasis. Recently, Atanasiu et al. reported that TRF2 might recruit ORC to OriP, the Epstein–Barr virus origin of replication, to stimulate its replication (Atanasiu et al. 2006). Considering our and their findings together, the virus appears to hijack the cellular mechanism to maintain telomere homeostasis for its own propagation.
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Results |
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To identify novel ORC-binding proteins, ORC was immunopurified and subjected to mass spectrometry analysis (Ohta et al. 2003). In addition to each ORC subunit (Ohta et al. 2003), we identified TRF2 and hRap1 (Fig. S1 in the Supporting Information). TRF2 is known to bind to OriP and stimulate its replication (Deng et al. 2002, 2003). Replication from OriP is dependent on the pre-RC components as well as a viral protein EBNA1, which might recruit ORC through physical interactions (Dhar et al. 2001; Schepers et al. 2001). We thus reasoned that TRF2 might physically interact with some of pre-RC components and first addressed this by Glutathione S-transferase (GST)-TRF2 pull-down assay with HeLa cell nuclear extracts (Fig. 1A). ORC subunits, ORC1, ORC2 and ORC3, were pulled down with GST-TRF2, with ORC1 being most efficiently enriched. Cdt1 and MCM7 did not bind to TRF2. We further confirmed in vivo physical interactions between transfected ORC and TRF2 by immunoprecipitation after cross-linking viable 293T cells (Fig. 1B). These data indicate that ORC physically interacts with TRF2. So far, we could not observe co-immunoprecipitation between endogenous ORC and TRF2. Therefore, their physical interaction might be relatively unstable. Nevertheless, as shown below, their interaction is specific and physiologically significant. TRF1 shares homology with TRF2, especially in the TTAGGG repeat binding factor homology (TRFH) domain/dimerization domain (Supporting Information Fig. S2; Smogorzewska & de Lange 2004). As shown in Fig. 1C, ORC was not efficiently pulled down by GST-TRF1.
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B lacking the amino terminal basic domain and GST-TRF2M lacking the Myb DNA-binding domain (Karlseder et al. 1999). We found that both mutants bind to ORC in similar efficiency with the full-length form (Fig. 2A), suggesting that the middle portion of TRF2, including the TRFH domain, is important for the interaction. Indeed, we found that GST-TRF2BM can bind to ORC, albeit to a lesser extent compared with the full length TRF2 (Supporting Information Fig. S2B). Whereas it is known that the TRFH domain of TRF2 is required for dimer formation, it is also involved in protein–protein interactions, as has been shown for several other proteins (Fairall et al. 2001; Freibaum & Counter 2006).
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B and GST-TRF2M (Fig. 2B), consistent with the observations on cellular ORC. We next tested whether the ORC2-5 complex lacking ORC1 can bind to TRF2 using nuclear extracts from S phase-arrested HeLa cells, where ORC1 protein is degraded but the ORC2-5 subcomplex remains (Fujita et al. 2002; Mendez et al. 2002; Ohta et al. 2003). The binding of ORC2 and ORC3 was weakened with the S phase cell extracts (Fig. 2C). These data indicate that ORC binding to TRF2 is largely mediated by ORC1. We found CDC6 binding to GST-TRF2 (Fig. 1A). However, we have not so far observed their co-immunoprecipitation, even after formaldehyde cross-linking (Fig. 1B). Rather, CDC6 binds to GST-TRF1 more efficiently (Fig. 1C). The physiological significance of CDC6 binding to TRF1 remains to be elucidated.
ORC and TRF2 partially co-localize in nuclei
TRF2 are localized to telomeres (van Steensel et al. 1998; Smogorzewska et al. 2000). We thus tested whether ORC co-localizes with TRF2. Asynchronous HeLa cells were immunostained with anti-ORC1 and anti-TRF2 antibodies and analyzed by confocal microscopy. The anti-ORC1 antibody stains numbers of dots in nuclei of G1 cells (Fujita et al. 2002; Tatsumi et al. 2003). In ORC1 positive cells, some of the dots were co-localized with TRF2 (Fig. 3A; white arrows) and some were observed side by side (yellow arrows). Approximately 50% of the TRF2 foci in HeLa cells co-localized (at least partially) with ORC1 foci (Fig. 3A). Thus, at least a part of TRF2 is likely to interact with ORC at telomeres and/or subtelomeric regions in human cells.
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The above data revealed a physical interaction between ORC and TRF2 around telomeres. Thus, ORC might be recruited around telomeres by TRF2 and thereby facilitate pre-RC assembly. Using ChIP assays with specific antibodies combined with dot-blot hybridization to detect telomeric TTAGGG repeats, we examined whether pre-RCs are assembled at telomeres. In agreement with the previous results, significant enrichment of telomeric DNA with anti-TRF2 antibody was observed in all of asynchronously growing 293T cells, human normal foreskin fibroblasts immortalized with telomerase (HFF2/T), and HeLa cells (Figs 3B, 5B and 6B, respectively). Interestingly, significant enrichment of telomeric DNA was also observed with anti-ORC2 and anti-MCM7 antibodies in all the cells tested (Figs 3B, 5B and 6B). For example, in 293T cells, 1.2% input, 0.27% input and 0.38% input of telomere-repeat DNA were enriched for TRF2, ORC2 and MCM7 respectively, whereas only 0.01% input was recovered with control IgG (Supporting Information Fig. S3). The association of TRF2 and ORC2 with telomeres appears specific because Alu-repeat DNA was not significantly enriched in ChIP with anti-ORC2 and anti-TRF2 antibodies (Fig. 3B and F, and Supporting Information Fig. S3). Furthermore, ORC2 silencing by siRNA treatment significantly reduced the enrichment of telomeric DNA with anti-ORC2 antibody in HeLa cells (Fig. 3E and F), also demonstrating specificity of the assay. For MCM7, Alu DNA was somewhat enriched in the ChIP assay (Fig. 3B and Supporting Information Fig. S3). This could be because multiple MCM complexes are relatively widely distributed on chromatin beyond each origin to ensure complete replication under replicative stress (Fujita et al. 2002; Ge et al. 2007). Taken together, these data suggest pre-RC formation around telomeres.
