Human Molecular Genetics, 2001, Vol. 10, No. 26 3093-3099
© 2001 Oxford University Press
Manipulating mitochondrial DNA heteroplasmy by a mitochondrially targeted restriction endonuclease
1Department of Cell Biology and Anatomy and 2Department of Neurology, University of Miami School of Medicine, 1095 NW 14th Terrace, Miami, FL 33136, USA
Received October 5, 2001; Revised and Accepted October 29, 2001.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Mutations in the mitochondrial DNA (mtDNA) can cause a variety of human diseases. In most cases, such mutations are heteroplasmic (i.e. mutated and wild-type mtDNA coexist) and a small percentage of wild-type sequences can have a strong protective effect against a metabolic defect. Because a genetic approach to correct mtDNA mutations is not currently available, the ability to modulate heteroplasmy would have a major impact in the phenotype of many patients with mitochondrial disorders. We show here that a restriction endonuclease targeted to mitochondria has this ability. A mitochondrially targeted PstI degraded mtDNA harboring PstI sites, in some cases leading to a complete loss of mitochondrial genomes. Recombination between DNA ends released by PstI was not observed. When expressed in a heteroplasmic rodent cell line, containing one mtDNA haplotype with two sites for PstI and another haplotype having none, the mitochondrial PstI caused a significant shift in heteroplasmy, with an accumulation of the mtDNA haplotype lacking PstI sites. These experiments provide proof of the principle that restriction endonucleases are feasible tools for genetic therapy of a sub-group of mitochondrial disorders. Although this approach is limited by the presence of mutation-specific restriction sites, patients with neuropathy, ataxia and retinitis pigmentosa (NARP) could benefit from it, as the T8399G mutation creates a unique restriction site that is not present in wild-type human mitochondrial DNA.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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It has been shown that most pathogenic mitochondrial DNA (mtDNA) mutations are present in a heteroplasmic state (i.e. co-existing with wild-type genomes) (1–3). In most cases, a very high percentage of mutated mtDNA is necessary to elicit phenotypic abnormalities, commonly associated with muscle and the central nervous system dysfunction. This has been exemplified by the variable clinical picture of different members of the same kindred (4,5) as well as by the different tissue involvement in patients (6). Intercellular phenotypic differences in a tissue have also been associated with small variations in the proportion of wild-type and mutated mtDNA in individual cells (7,8). This concept suggested that approaches to manipulate mtDNA heteroplasmy may have potential therapeutic applications (9). We attempted to manipulate mtDNA heteroplasmy by taking advantage of unique features of specific mtDNA haplotypes, namely the presence of a restriction endonuclease site that could differentiate two forms of the mitochondrial genome.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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We were interested in testing the ability of a restriction endonuclease to alter mtDNA heteroplasmy as well as in the potential role of double-strand breaks in mtDNA recombination. For this reason, we chose to express the restriction endonuclease PstI in mammalian mitochondria. The human and mouse mtDNA have two relatively close sites for PstI, which could promote intramolecular recombination and create deletion mutants. PstI could also distinguish between mtDNA haplotypes in rodent cell lines developed in our laboratory, as described below. Therefore, we synthesized a gene coding for the bacterial PstI endonuclease (NCBI P00640) altering the codon usage to optimize expression in mammalian cells. The recoded PstI gene was cloned downstream of a DNA fragment coding for a mitochondrial targeting sequence (Materials and Methods). The final construct was cloned into a mammalian expression vector (Fig. 1A) and transfected into human 293T and mouse NIH 3T3 cells. A polyclonal antibody, raised against the bacterial PstI, was able to detect a polypeptide with a migration that was indistinguishable from the bacterial PstI (Fig. 1B). This observation showed that the polypeptide was expressed and that the mitochondrial targeting sequence was removed from the mature polypeptide. Confocal microscopy showed that the PstI protein co-localized with the mitochondrial marker CMX-ROS MitotrackerTM (Fig. 1C). The antibody also detected nuclear structures in both transfected and untransfected cells (Fig. 1C). The same structures were also observed in 143B cells not subjected to transfection with pMtPstI (not shown).
