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Nucleic Acids Research 2004 32(19):5916-5927; doi:10.1093/nar/gkh926
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Published online 4 November 2004

Nucleic Acids Research, Vol. 32 No. 19 © Oxford University Press 2004; all rights reserved

Cis-acting regulatory sequences promote high-frequency gene conversion between repeated sequences in mammalian cells

Steven J. Raynard and Mark D. Baker*

Department of Molecular Biology and Genetics, College of Biological Science, University of Guelph, Guelph, Ontario, Canada N1G 2W1

* To whom correspondence should be addressed. Tel: +1 519 824 4120; Ext. 54788; Fax: +1 519 837 2075; Email: mdbaker{at}uoguelph.ca

Received August 6, 2004; Revised and Accepted October 19, 2004


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In mammalian cells, little is known about the nature of recombination-prone regions of the genome. Previously, we reported that the immunoglobulin heavy chain (IgH) µ locus behaved as a hotspot for mitotic, intrachromosomal gene conversion (GC) between repeated µ constant (Cµ) regions in mouse hybridoma cells. To investigate whether elements within the µ gene regulatory region were required for hotspot activity, gene targeting was used to delete a 9.1 kb segment encompassing the µ gene promoter (Pµ), enhancer (Eµ) and switch region (Sµ) from the locus. In these cell lines, GC between the Cµ repeats was significantly reduced, indicating that this ‘recombination-enhancing sequence’ (RES) is necessary for GC hotspot activity at the IgH locus. Importantly, the RES fragment stimulated GC when appended to the same Cµ repeats integrated at ectopic genomic sites. We also show that deletion of Eµ and flanking matrix attachment regions (MARs) from the RES abolishes GC hotspot activity at the IgH locus. However, no stimulation of ectopic GC was observed with the Eµ/MARs fragment alone. Finally, we provide evidence that no correlation exists between the level of transcription and GC promoted by the RES. We suggest a model whereby Eµ/MARS enhances mitotic GC at the endogenous IgH µ locus by effecting chromatin modifications in adjacent DNA.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Homologous recombination (HR) is the process by which genetic information is exchanged between two similar or identical DNA duplexes. In eukaryotes, recombination plays an important role in generating genetic diversity in meiosis, and in mitotic cells, represents a pathway for the repair of DNA damage. Nevertheless, these benefits are associated with the risk of aberrant rearrangements due to recombination between the myriad of repeated sequences in the eukaryotic genome. Germane to this risk is the knowledge that the frequency of recombination is not uniform, in that some loci exhibit relatively high frequencies of recombination (hotspots), while others exhibit relatively low frequencies of recombination (coldspots). This fact has important implications for the maintenance of genomic stability.

Relatively little is known about the nature of mitotic recombination hotspots in mammalian cells. However, since the primary role of HR in somatic cells is as a DNA repair mechanism, it stands to reason that regions of the genome that are prone to DNA damage would be active in HR. Current evidence suggests that errors that occur in the course of normal DNA metabolism, such as transcription (1), and replication (2), are a major source of recombinogenic lesions. It follows then, that DNA regulatory elements could act as recombination hotspots as an indirect by-product of their normal biological function. For example, the yeast recombination hotspot, HOT1, contains an initiation site and enhancer of transcription by RNA polymerase I (3). The stimulatory activity of HOT1 correlates with its capacity to promote transcription through the recombining sequences (4).

Other DNA motifs are thought to stimulate genomic rearrangement through their effects on the secondary structure of DNA. Regions of alternating purines and pyrimidines that can adopt the Z-DNA conformation (5–7), trinucleotide repeats (8) and inverted (palindromic) repeats that can extrude and form hairpin or cruciform structures (9,10) are all prone to rearrangement in eukaryotic cells.

Indirect evidence suggests higher order chromatin structure might also influence HR rates. It is plausible that a change in chromatin structure facilitates the access of recombination proteins, or possibly, leads to hypersensitivity to DNA-damaging agents. Hyper-recombination phenotypes were reported for certain yeast mutants defective in proteins involved in chromatin-mediated repression of transcription (11). A correlation between Sir2-mediated DNA silencing and a more ‘closed’ chromatin structure was shown by Fritze et al. (12). Since DNA silencing also correlates with reduced recombination (13), it was suggested that ‘closed’ chromatin has a double effect in repressing both transcription and recombination.

Our laboratory previously reported that the mouse immunoglobulin heavy-chain (IgH) µ locus acts as a hotspot for spontaneous mitotic gene conversion (GC) (14). The assay system monitors intrachromosomal GC events between closely linked direct repeats of the IgH µ gene constant (Cµ) region. In this paper, we report the identification of a 9.1 kb segment of DNA encompassing the IgH µ gene regulatory region, which stimulates GC between adjacent Cµ repeats both at the endogenous IgH locus, and when appended to the same Cµ repeats stably integrated at ectopic genomic sites. We also show that deletion of the IgH major intronic enhancer (Eµ) and flanking matrix attachment regions (MARs) from the regulatory region abolishes GC hotspot activity at the chromosomal IgH µ locus. However, the Eµ/MARs fragment by itself does not stimulate recombination at ectopic genomic sites. Finally, we provide evidence that there is no correlation between the level of transcription and the level of GC promoted by the IgH µ gene regulatory region. We suggest a model whereby the endogenous Eµ/MARs effects chromatin modifications in adjacent chromosomal regions that enhance spontaneous mitotic GC.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Hybridoma cell lines
The hybridoma cell line, Sp6/HL, bears a single copy of the trinitrophenyl (TNP)-specific chromosomal IgH µ chain gene and makes normal, cytolytic TNP-specific IgM ({kappa}-chain) (15,16). The following Sp6/HL-derived hybridoma cell lines were used in this study. Hybridoma igm10 is a mutant that has lost the TNP-specific chromosomal µ gene (16). Hybridoma Im/RCµ2A2 was constructed by gene targeting in the Cµ region of the haploid, TNP-specific chromosomal µ gene, as described previously (17). The conditions for hybridoma cell growth are described elsewhere (15,16).

