Foreign DNA integration is one of the most widely exploited cellular processes in molecular biology. Its technical use permits us to alter a cellular genome by incorporating a fragment of foreign DNA into the chromosomal DNA. This process employs the cell's own endogenous DNA modification and repair machinery. Two main classes of integration mechanisms exist: those that draw on sequence similarity between the foreign and genomic sequences to carry out homology-directed modifications, and the nonhomologous or ‘illegitimate’ insertion of foreign DNA into the genome. Gene therapy procedures can result in illegitimate integration of introduced sequences and thus pose a risk of unforeseeable genomic alterations. The choice of insertion site, the degree to which the foreign DNA and endogenous locus are modified before or during integration, and the resulting impact on structure, expression, and stability of the genome are all factors of illegitimate DNA integration that must be considered, in particular when designing genetic therapies.
The development of methods to manipulate and modify the genome is one key achievement of molecular biology. Foreign DNA integration is one of those widely used techniques, allowing us to modify a cell's genetic content. The integration process begins with the delivery of the DNA to the cell. Many transfection procedures have been developed that enable DNA to enter into the cytoplasm, but its passage into the nucleus is mainly mediated by cellular processes.1,2 After its entry into the nucleus, a large proportion of the foreign DNA is rapidly degraded or diluted among subsequent cell divisions.3 However, molecules containing an origin of replication (typically derived from viruses) can persist for long periods as extrachromosomally replicating episomes under certain conditions (such as expression of viral tumour antigens or association with the nuclear matrix).4 Alternatively, in approximately one cell per thousand, the introduced foreign DNA will integrate into chromosomal DNA (although this figure may vary somewhat depending on cell type).5,6 This phenomenon is not limited to laboratory transfections as many natural cellular processes, such as integration of DNA from apoptotic bodies,7 repair of chromosomal lesions by insertion of mitochondrial DNA,8 or retrotransposition events, give rise to de novo integration of DNA.9,10 Thus DNA integration may be seen as an ongoing natural process, which can be harnessed to artificially introduce modifications to a cell's genetic content.
DNA can become integrated into chromosomes by two main processes: homology-dependent means and illegitimate integration. Homology-dependent genome modifications rely on mechanisms that make use of sequence similarities between the incoming DNA and the targeted locus to induce homologous recombination. These processes usually give rise to predictable results, such that the configurations of the modified loci, and of the integrated DNA, can be predefined by the investigators. Techniques employing these mechanisms have numerous applications.11 Among others, small fragment homologous recombination (SFHR) employs short (400–800 bp) DNA fragments to modify any locus and can be used to correct punctual mutations.12 Other techniques, such as classic gene targeting, are routinely used to introduce DNA to a specific site or to knock-out genes.13 However, while these methods hold great promise for the field of gene therapy, and for the development of experimental tools to investigate genome dynamics, we must take into account the fact that a significant proportion of cells integrate DNA into their genomes via homology-independent means, collectively referred to as illegitimate integration. Illegitimate integration events are usually more frequent than homology-directed integration: the ratio of homology versus non-homology-dependent integration can be anywhere from 4:1 to 1:1 000 000, depending on experimental conditions and cell types, but illegitimate integration is typically 1000–10 000 times more frequent than targeted integration (see Smith14 and references therein). These mechanisms give rise to much less predictable integrated structures, as one usually cannot preselect the genomic site of integration, nor the resulting foreign DNA–chromosome structure. The conditions governing how the cell chooses which mode of integration to employ are unclear, but may be dictated by cell cycle-dependent availability of DNA-modifying machinery: homology-dependent machinery is available mainly in G2/S, while homology-independent means are present during most of the cell cycle (see below).15
It must therefore be stressed that DNA often integrates into the genome in ways that we do not fully understand, nor totally control. In the context of gene therapy, it is likely that local or systemic delivery of DNA to cells will eventually result in illegitimate integration of DNA molecules. This appears to happen at a low frequency.16,17 However, we will see in this review that the resultant integrated structure can be surprisingly complex and unpredictable compared to the expected structure of targeted integration, making it difficult to evaluate the risks inherent in these events (see Figure 1).
