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First published online January 23, 2008
doi: 10.1242/10.1242/jcs.003632
Research Article |
1 Molecular Biology Laboratory, Scuola Normale Superiore, AREA della Ricerca del CNR, Via Moruzzi 1, Pisa, Italy
2 Molecular Medicine Laboratory, International Centre for Genetic Engineering and Biotechnology (ICGEB), Padriciano 99, Trieste, Italy
3 Laboratory of Genetics, The Salk Institute for Biological Studies, La Jolla, CA 92037, USA
Author for correspondence (e-mail: giacca{at}icgeb.org)
Accepted 30 October 2007
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Summary |
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Key words: AAV, DNA repair, MRN complex, Viral vectors
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; Tratschin et al., 1984). Although the utilization of rAAV vectors in pre-clinical and clinical gene transfer applications is rising, several aspects of the life cycle of both the wild-type virus and, most notably, of the recombinant vectors remain largely obscure. In particular, limited information is available on the intracellular fate of rAAV DNA after internalization. This appears to be a topic of particular relevance considering that a relatively small number of tissues are permissive to rAAV transduction, despite the receptors for AAV internalization being widespread in most cell types in vivo.
In cultured cells, a marked increased in rAAV transduction efficiency is obtained by treating cells with agents that affect genomic DNA integrity or metabolism, such as UV irradiation, hydroxyurea (HU), topoisomerase inhibitors and several chemical carcinogens (Alexander et al., 1994; Russell et al., 1995; Zentilin et al., 2001; Zhou et al., 1999). A major effect of these treatments is the improved conversion of the input vector ssDNA genome into double-stranded (ds) DNA (Ferrari et al., 1996; Fisher et al., 1996), which represents an essential requisite for gene expression. These observations have raised the possibility that permissivity to rAAV transduction might be related to the induction of the cellular DNA-damage response (DDR) and, in particular, of the proteins involved in DNA-double-strand break (DSB) repair (Zentilin et al., 2001).
In eukaryotic cells, surveillance mechanisms have evolved to rapidly recognize DNA damage and signal its presence. In particular, the occurrence of DSBs in chromosomal DNA elicits a signaling response through the ataxia-telangiectasia mutated (ATM) protein kinase, which coordinates cell-cycle arrest, DNA repair and apoptosis (Shiloh, 2003). Recent evidence indicates that the MRE11-RAD50-NBS1 (MRN) complex is an essential mediator of ATM recruitment to, and activation by, DSBs, both by forming multiple protein-protein contacts with ATM (Lee and Paull, 2005) and by tethering damaged DNA, thereby increasing its local concentration (Dupre et al., 2006). The retention of MRN at the DSB sites requires direct binding of its NBS1 component to the mediator of DNA damage checkpoint protein 1 (MDC1) (Goldberg et al., 2003; Lukas et al., 2004; Stewart et al., 2003).
In response to DNA damage, several components of the DDR, normally diffuse in the nucleoplasm, are rapidly relocalized and concentrated into subnuclear compartments that are microscopically detected as foci. Although the exact nature of the molecular processes occurring at these foci is still not completely understood, experimental evidence indicates that at least some of these foci form in correspondence or in close proximity to the sites of actual DNA damage and that the increase local concentration of factors they determine is beneficial for genome surveillance and repair (Seno and Dynlacht, 2004; van den Bosch et al., 2003).
Essential to comprehend the mechanisms of rAAV genome processing is the understanding of the intracellular fate of vector genomes after cell transduction. In this respect, recent studies have demonstrated the feasibility of visualizing specific genomic regions within the nucleus by exploiting the interaction of sequence-specific DNA-binding proteins linked to the green fluorescent protein (GFP) with tandem repeats of cognate binding sites. In particular, different studies have employed a system based on the bacterial Lac operator-repressor (LacO/R) interaction to examine chromatin organization, separation of sister chromatids, chromosome dynamics and gene expression in living cells (Belmont and Straight, 1998; Straight et al., 1997; Tsukamoto et al., 2000; Tumbar et al., 1999). This procedure has also proved useful in the study of wild-type AAV replication in the presence of helper virus (Fraefel et al., 2004; Glauser et al., 2005).