DNA transfer
Linear plasmid vector DNA (8.7 pmol) was introduced into 2 x 107 hybridoma cells by electroporation as described previously (17). Individual transformants were recovered by limited dilution cloning procedures as described in (18,19).

Quantitative PCR (qPCR) assay
The conditions used for qPCR and details of the construction of the vector control have been reported previously (14).

DNA analysis
Preparation of genomic DNA was performed by the method of Gross-Bellard et al. (20) with the exception of DNA samples used in the qPCR assay, which were prepared with the QIAamp DNA mini kit (QIAGEN). For Southern analysis, restriction enzymes were purchased from New England Biolabs Inc. (Mississauga, ON, Canada) and used in accordance with the manufacturer's specification. Gel electrophoresis, transfer of DNA onto nitrocellulose membrane, 32P-labeled probe preparation and hybridization were all performed according to standard procedures (21). Primers used in this study were synthesized at Sigma (Oakville, ON, Canada), and their sequences have been reported previously (14). New primers used in this study are as follows: 23819, 5'-CAT CCT CCT CCT CAT CAT CGT CAT-3'; CµXbaRI, 5'-GAA TCT GTC TTC TTG CCT CCT GTC-3'; 24226, 5'-GCT GTG TAG AAG TAC TCG CCG ATA-3'.

RNA analysis
Hybridoma cells were grown to a density of 3–5 x 105 cells/ml. Total RNA was isolated using Trizol (Gibco) in accordance with the manufacturer's specification, and assayed by dot blotting for hybridization to a Cµ fragment (Probe F) and a ß-actin probe. The procedures of Baumann et al. (22) were adopted for RNA dot blotting, while hybridizations were performed according to standard methods (21).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Role of the IgH µ gene regulatory region in spontaneous HR hotspot activity
In this study, we investigated whether cis-acting DNA elements within the IgH µ gene regulatory region promote intrachromosomal GC. The haploid IgH µ locus in the hybridoma cell line, Im/RCµ2A2, bears a pair of Cµ region heteroalleles that exhibit high-frequency intrachromosomal GC (14). The upstream (5') Cµ region is derived from the wild-type Sp6/HL hybridoma and resides in its normal position to be expressed from the TNP-specific VH region. Vector pSV2neo sequences separate the 5' wild-type Cµ region from the downstream (3') Cµ region, which bears a 2 bp deletion in exon Cµ3 (referred to as the mutant igm482 Cµ3 region) (15,16).

The 9.6 kb omega ({Omega})-form vector, pVHCµKO, was used in the targeted modification of the Im/RCµ2A2 µ gene (Figure 1A). The pVHCµKO backbone consists of an enhancer-trap derivative of pSV2hyg in which the 372 bp NsiI/NdeI fragment encompassing the simian virus 40 (SV40) early region enhancer residing upstream of the hygromycin phosphotransferase (hyg) gene was deleted. Although not relevant to this study, the Herpes Simplex Virus-1 (HSV-1) thymidine kinase (tk) gene is also present. In pVHCµKO, the flanking arms consist of a 2.3 kb EcoRI/HpaI segment with homology 5' of the µ gene promoter (Pµ) and a 1.6 kb XbaI/MfeI Cµ region fragment, positioned to the left and right of pSV2hyg, respectively. Flanking arm alignment with isogenic regions of the Im/RCµ2A2 chromosomal µ gene is indicated by the dashed lines in Figure 1A.



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  		Figure 1. Construction of 2A2{Delta}VH cell lines. (A) The structure of the haploid, chromosomal µ locus in the recipient murine hybridoma cell line, Im/RCµ2A2, is presented along with the 9.7 kb replacement vector, pVHCµKO. Regions of alignment between the homologous arms of the vector and Im/RCµ2A2 chromosomal µ locus are indicated by the dashed lines. (B) The structure of the recombinant µ locus following targeted gene replacement by the vector pVHCµKO. In both panels, the mutant igm482 Cµ3 exon is indicated by an open box, while wild-type Cµ exons are indicated by filled boxes. Although not labeled, matrix attachment regions (MARs) are indicated by filled diamonds. The diagnostic fragment sizes generated following digestion with the indicated restriction enzymes are presented. DNA probe fragment F is an 870 bp XbaI/BamHI fragment while probe tk is a 1.3 kb fragment from the HSV-1 thymidine kinase (tk) gene. VH, TNP-specific µ-heavy-chain variable region; Pµ, µ-gene promoter; Eµ, major intronic enhancer; Sµ, µ-gene switch region; Cµ, µ-gene constant region; neo, neomycin phosphotransferase gene; tk, HSV-1 thymidine kinase gene; hyg, hygromycin resistance gene; B, BamHI; E, EcoRI; H, HpaI; M, MfeI; Sc, ScaI; X, XbaI. The figure is not drawn to scale.

 
Targeted gene replacement by pVHCµKO deletes all coding and regulatory DNA sequences required for expression of the TNP-specific µ heavy chain gene in Im/RCµ2A2 (Figure 1B) (23). Thus, following electroporation, culture supernatant from a total of 270 hygR transformants was tested for the presence of TNP-specific IgM, by complement-dependent lysis of TNP-coupled sheep erythrocytes in spot tests (15). The structure of the IgH µ locus in six IgM–, hygR transformants was examined by Southern analysis to identify those in which targeted gene replacement had occurred. The fragment sizes of the recipient µ locus in Im/RCµ2A2 (Figure 1A), and of correctly targeted transformants (Figure 1B), following digestion with BamHI and hybridization with Cµ-specific probe F are shown. Two independent hybridoma cell lines, 2A2{Delta}VH-82 and 2A2{Delta}VH-236 were identified as having the correctly targeted structure (data not shown).

