Preferential accessibility to specific genomic loci for the repair of
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《核酸研究医学期刊》
1 Molecular Biology Program and 2 Department of Pathology and Cellular Biology, 3 Montreal Cancer Institute, CHUM and 4 IRIC, Université de Montréal, Montréal, Québec, Canada
* To whom correspondence should be addressed. Tel: +1 514 343 6111, Ext. 3573; Fax: +1 514 343 7780; Email: pierre.chartrand@umontreal.ca
ABSTRACT
The dynamic organization of the human genome in the nucleus is gaining recognition as a determining factor in its functional regulation. In order to be expressed, replicated or repaired, a genomic locus has to be present at the right place at the right time. In the present study, we have investigated the choice of a double-strand break (DSB) repair partner for a given genomic loci in an ATM-deficient human fibroblast cell line. We found that partner choice is restricted such that a given genomic locus preferentially uses certain sites in the genome to repair itself. These preferential sites can be in the vicinity of the damage site or megabases away or on other chromosomes entirely, while potential sites closer to the break along the length of the chromosome can be ignored. Moreover, there can be more than a 10-fold difference in usage between repair sites located only 10 kb apart. Interestingly, arms of a given chromosome are less accessible to one another than to other chromosomes. Altogether, these results indicate that the accessibility between genomic sites in the human genome during DSB repair is specific and conserved in a cell population.
INTRODUCTION
The mammalian genome in the interphase nucleus is not randomly arranged but is highly organized in discrete chromosomal territories (1,2). This compartmentalization appears to be important for the regulation of cellular processes, such as replication, transcription, RNA processing and DNA repair (3). Also, there are a few indications that genomic loci located at different chromosomal sites can be physically positioned close together in the interphase nucleus in a tissue and cell-cycle-dependent manner (4–6).
When a double-strand break (DSB) occurs in the mammalian genome, a number of mechanisms can come into play to repair it. Some are local and involve the rejoining of the broken ends through a process referred to as non-homologous end joining (NHEJ) (7,8). However, others involve the physical contact with other regions of the genome that can be on the same or distinct chromosomes. Two categories can be distinguished. The first involves NHEJ of two DSBs that leads to chromosomal rearrangements (NHCR), such as deletions, inversions and translocations (9). The second involves recombination with homologous sequences located elsewhere in the genome (HR) (7,8). In the case of NHCR, it is assumed that any two loci from the same chromosome or distinct chromosomes can come into contact to join ends resulting from DSBs. However, it is not clear whether the genomic loci that interact are to start with in close physical proximity or are brought together as a result of the occurrence of the DSBs. Recent results in yeast and mammalian cells indicate that when multiple DSBs are produced, these can be relocated together presumably for repair (10,11). However, genomic loci that are involved in translocations have been shown to co-localize before the event (4–6,12). It is not clear whether NHCR involves two independent DSBs or that the initial DSB conditions the occurrence of a second one (13). In the case of HR, the homologous sequences have to be brought together, but again it is not clear whether this occurs before or after the occurrence of the DSB, which in this case involves only one of the two partners initially. Experiments, where two homologous sites were positioned in the mammalian genome with one sustaining a DSB, indicate that DSB repair through HR can occur between homologous sites on the same or distinct chromosomes, the later occurring at a much lower frequency than the former (14). In these experiments, a very limited number of potential genomic sites, however, were tested and not in direct competition with one another. In both cases of NHCR and HR, it has not been determined as to what extent the accessibility to other genomic sites plays a role in the process of DSB repair.
In the present work, we have developed an assay to compare the accessibility of a given genomic locus to other loci in the human genome for the repair of a DSB both by NHCR and HR. For HR repair, several thousand potential partners were present throughout the genome. We used an ATM-deficient human fibroblast cell line for this study because of its high incidence of genomic homologous recombination (15). The results indicate that the choice of partners is rather specific and discriminatory involving dominantly the immediate chromosomal surroundings of the DSB site. However, specific partners found distantly on the same chromosome arm or even on other chromosomes are also preferentially used for repair. On the opposite, there appears to be a restricted access between the arms of the same chromosome.
MATERIALS AND METHODS
Plasmid constructions
The 2.5 kb LINE-1 portion of the PL1Hs1V1.2 vector (16) was modified by replacement of the highly conserved NheI–BamHI 23 bp fragment by the rare endonuclease restriction site I-SceI (17), resulting in the pL1HsSceI vector (Figure 1A). PCBASceTcr expression vector was created by cloning the PCR-amplified pBR322 tetracycline-resistance gene (New England Biolabs) in the ScaI site of pCBASce (17), in order to inactivate the ampicillin-resistance gene.
Figure 1. Possible repair products resulting from DSB induction at the I-SceI site of the integrated pL1HsSceI vector. (A) Structure of the integrated pL1HsSceI vector containing origin of replication (ori), ampicillin gene resistance (amp) and neomycin gene selection (neo). The 2.5 kb L1 portion corresponds to the 3' segment (3390–5976 bp) of cD11 (cDNA of an active L1 element), inside which the 23 bp NheI–BamHI segment was replaced by an 18 bp I-SceI restriction site (black box). (B) Structure of a typical NHEJ repair product, characterized by the retention of the Sp (SpeI) and S (SacII) plasmidic sites, the loss of the I-SceI site and the absence of the N B (NheI–BamHI) segment. NHEJ junctions frequently showed deletion and/or insertion sequences at the break site. (C) Structure of a typical NHCR repair product, characterized by the loss of the SpeI and SacII plasmidic sites and the absence of the NheI–BamHI segment and the acquisition of a novel chromosomal junction (dotted line). (D) Structure of a typical gene conversion product, characterized by the retention of the SpeI and SacII plasmidic sites and the presence of the NheI–BamHI segment acquired from an endogenous L1 partner by HR. (E) Structure of a typical crossing-over/one sided invasion product, characterized by the presence of the NheI–BamHI segment acquired from an endogenous L1 partner by HR but extending beyond the homologous L1 sequences into adjacent chromosomal sequences (dotted line).