In this work, we adapted the LacO/R system for the visualization in real time of the sites of ssDNA to dsDNA conversion of rAAV vector genomes inside the nucleus of the transduced cell, in the absence of helper virus or of any other helper gene products. We observed that rAAV DNA accumulates in discrete foci inside the nucleus, which increase in size and are relatively immobile over time. These rAAV foci lie in close proximity to the foci in which proteins of the MRN complex and MDC1 accumulate after DNA damage. We also report that silencing of NBS1 and MDC1 by RNA interference markedly enhances rAAV transduction, indicating that MRN and MDC1 play an inhibitory role on rAAV genome processing. These findings support the conclusion that these cellular DDR proteins significantly restrict rAAV transduction.
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Time-lapse imaging studies performed in living infected cells in order to track the movement of individual foci indicated that these structures were relatively immobile and only displayed an apparently random movement in a relatively restricted area of the nucleus (supplementary material Fig. S1A-D). These experiments also revealed that the intensity of fluorescence of individual foci progressively increased over the first 8-10 hours p.i. (supplementary material Fig. S1E), suggestive of a progressive accumulation of dsDNA rAAV genomes into these structures.
Quantitative analysis of rAAV formation indicated that the percentage of cells showing rAAV foci increased during the first 24 hours p.i.; at all times, this number was higher after treatment with HU; 25.1±9.2% and 11.9±0.6% cells with foci in HU-treated and untreated cells, respectively, at 24 hours; Fig. 2A). The number of AAVLacO.14 foci per cell nucleus was also determined in 3D-images of 30-60 nuclei observed over time (Fig. 2B). The maximum number of foci was detected at 24 hours p.i. in HU-treated cells (23.9±19.1 foci per nucleus) and at 8 hours in untreated cells (12.1±9.6). Interestingly, the number of rAAV foci only modestly increased at these times compared with the earlier time points, suggesting that their number per cell was determined very soon after infection. Finally, rAAV foci progressively disappeared at later time points, even if some foci were found to persist as long as 6 days in both HU-treated and untreated cells (data not shown).
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-tubulin and nuclear poly (ADP-ribose) polymerase (PARP) proteins (supplementary material Fig. S2). We found that the number of rAAV genomes inside the nucleus progressively increased up to 12 hours in HU-treated cells, reaching a maximum of over 400 genomes/nucleus; in non-treated cells, the number of genomes at 12 hours p.i. was less than half this amount (Fig. 2C). Considering the number of foci per cell observed under similar conditions, and assuming that all rAAV DNA clusters form rAAV foci, these data indicate that, on average, each rAAV focus is composed of no more than 
20 viral genomes. In agreement with these results, through the comparison of the GFP fluorescence intensity of rAAV foci with that of GFP-tagged rotavirus [virus-like particles containing a known number of GFP molecules (Dundr et al., 2002)], we estimated that the great majority of the observed rAAV foci (>80%) contained one to ten vector genomes, with a range of estimated genomes per foci that varied from one to 250 (data not shown).
rAAV foci form in close contact with cellular MRN complex and MDC1
Given the ss nature of AAV genomes and the existence of regions of secondary DNA structure such as the viral ITRs within the vectors, we wanted to establish whether a relationship existed between the formation of rAAV foci and the localization of the proteins of the MRN complex, which is known to bind both ss and dsDNA and to have a pivotal role in sensing damaged or hairpin-structured DNA (D'Amours and Jackson, 2002). We observed that a vast proportion (>70%) of rAAV foci colocalized to the nuclear foci that NBS1, MRE11 and RAD50 form after HU-induced DNA damage (see Fig. 3A,C and D, respectively, for representative images and Fig. 3F for quantification). In addition, most rAAV foci colocalized with NBS1 or MRE11 foci in the few cells that showed a speckled NBS1 or MRE11 localization in the absence of DNA damage (Fig. 3A,C). Furthermore, in both treated and untreated cells, rAAV foci specifically colocalized with NBS1 phosphorylated at serine 343 (P-S343-NBS1; Fig. 3B), a marker of protein activation after DNA damage (Gatei et al., 2000; Lim et al., 2000; Wu et al., 2000). Similar findings were obtained in both HeLa/GFP-LacR and MRC5/GFP-LacR cells and upon cell treatment with HU or camptothecin (Fig. 3D,E and data not shown).
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AAVLacO.14 foci were also found in close proximity to the foci at which the MDC1 protein accumulates, in both untreated cells and in cells treated with camptothecin to induce DNA damage (Fig. 3E). MDC1 is known to control cellular responses to DNA damage, in part by interacting with the MRN complex and, more specifically, by mediating the transient interaction of NBS1 with DSBs and its phosphorylation by ATM (Goldberg et al., 2003; Lukas et al., 2004; Stewart et al., 2003; Xu and Stern, 2003).