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Transiently transfected 293T cells were analyzed for the presence of mtDNA fragmentation 48 h after transfection with the mitochondrial PstI construct (pMtPstI). The human mtDNA harbors two restriction sites for PstI (at positions 6914 and 9024) (10) (Fig. 2A). Therefore, we expected to observe the release of a 2.1 kb fragment from the mitochondrial genome that could be detected by a specific probe within this region (asterisk in Fig. 2A). We routinely have
30–40% of 293T cells transiently transfected, what should have been enough to show the presence of a mtDNA fragment. Southern blot analysis showed that most of the mtDNA molecules were present as full-length circles. We could observe the presence of an extremely faint band of 2.1 kb, suggesting that only a very small fraction of the mtDNA pool was readily digested by the mitochondrial PstI (Fig. 2B).
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To study the effect of long-term mtDNA exposure to mitochondrial PstI we obtained 143B human osteosarcoma cells stably expressing the mitochondrial PstI gene. Four independent G418-resistant clones were obtained 15 days after pMtPstI transfection. These clones were expanded, tested for PstI expression (Fig. 2C) and analyzed for the presence of intact or rearranged mtDNA. To detect the presence of mtDNA in these cells, we first amplified a fragment corresponding to the ND5 mitochondrial gene (Fig. 2A). The amplified product could be observed in two of the four clones, suggesting that a dramatic loss of mitochondrial genomes occurred in two of the mitochondrial PstI-expressing clones (Fig. 2D). We also searched for the presence of rearranged molecules that could have originated by the recombination of free staggered ends created by PstI digestion. This was performed by the amplification of a DNA fragment using oligonucleotide primers flanking the PstI restriction sites (Fig. 2A). Amplification products were observed only with DNA from the same two clones that showed amplification of the ND5 gene (Fig. 2E). A recombination involving free PstI-digested ends would give rise to amplification products of 200 bp, whereas amplification of the wild-type sequence would give rise to a 2.3 kb amplicon. The two clones that produced an amplicon showed exclusively the 2.3 kb fragment, suggesting that recombination between free PstI-digested sites did not occur at any significant level (Fig. 2E).
Southern blot analysis of the 143B clones stably expressing the mitochondrial PstI confirmed that clones 2 and 4 had lost their mtDNA. The remaining two clones (clones 1 and 3) showed extensive mtDNA degradation observed as a smear as well as discrete low molecular weight bands (Fig. 2F). This was observed when total DNA was digested with either PstI or PvuII. This experiment also showed that the mtDNA molecules in clones 1 and 3 retained their original PstI sites. Oxygen consumption assays confirmed the mtDNA depletion, as no detectable respiration was observed in clones 2 and 4 (not shown).
To test if the mitochondrial PstI could modulate mtDNA heteroplasmy in intact cells, we produced a hybrid cell line containing both mouse and rat mtDNA. The mouse mtDNA harbors two restriction sites for PstI (at positions 8420 and 12 232) (11) whereas the rat mtDNA harbors none. This hybrid cell line was produced by fusing a mouse LM(TK–) to a rat NRK cell as described by Dey et al. (12). The original percentage of rat mtDNA in this cell line (termed LMNRK#6) was 33%. Because heteroplasmy fluctuations are known to occur during clonal analyses, we transfected LMNRK#6 cells with either the pMtPstI or pCDNA3. Seventy-two hours after transfection, cells were subjected to selection with G418. After 15 days under selection, 12 individual clones were isolated from both pMtPstI and pCDNA3 transfections. PCR/RFLP analyses of mtDNA haplotypes showed that pCDNA3-transfected cells had an average of 25% rat mtDNA (Fig. 3A). Although heteroplasmy fluctuations were observed, a significant number of clones (6/12) had extremely low levels of rat mtDNA, suggesting a bias toward maintaining the mouse mtDNA. This bias may be related to the preferential loss of rat chromosomes in this cell line. On the other hand, clones transfected with pMtPstI had significantly higher levels of rat mtDNA (53%, Fig. 3A and B). However, three of the pMtPstI-transfected clones showed a predominance of mouse mtDNA. Western blot analysis of all clones showed that these three pMtPstI-transfected clones were the only ones with undetectable levels of PstI, demonstrating that mitochondrial PstI expression can effectively modulate mtDNA heteroplasmy in a predictable direction. The difference in percentage rat mtDNA between pCDNA3 and pMtPstI transfections, when the three non-expressing clones were removed from the pMtPstI group, was highly significant (P = 0.0008). The mean percent rat mtDNA was also increased to 62%. We did not observe a direct correlation between the levels of PstI expression and the percentage of rat mtDNA, possibly because heteroplasmy was not stable in these cells and showed some fluctuations, as observed in the pCDNA3-transfected cells.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The ability to manipulate the mitochondrial genome in mammalian cells would provide a powerful approach both to create mutants and modulate heteroplasmy. Unfortunately, the location of the mtDNA and its association with mitochondrial proteins and membranes make this task extremely difficult. Although modification of mitochondrial genes in yeast have been accomplished (13), no such successes have followed with mammalian cells. Because of this limitation, the field of mammalian mitochondrial genetics has relied on naturally occurring mutations for the study of altered genes. Moreover, the association of mtDNA mutations with human diseases have underscored the need for an approach that could lead to the manipulation of mitochondrial genomes.