For determination of GC frequencies, a quantitative PCR (qPCR) assay was used, as described previously (14). In brief, the assay makes use of four primers. Primers 1 and 2 bind upstream of the 5' and 3' Cµ regions, respectively. Primers 3 and 4 are specific for the wild-type and mutant igm482 Cµ3 regions, respectively. Two related hybridomas were used to standardize the assay, Im/RCµ3/1-7 (abbreviated 3/1-7) and Im/RCµD7-7 (abbreviated D7-7) (Figure 2A). In hybridoma cell line, 3/l-7, both the 5' and 3' Cµ regions are wild-type (designated, double wild-type), whereas, in hybridoma cell line, D7-7 they are both mutant igm482 Cµ3 regions (designated double mutant). Figure 2A indicates the primer binding sites and product sizes generated by PCR amplification of 3/1-7 and D7-7. To circumvent the problem of variability in efficiency of amplification, 10–9 pmol of a heterologous vector control, sharing only the primer binding sites with the target sequence, was included in each PCR reaction. Amplification of the vector generates a 1.3 kb product (Figure 2A). Genomic mixtures of 3/1-7 and D7-7 were prepared in ratios of 1:50, 1:200, 1:500 and 1:1000, and amplified using primer pair 2/3 for generation of the wild-type, 1.9 kb 3' Cµ region product (Figure 2B, lanes 2–5). Following gel electrophoresis, quantitation of the EtBr staining in the 1.9 kb target band divided by that in the 1.3 kb vector control band yields a ratio that, when plotted against the wild-type Cµ region copy number, generates a standard curve (Figure 2B, inset). In a similar manner, the qPCR assay was standardized with the primer pairs 1/3, 1/4 and 2/4 to detect the wild-type 5' Cµ, mutant 5' Cµ and mutant 3' Cµ regions, respectively.



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  		Figure 2. qPCR assay to measure gene conversion between the Cµ region repeats. (A) Hybridoma 3/1-7 has the wild-type sequences in both the 5' and 3' Cµ regions (double wild-type), while hybridoma D7-7 bears the mutant igm482 Cµ3 exon in both the 5' and 3' Cµ regions (double mutant). The PCR primers and the sizes of the amplified products they produce are indicated. Primer 1 binds upstream of the 5' Cµ region and outside the region of homology in the 3' Cµ region. Primer 2 binds within pSV2neo vector sequences upstream of the 3' Cµ region. Primers 3 and 4 bind specifically to the complementary strand of the wild-type and mutant igm482 Cµ3 exons, respectively. The vector control contains the primer 1 and 2 binding sites and primer 3 and 4 binding sites on opposite strands flanking a segment of the thymidine kinase (tk) gene. (B) Lanes 2–5, standard solutions of genomic DNA consisting of 3/1-7 and D7-7 prepared in ratios of 1:50, 1:200, 1:500, 1:1000 along with a constant amount of vector control (10–9 pmol) were amplified using primer pair 2/3 for generation of the 1.9-kb wild-type 3' Cµ region product and 1.3-kb vector control product. DNA from the double mutant, D7-7 cell line, was included as a negative control (lane 6). Quantitation of the EtBr staining in the target band divided by that in the vector control band yielded the standard curve (inset). Lanes 7–11, representative samples from 2A2{Delta}VH-82 subclone 3 subculture wells 1–5, amplified with primer pair 2/3 to detect gene conversion in the 3' Cµ region to the wild-type sequence. Positive signals are present in subcultures 2 and 4. Abbreviations are the same as in Figure 1. The figure is not drawn to scale.

 
To quantify the GC frequency in the cell lines, replicate genomic DNA samples, along with a constant amount of vector control, were amplified using primer pair 2/3. Following gel electrophoresis, and quantitation of the EtBr staining, the resulting target:vector ratio was used to determine the GC frequency from the standard curve. In cases where the GC frequency was below the sensitivity of the assay (<0.001), the culture was distributed at a density of 500 cells/well in 24-well plates. Following cell growth, genomic DNA was prepared from each culture well and qPCR analysis was performed on the separate DNA preparations using primer pair 2/3. As an example, Figure 2B (lanes 7–11) presents five representative genomic DNA samples for hybridoma 2A2{Delta}VH-82 subclone 3. From the fraction of negative wells in the qPCR assay (as examples, lanes numbered 1, 3 and 5 in Figure 2B) and the Poisson distribution, the mean GC frequency between the Cµ repeats was calculated.

Similar GC frequencies were detected previously for the 5' and 3' Cµ regions in Im/RCµ2A2 (14), and therefore, in cell lines, 2A2{Delta}VH-82 and 2A2{Delta}VH-236, the frequency of GC of the recipient, 3' mutant Cµ region by the donor, 5' wild-type Cµ region was determined. Three subclone cultures of 2A2{Delta}VH-82 and two subclone cultures of 2A2{Delta}VH-236 were each started from a single cell. Southern blot and PCR analysis revealed that the µ gene structure in each subclone was identical to that in the parent cell line (data not shown). Each subclone was grown for ~25 generations in medium supplemented with G418 (0.6 mg/ml) and hygromycin (0.6 mg/ml), and then assayed by qPCR using primer pair 2/3. This analysis revealed that the mean GC frequency between the Cµ repeats in 2A2{Delta}VH-82 and 2A2{Delta}VH-236 was ~0.2 x 10–3 recombinants/cell (Table 1). In comparison, GC between the same repeats integrated at the wild-type µ locus of hybridoma Im/RCµ2A2 was previously shown to occur at a frequency of 6.1 x 10–3 recombinants/cell (14). The procedures and conditions used for cell growth, and determination of GC frequencies were identical for both studies (14). The ~31-fold difference in the GC frequencies is highly significant (t-test of log-transformed data; P = 0.0002), suggesting that the IgH µ gene regulatory region is required for GC hotspot activity.


