Identification of Protein Cofactors Necessary for Sequence-specific Plasmid DNA Nuclear Import

original article

© The American Society of Gene & Cell Therapy

Identification of Protein Cofactors Necessary for Sequence-specific Plasmid DNA Nuclear Import Aaron M Miller1, Felix M Munkonge2, Eric WFW Alton2 and David A Dean1,3 Department of Medicine, Division of Pulmonary and Critical Care Medicine, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA; 2Department of Gene Therapy, Section of Molecular Medicine, National Heart & Lung Institute, Faculty of Medicine, Imperial College London, London, UK; 3Department of Pediatrics, Division of Neonatology, School of Medicine and Dentistry, University of Rochester, Rochester, New York, USA 1

Although transfections are routinely used in the laboratory, the mechanism(s) by which exogenous DNA is transported into the nucleus is poorly understood. By improving our understanding of how vectors circumvent the numerous cellular barriers to gene transfer, more efficient gene delivery methods can be devised. We have begun to design plasmid constructs that enter the nucleus of specific cell types in the absence of cell division, thereby enhancing levels of expression. We have shown that inclusion of specific DNA sequences in plasmid constructs mediates nuclear import both in vitro and in vivo. Here, we use plasmid affinity chromatography, mass spectrometry (MS), and live-cell pulldowns of transfected plasmid constructs to identify protein cofactors that interact in a sequence-specific manner with these DNA nuclear targeting sequences (DTSs). Importin β1, importin 7, and the small guanosine triphosphatase Ran all demonstrate DTS-specific interaction in both MS and pull-down assays, consistent with our model of plasmid nuclear import. In addition, knockdown of importin β1 with small interfering RNA (siRNA) abrogates plasmid nuclear import, indicating that it is a necessary cofactor. Our discovery that specific karyopherins mediate ­plasmid nuclear import can be used to design more effective ­vectors for gene delivery. Received 16 December 2008; accepted 12 May 2009; published online 16 June 2009. doi:10.1038/mt.2009.127

Introduction Despite a 30-year history of using transfections in mammalian cells, the mechanism of DNA transport through the cytoplasm and into the nucleus, following delivery by any methodology, remains unresolved. There are numerous extracellular and intra­cellular obstacles that preclude efficient gene delivery, including the cell membrane, the dense cytoskeletal network, and the nuclear membrane. Each of these obstacles presents a unique challenge to investigators hoping to maximize transfection efficiency.1–6 In ­quiescent cells, the rate-limiting barrier to gene delivery is the nuclear ­envelope across which plasmid vectors must translocate.2,7,8 Laboratory transfections largely circumvent this hurdle by using dividing cells in

which breakdown of the nuclear envelope ­during mitosis permits DNA to enter the nucleus and be expressed. However, in vivo most cells are nondividing and the nuclear envelope remains intact. In order to maximize levels of gene delivery to target tissues in vivo, the nuclear envelope must be circumvented. To achieve more efficient gene expression from nonviral ­vectors, our lab has designed plasmid constructs that are targeted to the nucleus.9,10 We have demonstrated that plasmids containing portions of specific DNA promoter sequences can achieve nuclear entry both in vitro and in vivo.11,12 Constructs lacking these DNA sequences, which we term DNA nuclear targeting sequences (DTSs), remain confined to the cytoplasm until cell division or until they are degraded.9 Initially, we demonstrated that inclusion of as little as 72 base pairs (bp) of the simian virus 40 (SV40) promoter/enhancer in plasmid vectors, downstream of the encoded transgene, mediates translocation of plasmid constructs into the nucleus of all cell types tested.9 We also identified a tissue-specific DTS that utilizes the 176 bp proximal portion of the smooth muscle gamma actin (SMGA) promoter to mediate nuclear import of plasmid DNA (pDNA) specifically in smooth muscle cells (SMCs).10,13 Due to the fact that both the SV40 DTS and SMGA DTS contain binding sites for ubiquitously expressed and SMC-specific transcription factors, respectively, we hypothesized that these factors bind to the DTSs resulting in a coating of areas of the plasmid with nuclear localization signals (NLSs) that interact with karyopherins (importins) to chaperone the vector–protein complex across the nuclear pore complex (NPC). Indeed, we and others have demonstrated that nuclear import of pDNA is sequence dependent, requires karyopherins, the small guanosine triphosphatase Ran and occurs through the NPC.14–18 Similar NLS-mediated nuclear import models have been proposed for viral genomes, including human immunodeficiency virus and adenovirus.19,20 In addition, we have shown that the SMC-specific transcription factors, serum response factor and NK3 (a mammalian bagpipe homologue), are necessary for plasmid nuclear import and likely serve as the adaptor proteins for karyopherin binding to pDNA.21 However, we have not yet identified which karyopherins are responsible for plasmid nuclear import. In this study, we have identified the protein cofactors that interact in a sequence-specific manner with the SV40 DTS