A brief note regarding the source of integrating DNA discussed in this review, since much of the early work done on illegitimate integration described the integration of viral DNA. Viral DNA per se appears to integrate into the genome in the same way as any foreign DNA. However, available data suggest that viral-encoded integrases (such as with retroviruses) may target incoming DNA to particular genomic loci, and influence (at least in part) the mechanisms of integration. Indeed, characterization of retroviral integration processes has shown only a partial dependence on host cell factors, suggesting that viral factors can also be involved in the integration.18,19,20 Thus, some viral DNA integration events appear to be identical in nature to any type of foreign DNA integration (for example SV40 DNA), while those depending on viral-encoded machinery are clearly different. These types of virus-specific events will not be considered here, but it should be noted that many of the mechanisms involved in the illegitimate integration of foreign DNA could also be involved, at one point or another, in viral DNA integration.
Extrachromosomal DNA is often modified before integration into chromosomal DNA
The first part of this article addresses the status of foreign DNA before, during or shortly after integration (see Figure 2a). As mentioned earlier, it has been shown that most transfected DNA is rapidly degraded upon introduction into cells. Molecules that are not degraded can be modified extrachromosomally in many different ways. Homologous recombination can modify extrachromosomal DNA very efficiently using either inter- or intramolecular sequence homology and can result in multiple products depending on the organization of the sequences sharing identity.21,22,23,24,25,26 The end products of extrachromosomal homologous recombination are similar to those obtained during chromosomal homologous recombination, suggesting that at least some mechanisms are shared by the two processes.27,28,29,30,31
Transfected DNA can also be mutated at high frequency (on the order of 1%) by homology-independent means including point mutations, deletions, and more complex rearrangements such as insertion of genomic DNA.32,33,34 (see also our unpublished work). Linear extrachromosomal DNA can also be efficiently circularized in the nucleus, and different molecules can be joined together to form concatamers35 by processes referred to as nonhomologous end joining (NHEJ). NHEJ often involves short sequence homology between the joined ends, and additions or deletions of approximately 25 nucleotides or less at the junctions.34,36,37 Similar processes have been shown to occur during chromosomal double-strand break (DSB) repair,27,29,38 which again suggests that extrachromosomal and chromosomal repair are mediated by similar cellular machinery.
Integrated DNA is modified in the same ways as extrachromosomal DNA
Studies aimed at characterizing integrated foreign DNA reveal modifications very similar to those observed in nonintegrating extrachromosomal DNA. However, it must be kept in mind that it is not known whether the integrated DNA was modified before or during integration, since the observations are typically made after the integration has taken place. Following introduction into mouse cells, transfected DNA often becomes genetically linked39,40 and forms concatamers, sometimes as large as 2000 kb, which are either amplified or deleted coordinately. It appears that exogenous DNA generally integrates at one, or very few, site(s) in the recipient genome, but often in multiple copies showing the presence of 1–6 bp of microhomology or slight degradation or addition of nucleotides at transgene-transgene junctions.41,42,43,44,45,46,47 The NHEJ activities capable of circularizing and concatemerizing extrachromosomal DNA have been suggested to account for randomly oriented transgene arrays, while the frequent appearance of tandem arrays comprised of direct repeats is best explained by homologous recombination between circular and linear molecules before or during the integration process.41,42
The organization and quantity of integrated DNA appear to vary according to cell type. A comparison of integrated transgenes between different cell lines suggested that the degree of genetic instability may be reflected in more complex integration patterns:48 ‘normal’ cell lines show simpler integration patterns than transformed ones. Indeed, integrated arrays can be quite complex, sometimes consisting of variably degraded exogenous DNA interspersed with endogenous genomic DNA.49,50,51,52 Cell type differences in the amount of integrated DNA are reflected in experiments with human cells showing their genomes are 30–100 times less inclined to integrate as much exogenous DNA as rodent cells.53,54,55,56 Finally, the transfection method can have an effect on the amount, and resulting form, of DNA integrating into a recipient genome. While calcium phosphate precipitation and microinjection result in relatively large amounts of DNA integrated in mouse cells,39,41 electroporation shows a strong tendency for low copy numbers of integrated foreign DNA, and usually not in a standard head-to-tail array, but instead in a randomly arrayed structure.57
The recipient chromosomal locus is often found modified after an integration event
Some integration events are relatively ‘harmless’ to the integrity of a genomic site and can sometimes leave the recipient locus completely unchanged (apart from the inclusion of the new sequence).44 However, more complex insertion patterns are frequently observed (see Figure 2b). The recipient genomic locus can undergo extensive physical rearrangements at the site of integration,45,50,51,58,59 including deletions (as much as tens of kb), duplications, and translocations, among others.43,52,60 Another consequence is that the integrated material can interrupt coding sequences.61 Indeed, an estimated 15% of all transgenic mouse lines studied had incurred mutations due to DNA integration.50,51 This is a remarkably high rate of induced change, considering that only about 2–3% of the most extensively sequenced mammalian genome (that of homo sapiens) is made up of coding sequences,62 whose disruption could give rise to a distinct phenotype. The high frequency of gene inactivation observed during integration could be due to gross disruptions in normal chromatin structure and function rather than simple interruption of coding sequences by the transgene. It may also be that expressed genomic regions are preferentially targeted by illegitimate integration (see below).