Cell growth and transfections
The GM05849 cell line is an ATM-deficient human fibroblast strain obtained from NIGMS Human Mutant Cell Repository (Coriell Institute for Medical Research). The cells were maintained in EMEM supplemented with 10% fetal bovine serum and 50 μg/μl gentamicin (Wisent) and kept in a 5% CO2 atmosphere at 37°C. A total of 5 x 106 cells were electroporated as described previously (18) with 1 μg of BglII-linearized pL1HsSce1 and selected 48 h after transfection in 200 μg/μl geneticin (Wisent). The resulting resistant cells were cloned and amplified.
Genomic DNA extraction and clones characterization
Genomic DNA was extracted from cells using lysis solution (50 mM Tris–HCl, pH 7.6, 100 mM EDTA, 100 mM NaCl, 1% SDS and 1.6 mg/ml proteinase K) for 18 h at 60°C with agitation. Genomic DNA was purified by RNAse treatment and phenol–chloroform extraction. Integrated plasmids were characterized by Southern blot analysis (19) using 13 μg of purified genomic DNA and 32P EagI–BamHI neomycin fragment of pMC1neo (Stratagene) as a probe.
DSB induction
Transient I-SceI expression was induced in clones by electroporation of 5 x 106 cells with 100 μg of circular pCBASce1Tcr as described previously (18). Surviving cells were harvested after 48 h for genomic DNA extraction and plasmid rescue.
Plasmid rescue
Total genomic DNA was first digested with SacI (ATA34) or BglII (ATA67) for plasmid excision from the genome of each clone. Plasmids were circularized by ligation (T4 DNA ligase, New England Biolabs) and digested by I-SceI meganuclease enzyme (New England Biolabs) to enrich for repair events having lost DSB site. Plasmids were electroporated as described previously (18) into DH10B electrocompetent bacteria (Invitrogen) and selection was performed with 50 μg/ml ampicillin. Colonies were individually picked and their plasmids were extracted by modified alkaline lysis miniprep (20).
Integration sites and recombinants analysis
PL1HsSce1 integration sites and DSB repair events were analysed by restriction analysis and sequencing using the Thermo Sequenase Radiolabeled Terminator Cycle Sequencing kit (Amersham). Plasmid integration sites were identified using TK4415R primer (5'-TTAACAGCGTCAACAGCGT-3'). HDF4683 (5'-GCTTACCTACAACTATCTGA-3') and HDR4885 (5'-GGGTTTTTATGGTTTTAGGT-3') primers were used to sequence repaired DSB sites resulting from recombination events. Plasmid integration sites, endogenous LINE-1 partners of HR and genomic NHCR sites were identified with GenBank database (National Center for Biotechnology Information BLAST Human genome software) (http://www.ncbi.nlm.nih.gov).
RESULTS
Experimental approach
To investigate the accessibility of a given genomic locus to the rest of the genome, we induced a DSB in a chromosomal LINE-1 (L1) element, which could then be repaired by NHEJ, NHCR or alternatively by HR using any of the 5 x 106 copies of L1 elements naturally scattered throughout the human genome (21). A tagged L1 element was introduced in the GM05849 ATM-deficient human cell line genome by transfection of the pL1HsSceI plasmid (Figure 1A). Two independent clones, ATA34 and ATA67, were selected for this study because they had integrated a complete copy of the vector at a single site on chromosomes 6q23.3 and 2p22.1, respectively. The position and organization of the genomic site of integration was determined by sequencing the genomic sequences flanking the integrated pL1HsSceI plasmids rescued from ATA34 and ATA67. In the integrated L1 element, the highly conserved 23 bp NheI–BamHI segment has been replaced by the rare I-SceI restriction site, allowing induction of a DSB at that specific genomic site. After repair of the I-SceI site, the pL1HsSceI vector was recovered from the genome by plasmid rescue and all repair events for which the I-SceI site had been lost were analysed. This approach allows the recovery of all types of DSB repair events (Figure 1B–E), resulting from NHEJ, NHCR or HR (both gene conversion and crossing-over).
Mechanism of DSB repair
The majority of repair events recovered from ATA34 and ATA67 resulted from NHEJ (Table 1), the main DSB repair pathway in mammalian cells (18,22,23). However, it should be noted that sister chromatid homologous repair events could not be scored in our assay. We also observed NHCR accounting for 2.3% of ATA34 and 12.3% of ATA67 repair events. HR accounted for 4% of ATA34 and 14.8% of ATA67 repair events. As previously observed (24), gene conversion was by far the most frequent HR repair mechanism, but we also detected three crossing-over/one one-sided invasion events (25), the later also referred to as long track gene conversion (24). Note that we cannot distinguish between crossing-over events and one-sided invasion events since the former could be one-sided invasion events that extended beyond the restriction site used for plasmid rescue. To evaluate the accessibility of ATA34 and ATA67 genomic loci to other genomic sites, we next determined the location of NHCR junction sites and endogenous L1 HR partners.