MRE11 associates with rAAV genomes
To directly assess the possibility that MRN proteins might physically interact with rAAV genomes, we performed chromatin immunoprecipitation (ChIP) experiments using specific antibodies to MRE11, NBS1, P-S343-NBS1 and the cellular transcription factor USF. Cross-linked and sonicated chromatin from uninfected cells (not shown) or cells transduced with AAV-LacZ at 8, 16 and 24 hours after transduction, either in the presence or absence of HU treatment, was immunoprecipitated with each antibody; the amount of cross-linked DNA specific for rAAV or for two cellular genomic loci was then assessed by real-time PCR. One of the analyzed genomic regions (B48) contains a binding site for USF and served as a positive control (Lusic et al., 2003). The anti-USF antibody was indeed found to specifically enrich for this region (25 times over background), irrespective of HU treatment, in agreement with previously published findings (Todorovic et al., 2005); no enrichment was detected for rAAV DNA (Fig. 4A). The antibody against MRE11, which represents the DNA binding component of MRN, was found to specifically enrich for rAAV DNA at 8 hours (approximately sixfold) and, most remarkably, at 16 hours and 24 hours after transduction (over 45-fold at both time points) in the absence of HU treatment (Fig. 4B). However, the extent of enrichment (2.5-, 14- and 15-fold at 8, 16 and 24 hours, respectively) was significantly lower after cell treatment with HU. The observation that MRE11 associates with rAAV genomes in the absence of HU and that the extent of binding decreases upon HU treatment is consistent with the possibility that MRN might be involved in rAAV DNA processing and then become displaced from rAAV DNA upon induction of global cellular DNA damage. In similar ChIP experiments, antibodies against both NBS1 and P-S343-NBS1 scored negative, suggesting either that NBS1 is not in such close proximity to DNA to allow effective cross-linking or it is not available to antibodies once embedded in the chromatin crosslinks.
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MRN proteins inhibit formation of rAAV foci
To test the functional significance of MRN and MDC1 protein interaction with rAAV genomes, we knocked down NBS1 and MDC1 expression by RNA interference prior to cell transduction with AAVLacO.14, or with control AAV-LacZ and AAV-GFP vectors. After NBS1 silencing to less than 10% of control (Fig. 5A), the number of cells with rAAV foci was markedly increased as compared with control cells treated with an unrelated siRNA against luciferase. This effect was very evident at both 16 hours and 24 hours after transduction and in the presence or absence of HU treatment (Fig. 5C). At 24 hours after transduction and in the absence of HU, there were 41.08±9.93% of cells with rAAV foci after treatment with the anti-NBS1 siRNA, compared with 10.35±4.75% of cells with foci upon treatment with the control siRNA (representative images are shown in Fig. 5B). Of interest, the effect of HU on the number of cells with rAAV foci was less pronounced at 24 hours after transduction as compared with 16 hours (1.26-fold increase in the number of foci upon HU treatment at 24 hours, 3.19-fold increase at 16 hours), showing that the mechanism by which HU induces rAAV foci formation is saturable when NBS1 is knocked down.
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NBS1 and MDC1 proteins inhibit rAAV transduction
Next we wanted to evaluate whether the increase in the number of cells with detectable rAAV foci upon NBS1 knock down correlated with improved rAAV vector efficiency. MRC5 and HeLa cells in which the NBS1 gene had been knocked down were significantly more permissive to transduction with the AAV-LacZ vector at different m.o.i.s, both in the presence and absence of HU (Fig. 6A,B). This finding further supports the conclusion that permissivity to rAAV transduction correlates with the number of cells with rAAV foci, and thus with the levels of dsDNA genomes inside the nucleus of the cell.
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). In agreement with the siRNA results, NBS1-ILB1 fibroblasts complemented with NBS1 were less permissive to rAAV transduction when compared with the non-complemented parental cells (Fig. 6C). In contrast to wild-type MRC5, NBS1 silencing was found to have no effect in AT5 (ATM–/–) cells, which are known to be highly permissive to rAAV transduction and to respond marginally to HU treatment (Zentilin et al., 2001) (Fig. 6D). This result is in keeping with the possibility that the negative role of NBS1 on AAV transduction might require functional ATM.