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  		Table 1. Frequency of gene conversion between Cµ region repeats

 
The IgH µ gene regulatory region stimulates intrachromosomal gene conversion between Cµ region repeats at ectopic sites in the hybridoma genome
Since deletion of the regulatory region from the endogenous IgH locus reduced the frequency of GC between adjacent Cµ repeats, it was of interest to determine whether the region stimulated GC when appended to the same Cµ repeats integrated outside the IgH locus. For these studies, we used a derivative of the vector pCµ-repeat described previously (Figure 3A) (14). It bears 4.3 kb segments encoding the mutant igm482 and wild-type Cµ regions flanking a pSV2neo vector backbone. The 10.8 kb EcoRI/MfeI fragment encompassing the VH region and the VH–Cµ intron (Figure 1A) was obtained from the cloned wild-type Sp6 µ gene (24), and inserted into pCµ-repeat in its correct position and orientation, upstream of the mutant igm482 5' Cµ region to create the vector, pVHCµRep (Figure 3B). The µ gene structure in pVHCµRep is isogenic to that in Im/RCµ2A2, with the exception of an ~2.8 kb deletion in the µ gene switch (Sµ) region that occurred during cloning of the genomic DNA in Escherichia coli, leaving an ~0.4 kb residual Sµ segment (24). The vector pVHCµRep was linearized at the unique NotI site and transferred by electroporation into the Sp6-derived hybridoma cell line igm10, which lacks the endogenous chromosomal µ gene (16). A total of 96 individual G418R transformants were isolated and screened by PCR analysis in conjunction with Southern blotting (Figure 3B) to determine the structure and copy number of the integrated vector. Five cell lines, designated VHCµRep-10, VHCµRep-12, VHCµRep-15, VHCµRep-19 and VHCµRep-21, were identified as having a single copy of the integrated vector with the structure shown in Figure 3B (data not shown). Each transformant was expanded from a single cell and grown for ~25 generations under G418 selection. Genomic DNA was prepared from each culture, and qPCR utilizing primer pair 1/3 was performed to measure the frequency of GC in the 5' Cµ region. It should be noted that randomly integrated constructs are susceptible to position effects, which could influence GC rates. As an unusually high or low frequency of GC in one of the cell lines could have a misleading effect on the mean GC frequency (due to the low sample size), we felt that comparison of median values was more appropriate for the ectopic data. When compared to transformants containing just the Cµ repeats integrated at ectopic genomic sites (R/CµRepeat cell lines in Table 1) (14), the frequency of GC was stimulated up to as much as 30-fold in the VHCµRep transformants (Table 1) (Mann–Whitney test, P = 0.036).



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  		Figure 3. Constructs integrated at ectopic sites in the R/CµRepeat and VHCµRep transformants. The structures of the linearized vectors transfected into hybridoma igm10, which lacks the endogenous chromosomal µ gene (16), are shown. (A) The vector pCµ-repeat bears the 4.3-kb Cµ region repeats inserted into a pSV2neo vector backbone in the tandem orientation (14). The upstream (5') Cµ region bears the 2-bp igm482 Cµ3 deletion (open box), but is otherwise isogenic with the downstream (3') Cµ region. (B) The vector pVHCµRep contains the EcoRI/MfeI VH-region fragment from the hybridoma Sp6/HL inserted into the MfeI site of pCµ-repeat in its correct position and orientation with respect to the Cµ regions. As described in the text, a residual ~0.4 segment remains of the Sµ region (denoted Sµ*) (24). The diagnostic fragment sizes generated following digestion with EcoRI and SpeI, and hybridization with probe F (Figure 1) and with probe Eµ, a 992-bp XbaI fragment, are presented. Southern blotting using both single cutters and noncutters was used to determine the copy number of the integrated vector (data not shown). N, NotI; Sp, SpeI; other abbreviations are the same as in Figure 1. The figure is not drawn to scale.

 
The µ gene switch region, Sµ, is not required for high-frequency gene conversion at the IgH locus
The frequency of GC promoted by the IgH µ gene regulatory region at ectopic sites was only ~20–30% of the mean value measured in Im/RCµ2A2 (Table 1). This might suggest a role for Sµ in stimulating GC, since as indicated above, it was largely deleted from the construct used in the ectopic studies. To investigate this, the frequency of GC was determined in two related hybridomas, Emut1-Sµ+ and Emut1-Sµ–, that were isolated in a previous study (14). As shown in Figure 4, both cell lines contain Cµ repeats positioned at the IgH µ locus: the 3' Cµ region is wild type, while the 5' Cµ region is mutant as a consequence of a 4 bp insertion in Cµ exon 1. Cell line Emut1-Sµ+ bears the chromosomal Sµ region upstream of both the 5' and 3' Cµ regions, whereas Emut1-Sµ– contains the ~2.8 kb Sµ deletion upstream of both Cµ regions (Figure 4). The Sµ deletion does not affect µ gene expression (24).



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  		Figure 4. Structure of the haploid, chromosomal IgH µ gene in the Emut1-Sµ+ and Emut1-Sµ– hybridoma cell lines. In each cell line, the mutant, recipient 5' Cµ region bears a BspHI marker in Cµ exon 1 and is separated from the wild-type, donor 3' Cµ region by the integrated pSV2neo vector sequences. In hybridoma Emut1-Sµ+, the endogenous µ gene switch region (denoted Sµ) is present upstream of both the 5' and 3' Cµ regions. Whereas, hybridoma Emut1-Sµ– bears an ~2.8 kb Sµ deletion (denoted Sµ*) upstream of both Cµ regions. The figure is not drawn to scale.