Table 1. Frequencies of DSB repair mechanisms
Characterization of non-homologous recombination events
Most of the NHEJ repair events resulting from the joining of the broken extremities (Figure 1B) were accompanied by a deletion and/or an insertion of few nucleotides at the DSB site. Recombinant plasmids having replaced the 3' sequences adjacent to the DSB with novel genomic sequences were identified as NHCR (Figure 1C), and chromosomal sites used for repair were identified by sequencing of the genomic junction next to the DSB and comparing these sequences with human genome databases (see Materials and Methods, Table 2). With clone ATA34, two out of the six sites analysed involved a genomic site located on the same chromosomal arm 6q as the integration site of pL1HsSceI, while the four others involved a genomic site from a different chromosome. Interestingly, two events obtained from separate experiments (34-t2a, 34-t2b) involved genomic sites separated by 1 bp on 1q24 .3. In the case of the ATA67 NHCR events, 12 of the 24 sites analysed were located on the same chromosomal arm 2p as the integration site of pL1HsSceI. There was a clear indication of clustering of some of the events around 2p22 and 2p23, including two events (67 t-23 and 67 t-24) that occurred 3 kb apart but 600 kb from the integration site (Figure 2B). The vast majority of the intrachromosomal NHCR sites were 3' of the DSB as could have been expected since 5' sites would have deleted the plasmidic sequences in most cases and would have not been recovered during the plasmid rescue procedure. However, the orientation of the novel chromosomal junction with regard to the integration site was sometimes inverted, which is indicative of an inversion event rather than a simple deletion. The 12 remaining events involved interchromosomal sites with two events occurring in rather close proximity on 14q (67 t-13, 67 t-14).
Table 2. Localization of chromosomal junction sites in NHCR repair events
Figure 2. Mapping of ATA34 (A) and ATA67 (B) integration region. Each HR partner is localized in relation to the integrated pL1HsSceI with their description (name, length, degree of homology and frequency). Other endogenous L1s having overlapping sequences with pL1HsSceI are indicated with their description (length and degree of homology). Arrows indicate the relative orientation of endogenous L1s or NHCR sites in comparison with the integrated pL1HsSceI.
Characterization of homologous recombination events
HR events with endogenous L1 partners were characterized by the reacquisition of the conserved NheI–BamHI L1 segment at the broken I-SceI site (Figure 1D and E). Since endogenous L1 elements vary somewhat in their sequence from one another, L1 sequences acquired by the integrated pL1HsSceI vector through HR were used to try to identify which endogenous L1 element had participated in the repair event (Table 3). The entire gene conversion track was used to identify the genomic L1 partner that had a matching sequence in the human genome database. With clone ATA34, specific L1 partners were identified by BLAST for 15 out of the 17 HR repair events. The remaining two could not be assigned to a single L1 partner. Some L1 partners were preferentially used (34-A, B, C and E). Out of the nine different L1 partners identified for HR repair, eight were located on the same chromosomal arm as the integration site on 6q. The other (34-A) was located on a distinct chromosome (4q24) but was used in three separate HR repair events. Out of the 96 HR repair events analysed for clone ATA67, we could assign 64 events to 9 different L1 endogenous partners (Table 3). Some events were assigned to specific partners on the basis of base changes common to several events (see Supplementary Material). Interestingly, in the case of partner 67-B, we initially did not find a good match by BLAST analysis to a single endogenous L1 partner. However, we recovered a crossing-over event that permitted to identify the genomic location of this specific endogenous L1 partner. It turned out that the L1 element at that site in the GM05849 cell line showed sequence variations compared to the L1 GenBank sequence for that site. Six additional events that had acquired endogenous L1 sequences could not be assigned to a single endogenous L1 partner (multiple matches). The remaining 26 events showed no sequence difference with pL1HsSceI except for the reacquisition of the 23 bp NheI–BamHI segment at the I-SceI site, which clearly necessitated a HR repair event. Thus, with ATA67, 15 different L1 partners were identified, with three of them (67-A, B and D) accounting for at least 50% of the total HR repair events. As was the case for clone ATA34, most of the identified L1 partners used for HR repair were located on the same chromosomal arm 2p. Three partners were on a different chromosome with two of them on chromosome 3q, including the one that was used on four separate occasions.
Table 3. Description of HR endogenous L1 partners
Length and homology of the endogenous L1 partner used for HR repair
Endogenous L1 partners used for HR repair with ATA34 and ATA67 shared 93–98% of homology with the L1 sequences present in the integrated pL1HsSceI vector. Their length varied from 354 bp to above the length of homology of 2586 bp present in the vector (Table 3). It has been reported that the degree and length of homology greatly influence the choice of a partner for homologous recombination (26–28). Indeed, we did not observe the use of a L1 partner with <93% homology, and most of the frequently used partners had homologies of 97% and above. However, less homologous partners were also used quite frequently while potential partners with a higher degree of homology that were in the vicinity of these preferential partners were not used at all. The same ambivalence was seen for the length of chosen versus ignored L1 partners. Thus, although the criteria of length and degree of homology could have played a role, they were certainly not the sole determinant in the preferential choice of specific L1 partners.
The relative position of the intrachromosomal L1 partners used for DSB repair
The distribution of all endogenous L1 elements present around the integration sites of ATA34 and ATA67 was analysed in detail (Figure 2). Interestingly, in the case of ATA34, a L1 element of 97% homology and 1280 bp in length located 123 kb away from the integration site was not used for repair, whereas an L1 element (34-C) of less homology (96%) and length 354 bp located 312 kb from the integration site was used three times (Figure 2A). In the case of ATA67, 750 kb separates the integration site from the first cluster of L1 repair partners (67-A, B, C and E) that were collectively used 45 times (Figure 2B). There are no other L1 elements more homologous and closer than these partners on this side of the integration site. Interestingly, 67-E, which is 95% homologous and 2017 bp in length, was used once for HR repair, whereas 67-A with similar homology and length, located 11 kb further away, was used 13 times.