The protective effect of low levels of wild-type mtDNA in patients with mtDNA mutations was first observed more than a decade ago (3,14,15). Pathogenic mtDNA mutations are usually heteroplasmic, and are associated with deleterious phenotypic changes only if their levels are extremely high (5,8,16). Therefore, the ability to manipulate mtDNA heteroplasmy could lead to therapeutic approaches to most mitochondrial diseases. Such approaches are being developed, including the use of antisense peptide nucleic acids (9). However, these have not yet been shown to work in intact cells. Taivassalo et al. (17) showed that patients performing concentric exercise training repopulated damaged muscle cells with regenerating satellite cells that contained lower levels of mutated mtDNA, an alternative approach that also operates by heteroplasmy shifting.
We explored the usefulness of expressing a polypeptide that could target specific mtDNA haplotypes. A bacterial restriction endonuclease has the required specificity to perform such a task. Although the expression of bacterial genes in mammalian cells have the potential to exert desirable biochemical functions, translational codon bias can be a difficult obstacle to overcome in order to obtain high levels of expression. Because of this, we chose to synthesize a gene coding for the Providencia stuartii PstI endonuclease that was optimized for translation in mammalian ribosomes. We chose this gene because we were also interested in the mechanisms associated with mtDNA recombination. The human (and the mouse) mtDNA has two relatively close sites for PstI, which could recombine and in theory create deletion mutants.
The modified PstI gene, engineered to have its polypeptide product targeted to mitochondria, was efficiently expressed in human and mouse cells and was correctly targeted to mitochondria. We did not observe any change in morphological or growth features associated with mitochondrial PstI expression. Mitochondrial targeting was efficient, and we could not detect PstI in the nucleus. The expression of this protein in mitochondria had an unequivocal biological effect, namely the degradation of human mtDNA, in some cases leading to its complete loss. It is interesting to note that cleaved mtDNA fragments seemed to be rapidly degraded by mitochondrial nucleases, mostly by exonucleases as we observed a smeary pattern of degradation. Discrete bands were clearer when the DNA was digested with PstI, suggesting that many fragmented molecules had one common end, possibly one of the PstI sites. This pattern of fragmentation contrasts with the Southern blot pattern of yeast mtDNA exposed to EcoRI targeted to mitochondria. Even though these cells were exposed for a shorter period of time to the endonuclease, the predicted fragmentation pattern was observed (18). It is also possible that mtDNA molecules are protected by the nucleoid structure (19) and became exposed to PstI only during specific periods (e.g. certain replicative or organelle division stages), but as soon as a endonucleolytic cleavage occurs, the molecule is rapidly degraded. The absence of recombined PstI sites suggested that these staggered ends were not available for long periods of time to engage in recombination/repair. However, recombined molecules would still be susceptible to PstI degradation, unless the site was somehow destructed. Therefore, the fact that we could not observe re-ligation of PstI sites does not exclude the possibility that these sites could be re-joined if PstI was not present, as observed in yeast (18).