Very similar results were also observed after knocking down the expression of MDC1. For these experiments, we took advantage of the availability of stable HeLa cell clones transduced with retroviral vectors expressing short hairpin RNAs against either MDC1 or against control β-galactosidase (Lukas et al., 2004). The cell clone in which MDC1 was knocked down was significantly more permissive to AAV-GFP transduction, both in the absence or following HU treatment (Fig. 6E). Similar results were also obtained when HeLa cells where transiently transfected with synthetic siRNAs against MDC1 (Fig. 6F).
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20. These genomes most likely correspond to the multimerized rAAV DNA forms detected by other studies that analyzed the molecular nature of transcription-competent AAV genomes in the transduced cells (Afione et al., 1996; Yang et al., 1999).
Treatment of cells with HU is known to significantly boost the efficiency of rAAV transduction (Russell et al., 1995; Zentilin et al., 2001). In our experiments, we observed that the same treatment also increased the number of cells with rAAV foci, thus showing that the formation of rAAV foci positively correlates with the efficiency of effective transduction. Analysis of the accumulation of rAAV genomes inside the nucleus in the presence and absence of HU treatment and at several time points suggests that the majority of the viral genomes are already inside the cell nucleus at 4 hours after infection. Although the number of viral genomes (ss and ds) reaches its maximum level at 12 hours p.i., the highest number of cells with foci is observed at 24 hours p.i., and then gradually decreases. This discrepancy is likely due to the fact that, at early time points, most of the viral genomes are present as ssDNA and possibly still protected by the viral capsids (Bartlett et al., 2000; Lux et al., 2005); at later time points, a proportion of these genomes are converted to dsDNA while the others are most likely targeted by cellular factors and eventually degraded.
Previous studies in adenovirus-infected cells, using immunofluorescence and in situ hybridization, have shown that, during infection, wt AAV DNA and proteins accumulate in discrete nuclear foci (Weitzman et al., 1996; Wistuba et al., 1997). Similar findings have been confirmed in live cells infected with HSV-1, using a rAAV vector containing 40 LacO repeats and a reporter protein similar to ours (Fraefel et al., 2004). The presence of these foci has been interpreted as an indication of the existence of subnuclear compartments in which AAV DNA replication takes place, by virtue of the colocalization, within the same replication centers, of the AAV Rep proteins, of the replicative proteins of the helper virus and of the helper virus DNA (Fraefel et al., 2004; Weitzman et al., 1996). The rAAV foci we detected in our experiments might well be coincident with these AAV and adenovirus replication centers. In this respect, however, it should be emphasized that the rAAV foci that we observed are not determined by the subnuclear localization of the replicative proteins of either AAV or helper virus, thus indicating that rAAV DNA is itself recognized by nuclear factors of cellular origin.
Indeed, we found that the rAAV foci colocalize with the nuclear compartments where the components of the MRN complex accumulate upon DNA damage. In support of the notion that these proteins are actively involved in a DNA repair process, we observed that the NBS1 foci were also detected by an antibody specific for a DNA-damage-activated form of NBS1, phosphorylated at serine 343, (Gatei et al., 2000; Lim et al., 2000; Wu et al., 2000). In keeping with the possibility that the DNA-double-strand-break repair pathway is involved in rAAV genome processing, we found that an upstream mediator, MDC1, known to increase the retention of NBS1 at the sites of DNA damage (Lukas et al., 2004), also formed foci that colocalized with rAAV foci.
What might be the role of the MRN proteins and of MDC1 in rAAV genome processing? Our RNAi experiments clearly suggest that the overall effect of these proteins is inhibitory in rAAV dsDNA formation. Silencing of NBS1 by RNAi increased the number of cells containing rAAV foci and significantly improved the permissivity of both HeLa and MRC5 cells to transduction, again indicating that generation of dsDNA, and thus formation of rAAV foci, positively correlates with the efficiency of transduction. Similar considerations also apply to the silencing of MDC1. Interestingly, the NBS1 knock down had a marked effect in increasing the number of cells with rAAV foci; however, in each of the positive cells we observed no significant changes in the average number of foci present per cell. This observation is consistent with an effect of MRN and related proteins on the overall rate of ss to dsDNA conversion (a reduction in the intensity of fluorescence of the individual rAAV foci determines the microscopic detection of a lower number of cells presenting rAAV foci over the diffuse EGFP-LacR background), rather than with a specific effect on the mechanism of rAAV foci formation. The conclusion that proteins of the MRN complex are inhibitory for rAAV transduction is further supported by our observation that NBS1-mutated fibroblasts are more permissive to rAAV transduction when compared with the same fibroblasts complemented with full length NBS1 cDNA, as well as by the findings of M. Weitzman and collaborators, who observed that degradation of MRN by the adenovirus E1b55K/E4Orf6 markedly increases both rAAV transduction and rAAV foci formation (Schwartz et al., 2007). In keeping with these observations, chromatin immunoprecipitation experiments clearly demonstrated that MRE11, the DNA binding component of MRN, directly interacts with the rAAV genomes and that this interaction is diminished when cells are treated with DNA-damaging agents. These findings are fully in agreement with the increase in transduction and foci formation observed after induction of DNA damage.