Figure 3 presents the relative position of all genomic intrachromosomal sites (HR and NHCR events) used for DSB repair, located on 6q for ATA34 events or 2p for ATA67 events. For ATA34, there is one major cluster of partners around the integration site and partners are located equally on both sides of the integration site (Figure 3A). For ATA67, a major cluster was found spreading 2 Mb on one side of the integration site. Another important cluster was found 12 Mb away towards the telomere (Figure 3B). Interestingly, both clusters include HR and NHCR repair events. Contrary to ATA34, the vast majority of the partners were located on the same side (telomeric) of the integration site. Thus, although partners proximal to the integration site were certainly favoured, other partners located quite distally were also used preferentially for repair, whereas potential partners located in between were ignored.
Figure 3. Localization of intrachromosomal partners for HR and NHCR repair events in clones ATA34 (A) and ATA67 (B). Distances from integration sites of the pL1HsSceI vector are indicated in parenthesis for each site. When an endogenous L1 partner was used more than once, the frequency is indicated at the right.
DISCUSSION
Understanding the principles that govern genomic organization is viewed as essential to understand genomic function (29). We have investigated the accessibility of given genomic loci to other loci in the genome of human cells during DSB repair. We have used in our assay an ATM-deficient cell line because of its high incidence of genomic homologous recombination (15) that made it possible to generate enough HR events for their comparison. This may have introduced some bias in the relative frequencies of repair events. The assay permitted the recovery of all types of repair products including NHEJ, NHCR and HR products. However, it should be noted that specific sub-categories of recombination events could not be recovered or scored in our assay. Indeed, we could not score for sister chromatid homologous repair events. Also, any event that deleted the plasmidic sequences could not be recovered. NHEJ was by far the most frequent DSB repair mechanism observed in our assay and this is possibly an underestimate considering that NHEJ events that reconstituted the I-SceI site could not be identified. For the first time to our knowledge, NHCR and HR were compared in direct competition for genomic sites that were basically side by side throughout the genome, albeit with the proviso mentioned above on the events that could not be scored. The results showed that they occurred at the same frequency for a given integration site and often involving partners from the same genomic location. Interestingly, there was a significant variation in NHCR and HR frequency relative to NHEJ between the two integration sites studied. This could suggest that the choice of repair mechanism, local versus distal, is influenced by the genomic site where the DSB occur, possibly as a consequence of relative accessibility to distal genomic sites.
In the case of the HR repair events, the potential L1 targets were dispersed all over the genome. Since the vast majority of HR events involved gene conversion without chromosomal rearrangement, there was no evident counter selection against certain chromosomal interactions that would result in non-functional chromosomal structures. The degree of homology of the targeted L1 elements, below a certain threshold, appeared to be a limiting factor in partner choice, but that still left the possibility of interacting with thousands of loci dispersed throughout the genome. Out of the 79 HR events that could be assigned to a specific L1 partner for ATA67 and ATA34, 70 were intrachromosomal and nine were interchromosomal, thus indicating about a 10-fold preference for intra versus inter chromosomal sites. Clearly, however, this was not due to a simple question of linear proximity on the same chromosome. Indeed, the immediate vicinity, within a couple of 100 kb, appears to be of the highest accessibility, in the case of ATA67 accounting for 70% of the HR events. But another partner located more than 10 Mb away accounts for 20% of HR events, bypassing numerous potential L1 partners closer in terms of chromosomal linearity. Even for partners of similar size and degree of homology located 10 kb apart (67A and 67E), there can be a difference of more than 10-fold in usage. Interestingly, the other chromosomal arm appears to be less accessible than other chromosomes. This lack of accessibility between the two arms of the same chromosome supports previous observations that, within a given territory, chromosomal arms are separated in distinct domains (30). The accessibility to other chromosomes also indicates specificity with one particular interchromosomal genomic site accounting for 6% (67-N) of HR events in ATA67 and 20% (34-A) in ATA34.
The fact that NHCR and HR events cluster to the same genomic sites suggests that the specificity in accessibility is not due to the repair mechanism but rather to a common situation in both processes. In the case of NHCR repair events, the choice of the partner site presumably involves the occurrence of a second DSB at that site. We did not find evidence of the presence of a pseudo-I-SceI site or fragile site in the vicinity of the breakpoints to explain the occurrence of a presumed second DSB. In the case of HR repair events, the choice of the partner involves the presence of a homologous element at that site. The coincidence of both types of events in close vicinity would suggest that these sites were in close physical proximity to the integration site to start with. This is supported by the observation of physical proximity for genomic sites involved in NHCR events (4–6,12) and the choice of partner in HR events being influenced by chromosomal topology (31). The chromatin structure could also influence the accessibility of a donor sequence. If physical proximity is the reason for the choice of repair partners, our results suggest that chromosomal territories have a specific structure that directs accessibility of a given genomic locus to preferential genomic sites. The fact that the same genomic sites used for repair are isolated repeatedly from a given cell population and also from separate cell passages indicates that the organization of chromosomal territories of interphase nuclei in a human cell population is conserved.
SUPPLEMENTARY MATERIAL
Supplementary Material is available at NAR Online.