Integrated DNA and recipient chromosomes can be modified in even more surprising ways. Some integrated DNA has been shown to acquire telomeric repeats at its ends,63 a modification capable of inducing a chromosomal breakage, with the foreign DNA taking the place of the normal telomere on the recipient chromosome. Transfection of a vector that already displays telomeric ends also induced similar processes.64 Aside from physical rearrangements, illegitimate DNA integration can alter other cellular characteristics, such as perturbation of genome-wide methylation patterns.65,66 The mechanisms involved are not fully understood, but the consequences for the recipient genome can be drastic. Since gene expression levels can be modified by promoter methylation,67 the integration of foreign DNA can give rise to diverse phenotypes. Chromatin compaction and methylation status of transgenes can also be influenced by copy number, a phenomenon known as repeat-induced gene silencing (RIGS). Increasing the number of transgene copies within a tandem array has been shown to correlate with decreased levels of transgene expression: this may be explained by the fact that integrated concatemers can incur increased methylation68 and reduced histone acetylation.69 Thus, illegitimate integration events can have dramatic repercussions on the content, organization, and functions of the recipient genome.
Recipient genomic loci are often unstable after illegitimate integration
One could ask whether the rearrangements are produced before, during or after integration. Since most integrations are investigated days or even weeks after the fact (via selection for expression of a marker transgene), any initial events, if they exist, could be missed. In some cases, however, instability appears to be restricted to the time of integration. Many studies carried out in cultured cells41,47,48,53 and transgenic mouse lines43 have shown that while vectors can be physically rearranged initially, expansion of cell or mouse lines reveals no further rearrangements. This leads to the conclusion that the deletions and rearrangements observed are sometimes early events in the integration process.
However, selective pressure for certain regions of the introduced exogenous DNA can prevent unstable or rearranged clones from being noticed.54 Indeed, a cell population will often lose most of the unselected foreign DNA during the first weeks of expansion, while the selected DNA appears much more stable.56,70 Many studies on SV40 transformed cell lines reported sustained instability involving the SV40 sequences and flanking cellular DNA upon continued culturing,56,71,72,73 suggesting that integrated structures are often unstable immediately after integration, and that this instability can persist in the absence of selection. While in some cases, tandem arrays of transgenes remain stable following integration,41 other groups have observed homologous recombination between copies within the array, which lead to changes in structure, expression, and transgene copy number74,75 (see also our unpublished results).
The fact that many integration sites display initial or sustained instability raises the question of a causal role for integration in instability. As some loci show no extensive rearrangements upon integration, the involvement of foreign DNA in every case of instability is doubtful. It should be noted, however, that the integrated DNA studied is often of viral or bacterial origin, and that this DNA is not necessarily ‘neutral’ in terms of stability. Bacterial and viral origins of replication, as well as plasmid sequences, often contain structures such as bent DNA that have been implicated in illegitimate rearrangements,76 and that seem to play a role in integration.77 Moreover, some foreign DNA sequences can by themselves induce instability: integration of a vector containing pericentromeric alphoid DNA can promote increases in chromosome number, changes in chromosome structure, higher frequencies of sister chromatid exchange, and the appearance of dicentric or ring chromosomes.78 Endogenous pericentromeric alphoid DNA is known to be somewhat unstable, but appears to have evolved some degree of stability in its natural context. However, its integration into nonpericentromeric DNA is highly destabilizing. Other transfected genes, such as DHFR or CAD, can be amplified if cells are treated with agents causing amplifications of the endogenous gene79,80 and, as we have seen earlier, transfection of a telomeric repeat-containing vector can cause chromosomal breakage63,64 and instability at internal chromosomal loci.81 Some exogenous sequences can thus be rearranged frequently and induce instability at an otherwise stable site.