Over the last few years, we and others have reported that cells lacking functional ATM are markedly more permissive to rAAV transduction (Sanlioglu et al., 2000; Zentilin et al., 2001). The observation that the knock down of NBS1 has no apparent effect in ATM-defective cells is consistent with the possibility that the ATM protein might mediate the negative regulation of MRN on the incoming rAAV genomes. In this respect, it has been observed that, after ionizing radiation, NBS1 is phosphorylated by ATM at serine 343 and that the S-phase checkpoint is abrogated in mutants lacking this ATM phosphorylation site (Lim et al., 2000). It is worth noting that ATM and NBS1 have been recently shown to be essential in the formation of the replication protein A-coated ssDNA microcompartments that follow cell irradiation with ionizing radiations (Bekker-Jensen et al., 2006). It might thus be envisioned that these proteins also participate in the processing of the ssDNA AAV genomes.
Together these results support the following model. On entering the nucleus, rAAV genomes, by virtue of both their ssDNA nature and the presence of the viral ITRs, are recognized by cellular DDR proteins, including MRN and MDC1. These proteins are inhibitory of ss to ds genome conversion, impeding rAAV dsDNA production or routing the genomes to aberrant processing or nucleolytic degradation, thus limiting transduction. Once this inhibition is overcome, the genomes are converted to dsDNA either by second strand DNA synthesis or direct annealing of the incoming vector complementary DNA strands (Fisher et al., 1996; Hauck et al., 2004). Cell treatment with DNA damaging agents and infection with adenovirus both have a positive effect on transduction; the former treatment diverts the inhibitory proteins away from the rAAV genomes, the latter triggers their degradation.
After ss to dsDNA conversion, the vector genome is known to undergo additional changes, mediated by host cell factors acting on the AAV ITR ends (McCarty et al., 2004). Recent work exploiting self-complementary rAAV vectors (which bypass the ss to dsDNA conversion step) has shown that ATM and MRN proteins participate in genome circularization (Choi et al., 2006). Thus, it might be envisioned that these proteins become positive factors when dsDNA conversion has already occurred. At this stage, resolution of the secondary structures in the ITRs by promoting circularization or multimerization might be essential to allow stable maintenance of the viral genomes inside the nucleus.
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Plasmids and transfection
The plasmid p3'ssdEGFP, which expresses GFP linked to the lac repressor (LacR) and containing a nuclear localization signal, and pSV2dhfr 8.32, which contains 10 kb of 292 bp lac operator repeat (LacO), were kindly provided by A. S. Belmont (University of Illinois, IL). The plasmid pCMV-MCS (Stratagene, La Jolla, CA, USA) was digested with NotI to excise the multiple cloning site and substitute it with a multiple cloning site containing NotI, SalI, EcoRI, XhoI, BamHI and NotI to obtain plasmid pMCS3'. The 292 bp LacO repeat, obtained by digesting pSV2dhfr 8.32 with EcoRI, was cloned into the EcoRI site of pMCS3'; the 8-mer repeat was then amplified by directional cloning (Robinett et al., 1996) to obtain a vector containing 14 LacO repeats, for a total of 112 LacR binding sites. The 14 LacO repeats were cloned into the NotI site of pAAV-MCS to generate pAAVLacO.14. To obtain stable clones expressing the LacR fused to GFP, HeLa and MRC5 cells were transfected with Polyfect transfection reagent (Qiagen GmbH, Hilden, Germany) and clones were selected with hygromycin (300 µg/ml for HeLa cells; 100 µg/ml for MRC5).