ACKNOWLEDGEMENTS
We thank M. Jasin for the generous gift of pCBASce. We also thank H. Wurtele and K. Little for their critical reading of the manuscript and their comments. This work was supported by a grant from the Canadian Institutes of Health Research to P.C.
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* To whom correspondence should be addressed. Tel: +1 514 343 6111, Ext. 3573; Fax: +1 514 343 7780; Email: pierre.chartrand@umontreal.ca
ABSTRACT
The dynamic organization of the human genome in the nucleus is gaining recognition as a determining factor in its functional regulation. In order to be expressed, replicated or repaired, a genomic locus has to be present at the right place at the right time. In the present study, we have investigated the choice of a double-strand break (DSB) repair partner for a given genomic loci in an ATM-deficient human fibroblast cell line. We found that partner choice is restricted such that a given genomic locus preferentially uses certain sites in the genome to repair itself. These preferential sites can be in the vicinity of the damage site or megabases away or on other chromosomes entirely, while potential sites closer to the break along the length of the chromosome can be ignored. Moreover, there can be more than a 10-fold difference in usage between repair sites located only 10 kb apart. Interestingly, arms of a given chromosome are less accessible to one another than to other chromosomes. Altogether, these results indicate that the accessibility between genomic sites in the human genome during DSB repair is specific and conserved in a cell population.
INTRODUCTION
The mammalian genome in the interphase nucleus is not randomly arranged but is highly organized in discrete chromosomal territories (1,2). This compartmentalization appears to be important for the regulation of cellular processes, such as replication, transcription, RNA processing and DNA repair (3). Also, there are a few indications that genomic loci located at different chromosomal sites can be physically positioned close together in the interphase nucleus in a tissue and cell-cycle-dependent manner (4–6).
When a double-strand break (DSB) occurs in the mammalian genome, a number of mechanisms can come into play to repair it. Some are local and involve the rejoining of the broken ends through a process referred to as non-homologous end joining (NHEJ) (7,8). However, others involve the physical contact with other regions of the genome that can be on the same or distinct chromosomes. Two categories can be distinguished. The first involves NHEJ of two DSBs that leads to chromosomal rearrangements (NHCR), such as deletions, inversions and translocations (9). The second involves recombination with homologous sequences located elsewhere in the genome (HR) (7,8). In the case of NHCR, it is assumed that any two loci from the same chromosome or distinct chromosomes can come into contact to join ends resulting from DSBs. However, it is not clear whether the genomic loci that interact are to start with in close physical proximity or are brought together as a result of the occurrence of the DSBs. Recent results in yeast and mammalian cells indicate that when multiple DSBs are produced, these can be relocated together presumably for repair (10,11). However, genomic loci that are involved in translocations have been shown to co-localize before the event (4–6,12). It is not clear whether NHCR involves two independent DSBs or that the initial DSB conditions the occurrence of a second one (13). In the case of HR, the homologous sequences have to be brought together, but again it is not clear whether this occurs before or after the occurrence of the DSB, which in this case involves only one of the two partners initially. Experiments, where two homologous sites were positioned in the mammalian genome with one sustaining a DSB, indicate that DSB repair through HR can occur between homologous sites on the same or distinct chromosomes, the later occurring at a much lower frequency than the former (14). In these experiments, a very limited number of potential genomic sites, however, were tested and not in direct competition with one another. In both cases of NHCR and HR, it has not been determined as to what extent the accessibility to other genomic sites plays a role in the process of DSB repair.
In the present work, we have developed an assay to compare the accessibility of a given genomic locus to other loci in the human genome for the repair of a DSB both by NHCR and HR. For HR repair, several thousand potential partners were present throughout the genome. We used an ATM-deficient human fibroblast cell line for this study because of its high incidence of genomic homologous recombination (15). The results indicate that the choice of partners is rather specific and discriminatory involving dominantly the immediate chromosomal surroundings of the DSB site. However, specific partners found distantly on the same chromosome arm or even on other chromosomes are also preferentially used for repair. On the opposite, there appears to be a restricted access between the arms of the same chromosome.
MATERIALS AND METHODS
Plasmid constructions
The 2.5 kb LINE-1 portion of the PL1Hs1V1.2 vector (16) was modified by replacement of the highly conserved NheI–BamHI 23 bp fragment by the rare endonuclease restriction site I-SceI (17), resulting in the pL1HsSceI vector (Figure 1A). PCBASceTcr expression vector was created by cloning the PCR-amplified pBR322 tetracycline-resistance gene (New England Biolabs) in the ScaI site of pCBASce (17), in order to inactivate the ampicillin-resistance gene.
Figure 1. Possible repair products resulting from DSB induction at the I-SceI site of the integrated pL1HsSceI vector. (A) Structure of the integrated pL1HsSceI vector containing origin of replication (ori), ampicillin gene resistance (amp) and neomycin gene selection (neo). The 2.5 kb L1 portion corresponds to the 3' segment (3390–5976 bp) of cD11 (cDNA of an active L1 element), inside which the 23 bp NheI–BamHI segment was replaced by an 18 bp I-SceI restriction site (black box). (B) Structure of a typical NHEJ repair product, characterized by the retention of the Sp (SpeI) and S (SacII) plasmidic sites, the loss of the I-SceI site and the absence of the N B (NheI–BamHI) segment. NHEJ junctions frequently showed deletion and/or insertion sequences at the break site. (C) Structure of a typical NHCR repair product, characterized by the loss of the SpeI and SacII plasmidic sites and the absence of the NheI–BamHI segment and the acquisition of a novel chromosomal junction (dotted line). (D) Structure of a typical gene conversion product, characterized by the retention of the SpeI and SacII plasmidic sites and the presence of the NheI–BamHI segment acquired from an endogenous L1 partner by HR. (E) Structure of a typical crossing-over/one sided invasion product, characterized by the presence of the NheI–BamHI segment acquired from an endogenous L1 partner by HR but extending beyond the homologous L1 sequences into adjacent chromosomal sequences (dotted line).