Correspondence: David A Dean, Department of Pediatrics, University of Rochester, MRBX 3-11126, Rochester, New York 14642, USA. E-mail: [email protected] Molecular Therapy vol. 17 no. 11, 1897–1903 nov. 2009

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Protein Cofactors for Plasmid Nuclear Import

and SMGA DTS using plasmid affinity chromatography, mass ­spectrometry (MS), and live-cell pulldowns of transfected ­plasmid constructs in smooth muscle. Importin β1, importin 7, and the small guanosine triphosphatase Ran form a complex with the SMGA DTS and SV40 DTS but not to control pDNA that lacks a DTS. When importin β1 is knocked down with small-interfering RNA (siRNA), nuclear import of both the SV40 DTS construct and SMGA DTS construct is eliminated. These results suggest that specific karyopherins form a complex with these DTSs to facilitate plasmid nuclear uptake.

Results Plasmid affinity chromatography purifies proteins that interact specifically with the SMGA DTS To purify and identify proteins that interact with the SMGA DTS to mediate SMC-specific nuclear import, we utilized an affinity chromatography strategy. In this approach, cyanogen bromide– activated Sepharose beads were covalently linked to peptide nucleic acid (PNA) clamps that recognize the origin of replication of all pUC-derived plasmids (Figure  1b).15 The Sepharose-PNA conjugate was then hybridized to either a plasmid containing

the SMGA DTS (pTOPO-DTS) or a control plasmid lacking the SMGA DTS (pTOPO). The Sepharose-PNA-pDNA beads were then incubated at 37 °C with whole-cell SMC extracts in column buffer along with an adenosine triphosphate–regenerating system to facilitate protein binding to the vector.22 Following incubation, the mixture was packed into a column, washed extensively with column buffer and the bound protein eluted using a two-step salt gradient. The advantage of this experimental approach is the use of the entire supercoiled plasmid construct (i.e., the form used for transfections and gene therapy) to purify the proteins that interact with the SMGA DTS. As shown in the chromatograms of the eluted protein, columns containing the SMGA DTS bound considerably more protein than control columns lacking the DTS (Figure 1a). Western blots reveal that import proteins, including Ran and importin β1, were present in the protein ­eluates from the plasmid affinity columns containing the SMGA DTS but were absent in eluates from columns charged with plasmids containing no DTS (the control for nonspecific interactions with the ­vector backbone) (Figure 1c). These results are consistent with our model of plasmid nuclear import.

a a 100

0.4 mol/l

1.0 mol/l

Nucleocytoplasmic Shuttling (6) DNA processing (6)

b

Chromatin assembly (7)

Cell structure (39)

Absorbance (mAU)

Cell adhesion (11)

Sepharose 4B

PNA DNA 5� DNA 3�

50

J J T J T TJ GGAGA AG CC T C T T C CC

TCT T

Transcription (13) Cell metabolism (13)

Hoogstein 3� WC 5�

Proteolysis (34) Cell cycle (13)

C RNA processing (14)

0

Total protein (µg)

35 SMGA DTS No DTS

25

c

Control extract

SMGA DTS

No DTS

Signal transduction (28)

Cellular transport (14)

Cellular chaperone (16)

Ran

Unclassified (24) Protein interactions (17)

15

Importion α1

5

Importion β1

b

Translation (19)

DNA processing (1) Cellular transport (2) Transcription (3)

Cell structure (8)

Eluted fractions

Figure 1  Purification of smooth muscle cell extracts that interact with the SMGA DTS. Sepharose beads were linked to peptide nucleic acid (PNA) clamps and hybridized to plasmids that contain or lack the SMGA DTS (pTOPO-DTS and pTOPO, respectively). Affinity chromato­ graphy was performed using whole-cell smooth muscle extracts to purify factors that interact with the plasmids. (a) A chromatogram showing the milli-absorbance units absorbance spectra of eluted protein and quantification of total eluted protein. A two-step salt elution (0.4 mol/l and 1.0 mol/l) was used. (b) Diagram of the Sepharose-PNA-pDNA phase. Sepharose beads are covalently linked to the PNA by an isourea bond. The amino-terminal half of the PNA hybridizes to the plasmid target sequence by Watson-Crick (WC) base pairs and the carboxy-terminal half uses Hoogstein base pairs to form a triplex. 8-amino-3,6-dioxaoctanoic acid linkers connect the two halves of the PNA (abbreviated a “O”). The displaced “D-loop” is shown bulging away from the complementary DNA strand. (c) Immunoblots of eluted protein showing that Ran and importin β1 (but not importin α1) are purified from affinity columns in a DTS-specific manner. DTS, DNA nuclear targeting sequence; SMGA, smooth muscle gamma actin.