 
The frameshift mutation in the expressed 5' Cµ region causes Emut1-Sµ+ and Emut1-Sµ– to produce a truncated µ heavy chain that cannot form pentameric IgM and activate complement-dependent lysis of TNP-coupled sheep erythrocytes (14). During growth under G418-selective conditions, conversion of the recipient, 5' mutant Cµ region by the donor, 3' wild-type Cµ region can restore normal, TNP-specific IgM production in the hybridoma cells, allowing them to be detected as plaque-forming cells (PFC) in a TNP-specific plaque assay (17). Previously, we showed that there was no difference in the frequency of GC of the 4 bp mutation when inserted at different sites spanning the Cµ region (14). Furthermore, the frequency of GC of the 4 bp insertion was similar to that measured for the 2 bp deletion in Im/RCµ2A2 (14). Three subclone cultures of the Emut1-Sµ+ and Emut1-Sµ– mutant hybridoma cell lines were started from a single cell. Southern analysis of each subclone revealed the same µ gene structure in the subclones and parental cultures (data not shown). Each subclone culture was grown for ~24 generations and then subjected to the TNP-specific plaque assay. As shown in Table 1, the mean frequency of generating TNP-specific PFC in each cell line was not significantly different (t-test, P = 0.30). This suggests that the Sµ region is not required for high-frequency intrachromosomal GC.

The IgH major intronic enhancer, Eµ, is required for stimulation of gene conversion by the IgHµ gene regulatory region
To determine whether the major intronic enhancer (Eµ) and/or the flanking matrix attachment regions (MARs) play a role in HR hotspot activity, a ‘hit-and-run’ gene targeting technique (25,26) was used to delete these elements from the endogenous IgH µ locus in the hybridoma, Im/RCµ2A2. The ‘hit’ step takes advantage of the 10.8 kb enhancer-trap, insertion vector pVH{Delta}Eµ (Figure 5A). pVH{Delta}Eµ contains the 8.9 kb XbaI fragment encompassing the VH region exon and VH–Cµ intron, inserted into the enhancerless pSV2hyg backbone bearing the HSV-1 tk gene. The region of homology was modified by deleting the 992 bp XbaI Eµ/MARs-containing fragment. The vector was linearized at the unique AfeI site, which provides 3.0 and 2.2 kb of homology on the 5' and 3' sides of the cut site, respectively, and transferred by electroporation into Im/RCµ2A2 (17). A total of 446 independent hygR transformants were isolated, 106 of which were screened by PCR using primers 23819 and CµXbRI for the 9.5 kb product that identifies the endogenous, chromosomal IgH region (Figure 5A). Southern analysis was performed on five cell lines from which the 9.5 kb PCR product was not amplified to identify those in which a single copy of the vector had correctly integrated at the IgH locus. The fragment sizes of the recipient µ locus of cell line Im/RCµ2A2 (Figure 5A), and of correctly targeted transformants (Figure 5B), following digestion with BamHI and hybridization with Probe N, are shown. Two hybridoma cell lines, Im/2VHRCµ2A2-17 and Im/2VHRCµ2A2-26 were identified as having the correctly targeted structure (data not shown).



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  		Figure 5. Construction of 2A2Eµ+ and 2A2Eµ– cell lines by ‘Hit’ and ‘Run’ gene targeting at the chromosomal µ locus. (A) The ‘hit’ step involves targeted integration of the enhancer-trap insertion vector pVH{Delta}Eµ into the chromosomal µ gene of the hybridoma Im/RCµ2A2. pVH{Delta}Eµ contains a cloned segment of theSp6/HL µ gene regulatory region from which the 992 bp Eµ/MARs-containing XbaI fragment (indicated by an inverted ‘V’) was deleted. Regions of alignment between the vector and the Im/RCµ2A2 chromosomal µ locus are indicated by the dashed lines. (B) The structure of the Im/RCµ2A2 chromosomal µ gene following targeted integration of a single copy of the pVH{Delta}Eµ vector. (C) The ‘run’ step involves excision of the integrated vector and one copy of homologous DNA by intrachromosomal HR, an event that exploits the ability to select against the HSV-1 tk gene with gancyclovir (Ganc). Intrachromosomal reciprocal crossover that occurs in the region of homology 5' or 3' of the Eµ/MARs deletion generates the µ gene structure depicted in (D) and (E), respectively. The diagnostic fragment sizes generated following digestion with BamHI are presented. DNA probe N consists of adjacent 475 and 495 bp NheI fragments. The positions of primers 23819 and CµXbRI are indicated. Af, AfeI; other abbreviations are the same as in Figure 1. The figure is not drawn to scale.

 
The ‘run’ step involves excision of the integrated vector by intrachromosomal HR between the duplicated region of homology that removes the integrated vector, hyg and tk genes along with one copy of homologous DNA (Figure 5C). Duplicate subcultures of one cell line (Im/2VHRCµ2A2-17) were plated in media lacking hygromycin to allow for growth of hygS hybridomas in which vector excision had occurred. Following growth, each subculture was distributed at low cell density in 480 individual culture wells in media supplemented with gancyclovir (Ganc) to select against the tk gene (25). A total of 71 and 141 GancR colonies were recovered from the duplicate subcultures, respectively. As shown in Figure 5D and E, and outlined in the figure legend, multiple IgH µ gene structures are possible following vector excision depending on the location of the crossover. Genomic DNA was prepared from the GancR colonies and screened by PCR using primer pair 23819–CµXbRI (Figure 5A) (data not shown). From each of the duplicate subcultures, two GancR cell lines that contained the 8.5 kb PCR product diagnostic of the µ gene excision product that is deleted for Eµ/MARs (Figure 5D) (designated, 2A2Eµ–#1–5, 2A2Eµ–#1–6, 2A2Eµ–#2–4 and 2A2Eµ–#2–10), and two GancR cell lines that contained the 9.5 kb PCR product diagnostic of the normal µ gene locus (Figure 5E) (designated, 2A2Eµ+#1–4, 2A2Eµ+#1–8, 2A2Eµ+#2–16 and 2A2Eµ+#2–20) were isolated. Southern analysis using BamHI and Probe N (Figure 5D and E) confirmed the IgH µ gene structures in the eight cell lines (data not shown).