We compared telomere binding of ORC2 and MCM7 in 293T cells asynchronously growing (50% in G1 phase), treated with hydroxyurea (early S phase), and released for 9 h after hydroxyurea arrest (mid S to G2/M phase) (Fig. 3B–D). The binding of ORC2 was significantly reduced in early S phase cells, in which ORC1 is degraded, and reduction was also observed in mid S to G2/M phase cells, suggesting that ORC binding to telomeres is promoted by ORC1. Decrease in MCM7 binding to telomeres was also observed in early S phase cells and mid S to G2/M phase cells. Overall, the observed changes in patterns of ORC2 and MCM7 binding are consistent with cell cycle regulation of pre-RC (Bell & Dutta 2002; Diffley 2004; Fujita 2006). By analyzing 293T cells expressing Flag-ORC1, we could also observe the similar cell cycle-dependent binding of ORC1 to telomeres (data not shown). Interestingly, TRF2 binding to telomeres was also decreased in both early S phase and mid S to G2/M phase cells (Fig. 3B and C), implying a close relationship between the ORC and TRF2 binding.
TRF2 facilitates ORC recruitment and pre-RC formation at telomeres
We investigated whether TRF2 affects ORC binding and pre-RC assembly at telomeres by a genetic approach using HFF2/T cells. We first generated HFF2/T cells over-expressing HA-TRF2 and examined whether over-expressed TRF2 might promote pre-RC assembly by chromatin binding assay (Fujita et al. 1997). Almost all of the endogenous TRF2 and approximately 70% of the over-expressed TRF2 was found in the chromatin/nuclear matrix fraction (Fig. 4A). In the HA-TRF2 over-expressing cells, levels of the chromatin/nuclear matrix-bound ORC2, ORC3 and MCM7 proteins were increased (Fig. 4A and B). As the cell cycle distribution was not changed by TRF2 over-expression (Fig. 4C), the increase was not as result of accumulation of a G1 population. We further tested whether the increase in pre-RC assembly occurs at telomeres by ChIP assay. Although the total level of over-expressed TRF2 was 10 times greater than the endogenous level, only twofold increase was observed at telomeres (data not shown), suggesting that TRF2 binding to telomeres might already have been saturated in the parental cells. Reflecting this, no significant increase in ORC binding to telomere was observed in TRF2-over-expressing cells (data not shown). Thus, over-expressed TRF2 could bind to chromatin regions other than telomeres and promote the pre-RC assembly. In contrast, such saturated TRF2 binding to telomeres could explain why the rate of telomere elongation upon TRF2 silencing (see below) appears exceeding the rate of telomere shortening upon TRF2 over-expression (Smogorzewska et al. 2000).
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We next wanted to investigate the effect of genetic manipulations of ORC1 on telomeres in the HFF2/T cells and first tried to establish ORC1-over-expressing cells. However, so far we have failed to generate such cells. As we could achieve transient over-expression of ORC1 (data not shown), such failure might be because of toxicity of ORC1 over-expression (Saha et al. 2005). We thus examined the effect of ORC1 knockdown in HFF2/T cells. Significant growth retardation was observed in HFF2/T cells expressing an shRNA against ORC1 (Supporting Information Fig. S4E), as expected from essential roles of ORC1. We expanded the slowly growing cells and analyzed them (Fig. 5E). As expected, reduction of ORC1 to approximately 10% was observed. Unexpectedly, TRF2 protein levels were remarkably up-regulated in the ORC1-depleted cells. The finding was observed repeatedly in independent experiments. Although the molecular mechanism is currently unknown, it is possible that TRF2 up-regulation is required to compensate for the growth defect caused by ORC1 depletion. Thus, there might be some difficulties in analyzing these cells and interpreting the obtained results. Nevertheless, we investigated telomere length by Southern blot analysis and found it to be elongated in ORC1-silenced HFF2/T cells, although to a lesser extent as compared with TRF2-silenced cells (Fig. 5I, left panel). Such findings were repeatedly obtained. ChIP assays showed reduced telomere binding of ORC2 and MCM7 in ORC1-depleted cells (Fig. 5F–H). Increased TRF2 binding to telomeres might simply reflect an increase in its total amounts. Taken together, these data suggest that ORC binding to telomeres, and possibly subsequent pre-RC formation, plays a role in telomere length control.