1898

Proteolysis (3)

Protein interaction (3) Cell adhesion (7)

Cell metabolism (3)

Cell cycle (4)

Unclassified (7)

Figure 2 Classification of proteins purified from plasmid DNA ­affinity columns. The functional classes of proteins identified by mass spectrometry analysis of column eluates from (a) SMGA DTS columns and (b)  No DTS columns are shown as the total number of identified proteins. Classifications are based on data acquired through National Center for Biotechnology Information.

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Protein Cofactors for Plasmid Nuclear Import

MS of column eluates identifies proteins that interact with the SMGA DTS We employed MS to identify the proteins in the column ­eluates. Unlike the immunoblotting approach, MS provides an ­unbiased means of identifying the proteins eluted from each plasmid affinity column condition. Protein samples from each experimental condition were separated using sodium dodecyl sulfate– polyacrylamide gel electrophoresis, excised into equal bands and then subjected to in-gel proteolytic digestion with trypsin. Samples were then analyzed by nano LC/MS/MS and the MS/­MS data were annotated using the Mascot database. A total of 274 unique proteins were identified within the pTOPO-DTS ­eluates, whereas only 41 unique proteins were identified within the pTOPO pool (Supplementary Table S1 and Supplementary Table S2, respectively). This data reveal that the SMGA DTS is responsible for the majority of protein interactions with the plasmid constructs and purifies unique classes of proteins when compared to the control DNA (Figure 2). Consistent with our model of karyopherin/NLS-mediated plasmid nuclear import, a number of proteins that play a role in signal-mediated protein nuclear import, including importin β1, importin 7, Ran, RanBP1, and RanBP3, were purified from plasmid affinity ­columns in a DTS-specific manner (Table  1). This data suggest that specific

karyopherins complex with DTS-containing ­plasmids to ­facilitate nuclear import.

Pulldown of plasmid constructs transfected into living cells validates DTS-specific docking of import proteins We next employed a pull-down strategy to assess whether import complex proteins are part of the plasmid vector–protein complex in living cells. In the pull-down assay, plasmid constructs were hybridized to a biotinylated PNA clamp and then electroporated into human pulmonary artery SMCs (hSMCs). At various time points post-transfection, the cells were treated with formaldehyde to cross-link protein-pDNA interactions and lysed, and streptavidin-coated beads were used to pellet pDNA-­protein conjugates. To verify that each construct was labeled with PNAbiotin at equivalent levels and to determine the threshold of pDNA detection, known amounts of labeled DNA were transferred onto membranes and probed for biotin with horseradish peroxidase–streptavidin (Supplementary Figure S1). Plasmids containing either the SV40 DTS or the SMGA DTS bound equivalent amounts of horseradish peroxidase–streptavidin, whereas a plasmid containing no DTS bound roughly half the amount of the other two. Using this strategy, we performed pulldowns on the lysates of SMCs electroporated with biotinylated pDNA containing either the SV40 DTS, the SMGA DTS, or no DTS. Importin β1, importin 7, and Ran were in the pellet of SV40 DTS pulldowns at 60 minutes post-transfection but not at 120 minutes (not shown) or 240 minutes (Figure  3a). DNA Slot blots verified that equal levels of transfection efficiency were obtained at all times in all samples by detection of biotin-pDNA with horseradish perox­ idase–streptavidin (Figure 3b). The faint signal present in the no DNA control lanes likely reflects endogenous biotin present in the cells. Importin β1, importin 7, and Ran were present in the pellet of SMGA-DTS pulldowns at 240 minutes post-transfection, but not at earlier times (Figure 3a). Control pBR322 DNA (devoid of any eukaryotic sequences) did not pull down these factors in the pellet, ­suggesting that the docking of importin β1, importin 7, and Ran to the DNA–protein complex is temporally and sequence‑specific.