We showed that PstI could effectively reduce the levels of a mtDNA haplotype harboring PstI sites. In a heteroplasmic environment, a mtDNA haplotype lacking PstI sites was preferentially maintained, causing a significant shift in heteroplasmy. Although there was a highly significant correlation between the presence of PstI and the percent of rat mtDNA (P = 0.0008), we did not observe a strict correlation between the relative levels of PstI expression and the percentage of rat mtDNA. However, none of the expressing clones showed an accumulation of mouse mtDNA, the most common outcome of pCDNA3-transfected cells (6/12 of pCDNA3-transfected clones). This latter observation can be explained by the fact that chromosomal changes in these hybrid cells may confer a preferential maintenance to different haplotypes. In fact, although most of the pCDNA3-transfected clones showed a strong bias towards maintaining the mouse mtDNA, a few clones showed an accumulation of rat mtDNA. We believe that, even though this fluctuation would prevent a strict correlation between PstI levels and rat mtDNA percentage, it would make it harder to obtain clones with predominantly rat mtDNA as observed in the pMtPstI-transfected cells.
The usefulness of mitochondrially targeted restriction endonucleases in modulating mtDNA heteroplasmy is limited by the presence of an appropriate restriction site. However, a mitochondrial disease caused by a T
G transversion in the mitochondrial ATP6 gene (at mtDNA position 8399) creates a unique SmaI–XmaI site that could be the target for such approach. This mutation has been associated with neuropathy, ataxia and retinitis pigmentosa (NARP) and maternal inherited leigh syndrome (MILS) (2,5,20,21). In most patients, the mutation is heteroplasmic and the severe MILS phenotype differs from the milder NARP by the presence of relatively higher levels of wild-type mtDNA. Family members with mtDNA harboring the T8399G mutation in less than 60–90% of their mtDNA can be completely asymptomatic (5,16,21). Therefore, the approach described here may be applicable to genetic therapy of a sub-group of patients with mitochondrial diseases. Potential problems associated with the expression of a bacterial protein in human mitochondria are still unknown, but inducible promoters may help minimize this problem.
In conclusion, we believe that mitochondrially targeted restriction endonucleases can provide unique tools to modulate mtDNA heteroplasmy, with applications to both basic research and genetic therapy.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Construction of a mitochondrial PstI gene
A synthetic gene coding for the PstI restriction endonuclease (NCBI P00640) was synthesized by IDT Technologies (Coralville, IA). The gene was constructed with a codon bias towards mammalian ribosome usage. The following codons were used: Ala (GCC), Arg (CGC), Asn (AAC), Asp (GAC), Cys (TGC), Gln (CAG), Glu (GAG), Gly (GGC), His (CAC), Ile (ATC), Leu (CTG), Lys (AAG), Met (ATG), Phe (TTC), Pro (CCC), Ser (TCC), Thr (ACC), Trp (TGG), Tyr (TAC) and Val (GTG). We included a unique HindIII site flanking the methionine start codon for cloning downstream of a cytochrome oxidase subunit VIII mitochondria targeting sequence (22). The final construct was cloned into EcoRI–XbaI sites of pCDNA3 (Invitrogen, Carlsbad, CA), sequence verified and named pMtPstI.
Cell lines and culture conditions
Human osteosarcoma 143B, human embryonal kidney 293T, mouse LM(TK), NIH 3T3 and rat NRK (normal rat kidney) were obtained from the American Type Culture Collection (ATCC CRL-8303, CCL1.3, CRL1658 and CRL6509, respectively). HeLa cells were also used for co-localization studies. Cells were cultured in high glucose Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 100 µg/ml sodium pyruvate and 50 µg/ml uridine. The heteroplasmic hybrid cell LMNRK#6 was obtained as described previously and characterized for nuclear and mitochondrial markers (12).