Production of rAAV stocks
The rAAV vectors used in this study were produced from pTR-UF5 (AAV-GFP), kindly provided by N. Muzyczka (University of Florida College of Medicine, FL), pAAV-LacZ (Stratagene) and pAAVLacO.14. Cloning and propagation of AAV plasmids was carried in XL-10 Gold E. coli strain (Stratagene, La Jolla, CA, USA). Infectious AAV vector particles were generated by the AAV Vector Unit (AVU) at ICGEB Trieste (http://www.icgeb.org/RESEARCH/TS/COREFACILITIES/AVU.htm) in HEK293 cells, using dual plasmid cotransfection procedure with pDG as packaging helper plasmid (kindly provided by J. A. Kleinschmidt, DKFZ, Germany), as previously described (Zentilin et al., 2001). Titration of AAV-GFP and AAV-LacZ viral particles was performed by real-time PCR quantification of the number of viral genomes, as described previously (Zentilin et al., 2001); the viral preparations used in this work had titers between 1x1011 and 1x1012 viral genome particles (vgp) per ml. The titers of AAVLacO.14 were measured by Southern blotting using serial dilutions of the input pMCS3'lacO.14 plasmid as a standard; the preparations of this vector had titers between 1x108 and 1x109 vgp/ml.
Vector transduction experiments
Transduction with AAV-GFP was performed by plating 4x105 cells in 24-well plates. In the experiments with AAVLacO.14, 2x104 cells were seeded 24 hours before vector addition in eight-well chamber slides (Labtech International, Woodside, UK) or 5x105 cells in 6 cm glass bottom dishes). Transduction with AAV-LacZ was performed in 96-well plates with 3x103 cells per well. When indicated, cells were treated for 12-16 hours with 1 mM HU, for 6 hours with 1 nM camptothecin (Sigma-Aldrich, St Louis, MO, USA) or for 6 hours with 0.15 IU/ml bleomycin (Calbiochem) from 15 IU/ml stocks dissolved in water, before the addition of vector. Transduction with the different vectors was performed in DMEM supplemented with 10% FCS. After 3 hours incubation, cells were washed in PBS and fresh culture medium was added. Cellular GFP fluorescence was analyzed by flow cytometry using a FACSCalibur (Becton Dickinson, San Jose, CA, USA) 36 hours after AAV-GFP addition. In the case of AAV-LacZ transduction, β-galactosidase activity was determined 36 hours post-infection by measuring o-nitrophenyl-D-galacto-pyranoside (ONPG) cleavage using a photometric assay, as described elsewhere (Brown et al., 2000). Relative light units (RLU) were expressed as the ratio between ONPG absorbance at 405 nm and the protein concentration (in ng/ml) of the sample, multiplied by an arbitrary factor of 1000.
Confocal microscopy
Live or fixed cultures were analyzed by confocal microscopy using a Leica TCS-SL or a Zeiss LSM 510 laser scanning microscope. The different channels were detected sequentially, and the laser power and detection windows were adjusted for each channel to exclude overlap between different fluorochromes and to avoid signal saturation. rAAV foci location was monitored through a z-series of images. For live cell recording, cells plated on 6 cm glass bottom dishes were placed in a humidified Plexiglas chamber and maintained at 37°C and 5% CO2 throughout the experiment. The fluorescence intensity of DNA foci was measured by calculating the mean fluorescence intensities of pixels of maximum intensity projection of x,y,z,t scan and subtracting the background intensity calculated as mean fluorescence intensity outside the cell, at each time point.
Foci tracking analysis
Tracking of rAAV foci was performed as already described (Ferrari et al., 2003). Briefly, nuclei were scanned with x,y,z,t scan mode (3D + time lapse) with a Leica TCS-SL laser scanning microscope. Movement information was obtained from 2D images obtained, at each time point, from maximum intensity projections of 0.24 µm (supplementary material Fig. S1A) or 0.45 µm (supplementary material Fig. S1B-D) -thick optical z sections spanning the full depth of the nucleus. Starting from each frame, the images of the nucleus were cropped and aligned. The relative movement of each rAAV focus inside the nucleus was measured according to its x and y coordinates at each frame using the MetaMorph software and plotted with Origin Pro v7.03 (OriginLab Corp., Northampton, MA, USA). The mean velocity was calculated from the observed trajectories.