Cell growth and transfections
The GM05849 cell line is an ATM-deficient human fibroblast strain obtained from NIGMS Human Mutant Cell Repository (Coriell Institute for Medical Research). The cells were maintained in EMEM supplemented with 10% fetal bovine serum and 50 μg/μl gentamicin (Wisent) and kept in a 5% CO2 atmosphere at 37°C. A total of 5 x 106 cells were electroporated as described previously (18) with 1 μg of BglII-linearized pL1HsSce1 and selected 48 h after transfection in 200 μg/μl geneticin (Wisent). The resulting resistant cells were cloned and amplified.
Genomic DNA extraction and clones characterization
Genomic DNA was extracted from cells using lysis solution (50 mM Tris–HCl, pH 7.6, 100 mM EDTA, 100 mM NaCl, 1% SDS and 1.6 mg/ml proteinase K) for 18 h at 60°C with agitation. Genomic DNA was purified by RNAse treatment and phenol–chloroform extraction. Integrated plasmids were characterized by Southern blot analysis (19) using 13 μg of purified genomic DNA and 32P EagI–BamHI neomycin fragment of pMC1neo (Stratagene) as a probe.
DSB induction
Transient I-SceI expression was induced in clones by electroporation of 5 x 106 cells with 100 μg of circular pCBASce1Tcr as described previously (18). Surviving cells were harvested after 48 h for genomic DNA extraction and plasmid rescue.
Plasmid rescue
Total genomic DNA was first digested with SacI (ATA34) or BglII (ATA67) for plasmid excision from the genome of each clone. Plasmids were circularized by ligation (T4 DNA ligase, New England Biolabs) and digested by I-SceI meganuclease enzyme (New England Biolabs) to enrich for repair events having lost DSB site. Plasmids were electroporated as described previously (18) into DH10B electrocompetent bacteria (Invitrogen) and selection was performed with 50 μg/ml ampicillin. Colonies were individually picked and their plasmids were extracted by modified alkaline lysis miniprep (20).
Integration sites and recombinants analysis
PL1HsSce1 integration sites and DSB repair events were analysed by restriction analysis and sequencing using the Thermo Sequenase Radiolabeled Terminator Cycle Sequencing kit (Amersham). Plasmid integration sites were identified using TK4415R primer (5'-TTAACAGCGTCAACAGCGT-3'). HDF4683 (5'-GCTTACCTACAACTATCTGA-3') and HDR4885 (5'-GGGTTTTTATGGTTTTAGGT-3') primers were used to sequence repaired DSB sites resulting from recombination events. Plasmid integration sites, endogenous LINE-1 partners of HR and genomic NHCR sites were identified with GenBank database (National Center for Biotechnology Information BLAST Human genome software) (http://www.ncbi.nlm.nih.gov).
RESULTS
Experimental approach
To investigate the accessibility of a given genomic locus to the rest of the genome, we induced a DSB in a chromosomal LINE-1 (L1) element, which could then be repaired by NHEJ, NHCR or alternatively by HR using any of the 5 x 106 copies of L1 elements naturally scattered throughout the human genome (21). A tagged L1 element was introduced in the GM05849 ATM-deficient human cell line genome by transfection of the pL1HsSceI plasmid (Figure 1A). Two independent clones, ATA34 and ATA67, were selected for this study because they had integrated a complete copy of the vector at a single site on chromosomes 6q23.3 and 2p22.1, respectively. The position and organization of the genomic site of integration was determined by sequencing the genomic sequences flanking the integrated pL1HsSceI plasmids rescued from ATA34 and ATA67. In the integrated L1 element, the highly conserved 23 bp NheI–BamHI segment has been replaced by the rare I-SceI restriction site, allowing induction of a DSB at that specific genomic site. After repair of the I-SceI site, the pL1HsSceI vector was recovered from the genome by plasmid rescue and all repair events for which the I-SceI site had been lost were analysed. This approach allows the recovery of all types of DSB repair events (Figure 1B–E), resulting from NHEJ, NHCR or HR (both gene conversion and crossing-over).
Mechanism of DSB repair
The majority of repair events recovered from ATA34 and ATA67 resulted from NHEJ (Table 1), the main DSB repair pathway in mammalian cells (18,22,23). However, it should be noted that sister chromatid homologous repair events could not be scored in our assay. We also observed NHCR accounting for 2.3% of ATA34 and 12.3% of ATA67 repair events. HR accounted for 4% of ATA34 and 14.8% of ATA67 repair events. As previously observed (24), gene conversion was by far the most frequent HR repair mechanism, but we also detected three crossing-over/one one-sided invasion events (25), the later also referred to as long track gene conversion (24). Note that we cannot distinguish between crossing-over events and one-sided invasion events since the former could be one-sided invasion events that extended beyond the restriction site used for plasmid rescue. To evaluate the accessibility of ATA34 and ATA67 genomic loci to other genomic sites, we next determined the location of NHCR junction sites and endogenous L1 HR partners.