Table 1  Import proteins that exhibit DTS-specific interaction with plasmid DNA in affinity purifications Accession number

Unique peptides

Karyopherin (importin) β1, isoform 1

gi|73966186

16

Ipo7

RanBP7/importin 7

gi|11342591

3

Ran

RAN, member RAS oncogene family

gi|119618923

8

RanBP1

Ran binding protein 1

gi|114685193

3

RanBP3

Ran binding protein 3

gi|109123082

3

Symbol

Protein name

Kpnβ1

Abbreviation: DTS, DNA nuclear targeting sequence. Protein name, accession number, and functional classification were derived from National Center for Biotechnology Information.

a

pBR322 C Sup Pel

SV40 DTS SMGA DTS Sup

Pel

Sup

Pel

C

pBR322

SV40 DTS SMGA DTS

Sup

Sup

Pel

Pel

Sup

Pel Importin α1 Importin β1 Importin 7 Ran

b

No DNA pBR322 SV40 DTS SGMA DTS

60 minutes

No DNA pBR322 SV40 DTS SGMA DTS

240 minutes

Figure 3  Precipitation of the protein–pDNA import complex in living cells. (a) Human smooth muscle cells were electroporated with plasmid DNA (pDNA) that was hybridized to a biotinylated peptide nucleic acid clamp. At 60 and 240 minutes post-transfection, cells were treated with formaldehyde to cross-link proteins bound to the pDNA, lysed and precipitated using streptavidin-agarose. Unbound fractions (Sup) and precipitated fractions (Pel) were probed for putative members of the import complex using western blots. (b) To verify equal levels of transfection efficiency, crude lysates from transfected cells were Slot blotted onto membranes, UV cross-linked and probed for biotinylated pDNA using horseradish peroxidase– streptavidin. DTS, DNA nuclear targeting sequence; SMGA, smooth muscle gamma actin; SV40, simian virus 40.

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Protein Cofactors for Plasmid Nuclear Import

a

pDNA-Cy3

FI-M9 NLS

Merge

No import

Import

DAPI

b

c

70

Plasmid nuclear import (% of total cells with signal)

60

siRNA

siRNA NT

+

NT

Importin β1

Importin 7

Actin

Actin

+

pBR322 SMGA DTS SV40 DTS

50 40 30

*

20

*

10 0

NT

Importin β1

Importin 7

Figure 4  Importin β1 is required for DTS-mediated plasmid nuclear import. (a) For microinjection experiments, Cy3-labeled plasmid DNA (pDNA) (0.5 mg/ml) and fluorescein-labeled (Fl) M9 NLS ­peptide (0.5 mg/ml) were microinjected into the cytoplasm of adherent cells and their localization was determined 8 hours later using fluore­scence microscopy. Representative examples of import and no import are shown. (b) Immunoblots of human smooth muscle cells (hSMCs) transfected with nontargeting (NT) small interfering RNA (siRNA) or siRNA targeting importin β1 and importin 7 reveal successful knockdown of each factor at 48 hours. (c) Quantification of levels of nuclear import of plasmid constructs cytoplasmically microinjected into hSMCs that were treated with siRNA for 48 hours. At least 100 cells were injected for each condition and the experiment was repeated three times (mean % nuclear import ± SD). *P < 0.001 versus respective NT control. DAPI,  4,6-diamidino-2-­phenylindole; DTS, DNA nuclear targeting sequence; NLS, nuclear localization signal; SMGA, smooth muscle gamma actin; SV40, simian virus 40.

As an additional control, we probed the membranes for ­importin α1 (which was not identified as a DTS-specific interacting protein in either the western blots of column eluates or the MS analysis) to verify that this factor is not present in protein–pDNA complexes in living cells. At the time points where importin β1, importin 7, and Ran exhibit SMGA– and/or SV40 DTS–specific binding in the pulldowns, importin α1 was absent from the pellet, providing another line of evidence that it does not play a role in DTSmediated plasmid import. The sequence-specific docking of importin β1, importin 7, and Ran to the DTS is consistent with the results from the affinity chromatography experiments that purified these same factors. Finally, it is interesting to note that plasmids containing the SMGA DTS or the SV40 DTS associated with import proteins at different rates, reflecting variable kinetics 1900

of nuclear import. This finding has been validated by live-cell ­tracking of fluorescently labeled versions of these vectors.21,23