We also sought to inhibit ORC1 in HeLa cells. ORC1 protein levels in HeLa cells are approximately 10 times greater than in non-transformed cells (Tatsumi et al. 2006) and thus we could examine the effect of ORC1 silencings on telomeres without inducing growth suppression. We generated HeLa cells in which levels of ORC1 proteins were reduced by approximately 80% with the shRNA (Supporting Information Fig. S5A). In contrast to HFF2/T cells, ORC1 knockdown at this level did not affect cell cycle progression and cell growth (Supporting Information Fig. S5B; and data not shown). Nevertheless, the TTAGGG signals with input DNA were increased in the ORC1-reduced HeLa cells (Supporting Information Fig. S5C and D), although to a lesser extent compared with that in ORC1-reduced HFF2/T cells. This could be because the residual ORC1 proteins in HeLa cells are relatively sufficient for telomere homeostasis. We then carried out telomere ChIP assays (Supporting Information Fig. S5C and E). Binding of ORC2 and MCM7 was significantly reduced in ORC1-depleted cells. Overall, these data are consistent with findings for HFF2/T cells.
Disruption of telomere protection functions often increases chromosomal end-to-end fusion. When the fused chromosomes are broken, translocations and deletions are generated. To test the effects of ORC1 and TRF2 silencing on telomere capping functions, we investigated the frequency of such abnormal chromosomes in HFF2/T cells. At 3–5 population doublings post-infection, cells were subjected to karyotypic analysis. As shown in Supporting Information Fig. S6, no significant increase in the frequencies of such abnormalities was observed in either ORC1- or TRF2-depleted HFF2/T cells.
Reduction in ORC binding to telomeres weakens TRF2 binding in HeLa cells
In the ORC1-depeleted HeLa cells, a tendency for decreased binding of TRF2 to the telomeric region was observed, albeit without statistically significance (Supporting Information Fig. S5E). We reasoned that we could more clearly observe effects of ORC1 reduction on TRF2 binding to telomeres by acute depletion of ORC1 with siRNA. Accordingly, HeLa cells were transfected with a siRNA targeting ORC1 or a control siRNA, and harvested 48 h after transfection. The ORC1 siRNA remarkably reduced the protein levels (Fig. 6A). Simultaneously, cells were subjected to telomere ChIP assays (Fig. 6B and C). Binding of ORC2 and MCM7 was significantly reduced in ORC1-depleted cells, as expected. Interestingly, we found that binding of TRF2 to telomeres is also reduced by approximately a half in ORC1-silenced cells. As the cell cycle distribution and cell growth was not changed remarkably by ORC1 silencing at this level (Fig. 6D and data not shown), the observed changes might not be as a result of the cell cycle effects. In addition, similar reduction in TRF2 binding to telomeres was also observed in ORC2-silenced HeLa cells (Fig. 3E and F). Taken together, these data suggest that TRF2 binding might be stabilized by ORC.
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Discussion |
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Atanasiu et al. reported that (i) ORC2 in the nuclear extracts binds to GST-TRF2; (ii) ORC2 co-immunoprecipitates with TRF2; (iii) TRF2 binds to ORC1; and (iv) reduction in TRF2 suppresses pre-RC formation at OriP (Atanasiu et al. 2006). Based on these data, they suggested that ORC binds to TRF2. In the present study, we found that three ORC subunits, ORC1, ORC2 and ORC3, all bind to GST-TRF2 and are retained with the TRF2 immunoprecipitates, providing further convincing data for ORC binding to TRF2. More importantly, we found that their interaction is largely mediated by the largest subunit ORC1. In line with this, ORC2 association with telomeres is inhibited by ORC1 silencing. In vivo associations between ORC and TRF2 are further supported by the finding that ORC1 and TRF2 are partially co-localized in nuclei. GST pull-down assays with TRF2 mutants have indicated that neither the myb DNA-binding domain nor the amino terminal basic domain are necessary for ORC binding, apparently differing from the previous report that the basic domain is responsible (Atanasiu et al. 2006). The reason for the difference is not clear at present. One possibility is that TRF2 has at least two binding domains for ORC and the basic domain has higher affinity so that its requirement becomes apparent when the binding is examined under more stringent conditions.
Telomere ChIP assays showed that pre-RC is assembled at telomeres. ORC2 binding to telomeres is reduced in S phase when ORC1 is degraded and MCM7 binding is also reduced, features consistent with pre-RC assembly during the cell cycle. In addition, ORC1 silencing leads to reduction in ORC2 and MCM7 binding to telomeres. It is of interest to ascertain how pre-RCs are generally assembled onto telomeres. However, current technical limitations in controlling cross-linking and immunoprecipitation efficiencies make this difficult.
Suppression of TRF2 by RNAi resulted in decreased ORC binding and pre-RC formation at telomeres and TRF2 over-expression enhanced ORC and MCM recruitment to nuclear matrix/chromatin fractions. The data are well compatible with the previous finding that TRF2 inhibition leads to reduction in pre-RC assembly at the OriP (Atanasiu et al. 2006). Although TRF2 silencing significantly reduces pre-RC formation at telomeres, the extent appears relatively low compared with the extent of reduction in TRF2 binding itself. Thus, whereas TRF2 is an important factor for facilitating ORC recruitment and pre-RC assembly at telomeres, there might also be other mechanisms.