For determination of GC frequencies, each cell line was expanded from a single cell for ~25 generations under G418 selection. Genomic DNA was prepared from each culture, and qPCR utilizing primer pair 1/4 was performed to measure the frequency of GC in the 5' Cµ region. This analysis revealed a mean GC frequency of <0.11 x 10–3 recombinants/cell in the Eµ/MARs deleted cell lines (Table 1). This value is 30-fold lower than the mean GC frequency measured in the cell lines with an intact µ gene regulatory region (3.4 x 10–3 recombinants/cell) (Table 1) (t-test of log-transformed data; P = 4 x 10–6). This suggests a role for the Eµ/MARs fragment in promoting high-frequency GC between the Cµ regions at the IgH µ locus.

The Eµ/MARs segment alone is not sufficient to stimulate gene conversion between Cµ region repeats at ectopic sites in the hybridoma genome
Since the DNA fragment containing the entire IgH µ gene regulatory region was able to stimulate GC between Cµ region repeats at ectopic sites in the genome, it was of interest to determine whether the Eµ/MARs fragment alone also stimulated GC outside the IgH µ locus. To examine this, the 992 bp XbaI fragment containing Eµ/MARs was inserted into pCµ-repeat, in its correct orientation, upstream of the mutant 5' Cµ region (Figure 6). The vector was linearized at the unique NotI site and transferred by electroporation into the hybridoma, igm10 (16). A total of 88 individual G418R transformants were isolated and screened by PCR analysis in conjunction with Southern blotting using Cµ probe F (Figure 6) to determine the structure and copy number of the integrated vector (data not shown). From this screening, five cell lines were recovered that contained a single, intact vector integrated in the genome, designated EµCµRep-17, EµCµRep-46, EµCµRep-61, EµCµRep-75 and EµCµRep-81. Each of the transformants was expanded from a single cell and grown for ~25 generations under G418 selection. Genomic DNA was prepared from each culture, and qPCR analysis utilizing primer pair 1/3 was performed to measure the frequency of GC in the 5' Cµ region. The median GC frequency was 0.10 x 10–3 recombinants/cell, which is similar to the median GC frequency of 0.07 x 10–3 recombinants/cell measured in the R/CµRepeat cell lines (Table 1). Thus, there was no significant difference in the frequency of GC between Cµ repeats at ectopic genomic sites, with or without the Eµ/MARs fragment (Mann–Whitney test, P = 0.24).



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  		Figure 6. Influence of Eµ on gene conversion between ectopic Cµ repeats. The structure of hybridoma cell lines bearing the linearized vector, pEµCµRep, as determined by Southern blot and PCR analysis is shown. The vector was derived from pCµ-repeat (Figure 3A), by inserting the 995-bp Eµ/MARs-containing XbaI fragment into the MfeI site, in its correct orientation 5' of the Cµ regions. The diagnostic fragment sizes generated following digestion with XbaI and SpeI, and hybridization with probe F (Figure 1), are presented. PCR amplification with primer pair 24226–CµXbRI generates a 1.9 kb PCR product. Southern blotting using both single cutters and noncutters was used to determine the copy number of the integrated vector (data not shown). Abbreviations are the same as in Figure 1. The figure is not drawn to scale.

 
Evidence that transcription initiated at the µ gene promoter is not necessary for the stimulation of gene conversion by the IgH µ gene regulatory region
It remained a possibility that the reduced GC frequency in the absence of the endogenous Eµ/MARs fragment was an indirect consequence of a reduced level of µ gene transcription. To determine what effect the Eµ/MARs deletion had on µ gene transcript level, the amount of µ-specific RNA in the cell lines was examined by RNA dot blot analysis (Figure 7) (16,22,27). Previously, deletion of the majority of the VH–Cµ intron, including the Eµ/MARs elements, was shown to cause equivalent changes in both the levels of µ mRNA and nuclear run-on activity, suggesting that no elements are present within this region that affect RNA stability or processing efficiency (28). Therefore, we suggest that the µ-specific RNA levels measured in our cell lines is probably a good indicator of the level of transcription through the Cµ regions. Densitometric analysis revealed that the µ-specific RNA levels in the Eµ/MARs-deleted cell lines, 2A2Eµ–#1–5 and 2A2Eµ–#2–10, were 25% and 10% of that in Im/RCµ2A2, respectively (Table 2). In comparison, µ RNA levels in the transformants bearing the intact Eµ/MARs fragment (2A2Eµ+ #1–4 and 2A2Eµ+ #2–20), were 144% and 92% of that in Im/RCµ2A2, respectively.



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  		Figure 7. RNA dot blot analysis of µ RNA. Serial 1:4 dilutions of total RNA were applied to a nitrocellulose filter starting with 10 µg as described in (22). Relative levels of µ RNA were determined by hybridization with 32P-labeled Cµ-specific Probe F (Figure 1), and separately with a 32P-labeled 2.0-kb PstI fragment containing the chicken ß-actin cDNA (27). The cell line igm10 is an Sp6-derived mutant that lacks the endogenous chromosomal µ gene (16).

 

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  		Table 2. Cµ-specific RNA levels

 
To investigate whether µ-specific RNA levels correlated with the frequency of GC between the Cµ repeats, a 154 bp XbaI/NcoI fragment encompassing the Pµ TATA box, and octamer motif, 5'-ATTTGCAT-3', was deleted from the vector pVHCµRep to generate pVHCµRep{Delta}Pµ (Figure 8). Protein binding to the octamer motif of Pµ is required for high-level expression of the IgH gene in B-cells (29,30). The vector pVHCµRep{Delta}Pµ was linearized at the unique NotI site and transferred by electroporation into the igm10 hybridoma (16). Southern blot and PCR analysis of genomic DNA from 84 individual G418R transformants identified five cell lines that contained a single copy of the integrated vector (Figure 8), designated VHCµRep{Delta}Pµ-1, VHCµRep{Delta}Pµ-5, VHCµRep {Delta}Pµ-7, VHCµRep{Delta}Pµ-15 and VHCµRep{Delta}Pµ-26 (data not shown). Next, the amount of µ-specific RNA in the Pµ-deleted cell lines was compared to those with an intact promoter region (the VHCµRep cell lines reported in Table 1) (Figure 9). As shown in Table 2, the µ RNA levels in the VHCµRep cell lines ranged between 21 and 183% of that in Im/RCµ2A2. In comparison, the µ RNA in the VHCµRep{Delta}Pµ cell lines was between 6 and 20% of that in Im/RCµ2A2 (Table 2).