Importin β1 is required to mediate plasmid nuclear import To ascertain whether the import proteins exhibiting specific interactions with the SMGA DTS in both the chromatography purifications and the live-cell pulldowns play a functional role in mediating plasmid nuclear import, we used siRNA to knock down their expression in hSMCs and determined the effect on the nuclear import of cytoplasmically microinjected pDNA. We selected importin β1 and importin 7 for siRNA analysis due to their known roles in facilitating the nuclear import of proteins across the NPC during interphase.24–26 In addition, we chose to knock down importin α1 because it is known to function as an adaptor protein that links importin β1 to classical NLS-containing cargo during nuclear import. However, knockdown of importin α1 was lethal in our cell line, likely due to it playing a vital role in regulating the nucleocytoplasmic shuttling of proteins essential to cell function. In contrast, immunoblots of total cell lysates from hSMCs transfected with siRNAs against importin β1 and importin 7 were not lethal to the cells and revealed successful knockdown (between 70 and 90%) of each factor at 48 hours (Figure 4b and data not shown). Cells were treated with siRNA for 48 hours and then cytoplasmically microinjected with pBR322 (negative control for plasmid import), pCMV-GFP-SV40 DTS, or pCMV-GFPSMGA404 DTS that had been labeled with a Cy3-PNA clamp. To rule out the possibility that knockdown of importin β1 or importin 7 might inhibit plasmid nuclear import by an indirect mechanism (for example, by altering nuclear pore structure), constructs were co-injected with purified fluorescein-labeled M9 NLS-GST. The M9 NLS peptide fused to GST is bound by importin β2 to facilitate its nuclear import through the NPC and therefore should not be affected by the knockdown of either importin β1 or importin 7.27 Eight hours later, the cellular localization of the pDNA and M9 NLS peptide was determined using fluorescence microscopy, and cells were only scored for levels of plasmid nuclear import if the M9 NLS peptide successfully translocated into the nucleus (Figure  4a). Control cytoplasmic injections with pBR322 DNA (which lacks a DTS) established baseline levels of plasmid import that resulted from cell division or inadvertent microinjection of pDNA directly into nuclei (Figure  4c, open bars). Cells treated with nontargeting siRNAs that were microinjected with pCMVGFP-SV40 DTS (Figure  4c, gray bars) or pCMV-GFP-SMGA404 DTS (Figure  4c, black bars) had significantly elevated levels of nuclear import (51.2 ± 3.2% and 47.9 ± 4.8%, respectively) compared to the pBR322 control (2.9 ± 2.4%), demonstrating that the SV40 DTS and SMGA DTS successfully mediates import in these cells following scrambled siRNA transfection. In contrast, cells treated with importin β1 siRNA exhibited significantly reduced levels of import of cytoplasmically microinjected SV40 DTS constructs (11.0 ± 4.5%) and SMGA DTS constructs (12.7 ± 8.0%) compared to the nontargeting control. For cells treated with importin-7 siRNA, import of either the SV40 DTS construct or SMGA DTS construct was not affected (55.2 ± 7.0% and 44.9 ± 3.6%, respectively), suggesting that the importin 7 does not play a direct role in facilitating import or that levels of importin-7 www.moleculartherapy.org vol. 17 no. 11 nov. 2009

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knockdown were insufficient to yield an observable effect on plasmid nuclear import. Taken together with our previous results demonstrating that recombinant importin β1 can support ­plasmid nuclear import in permeabilized cells, these results indicate that importin β1 is necessary and sufficient as the NLS-receptor to drive plasmid nuclear import of SMGA DTS and SV40 DTS ­plasmid constructs in SMCs.15

Discussion Transfections of mammalian cells with plasmid constructs are a common practice. Unfortunately, despite routine use of trans­ fections in the laboratory, our understanding of how DNA trans­ locates through the cytoplasm and into the nucleus is quite limited. In this study, we have used pDNA affinity chromato­graphy and MS to purify and identify protein cofactors that interact in a sequence-specific manner with the SMGA DTS. Previously, this DTS was shown to greatly enhance plasmid nuclear import and expression, specifically in smooth muscle, both in vitro and in vivo.10,13,21 By identifying the proteins that interact with this sequence, our aim was to discover the mechanism employed by the cell to transfer constructs containing this sequence into the nuclear compartment. Among the list of proteins that exhibited SMGA DTS–specific interactions were a number of proteins, including importin β1, importin 7, the small guanosine triphosphatase Ran, RanBP1, and RanBP3, that are involved in nucleocytoplasmic shuttling of ­proteins across the NPC during interphase.25,26,28,29 Given that mammals possess at least six karyopherin α isoforms and 20 isoforms of karyopherin β (10 of which have a proven role in nuclear import), the use of plasmid affinity chromatography combined with MS allowed us to identify the few potential mediators of ­plasmid nuclear import.24,30 In addition to the import proteins, there were a number of other proteins uniquely purified from DTS columns. These proteins included chaperone proteins, cytoskeletal elements, nucleic acid–binding proteins, and proteins involved in cellular transport. Factors known to bind directly to nucleic acids (including chromobox homologue 1, chromobox homologue 3, histone H1, PCNA, and a number of heterogeneous nuclear ribonucleoproteins) and indirectly as ternary members of the protein–pDNA complex (including transcriptional machinery, histone-binding proteins, and translational machinery) all exhibited DTS-specific purification.31–34 Although some of these factors were likely purified due to nonspecific interactions with the negatively charged DNA column, some likely play a role in facilitating plasmid transport. For example, we and others have shown that plasmids utilize the microtubule network and its associated motor proteins to traffic toward the nucleus.23,35,36 Consistent with this observation, α- and β-tubulin, several microtubule-associated proteins, and the molecular motor protein kinesin were all purified from DTS columns. In fact, a number of candidate proteins were identified for all aspects of DNA trafficking within the cell. Unfortunately, some candidates were not identified using this technique. These include serum response factor and NK3, which we have previously shown to be necessary and sufficient for SMGA DTS–mediated plasmid nuclear import.21 This illustrates the point that although plasmid chromatography is an effective tool for purifying cofactors for intracellular plasmid trafficking, it Molecular Therapy vol. 17 no. 11 nov. 2009