Certain genomic integration sites, however, are more prone to instability than others. One report examined an unstable clone in which the foreign DNA had integrated into pericentromeric satellite DNA, making this locus unstable.82 Disruption of this locus' natural organization with foreign DNA was proposed to have enhanced the site's inherent instability. Other investigations characterized vectors that had integrated into genomic fragile sites.83,84 These sites can be induced to undergo rearrangement by treating the cells with various chemicals.85 Induction of fragile sites resulted in frequent local vector integration, suggesting that an intrinsically unstable site is more prone to integrate DNA. However, the fragile sites into which foreign DNA had integrated were more prone to gross chromosomal rearrangements when reinduced. These findings, and the fact that it is sometimes impossible to isolate the source of instability of one particular integrated locus, point to an interplay between chromosomal and exogenous sequences during the genesis of sustained instability at the integrated locus.86,87,88 Taken together, these observations suggest that integration involves a degree of transient, and sometimes permanent, instability at the integration site.
Illegitimate integration sites are not totally random
The fact that unstable genomic sites might be exceptional candidates for illegitimate DNA integration raises the issue of the apparent lack of site specificity in these events. Historically, illegitimate integration has been considered an essentially random process, but relatively little data exist that demonstrate this randomness. Fluorescence in situ hybridization (FISH) analysis on pools of transfected clones has shown that no particular bias existed for integration in mouse cells with respect to chromosomal morphology or chromosomal position.89 However, more direct characterization of some integration sites reveals that not all chromosomes are subject to an equal degree of illegitimate DNA integration. A review of 35 different insertional mutants generated in transgenic mouse lines revealed that some chromosomes are selected more often for illegitimate integration than others, at least in this limited subset.61 For example, 5/35 lines examined had integrated DNA into chromosome 10, and in four other lines into chromosome 6. These high frequencies suggest that these particular chromosomes might have features that make them more likely to integrate DNA. Particular cell types seem to be extremely restrictive in the choice of foreign DNA integration site. For instance, several studies have shown that spermatozoa integrate DNA at one, or a few, specific site(s), and multiple instances of integration at the same site were observed.90,91,92 The authors suggested that there may be relatively few sites accessible for DNA integration, as the probability of DNA integrating in the same locus in two separate clones should be extremely low. In support of this, a preintegrated locus was shown to be 75- to 470-fold more likely to receive another incoming vector than any randomly assigned locus.46 This implies that the genomic locus in which the first vector was integrated was itself an integration ‘hotspot’, or that the integration of a vector renders a genomic site a hotspot for integration.
More detailed analyses of integration sites reveal some interesting trends that could be instrumental in making a site a ‘hotspot’.93 A review of multiple illegitimate integration sites found that in 93% of the cases these sites were only 10 bp away from a potential topoisomerase I (Topo I) cleavage site. The association of Topo I sites with runs of purines and AT-rich regions with the site of integration was also significant. Purine tracts can adopt non-B-DNA conformations which may be recombinogenic: these sequences are found in centromeres and may promote recombination of satellite DNA. Such findings are a recurrent theme: many other investigators have found Topo I consensus sequences, and other sequences such as Escherichia coli CHI element and minisatellite sequences, near transgene–chromosome junctions.43,44,59,94,95 It has been demonstrated that integration of a circular bent DNA-containing vector preferentially occurs at the bent site, while bent DNA structures are often found at illegitimate recombination junctions and rearrangement breakpoints.76 This suggests that such bent DNA sites in the genome (which are numerous) might be involved in integration.77 As mentioned earlier, fragile sites are also frequently involved in illegitimate integration events when cells are treated with the appropriate inducing chemicals. Hence, the frequency of integration at one particular site can be related to its base composition, and with which factors it can interact.