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  		Figure 8. Construction of µ gene promoter (Pµ)-deleted cell lines. The structure of the linearized vector, pVHCµRep, is shown. The vector is identical to pVHCµRep (Figure 3B) with the exception of a 154 bp XbaI/NcoI deletion encompassing the µ gene promoter, Pµ, as explained in the text. The diagnostic fragment sizes generated following digestion with EcoRI and SpeI, and hybridization with probe F (Figure 1) and probe Eµ (Figure 3), are presented. Southern blotting using both single cutters and noncutters was used to determine the copy number of the integrated vector (data not shown). N, NotI; Sp, SpeI; other abbreviations are the same as in Figure 1. This figure is not drawn to scale.

 


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  		Figure 9. RNA dot blot analysis of µ RNA. Serial 1:4 dilutions of total RNA were applied to a nitrocellulose filter starting with 10 µg as described (22). Relative levels of µ RNA were determined by hybridization with 32P-labeled Cµ-specific Probe F (Figure 1). To verify loading, the blot was reprobed with a 32P-labeled 2.0 kb PstI fragment containing the chicken ß-actin cDNA (27). The cell line igm10 is an Sp6-derived mutant that lacks the endogenous chromosomal µ gene (16).

 
Each of the VHCµRep{Delta}Pµ transformants was expanded from a single cell and grown for ~25 generations under G418 selection. Genomic DNA was prepared from each culture, and qPCR utilizing primer pair 1/3 was performed to measure the frequency of GC in the 5' Cµ region. This analysis revealed a median GC frequency of 1.7 x 10–3 recombinants/cell (Table 3). This value is not significantly different from the median value measured in the VHCµRep transformants (1.5 x 10–3 recombinants/cell) (Table 1) (Mann–Whitney test, P = 0.46). Importantly, no significant correlation exists between µ RNA levels and GC frequencies in the VHCµRep and VHCµRep{Delta}Pµ transformants (Pearson's correlation, P = 0.36).


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  		Table 3. Frequency of gene conversion at ectopic genomic sites

 
Interestingly, the GC frequency in transformant VHCµRep{Delta}Pµ-5 was 5.3% (Table 3), a value that is 761-fold higher than the median frequency measured in the R/CµRepeat transformants (Table 1). To determine whether this exceptionally high frequency resulted from an early recombination event during expansion of the culture (i.e. a ‘jackpot’), three subclone cultures of VHCµRep{Delta}Pµ-5, each started from a single cell, were grown for ~25 generations and the frequency of GC in the 5' Cµ region was determined by qPCR. As shown in Table 3, the frequency of GC in the subclone cultures ranged from 1.9 to 10%, suggesting that the vector may have fortuitously integrated into an unusually recombinogenic region of the genome.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Previously, we showed that the IgH µ locus behaved as a hotspot for spontaneous, mitotic intrachromosomal GC between direct repeats of the Cµ region in murine hybridoma cells (14). Here, we report the identification of a 9.1 kb segment of the µ locus regulatory region [referred to as the ‘recombination-enhancing sequence’ (RES)] that confers GC hotspot activity in this system: deleting RES from the endogenous IgH locus abolished GC between the Cµ repeats, while a stimulation of GC was observed when RES was appended to Cµ repeats in ectopic positions in the hybridoma genome. The RES encompasses DNA sequences beginning 5' of the VH region and extending to immediately 5' of the first Cµ region exon. Several regulatory elements that function in the assembly, expression and replication of the IgH µ gene reside within this fragment. This includes the µ gene promoter (Pµ), the major intronic µ gene enhancer (Eµ) (23) and the µ gene switch (Sµ) region (31). Also, associated with Eµ are two matrix attachment regions (MARs) (32), a promoter of sterile transcripts (Iµ) (23,33) and a putative origin of replication (34).

It is noteworthy that RES-stimulated GC between Cµ repeats in ectopic genomic positions is only ~20–30% of the mean value measured at the endogenous µ locus. As the RES fragment used in the ectopic studies contained an ~2.8 kb deletion encompassing most of the Sµ region, this might suggest a role for Sµ sequences in RES activity. The role of Sµ in class switch recombination (CSR) is well documented (31). In addition, Sµ is frequently involved in translocations observed in B-cell lymphomas (35), and is a preferred site for insertion of transfected DNA (36). However, the failure to observe a reduction in the frequency of GC between Cµ repeats positioned at the IgH locus, which contained the same ~2.8 kb Sµ deletion, argues against a role for Sµ in RES activity.

Rather, the reduced RES activity at ectopic sites might suggest that DNA sequences or elements at or near the IgH locus, which are not included within the RES fragment, are necessary to generate the optimal environment. A possible candidate is the 3' {alpha} enhancer (E{alpha}) complex that resides ~200 kb 3' of the Cµ region (37). E{alpha} has previously been shown to be essential for CSR (38), and is believed to function with Eµ to maintain high levels of expression of the IgH locus in fully differentiated B-cells (39). A second possibility is that elements within the RES itself might generate a domain that is conducive to HR at the endogenous µ locus, but at ectopic sites, cannot fully recreate this environment due to the influence of neighboring DNA sequences or chromatin structure at the site of chromosomal integration. That chromatin structure can influence spontaneous HR has been suggested previously (11,13).