Protein Cofactors for Plasmid Nuclear Import

has limitations and must be supplemented with more traditional hypothesis-driven testing of candidates. To this end, we decided to focus our analysis on validating whether Ran, importin β1, and importin 7 form a complex with DTS-containing plasmids in living cells and determining which (if any) of these factors are required for plasmid nuclear import. We show that Ran, importin β1, and importin 7 (but not importin α1 interact with DTS-containing constructs at the time points post-transfection that these constructs are known to translocate into the nucleus, as previously determined by live-cell tracking of fluorescently labeled plasmids.21,23 These results suggest that a complex of Ran, importin β1, and importin 7 forms on DTSs in living cells to facilitate plasmid nuclear import. Although the heterodimer of importin β1 and importin 7 has been described and is known to act as an import receptor for HIV-1 intracellular reverse transcription complexes and histone H1, a complex of Ran with these karyopherins has not been accounted for in current models of nucleocytoplasmic import.25,26 However, it has recently been suggested that a significant portion of cytoplasmic RanGDP might exist in such complexes.37 In fact, the existence of a Ran– importin β1–RanBP1 complex has previously been characterized in vitro, although its role in nuclear import is not understood.37–39 Plafker and Macara performed an elegant study using fluorescence resonance energy transfer biosensors to demonstrate that this complex also exists in vivo.37 In their study, they cytoplasmically microinjected a bipartite biosensor consisting of Ran-Alexa 546 and GST-GFP-RanBP1 into BHK21 cells and showed that a robust cytoplasmic fluorescence resonance energy transfer signal was detectable in the cytoplasm after equilibration, suggesting that a ternary complex of RanGDP, RanBP1, and karyopherins exists in living cells. A potential implication of our data and the findings of the Macara group is that Ran might complex with karyopherins to traverse the NPC with specific cargos. Finally, we performed siRNA-mediated knockdowns of ­importin β1 and importin 7 to ascertain whether these factors are required for plasmid nuclear import. We show that knockdown of importin β1, but not importin 7, abrogated nuclear import of plasmids containing either the SMGA DTS or SV40 DTS. Although it is possible that the level of importin-7 knockdown was not sufficient to achieve an observable effect on plasmid import, it may be that importin 7 merely complexes with DTS constructs due to its high affinity for importin β1 and is not actually required for plasmid import. Indeed, both the importin β1 and importin-7 transport receptors are ­present in equally high abundance in the cell and have been shown to form a cytoplasmic complex that is also quite abundant.35 In fact, importin β1 dimerizes with a number of cellular adaptors, including both importin α1 and other karyopherin β ­family members, and is therefore most likely found in such complexes in the cell.40–42 Alternatively, the NLSs present on transcription factors and other DNA-binding proteins at the DTS may interact with both importin β1 homodimers and importin-β1–importin-7 heterodimers, with the β1 homodimers playing a dominant role in nuclear import such that they compensate for loss of importin 7. Regardless of the role of importin 7 in the complex identified in this study, importin β1 is clearly vital to plasmid nuclear import. In this study, we have elucidated the mechanism employed by the cell to deliver exogenous DNA through the NPC and into the 1901

Protein Cofactors for Plasmid Nuclear Import

nucleus. We have shown that a complex of Ran, importin β1, and importin 7 interacts with pDNA in a DTS-specific manner both in vitro and in living cells that importin β1 is required for nuclear import of these constructs. These data should aid in improving the efficacy of gene delivery to target tissues by allowing investigators to design vectors that utilize this pathway of nuclear import. In fact, a number of laboratories have already begun to devise ­methods to enhance levels of gene delivery by complexing plasmids with ­proteins, such as NF-kB or histone proteins, or with peptide nucleus acids (PNAs) that contain NLSs, to facilitate interaction with the karyopherin system.35,43–45 These findings should facilitate the rational design of more efficient vectors for gene delivery.