Table 1. Frequencies of DSB repair mechanisms
Characterization of non-homologous recombination events
Most of the NHEJ repair events resulting from the joining of the broken extremities (Figure 1B) were accompanied by a deletion and/or an insertion of few nucleotides at the DSB site. Recombinant plasmids having replaced the 3' sequences adjacent to the DSB with novel genomic sequences were identified as NHCR (Figure 1C), and chromosomal sites used for repair were identified by sequencing of the genomic junction next to the DSB and comparing these sequences with human genome databases (see Materials and Methods, Table 2). With clone ATA34, two out of the six sites analysed involved a genomic site located on the same chromosomal arm 6q as the integration site of pL1HsSceI, while the four others involved a genomic site from a different chromosome. Interestingly, two events obtained from separate experiments (34-t2a, 34-t2b) involved genomic sites separated by 1 bp on 1q24 .3. In the case of the ATA67 NHCR events, 12 of the 24 sites analysed were located on the same chromosomal arm 2p as the integration site of pL1HsSceI. There was a clear indication of clustering of some of the events around 2p22 and 2p23, including two events (67 t-23 and 67 t-24) that occurred 3 kb apart but 600 kb from the integration site (Figure 2B). The vast majority of the intrachromosomal NHCR sites were 3' of the DSB as could have been expected since 5' sites would have deleted the plasmidic sequences in most cases and would have not been recovered during the plasmid rescue procedure. However, the orientation of the novel chromosomal junction with regard to the integration site was sometimes inverted, which is indicative of an inversion event rather than a simple deletion. The 12 remaining events involved interchromosomal sites with two events occurring in rather close proximity on 14q (67 t-13, 67 t-14).
Table 2. Localization of chromosomal junction sites in NHCR repair events
Figure 2. Mapping of ATA34 (A) and ATA67 (B) integration region. Each HR partner is localized in relation to the integrated pL1HsSceI with their description (name, length, degree of homology and frequency). Other endogenous L1s having overlapping sequences with pL1HsSceI are indicated with their description (length and degree of homology). Arrows indicate the relative orientation of endogenous L1s or NHCR sites in comparison with the integrated pL1HsSceI.
Characterization of homologous recombination events
HR events with endogenous L1 partners were characterized by the reacquisition of the conserved NheI–BamHI L1 segment at the broken I-SceI site (Figure 1D and E). Since endogenous L1 elements vary somewhat in their sequence from one another, L1 sequences acquired by the integrated pL1HsSceI vector through HR were used to try to identify which endogenous L1 element had participated in the repair event (Table 3). The entire gene conversion track was used to identify the genomic L1 partner that had a matching sequence in the human genome database. With clone ATA34, specific L1 partners were identified by BLAST for 15 out of the 17 HR repair events. The remaining two could not be assigned to a single L1 partner. Some L1 partners were preferentially used (34-A, B, C and E). Out of the nine different L1 partners identified for HR repair, eight were located on the same chromosomal arm as the integration site on 6q. The other (34-A) was located on a distinct chromosome (4q24) but was used in three separate HR repair events. Out of the 96 HR repair events analysed for clone ATA67, we could assign 64 events to 9 different L1 endogenous partners (Table 3). Some events were assigned to specific partners on the basis of base changes common to several events (see Supplementary Material). Interestingly, in the case of partner 67-B, we initially did not find a good match by BLAST analysis to a single endogenous L1 partner. However, we recovered a crossing-over event that permitted to identify the genomic location of this specific endogenous L1 partner. It turned out that the L1 element at that site in the GM05849 cell line showed sequence variations compared to the L1 GenBank sequence for that site. Six additional events that had acquired endogenous L1 sequences could not be assigned to a single endogenous L1 partner (multiple matches). The remaining 26 events showed no sequence difference with pL1HsSceI except for the reacquisition of the 23 bp NheI–BamHI segment at the I-SceI site, which clearly necessitated a HR repair event. Thus, with ATA67, 15 different L1 partners were identified, with three of them (67-A, B and D) accounting for at least 50% of the total HR repair events. As was the case for clone ATA34, most of the identified L1 partners used for HR repair were located on the same chromosomal arm 2p. Three partners were on a different chromosome with two of them on chromosome 3q, including the one that was used on four separate occasions.
Table 3. Description of HR endogenous L1 partners
Length and homology of the endogenous L1 partner used for HR repair
Endogenous L1 partners used for HR repair with ATA34 and ATA67 shared 93–98% of homology with the L1 sequences present in the integrated pL1HsSceI vector. Their length varied from 354 bp to above the length of homology of 2586 bp present in the vector (Table 3). It has been reported that the degree and length of homology greatly influence the choice of a partner for homologous recombination (26–28). Indeed, we did not observe the use of a L1 partner with <93% homology, and most of the frequently used partners had homologies of 97% and above. However, less homologous partners were also used quite frequently while potential partners with a higher degree of homology that were in the vicinity of these preferential partners were not used at all. The same ambivalence was seen for the length of chosen versus ignored L1 partners. Thus, although the criteria of length and degree of homology could have played a role, they were certainly not the sole determinant in the preferential choice of specific L1 partners.
The relative position of the intrachromosomal L1 partners used for DSB repair
The distribution of all endogenous L1 elements present around the integration sites of ATA34 and ATA67 was analysed in detail (Figure 2). Interestingly, in the case of ATA34, a L1 element of 97% homology and 1280 bp in length located 123 kb away from the integration site was not used for repair, whereas an L1 element (34-C) of less homology (96%) and length 354 bp located 312 kb from the integration site was used three times (Figure 2A). In the case of ATA67, 750 kb separates the integration site from the first cluster of L1 repair partners (67-A, B, C and E) that were collectively used 45 times (Figure 2B). There are no other L1 elements more homologous and closer than these partners on this side of the integration site. Interestingly, 67-E, which is 95% homologous and 2017 bp in length, was used once for HR repair, whereas 67-A with similar homology and length, located 11 kb further away, was used 13 times.