Immunofluorescence
At the indicated time points after infection, cells were washed twice with PBS and fixed and permeabilized either with 2% paraformaldehyde in PBS for 15 minutes at room temperature followed by two washes with 0.1% Triton X-100 in PBS or with 100% methanol at –20°C for 20 minutes followed by 100% acetone at –20°C for 20 seconds. After fixing, the cells were washed twice, and incubated with primary antibody for 1.5 hours in PBS plus 0.15% glycine and 0.5% BSA (PBS+) in a moist chamber at room temperature. The following antibodies were used: mouse anti-human RAD50 (GeneTex, San Antonio, TX, USA, Ab89) diluted 1:200; mouse anti-human MRE11 (GeneTex, Ab214, 12D7) and rabbit anti-human NBS1 (Novus Biologicals, Littleton, CO, USA, Ab398, NB 100-143), rabbit anti-phospho-NBS1 (Ser343; Novus Biologicals, 100-284A3) diluted 1:500 and anti-MDC1 rabbit polyclonal serum (kindly provided by S. P. Jackson, The Wellcome Trust/Cancer Research UK Gurdon Institute, University of Cambridge, UK) diluted 1:200. Cells were incubated with the secondary antibodies for 1 hour in PBS+ in a moist chamber at room temperature. The secondary antibodies used were goat anti-rabbit Alexa Fluor 594-conjugated and goat anti-mouse Alexa Fluor 594-conjugated (Molecular Probes, Eugene, OR, USA) both diluted 1:1000. Cells were mounted on chamber slides in Vectashield Mounting Medium (Vector Laboratories, Burlingame, CA, USA) containing 4'-6'-diamino-2-phenylidole (DAPI). Fixed cells were analyzed with a Leica DMLB fluorescence microscope connected to Leica DC camera or by confocal microscopy.
Cell fractionation and quantification of nuclear rAAV genomes
Transduction with AAV-LacZ (m.o.i. 10,000 vgp) was performed in 6 cm dishes containing 8x105 HeLa cells. When indicated, cells were treated for 12-16 hours with 1 mM HU before the addition of the vector. At the indicated times after infection, cytoplasmic and nuclear fractions were isolated, as described by Mendez and Stillman (Mendez and Stillman, 2000). Briefly, cells were washed once with PBS, treated with trypsin and further washed with PBS. Cell pellets were resuspended in 150 µl buffer A (10 mM Hepes pH 7.9, 10 mM KCl, 1.5 mM MgCl2, 0.34 M sucrose, 10% glycerol, 1 mM DTT, protease inhibitors) after which 150 µl of buffer A containing 0.2% Triton X-100 was added. Samples were incubated for 5 minutes on ice and then centrifuged for 5 minutes at 1300 g at 4°C. Nuclear fractions (pellets) were further washed with buffer A containing 0.1% Triton X-100 and resuspended in 200 µl PBS. Total DNA was purified from nuclear fractions using DNeasy Blood and Tissue kit (Qiagen), according to the manufacturer's instructions. The amount of rAAV genome present in nuclear fractions was quantified by real-time PCR using serial dilutions of pAAV-LacZ (Stratagene) as standards, essentially as performed for the quantification of rAAV stocks. Values from the real-time PCR quantification of B13 DNA segment of the lamin B2 genomic region were used as an internal control to normalize the total DNA concentration (number of nuclei) of the different samples. Purity of nuclear fractions was confirmed by western blotting to determine the absence of -tubulin and presence of the nuclear protein PARP (supplementary material Fig. S2).
RNA interference
Cells (4x105) were plates in 3.5 cm dishes and transfected using GeneSilencer (Gene Therapy Systems, San Diego, CA, USA) according to the manufacturer's recommendations. The sequence of the siRNAs against NBS1 (siNBS1), MDC1 (siMDC1), and luciferase (siLuc) were GGAAGAAACGUGAACUCAA (dTdT), CAACAAGAAGACGCGAAUC (dTdT) (Stewart et al., 2003), and CGUACGCGGAAUACUUCGA (dTdT) (Dharmacon, Lafayette, CO, USA), respectively. Twenty-four hours after siRNA transfection, cells well trypsinized and replated in 24-well plates, 96-well plates or eight-well-chambered slides and transduced with rAAV.