Materials and Methods Plasmid affinity chromatography. One gram of cyanogen bromide–­

activated Sepharose 4B beads (Amersham, Piscataway, NJ) were resuspended in 1 mmol/l HCl and washed with 15 volumes each of 1 mmol/l HCl and chilled coupling buffer (0.1 mol/l NaHCO3, 0.5 mol/l NaCl, pH 8.3). The activated beads were then coupled to PNA (1 nmol), targeting the pUC origin of replication, in 1-ml coupling buffer overnight at 4 °C with end-over-end mixing. Unreacted amide groups were blocked with 1 mol/l Tris-HCl, 0.5 mol/l NaCl, pH 8.0 for 2 hours at room temperature. The Sepharose-PNA beads were then washed and stored in ­hybridization buffer (TE, pH 6.5) at 4 °C until use. pDNA (500 µg) was ­hybridized to the Sepharose-PNA at 37 °C for 4 hours followed by end-over-end mixing at 4 °C overnight. A 50–70% slurry of Sepharose-PNA-pDNA was then incubated with sheep aorta whole-cell extract (1 mg) at 37 °C for 1 hour in column/binding buffer (20 mmol/l 4-(2-hydroxyethyl)-1­piperazineethansulfonic acid, 110 mmol/l potassium acetate, 2 mmol/l magnesium acetate, 1 mmol/l [ethylenebis(oxyethylenenitrilo)]tetraacetic acid, 2 mmol/l dithiothreitol, 1 mmol/l zinc sulfate, pH 7.3) containing an adenosine triphosphate–regenerating system (1 mmol/l guanosine triphosphate, 2 mmol/l adenosine triphosphate, 10 mmol/l phosphocreatine, and 20 units/ml of creatine phosphokinase). The slurry was then packed into a Tricorn 10/50 column (Amersham), connected to a ÄKTAFPLC (Amersham), and washed with 5 column volumes of column buffer at 0.5 ml/minute to remove proteins not specifically bound to the pDNA. A two-step salt elution (0.4 mol/l followed by 1.0 mol/l NaCl) spanning 4 column volumes was used to elute bound proteins from the pDNA. Eluted fractions (500 µl) were collected and stored at −80 °C until MS or immunoblotting analysis was performed.

MS. Pools of protein eluted from plasmid affinity columns were sent to NextGen Sciences (Ann Arbor, MI) for MS analysis. Samples were run on a NuPAGE Novex 4–12% Bis-Tris Gel (Invitrogen, Carlsbad, CA) in MOPS buffer system. Lanes were excised into 24 equal bands using a custom grid system, and each band was subjected to in-gel proteolytic digestion on a ProGest (Genomic Solutions, Ann Arbor, MI) workstation by reduction in 10 mmol/l dithiothreitol at 60 °C, cooling to room temperature, alkylation with 100 mmol/l iodoacetamide and incubation at 37 °C for 4 hours using 250 ng of sequencing grade modified trypsin (Promega, Madison, WI) per gel slice. Finally, formic acid was added to 0.1% final concentration to halt the reaction and protonate peptides for subsequent LC/MS/MS. Samples were analyzed by nano LC/MS/MS on a Thermo Fisher LTQ Orbitrap XL (Thermo Fisher Scientific, Waltham, MA). Thirty microliters of hydrolysate were loaded on a 75 µm C12 vented ­column at a flow rate of 10 µl/minute and eluted at 300 nl/minute over 1 hour. The MS/MS data were then annotated using a local copy of Mascot (Matrix Science, Boston, MA). Plasmids and plasmid labeling. Plasmids pCMV-GFP-SV40 DTS andt pCMV-GFP-SMGA404 DTS express green fluorescent protein from the CMV immediate early promoter and contain the 72 bp SV40 DTS or the

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404 bp SMGA DTS (respectively) downstream of the reporter gene.46,47 Derivatives of pBR322, pCMV-GFP-SV40 DTS, and pCMV-GFP-SMGA404 DTS that contain tandem binding sites for the GeneGrip biotin or Cy3labeled PNA (PNA; Gene Therapy Systems, San Diego, CA) were used for electroporations and microinjection studies and were labeled with biotin or Cy3-labeled PNAs as previously described.48 The pTOPO-DTS construct was created by insertion of the 2,294 bp SMGA DTS into the HindIII site of pCRII-TOPO (pTOPO) plasmid (Invitrogen). pGST-M9 was a generous gift from Mark Hannink (University of Missouri, Columbia, MO).49 All plasmids were purified from E. coli using Promega Maxiprep kits as described by the manufacturer (Promega). Preparation of tissue extracts. Whole-cell smooth muscle extracts were

derived from freshly acquired sheep abdominal aorta specimens as ­previously described.50