In the IgH µ locus, Eµ and the flanking MARs generate a domain of chromatin accessibility, as suggested by increased sensitivity to DNA-damaging agents (40,41). Analysis of Eµ/MARs deleted cell lines revealed a 30-fold decrease in the frequency of GC at the IgH µ locus, indicating that the Eµ/MARs segment is required for RES activity. However, by itself, the Eµ/MARs fragment did not have a stimulatory effect on adjacent Cµ repeats when integrated at ectopic genomic sites. This finding might suggest that other elements present in the larger RES segment, but not included within the smaller Eµ/MARs fragment, are necessary for GC-stimulating activity; or that the context of the Eµ/MARs elements, with respect to the adjacent nucleotide sequences, is important for its function.

Transcription has previously been shown to stimulate mitotic HR in mammalian cells (42–44). In comparison to the hybridomas with an intact regulatory region, a substantial reduction in µ-specific RNA levels was observed in the Eµ-deleted cell lines. This raised the possibility that the Eµ/MARs complex is indirectly related to RES activity through its role as a transcriptional activator. In contrast to our results, several B-cell lines that lack both Eµ and the MARs at the endogenous IgH locus, have been shown to produce nearly normal levels of Ig mRNA or protein (45,46). This is generally ascribed to the presence of functionally redundant elements, possibly the 3' {alpha} enhancers (39). The apparent discrepancy, however, can be resolved by the finding that introduction of a gpt cassette 3' of the endogenous IgH µ gene renders expression dependent on the Eµ/MARs elements (28). In that study, deletion of Eµ and the MARs depressed µ expression to ~2% the normal level. Our findings suggest that the IgH locus is similarly insulated from the effects of any redundant elements, quite possibly by the integrated pSV2neo vector sequences located downstream of the 5' Cµ region.

To directly determine whether GC between the Cµ repeats correlated with the high level of IgH µ gene transcription, Pµ was crippled in the otherwise intact RES fragment by deleting two highly conserved elements, the TATA box and an octamer motif. The mean µ RNA level in the Pµ-deleted transformants was reduced ~80% compared to transformants bearing the intact RES fragment, a result which agrees with previous studies showing that mutation of either motif drastically reduces µ transcription (29,30). More importantly however, the reduction in µ RNA levels was not associated with a corresponding decrease in the frequency of GC promoted by the RES. The lack of any correlation between the GC frequency and µ RNA levels suggests that transcriptional activity is unlikely to be responsible for RES-stimulated recombination in this system. However, since a residual level of µ RNA persisted in the stable transformants, possibly as a result of sterile transcripts initiating at Iµ (33), we cannot discount the possibility that a low level of transcription may still account for the RES activity.

Assuming the Eµ/MARs are the important elements, what role do they play in GC hotspot activity at the IgH µ locus? The simplest hypothesis is that enhancer-mediated chromatin changes within the Cµ repeats increases their susceptibility to nicks or breaks, which invoke HR for repair. This notion is consistent with previous results that suggest GC events initiate at random sites across the entire Cµ region (14). Also, a high frequency of mutation in the chromosomal Ig µ and {kappa} genes has been reported for hybridoma and myeloma cell lines (15,47). It would be interesting to know whether the high rate of mutation is also dependent on the enhancer elements.

Alternatively, chromatin remodeling mediated by the Eµ/MARs elements might facilitate the adoption of a non-B-DNA conformation within the Cµ repeats. Regions of alternating purines and pyrimidines readily form left-handed Z-DNA when contained in negatively supercoiled DNA (48). Interestingly, a potential Z-DNA forming run of 28 repeats of the dinucleotide, GT, is present within the Cµ region of homology (49). Smaller regions of alternating purines and pyrimidines are also present throughout the repeated Cµ regions (49). Sequences capable of forming Z-DNA have previously been shown to stimulate mitotic HR (5–7).

In summary, we have engineered various mouse hybridoma cell lines to investigate the importance of IgH µ gene regulatory elements in promoting GC in a recombination reporter consisting of a pair of homologous, repeated Cµ region segments. Our results suggest that a region we refer to as the RES, probably, through a main contribution from the internal Eµ/MARs fragment, promotes GC hotspot activity between the Cµ repeats at the endogenous IgH µ locus. While the exact mechanism is unknown, it is possible that Eµ/MARs exerts its effect through changes in chromatin accessibility or chromatin remodeling rendering the adjacent Cµ repeats more susceptible to DNA breaks, which invoke GC for repair. One could imagine that in the absence of proper repair by HR, a similar mechanism acting at the endogenous IgH µ locus in normal B cells might promote genomic instability, e.g. by stimulating chromosomal translocations into the IgH µ locus (35). While our results provide support for cis-acting regulatory elements promoting GC at the IgH µ locus, the analysis of recombination in cell line, VHCµRep{Delta}Pµ-5, suggests that GC hotspot activity can also be a feature of other loci in the mammalian genome. Finally, it is possible that the high frequency, Eµ/MAR-stimulated GC that we observe in this model system bears some mechanistic relationship to DNA transactions that are required to generate antibody diversity in normal B cells (50). In this regard, it is worth noting that the high frequency of GC that we observe in our system is known to be an important mechanism of antibody diversification in chickens and rabbits (51,52), and there is evidence that GC might play a role in some murine antibody responses as well (53–55).


   ACKNOWLEDGEMENTS
 
This research was supported by a PhD studentship from the Canadian Institutes of Health Research (CIHR) to S.J.R., and a CIHR operating grant to M.D.B.


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E. Schildkraut, C. A. Miller, and J. A. Nickoloff
Gene conversion and deletion frequencies during double-strand break repair in human cells are controlled by the distance between direct repeats
Nucleic Acids Res., March 14, 2005; 33(5): 1574 - 1580.
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