Interestingly, in HeLa cells, acute reduction in ORC1 or ORC2 levels with siRNA resulted in decreased binding of TRF2 to telomeres. In addition, TRF2 binding to telomeres was destabilized in cells in S phase, when ORC1 is degraded. Thus, ORC might play some role in stabilization of TRF2 binding. Reduced telomere binding of TRF2 was not observed in ORC1-silenced HFF2/T cells but this might have been because of unexpected up-regulation of TRF2 expression. At the OriP, TRF2 binds to TTAGGGTTA sequences preferentially in G1 phase and is replaced by TRF1 in G2 (Deng et al. 2003). Interdependent binding of ORC and TRF2 could fit this result. Verdun et al. investigated cell cycle changes with TRF2 binding to telomeres (Verdun et al. 2005). In their experiments, cells were first arrested in early S phase by aphidicolin and were chased for 16 h after release, when cells are in early G1 phase with partially accumulated ORC1 (Ohta et al. 2003; Tatsumi et al. 2003). Nevertheless, TRF2 binding appeared to gradually decrease as cells passed through S and G2 phases and to recover in early G1 (Verdun et al. 2005), consistent with the notion that TRF2 binding is destabilized after S phase. Given that the binding of TRF2 to telomeres is cell cycle regulated, what is the biological meaning? Although telomere ends should be protected and TRF2 has a major role in this context, telomere ends also have to be timely recognized by proteins involved in the DNA damage response to be appropriately processed (Verdun et al. 2005; Francia et al. 2007). Partial destabilization of TRF2 binding during S phase could be associated with such regulation.
Silencing of ORC1 resulted in decreased pre-RC formation at telomeres. Interestingly, ORC1 silencing induced elongated telomeres, although to a lesser extent compared with TRF2 silencing, suggesting that ORC plays a role in telomere length homeostasis. We could not find significant increase in the frequency of end-to-end chromosomal fusions by ORC1 suppression. Further depletion of ORC might reveal functions in telomere protection but we cannot readily address this question because of its essential requirement. There are several possible non-exclusive explanations for how ORC influences telomere length homeostasis. One is that efficient pre-RC assembly at telomeric regions is involved. For example, hypofunction of polymerase 
results in telomere elongation (Nakamura et al. 2005) and pre-RC assembly at telomeres could contribute to efficient recruitment of polymerase . As discussed above, telomere ends should be recognized appropriately by proteins involved in DNA damage responses. Activation of the ATR pathway requires prior pre-RC assembly and it is also suggested that interactions between MCM7 and Rad17 are needed for activation of the ATR pathway (Tsao et al. 2004). Thus, efficient pre-RC assembly at telomeres could regulate end processing by proteins involved in the DNA damage responses. Potential roles of CDC6 and MCM proteins in telomere regulation should be a focus for future research. Another possibility is that ORC binding itself is involved in telomere homeostasis. TRF2 plays a role in length control and its binding to telomeres is stabilized by ORC as shown here. It has been observed that depletion of ORC2 impairs heterochromatin formation (Pak et al. 1997; Prasanth et al. 2004). Interestingly, reduced histone H3-K9 trimethylation also impairs heterochromatin formation at telomeres and leads to aberrant telomere elongation (García-Cao et al. 2004).
TRF2 has been implicated in telomere length control. In human cells, over-expression of TRF2 results in telomere shortening (Smogorzewska et al. 2000). As a possible molecular mechanism, it has been suggested that the over-expressed TRF2 prevents access of telomerase. However, in murine skin cells over-expressing TRF2, genetic data indicate that telomere loss is because of inappropriate digestion of G-strand overhang by XPF nuclease (Muñoz et al. 2005). It is thus conceivable that reduced TRF2 function might conversely lead to telomere elongation. However, this has not been directly addressed. Knockout of TRF2 induces loss of the telomere protection function and consequent acute cell cycle arrest (Celli & de Lange 2005). In the present study, we found that reduction in TRF2 levels by RNAi did in fact induce telomere elongation, although it did not appear to impair the capping and protection function. Presumably, residual TRF2 might be sufficient for protecting telomere ends. In addition, our data indicate that TRF2 promotes ORC recruitment to telomeres and that ORC contributes to the length homeostasis. Some aspects of molecular mechanisms for telomere regulation are conserved, whereas even between human and mouse cells, there is divergence in telomere regulation by Pot1 proteins (Hockemeyer et al. 2006; Wu et al. 2006). It remains to be elucidated whether our observations in human cells are applicable to telomere regulation in other species.
During preparation of this manuscript, it was reported that ORC is recruited to telomeres by TRF2 (Deng et al. 2007). Our findings are essentially in agreement. In addition, our data indicate that the TRF2–ORC interaction is primarily mediated by ORC1 and that pre-RCs are indeed assembled on telomere-repeat DNA in a cell cycle–regulated manner. Furthermore, it was found that TRF2 binding to telomeres is influenced by ORC. In the paper, Deng et al. (2007) showed partial co-localization of TRF2 and ORC2 foci but the staining pattern of ORC2 appeared different from that for ORC1 presented here; the ORC2 foci appear larger and fewer than ORC1 foci. It has been previously reported that ORC2 localization in nuclei differs with the cell line tested (Prasanth et al. 2004). In some cell lines such as MCF7, ORC2 forms discrete large foci with a relatively small number and well co-localizes with HP1. However, in other cell lines such as HeLa, ORC2 is observed as numerous, relatively small dots with a small number of large foci co-localizing with HP1 (Fujita et al. 2002; Prasanth et al. 2004). The primary role of ORC is in pre-RC formation and there are many replication origins in the nucleus. Thus, the former large foci might not represent pre-RC foci in which multiple pre-RCs are assembled but rather might mainly represent heterochromatic foci. In HeLa cells, a significant fraction of the numerous ORC2 dots co-localize with ORC1, which might be pre-RC foci (Fujita et al. 2002). In the former cells, relatively weak signals away from large foci could represent pre-RC foci. The relatively small number of ORC2 foci in HCT116 and U2OS cells detected by Deng et al. (2007) could represent heterochromatic foci. However, when we examined ORC1 localization in HeLa cells, we observed numerous ORC1 dots, as reported previously (Fujita et al. 2002; Tatsumi et al. 2003). Nevertheless, approximately 50% of TRF2 foci co-localize with ORC1 foci. The most striking difference between the reported findings and ours concerns the effects of ORC inhibition on telomeres: Deng et al. (2007) suggested that ORC2 depletion results in telomere loss, dysfunctional telomeres, and telomere-circle formation, whereas our data indicate that ORC1 depletion leads to gradual telomere elongation. The reason for the difference is unclear at present, but there are several possible explanations. One is that they have used cancer-derived HCT116 and U2OS cells, whereas we employed normal human fibroblast immortalized with telomerase. In addition, they silenced ORC2, which is implicated also in centrosomal duplication, whereas we used retrovirus-mediated expression of an shRNA against ORC1. It is undoubtedly the case that telomere regulation by ORC and TRF2 is complicated and might be somewhat different among cells (e.g. between non-transformed and transformed cells) and further extensive studies will clearly be required to reach complete understanding.