The pSR-MDC1 cell line is a HeLa cell derivative transduced with the pSUPER-retro retroviral vector (Brummelkamp et al., 2002) stably expressing an anti-MDC1 shRNA (insert sequence: GATCCCCGTC TCCCAGAAGA CAGTGATTCA AGAGATCACT GTCTTCTGGG AGACTTTTTG GAAA). The control pSR-LacZ cell line expresses an anti-β-galactosidase shRNA (insert sequence: AGCTTTTCCA AAAAGTCTCC CAGAAGACAG TGATCTCTTG AATCACTGTC TTCTGGGAGA CGGG) from the same vector. Both cell lines were kindly provided by Y. Shiloh (Sackler School of Medicine, Tel Aviv University, Israel).
Western blotting
Total cell lysates were prepared from cells treated with siRNA. Cells were lysed with sample buffer (20 mM Tris-HCl pH 8, 20 mM NaCl, 10% glycerol, 1% NP40, 10 mM EDTA, 2 mM PMSF, leupeptin 2.5 µg/ml, pepstatin 2.5 µg/ml) followed by heating to 100°C for 5 minutes. Protein concentration was determined by the Bradford method (BioRad, Richmond, CA); 10 µg of protein per lane were loaded on 12% SDS-PAGE minigels, and transferred to nitrocellulose (Amersham Biosciences, Bucks, UK). Immunoblots were blocked in 5% non-fat dry milk in TBST (50 mM Tris-HCl pH 7.4, 200 mM NaCl, 0.04% Tween 20). Primary antibodies were rabbit anti--tubulin (Sigma; diluted 1:10,000), anti-MDC1 (diluted 1:10,000), anti-NBS1 (Novus Biologicals), 100-143 (diluted 1:5,000), and anti-PARP (Santa Cruz Biotechnology, Santa Cruz, CA; sc-7150, diluted 1:200) antibodies. Horseradish peroxidase-labeled anti-rabbit antibody (Santa Cruz Biotechnology), 1:1000 was used as a secondary antibody. Antibody staining was detected using the ECL chemiluminescence system (Amersham Biosciences).
Chromatin immunoprecipitation (ChIP)
HeLa cells were transduced with AAV-LacZ at an m.o.i. of 10,000 vgp/cell. Where indicated, cells were pre-treated with 1 mM HU overnight before infection. ChIP was essentially performed as already described (Lusic et al., 2003; Todorovic et al., 2005). At 8, 16 and 24 hours after infection, cells were fixed by adding fixing solution (11% formaldehyde, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA pH 8.0, 50 mM Tris-HCl pH 8.0) directly to the cell culture medium at 1% final concentration. Cross-linking was allowed to proceed for 10 minutes at 37°C and was stopped by the addition of glycine at a final concentration of 0.125 M. Fixed cells were washed once in ice-cold PBS, once in buffer B1 (0.25% Triton X-100, 10 mM EDTA, 0.5 mM EGTA, 10 mM Tris-HCl pH 8.0) and once in buffer B2 (1 mM EDTA, 0.5 mM EGTA, 200 mM NaCl, 10 mM Tris-HCl pH 8.0). Cells were pelletted by centrifugation and resuspended in RIPA 50 buffer [50 mM NaCl, 20 mM Tris-HCl pH 7.4, 1 mM EDTA, 0.5% Nonidet P-40 (NP-40), 0.5% deoxycholic acid, 0.1% sodium dodecyl sulfate (SDS), protease inhibitors]. Chromatin was sheared by sonication (20 pulses, 30 seconds each) on ice and centrifuged to pellet debris. Immunoprecipitations were carried out by adding 5 µl anti-USF or anti-MRE11 antibodies (Santa Cruz Biotechnology) at 4°C overnight. Immune complexes were collected with protein A-Sepharose CL-4B (Pharmacia), and beads were washed three times with 1 ml RIPA 150 buffer (same as RIPA 50 but with 150 mM NaCl). Protein-DNA complexes were resuspended in 300 µl of TE buffer and digested with 5 IU of DNase-free RNase (Roche) for 30 minutes at 37°C. Samples were then treated for 3 hours at 56°C with 300 g/ml proteinase K (Sigma) in 0.5% SDS, 100 mM NaCl, and were incubated overnight at 65°C to revert crosslinks. DNA was extracted with phenol-chloroform-isoamyl alcohol, ethanol precipitation and resuspended in water for real-time PCR quantification of two regions in the cellular lamin B2 region (B13 and B48), using already described primer pairs and TaqMan probes (Lusic et al., 2003; Todorovic et al., 2005) and for rAAV DNA (using the same primer pair and probe used for AAV titration).
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Acknowledgments |
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Footnotes |
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* These authors contributed equally to this work 
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