Cell culture and pull-down experiments. hSMCs were grown in

Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (FBS) and 2 mmol/l l-glutamine. For electroporations, cells were grown to confluency in 6-well dishes, and rinsed twice in serum- and ­antibio­tic-free media. Ten micrograms of biotinylated-PNA-labeled pDNA in 500 µl of serum- and antibiotic-free Dulbecco’s modified Eagle’s medium were added to each well, and one 165 mV square wave electric pulse was applied using a Petri Pulser electrode (BTX, San Diego, CA), as previously described.23 Electroporated cells were incubated at 37 °C and 5% CO2 to permit pDNA–protein complexes to form in the cell. To cross-link pDNA–protein complexes, formaldehyde was added directly to each well to a concentration of 1% for 10 minutes at 37 °C. Cells were washed twice with ice-cold PBS, scraped into lysis buffer (10 mmol/l Tris-HCl, 1 mmol/l EDTA, 2 mmol/l NaCl, 1% NP40, and 0.025% sodium deoxycholate) and incubated for 10  minutes on ice. To assess transfection efficiency, 20 µl of crude lysate was transferred to a Zeta-Probe membrane (Bio-Rad Laboratories, Hercules, CA) by vacuum filtration and UV cross-linked to the ­membrane. Membranes were probed with streptavidin–horseradish peroxidase and levels of biotinylated-pDNA gene transfer determined by chemiluminescence. The remaining crude lysates were incubated with streptavidin-agarose beads (Chemicon, Temecula, CA) for 30 minutes at 4 °C with end-over-end mixing and centrifuged to precipitate bead complexes. The unbound supernatant fraction was stored at −20 °C for later immuno­blotting analysis. The precipitated bead complexes were washed three times with 500 µl of lysis buffer. Beads were boiled in 100 µl of Laemmli sample buffer for 10 minutes to release protein bound to the beads for immunoblotting analysis and to reverse cross-links and unbound, and pelleted fractions were then subjected to SDS-PAGE, ­transferred to ­nitrocellulose membranes and immunoblotting. Expression, purification, and fluorescent labeling of bacterially-­expressed GST-M9 NLS. GST-M9 protein was expressed in bacteria, purified, and

fluorescently labeled as previously described.49

siRNA methods. Pools of siRNAs for human importin β1, importin 7, importin α, and nontargeting siRNAs were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). According to manufacturer’s instructions, hSMCs were plated at 30% confluency 24 hours prior to transfection, and transfection reagent was used to transfect cells with 100 nmol/l of each siRNA. Transfected knockdown cells were allowed to grow for 48 hours prior to microinjection. Microinjection. hSMCs were grown on cover slips and cytoplasmically

microinjected with plasmid constructs (0.5 mg/ml in phosphate-buffered saline) along with recombinant protein (0.5 mg/ml) using an Eppendorf FemtoJet system (Eppendorf, Westbury, NY) as previously described.48

Microscopy and image analysis. Nuclei of live cells were stained with

Hoescht 33258, and images were captured on a Leica DMIRB inverted

www.moleculartherapy.org vol. 17 no. 11 nov. 2009

© The American Society of Gene & Cell Therapy

microscope with a ×40 objective. Fixed cells and in situ hybridizations were observed under a Leica DMRXA2 epifluorescence microscope with a ×100 objective (NA 1.35; Leica Microsystems, Bannockburn, IL). Images were acquired using a Hamamatsu Orca-ER 12-bit, cooled CCD camera (Hamamatsu Photonics, Bridgewater, NJ) using OpenLab software (Improvision, Lexington, MA). Statistical analysis. Results are expressed as mean ± standard deviation

of the mean. Differences between multiple groups were analyzed using one-way analysis of variance with the Student Newman–Keuls post hoc test for all pairwise comparisons using SigmaStat software (SPSS, Chicago, IL).

Supplementary Material Figure S1.  DNA slot blot showing equivalent levels of plasmid ­labeling with PNA-biotin. Table S1.  LC/MS/MS identification of pTOPO-DTS column eluates. Table S2.  LC/MS/MS identification of pTOPO column eluates.

Acknowledgments We thank Melina Kibbe for insightful discussions and technical ­advice. This work was supported in part by grants HL59956 and HL81148 from the National Institutes of Health and by a predoctoral ­fellowship from the American Heart Association, Greater Midwest Affiliate (AMM).

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