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Experimental procedures |
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293T HeLa, and HFF2/T (human foreskin fibroblasts immortalized with telomerase; population doublings approximately 130) cells were grown in Dulbecco's modified Eagle's medium with 8% fetal calf serum. Cells synchronized in early S-phase were obtained with 2.5 mM hydroxyurea treatment for 16 h.
Plasmids, retroviral vectors and gene transfer
TRF2 cDNA was synthesized with the published sequences with codon optimization and confirmed by sequencing (GenScript, Scotch Plains, NJ). For convenience, TRF2 was tagged with the HA at its N-terminus, some artificial restriction enzyme sites were created in the coding sequences without changing codon usage, and recombination signals for the Gateway technology (Invitrogen, Carlsbad, CA) were also added to non-coding regions. The cDNA was inserted into pGEX6P-1 for bacterial expression of GST-TRF2, and into a retroviral vector pCLMSCVhyg (Tatsumi et al. 2006) for expression in mammalian cells. pGEX6P-1-TRF2M was created from pGEX6P-1-TRF2 by introducing a stop codon at amino acid 455 using oligonucleotide-directed mutagenesis (QuikChange Site-directed Mutagenesis Kit; Stratagene, La Jolla, CA) with 5'-CCAGATGAAGACAGCACCACCAACATTAC CTAGGAGCAGAAGTGGACCG-3' and a complementary oligonucleotide. pGEX6P-1-TRF2B was created by digesting pGEX6P-1-TRF2 with Nae I and self-ligation.
TRF1 cDNA in plasmid pENTR221 was purchased from Invitrogen (Ultimate Human ORF CLONE, IOH36709). For bacterial expression of GST-TRF1, TRF1 cDNA was inserted into pDEST15 (Invitrogen) from pENTR221-TRF1 by Gateway technology.
pcDNA-ORC1-FLAG expressing ORC1 tagged with the Flag epitope at its C-terminus was described previously (Tatsumi et al. 2003).
Retroviral vectors expressing shRNAs were produced as described previously (Tatsumi et al. 2006). Target sequences (sense strand) used were: shTRF2-3, 5'-AGCAGAAGTGGACTGTAGA-3'; shTRF2-4, 5'-GTGTCTGTCGCGGATTGAA-3'; and ORC1, 5'-GACTGCCACTGTTCATGAA-3'. Retrovirus production and infection were carried out as described previously (Tatsumi et al. 2006). Transient transfection into 293T cells was also carried out as described previously (Tatsumi et al. 2006) and 48 h after transfection, cells were subjected to analyses.
GST pull-down assay
GST fusion proteins were bacterially produced and purified as described previously (Sugimoto et al. 2004). Nuclear extracts were prepared as follows. Cells were first extracted with ice-cold modified CSK buffer (10 mM PIPES, pH 6.8, 100 mM NaCl, 300 mM sucrose, 1 mM EGTA, 1 mM MgCl2) containing 0.1% Triton X-100 and a multiple protease inhibitor cocktail (Fujita et al. 1997), and the remnant nuclear pellet was resuspended in mCSK buffer containing 0.1% Triton X-100, 0.5 M NaCl, and protease inhibitors. After centrifugation, the soluble nuclear fraction was obtained. In some experiments, whole cell extracts were prepared by directly adding mCSK buffer containing 0.5 M NaCl to cells. GST fusion proteins were bound to glutathione beads, and the beads were mixed with the diluted nuclear extracts. After washing three times with buffer A (25 mM Tris–HCl, pH 7.4, 0.01% Nonidet P-40 [NP-40], 1 mM dithiothreitol [DTT], 0.5 mM PMSF, 10% glycerol) containing 0.15 M NaCl, the bound proteins were eluted with 20 mM glutathione. Immunoblotting was carried out as described previously (Sugimoto et al. 2004) and antibody binding was visualized using the ECL system (Amersham, Buckinghamshire, UK).
ORC1, ORC2 and ORC3 proteins were synthesized by in vitro transcription–translation from pBluescript carrying the cDNAs (Fujita et al. 2002; Ohta et al. 2003) with rabbit reticulocyte lysate (TNT T7 quick coupled transcription–translation system, Promega, Madison, WI). The synthesized proteins were subjected to pull-down assay as detailed above.