Figure 3 presents the relative position of all genomic intrachromosomal sites (HR and NHCR events) used for DSB repair, located on 6q for ATA34 events or 2p for ATA67 events. For ATA34, there is one major cluster of partners around the integration site and partners are located equally on both sides of the integration site (Figure 3A). For ATA67, a major cluster was found spreading 2 Mb on one side of the integration site. Another important cluster was found 12 Mb away towards the telomere (Figure 3B). Interestingly, both clusters include HR and NHCR repair events. Contrary to ATA34, the vast majority of the partners were located on the same side (telomeric) of the integration site. Thus, although partners proximal to the integration site were certainly favoured, other partners located quite distally were also used preferentially for repair, whereas potential partners located in between were ignored.
Figure 3. Localization of intrachromosomal partners for HR and NHCR repair events in clones ATA34 (A) and ATA67 (B). Distances from integration sites of the pL1HsSceI vector are indicated in parenthesis for each site. When an endogenous L1 partner was used more than once, the frequency is indicated at the right.
DISCUSSION
Understanding the principles that govern genomic organization is viewed as essential to understand genomic function (29). We have investigated the accessibility of given genomic loci to other loci in the genome of human cells during DSB repair. We have used in our assay an ATM-deficient cell line because of its high incidence of genomic homologous recombination (15) that made it possible to generate enough HR events for their comparison. This may have introduced some bias in the relative frequencies of repair events. The assay permitted the recovery of all types of repair products including NHEJ, NHCR and HR products. However, it should be noted that specific sub-categories of recombination events could not be recovered or scored in our assay. Indeed, we could not score for sister chromatid homologous repair events. Also, any event that deleted the plasmidic sequences could not be recovered. NHEJ was by far the most frequent DSB repair mechanism observed in our assay and this is possibly an underestimate considering that NHEJ events that reconstituted the I-SceI site could not be identified. For the first time to our knowledge, NHCR and HR were compared in direct competition for genomic sites that were basically side by side throughout the genome, albeit with the proviso mentioned above on the events that could not be scored. The results showed that they occurred at the same frequency for a given integration site and often involving partners from the same genomic location. Interestingly, there was a significant variation in NHCR and HR frequency relative to NHEJ between the two integration sites studied. This could suggest that the choice of repair mechanism, local versus distal, is influenced by the genomic site where the DSB occur, possibly as a consequence of relative accessibility to distal genomic sites.
In the case of the HR repair events, the potential L1 targets were dispersed all over the genome. Since the vast majority of HR events involved gene conversion without chromosomal rearrangement, there was no evident counter selection against certain chromosomal interactions that would result in non-functional chromosomal structures. The degree of homology of the targeted L1 elements, below a certain threshold, appeared to be a limiting factor in partner choice, but that still left the possibility of interacting with thousands of loci dispersed throughout the genome. Out of the 79 HR events that could be assigned to a specific L1 partner for ATA67 and ATA34, 70 were intrachromosomal and nine were interchromosomal, thus indicating about a 10-fold preference for intra versus inter chromosomal sites. Clearly, however, this was not due to a simple question of linear proximity on the same chromosome. Indeed, the immediate vicinity, within a couple of 100 kb, appears to be of the highest accessibility, in the case of ATA67 accounting for 70% of the HR events. But another partner located more than 10 Mb away accounts for 20% of HR events, bypassing numerous potential L1 partners closer in terms of chromosomal linearity. Even for partners of similar size and degree of homology located 10 kb apart (67A and 67E), there can be a difference of more than 10-fold in usage. Interestingly, the other chromosomal arm appears to be less accessible than other chromosomes. This lack of accessibility between the two arms of the same chromosome supports previous observations that, within a given territory, chromosomal arms are separated in distinct domains (30). The accessibility to other chromosomes also indicates specificity with one particular interchromosomal genomic site accounting for 6% (67-N) of HR events in ATA67 and 20% (34-A) in ATA34.
The fact that NHCR and HR events cluster to the same genomic sites suggests that the specificity in accessibility is not due to the repair mechanism but rather to a common situation in both processes. In the case of NHCR repair events, the choice of the partner site presumably involves the occurrence of a second DSB at that site. We did not find evidence of the presence of a pseudo-I-SceI site or fragile site in the vicinity of the breakpoints to explain the occurrence of a presumed second DSB. In the case of HR repair events, the choice of the partner involves the presence of a homologous element at that site. The coincidence of both types of events in close vicinity would suggest that these sites were in close physical proximity to the integration site to start with. This is supported by the observation of physical proximity for genomic sites involved in NHCR events (4–6,12) and the choice of partner in HR events being influenced by chromosomal topology (31). The chromatin structure could also influence the accessibility of a donor sequence. If physical proximity is the reason for the choice of repair partners, our results suggest that chromosomal territories have a specific structure that directs accessibility of a given genomic locus to preferential genomic sites. The fact that the same genomic sites used for repair are isolated repeatedly from a given cell population and also from separate cell passages indicates that the organization of chromosomal territories of interphase nuclei in a human cell population is conserved.
SUPPLEMENTARY MATERIAL
Supplementary Material is available at NAR Online.
ACKNOWLEDGEMENTS
We thank M. Jasin for the generous gift of pCBASce. We also thank H. Wurtele and K. Little for their critical reading of the manuscript and their comments. This work was supported by a grant from the Canadian Institutes of Health Research to P.C.
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