Mitochondrial DNA analyses
Human cell lines expressing the mitochondrial PstI were analyzed by PCR and Southern blot. For Southern blot analysis 10 µg of total DNA from each cell line was digested with PstI or PvuII, electrophoresed through a 0.8% agarose gel, and transferred to a Zeta-Probe nylon membrane (Bio-Rad, Hercules, CA). For a probe, we used [
-32P]dCTP-labeled PCR fragment corresponding to the mtDNA small region between PstI sites (Fig. 2A). The presence of mtDNA in pMtPstI-transfected cells was also investigated by PCR using the following two sets of primers: ND5 (12 400–12 422F and 12 588–12 566B) and flanking PstI sites (6800–6823F and 9110–9087B).
To determine mtDNA haplotypes in rodent cells, we amplified a mtDNA region containing the COX I gene, using primers Rod-F [corresponding to nucleotides 5559–5578 in the mouse mtDNA (11) and nucleotides 5540–5559 in the rat mtDNA (23)] and Rod-B (nucleotides 6259–6278 in the mouse mtDNA and nucleotides 6240–6259 in the rat mtDNA). We labeled the PCR products with [-32P]dCTP in the last cycle of the reaction to avoid the detection of heteroduplexes (i.e. the formation of duplexes containing one strand from mouse and one from rat). The details of this procedure, termed ‘last cycle hot PCR’, have been reported previously by Moraes et al. (24). The radiolabeled fragments were digested with MspI, electrophoresed through a 12% polyacrylamide gel and analyzed in a Cyclone Phosphor Storage system (Packard Instruments, Meriden, CT).
DNA transfections
The cell line LMNRK#6 was plated at 70% confluence in six-well tissue culture plates (Costar, Cambridge, MA) and transfected (with pMtPstI or pCDNA3) using the cationic lipid LipofectAMINE PlusTM (Life Technologies) as described by the manufacturer, using 1.5 µg of plasmid DNA, 4 µl of LipofectAMINE reagent and 6 µl of PLUS reagent. After 72 h, cells were selected in medium containing 1 mg/ml GeneticinTM (G418; Life Technologies). Stable transfectants were isolated 15 days after selection was applied. HeLa cells were transfected by the same procedure.
Immunodetection of mitochondrial PstI
Approximately 20 µg of total cell proteins were resolved by SDS–PAGE (15% gels) and transferred onto polyvinylidene difluoride membranes (NEN Life Science Products, Carlsbad, CA). Blots were blocked with 5% milk and probed with an anti-PstI polyclonal antibody. The antibody was produced by injecting a purified PstI protein (a kind gift from New England Biolabs, Beverly, MA) with Freund’s complete adjuvant in rabbits. The antibody production was performed by Pocono Farms Inc. (Canadensis, PA). After washes, the membrane was treated with a secondary anti-rabbit antibody conjugated to horseradish peroxidase. Detection was performed using a Phototope-horseradish Peroxidase Western Blot Detection Kit (New England Biolabs).
Transiently transfected HeLa cells were used for subcellular localization of PstI. Cells were grown on coverslips to 60% confluency and transfected as described above. After 48 h cells were incubated with 200 nM CMX-ROS MitotrackerTM (Molecular Probes, Eugene, OR) for 20 min. The cells were washed with PBS and fixed with 4% paraformaldehyde. After washes, the preparations were permeabilized with –20°C methanol for 5 min and incubated with the polyclonal anti-PstI antiserum (1:100) for 12 h at 4°C. After washes with PBS the coverslips were incubated with anti-rabbit FITC (1:200, Sigma) for 2 h. After washes, the coverslips were mounted with ProLongTM (Molecular Probes) and imaged on a confocal Axiovert 100M microscope with a LSM510 scanning module (Carl Zeiss).
Computer and statistical analysis
Experimental data were analyzed using the Excel Statistical Package (Microsoft). The Student’s t-test was used for comparison of two groups. Results are expressed as mean ± SEM.
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ACKNOWLEDGEMENTS |
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We are grateful to New England Biolabs for the PstI donation for antibody production. This work was supported by National Institutes of Health Grant GM55766 and EY10804 and by the Muscular Dystrophy Association.
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FOOTNOTES |
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+ To whom correspondence should be addressed. Tel: +1 305 243 5858; Fax: +1 305 243 3914; Email: cmoraes@med.miami.edu
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REFERENCES |
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