Immunoprecipitation
293T cells transiently expressing HA-TRF2 and Flag-ORC1 were first cross-linked with 1% formaldehyde in phosphate-buffered saline (PBS) for 10 min at room temperature. After washing with PBS, cells were lysed on ice in 250 µL RIPA buffer (50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 0.5% deoxycholic acid) containing multiple protease inhibitors, sonicated, and the soluble fraction was separated by centrifugation. Separate aliquots of the extracts were then immunoprecipitated with anti-HA antibody and protein G-Sepharose beads (Amersham Bioscience). The beads were washed three times with 1 mL of NET gel buffer (50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 0.1% Triton X-100, 1 mM EDTA).
Cell fractionation
Cell fractionation to prepare chromatin/nuclear matrix-bound fractions was carried out with modified CSK buffer containing 0.1% Triton X-100 (0.1% TX–100 m CSK buffer) as described previously (Fujita et al. 1997).
siRNA experiments
HeLa cells were transfected with siRNA duplexes using HiPerFect (Qiagen, Venlo, the Netherlands) according to the manufacturer's instructions. siRNA oligonucleotides were synthesized (Dharmacon and IDT) with the following sequences (sense strand): ORC1 (5'-CUGCACUACCAAACCUAUAdTdT-3'), control GL2 (5'-CGUACGCGGAAUACUUCGAdTdT-3'), ORC2-1 (5'-AGA UUCAAGCUCAGAAUAGAGUAdGdT-3'), ORC2-2 (5'-GGA CACUAAUGCAGUCAUAUUCAdGdC-3'), ORC2-3 (5'-GGAAC AAUACGUCAUAUAUUUGGdAdA-3'), and control scrambled (5'-CUUCCUCUCUUUCUCUCCCUUGUdGdA-3').
Chromatin immunoprecipitation (ChIP) and dot-blot analysis
ChIP assays were carried out essentially as described previously (Yugawa et al. 2007). Briefly, 5 x 106 cells were fixed with 1% formaldehyde and lysed in SDS lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris–HCl, pH 8.0). Chromatin was sonicated to an average fragment size of 0.3 kb. Each sample was incubated overnight at 4 °C with 5 µg of the indicated antibodies or control IgGs and collected with protein G-Sepharose. After formaldehyde reversal, phenol–chloroform extraction and ethanol precipitation, the DNAs were dissolved in TE.
Input and immunoprecipitated DNAs were denatured by NaOH and spotted onto nylon membranes (GENESCREEN PLUS, Perkin Elmer, Waltham, MA). After baking, the membranes were hybridized with oligonucleotide probe 3xCCCTAA. Hybridization and detection of the labeled-probe were carried out with the Telo TAGGG Telomere Length Assay (Roche, Nutley, NJ). Alu sequence was excised from pUCAlu (RIKEN BioResource Center, Tsukuba, Japan), labeled with [-33P]-dCTP, and hybridized with the membranes.
Southern blot terminal restriction fragment analysis
Total DNAs were digested with Hinf I and Rsa I, separated on 0.9% agarose gel, and alkaline transferred onto nylon membranes. Detection of telomere sequences was carried out as above.
Cell cycle analysis and karyotype analysis
Cells were harvested with trypsin, stained with propidium iodide, and analyzed with a Becton Dickinson FACS Calibur. HFF2/T cells were treated with colcemid (20 ng/mL) for 2 h, incubated in hypotonic buffer, and fixed with Carnoy's fixative. The chromosomes were then stained with Giemsa.
Immunostaining
HeLa cells were washed with ice-cold PBS and extracted with modified CSK buffer containing 0.1% Triton X-100 at 4 °C for 10 min. Cells were then fixed with formaldehyde for 20 min on ice and incubated overnight at 4 °C with rabbit anti-ORC1 and mouse anti-TRF2 antibodies. The samples were further incubated for 2 h with the secondary goat anti-mouse IgG antibody conjugated with AlexaFluor 594 and goat anti-rabbit IgG antibody conjugated with AlexaFluor 488 (Molecular Probes, Eugene, OR).
Antibodies
Preparation of polyclonal rabbit antibodies against human Cdt1, ORC1, ORC2, CDC6 and MCM7 was described previously (Fujita et al. 2002; Ohta et al. 2003; Tatsumi et al. 2003, 2006; Sugimoto et al. 2004). Other antibodies were purchased from different companies: ORC2 (3B7, MBL), ORC3 (1D6, Santa Cruz), HA-tag (3F10, Roche), Flag-tag (M2, Sigma), TRF2 (clone 4A794, Upstate).
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Acknowledgements |
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Footnotes |
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aPresent address: Division of Biochemistry, Chiba Cancer Center Research Institute, Chiba 260-8717, Japan.
bPresent address: Department of Biochemistry, Jichi Medical University, 3311-1 Yakushiji, Shimotsuke-shi, Tochigi 329-0498, Japan.
These authors equally contributed to this work.
* Correspondence: mafujita{at}ncc.go.jp |
References |
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TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
|---|
Bell, S.P. & Dutta, A. (2002) DNA replication in eukaryotic cells. Annu. Rev. Biochem. 71, 333–374.[CrossRef][Medline]
Bell, S.P. & Stillman, B. (1992) ATP-dependent recognition of eukaryotic origins of DNA replication by a multiprotein complex. Nature 357, 128–134.[CrossRef][Medline]
Celli, G.B. & de Lange, T. (2005) DNA processing is not required for ATM-mediated telomere damage response after TRF2 deletion. Nat. Cell Biol. 7, 712–718.[CrossRef][Medline]
Danis, E., Brodolin, K., Menut, S., Maiorano, D., Girard-Reydet, C. & Méchali, M. (2004) Specification of a DNA replication by a transcription complex. Nat. Cell Biol. 6, 721–730.[CrossRef][Medline]
Deng, Z., Atanasiu, C., Burg, J.S., Broccoli, D. & Lieberman, P.M. (2003) Telomere repeat binding factors TRF1, TRF2, and hRAP1 modulate replication of Epstein–Barr virus OriP. J. Virol. 77, 11992–12001.