Proposed mechanisms of illegitimate DNA integration
From all of the above data, a mechanism can be presented for the integration of foreign DNA by illegitimate integration: a DNA repair-mediated integration process. This mechanism has been suggested by numerous investigators, and this model receives the most support from the reviewed data.42,58 According to this model, a genomic lesion, either single- or double-stranded, would attract repair factors. Any DNA with free ends, which happened to be in the vicinity or be recruited to the lesion by repair factors, could become part of the ‘healing’ process and be integrated (see Figure 3). As mentioned earlier, genomic DSBs can be repaired with high efficiency by NHEJ. One could thus envision that a foreign DNA in linear form (with free ends) could get ligated to the ends generated by genomic DNA damage. Indeed, it is demonstrated that DSBs and other types of DNA damage induce many types of recombination processes.96,97 Another mechanism of DSB repair that is often implicated in DNA integration is homologous recombination (HR). HR has been demonstrated to play an important role in concatamer formation,41,42 and some studies have shown that cells that participate in an extrachromosomal HR event are more likely to integrate DNA.29,30,31 In support of a link between both types of DNA repair and integration, cells that incorporated DNA by illegitimate integration were also 80-fold more likely to integrate additional DNA by gene targeting, a process dependent upon homologous recombination.98
Induction of genomic DNA damage by various means, such as treatment of cells with various Topo I and II inhibitors,85 restriction enzymes, crosslinking agents (eg psoralen), or UV damage, often leads to an increased frequency of illegitimate DNA integration, supporting the above model.85,97,99,100,101,102,103 Interestingly, it was shown that the effect of DSB-inducing restriction enzymes on integration was dependent upon the presence of Ku80, an essential end-binding component of the NHEJ pathway. NHEJ and HR (BRCA1) proteins were also shown to have an effect on integration frequencies in other systems: NHEJ deficiency was shown to inhibit integration in yeast, and BRCA1−/−cells had a 5- to 10-fold reduction in retroviral DNA integration.104,105 Other types of repair machinery might influence integration as well. For example, a deficiency of mismatch repair proteins (known to be involved in the disruption and correction of mismatched heteroduplex DNA that can arise during NHEJ and HR DNA repair) can increase the frequency of illegitimate integration 15-fold.106 Some evidence suggests that transcribed regions are more prone to damage than other regions,85 which could explain the frequent disruption of gene expression by illegitimate integration. On the other hand, homology-directed integration is also positively influenced by transcription,107 which again draws a link between both types of DNA repair processes, DNA damage and DNA integration. Damage to the exogenous DNA by UV or psoralen photo-adducts also results in enhanced integration in human cell lines.97,103 However, the clearest demonstration of the link between DNA repair mechanisms and illegitimate integration comes from studies that find that inducing a chromosomal DSB at a defined genomic locus (by expression of a site-specific endonuclease) promotes illegitimate integration at that site.29,108 These experiments clearly show that most integration events are probably mediated by the cellular DNA repair machinery.
Conclusions and perspectives
While questions still remain, some general themes emerge from studies of illegitimate DNA integration. Involvement of DNA damage at the site of integration seems likely, which explains why some genomic sites are involved more often than others in such events. In turn, this phenomenon could select for or induce genomic rearrangements and instability. Moreover, the introduced foreign DNA can be modified in many ways before, during, and even after the integration process. These facts must be kept in mind when designing gene therapy experiments.
Many aspects of illegitimate integration demand further investigation. While the disparate data available suggest that integration site choice is not random, a more systematic characterization of integration sites at the sequence level is warranted. The recent availability of the human genome sequence will allow more detailed analysis of transgene–genome junctions. This should also help to elucidate mechanisms and cellular processes involved in integration, as the extent of rearrangement at the integrated locus can now be evaluated more thoroughly. These analyses could prove valuable for investigating the global structure of the genome, a major challenge of the genome project era. Indeed, if the link between genetic instability and integration continues to be reinforced, one could use illegitimate integration sites as telltale indicators of unstable loci in the genome. Finally, while some experiments have investigated the genetic components necessary for efficient illegitimate integration in some systems,100,104,105 detailed knowledge about the mammalian cellular factors necessary for these processes is also lacking. A better understanding of the mechanisms and factors governing illegitimate DNA integration would allow for better control of these events and, ultimately, an improved and safer use of DNA-mediated genome modification in laboratory and clinical settings.
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Würtele, H., Little, K. & Chartrand, P. Illegitimate DNA integration in mammalian cells. Gene Ther 10, 1791–1799 (2003). https://doi.org/10.1038/sj.gt.3302074
- illegitimate DNA integration
- DNA repair
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