Deng, Z., Dheekollu, J., Broccoli, D., Dutta, A. & Lieberman, P.M. (2007) The origin recognition complex localizes to telomere repeats and prevents telomere-circle formation. Curr. Biol. 17, 1989–1995.[CrossRef][Medline]
Deng, Z., Lezina, L., Chen, C.J., Shtivelband, S., So, W. & Lieberman, P.M. (2002) Telomeric proteins regulate episomal maintenance of Epstein–Barr virus origin of plasmid replication. Mol. Cell 9, 493–503.[CrossRef][Medline]
Dhar, S., Yoshida, K., Machida, Y., Khaira, P., Chaudhuri, B., Wohlschlegel, J., Leffak, M., Yates, J. & Dutta, A. (2001) Replication from oriP of Epstein–Barr virus requires human ORC and is inhibited by geminin. Cell 106, 287–296.[CrossRef][Medline]
Diffley, J.F. (2004) Regulation of early events in chromosome replication. Curr. Biol. 14, R778–R786.[CrossRef][Medline]
Fairall, L., Chapman, L., Moss, H., de Lange, T. & Rhodes, D. (2001) Structure of the TRFH dimerization domain of the human telomeric proteins TRF1 and TRF2. Mol. Cell 8, 351–361.[CrossRef][Medline]
Fox, C.A. & McConnell, K.H. (2005) Toward biochemical understanding of a transcriptionally silenced chromosomal domain in Saccharomyces cerevisiae. J. Biol. Chem. 280, 8629–8632.
Francia, S., Weiss, R.S. & d’Adda di Fagagna, F. (2007) Need telomere maintenance? Call 911. Cell Div. 2, 3.[CrossRef][Medline]
Freibaum, B.D. & Counter, C.M. (2006) hSnm1B is a novel telomere-associated protein. J. Biol. Chem. 281, 15033–15036.
Fujita, M. (2006) Cdt1 revisited: complex and tight regulation during the cell cycle and consequences in deregulation in mammalian cells. Cell Div. 1, 22.[CrossRef][Medline]
Fujita, M., Ishimi, Y., Nakamura, H., Kiyono, T. & Tsurumi, T. (2002) Nuclear organization of DNA replication initiation proteins in mammalian cells. J. Biol. Chem. 277, 10354–10361.
Fujita, M., Kiyono, T., Hayashi, Y. & Ishibashi, M. (1997) In vivo interaction of human MCM heterohexameric complexes with chromatin: possible involvement of ATP. J. Biol. Chem. 272, 10928–10935.
García-Cao, M., O'Sullivan, R., Peters, A.H., Jenuwein, T. & Blasco, M.A. (2004) Epigenetic regulation of telomere length in mammalian cells by the Suv39h1 and Suv39h2 histone methyltransferases. Nat. Genet. 36, 94–99.[CrossRef][Medline]
Ge, X.-Q., Jackson, D.A. & Blow, J.J. (2007) Dormant origins licensed by excess Mcm2–7 are required for human cells to survive replicative stress. Genes Dev. 21, 3331–3341.
Hockemeyer, D., Daniels, J.P., Takai, H. & de Lange, T. (2006) Recent expansion of the telomeric complex in rodents: two distinct POT1 proteins protect mouse telomeres. Cell 126, 63–77.[CrossRef][Medline]
Huang, D.W., Fanti, L., Pak, D.T., Botchan, M.R., Pimpinelli, S. & Kellum, R. (1998) Distinct cytoplasmic and nuclear fractions of Drosophila heterochromatin protein 1: their phosphorylation levels and associations with origin recognition complex proteins. J. Cell Biol. 142, 307–318.
Karlseder, J., Broccoli, D., Dai, Y., Hardy, S. & de Lange, T. (1999) p53- and ATM-dependent apoptosis induced by telomeres lacking TRF2. Science 283, 1321–1325.
Karlseder, J., Hoke, K., Mirzoeva, O.K., Bakkenist, C., Kastan, M.B., Petrini, J.H. & de Lange, T. (2004) The telomeric protein TRF2 binds the ATM kinase and can inhibit the ATM-dependent DNA damage response. PLoS Biol. 2, E240.[CrossRef][Medline]
Karlseder, J., Smogorzewska, A. & de Lange, T. (2002) Senescence induced by altered telomere state, not telomere loss. Science 295, 2446–2449.
Mendez, J., Zou-Yang, X.H., Kim, S. Y., Hidaka, M., Tansey, W.P. & Stillman, B. (2002) Human origin recognition complex large subunit is degraded by ubiquitin-mediated proteolysis after initiation of DNA replication. Mol. Cell 9, 481–491.[CrossRef][Medline]
Minami, H., Takahashi, J., Suto, A., Saitoh, Y. & Tsutsumi, K. (2006) Binding of AlF-C, an Orc1-binding transcriptional regulator, enhances replicator activity of the rat aldolase B origin. Mol. Cell. Biol. 26, 8770–8780.
Muñoz, P., Blanco, R., Flores, J.M. & Blasco, M.A. (2005) XPF nuclease-dependent telomere loss and increased DNA damage in mice overexpressing TRF2 result in premature aging and cancer. Nat. Genet. 37, 1063–1071.[CrossRef][Medline]
Nakamura, M., Nabetani, A., Mizuno, T., Hanaoka, F. & Ishikawa, F. (2005) Alterations of DNA and chromatin structures at telomeres and genetic instability in mouse cells defective in DNA polymerase . Mol. Cell. Biol. 25, 11073–11088.
Ohta, S., Tatsumi, Y., Fujita, M., Tsurimoto, T. & Obuse, C. (2003) The ORC1 cycle in human cells: II. Dynamic changes in the human ORC complex during the cell cycle. J. Biol. Chem. 278, 41535–41540.
Pak, D., Pflumm, M., Chesnokov, I., Huang, D., Kellum, R., Marr, J., Romanowski, P. & Botchan, M. (1997) Association of the origin recognition complex with heterochromatin and HP1 in higher eukaryotes. Cell 91, 311–323.[CrossRef][Medline]
Prasanth, S.G., Prasanth, K.V. & Stillman, B. (2002) Orc6 involved in DNA replication, chromosome segregation, and cytokinesis. Science 297, 1026–1031.
Prasanth, S.G., Prasanth, K.V., Siddiqui, K., Spector, D.L. & Stillman, B. (2004) Human Orc2 localizes to centrosomes, centromeres and heterochromatin during chromosome inheritance. EMBO J. 23, 2651–2663.[CrossRef][Medline]
Saha, T., Ghosh, S., Vassilev, A. & DePamphilis, M.L. (2005) Ubiquitylation, phosphorylation and Orc2 modulate the subcellular location of Orc1 and prevent it from inducing apoptosis. J. Cell Sci. 119, 1371–1382.[CrossRef]
Schepers, A., Ritzi, M., Bousset, K., Kremmer, E., Yates, J.L., Harwood, J., Diffley, J.F. & Hammerschmidt, W. (2001) Human origin recognition complex binds to the region of the latent origin of DNA replication of Epstein–Barr virus. EMBO J. 20, 4588–4602.[CrossRef][Medline]
Smogorzewska, A. & de Lange, T. (2004) Regulation of telomerase by telomeric proteins. Annu. Rev. Biochem. 73, 177–208.[CrossRef][Medline]
Smogorzewska, A., van Steensel, B., Bianchi, A., Oelmann, S., Schaefer, M.R., Schnapp, G. & de Lange, T. (2000) Control of human telomere length by TRF1 and TRF2. Mol. Cell. Biol. 20, 1659–1668.
van Steensel, B., Smogorzewska, A. & de Lange, T. (1998) TRF2 protects human telomeres from end-to-end fusions. Cell 92, 401–413.[CrossRef][Medline]
Sugimoto, N., Tatsumi, Y., Tsurumi, T., Matsukage, A., Kiyono, T., Nishitani, H. & Fujita, M. (2004) Cdt1 phosphorylation by cyclin A-dependent kinases negatively regulates its function without affecting geminin binding. J. Biol. Chem. 279, 19691–19697.
Tatsumi, Y., Ohta, S., Kimura, H., Tsurimoto, T. & Obuse, C. (2003) The ORC1 cycle in human cells I. Cell cycle-regulated oscillation of human ORC1. J. Biol. Chem. 278, 41528–41534.
Tatsumi, Y., Sugimoto, N., Yugawa, T., Narisawa-Saito, M., Kiyono, T. & Fujita, M. (2006) Deregulation of Cdt1 induces chromosomal damage without rereplication and leads to chromosomal instability. J. Cell Sci. 119, 3128–3140.
Tsao, C.C., Geisen, C. & Abraham, R.T. (2004) Interaction between human MCM7 and Rad17 proteins is required for replication checkpoint signaling. EMBO J. 23, 4660–4669.[CrossRef][Medline]
Vashee, S., Cvetic, C., Lu, W., Simancek, P., Kelly, T.J. & Walter, J.C. (2003) Sequence-independent DNA binding and replication initiation by the human origin recognition complex. Genes Dev. 17, 1894–1908.
Vashee, S., Simancek, P., Challberg, M. D. & Kelly, T.J. (2001) Assembly of the human origin recognition complex. J. Biol. Chem. 276, 26666–26673.
Verdun, R.E., Crabbe, L., Haggblom, C. & Karlseder, J. (2005) Functional human telomeres are recognized as DNA damage in G2 of the cell cycle. Mol. Cell 20, 551–561.[CrossRef][Medline]
Wu, L., Multani, A.S., He, H., Cosme-Blanco, W., Deng, Y., Deng, J. M., Bachilo, O., Pathak, S., Tahara, H., Bailey, S.M., Deng, Y., Behringer, R.R. & Chang, S. (2006) Pot1 deficiency initiates DNA damage checkpoint activation and aberrant homologous recombination at telomeres. Cell 126, 49–62.[CrossRef][Medline]
Yugawa, T., Handa, K., Narisawa-Saito, M., Ohno, S., Fujita, M. & Kiyono, T. (2007) Regulation of Notch1 gene expression by p53 in epithelial cells. Mol. Cell. Biol. 27, 3732–3742.
Received: 24 March 2008
Accepted: 13 July 2008
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