Chromosomal fragile sites are points on chromosomes that usually appear as nonstaining chromosome ... site expression and support the view that fragile site.
Am. J. Hum. Genet. 43:265-273, 1988
Chromosome Breakage and Recombination at Fragile Sites Thomas W. Glover,* t and Constance K. Stein* *Department of Pediatrics and Communicable Diseases and tDepartment of Human Genetics, University of Michigan, Ann Arbor
Summary Chromosomal fragile sites are points on chromosomes that usually appear as nonstaining chromosome or chromatid gaps. It has frequently been suggested that fragile sites may be involved in chromosome breakage and recombination events. We and others have previously shown that fragile sites predispose to intrachromosomal recombination as measured by sister-chromatid exchanges. These findings suggested that fragile site expression often, if not always, is accompanied by DNA strand breakage. In the present report, fragile sites are shown to predispose to deletions and interchromosomal recombination. By use of somatic cell hybrids containing either human chromosome 3 or the fragile X chromosome, deletions and translocations were induced by FUdR or aphidicolin with breakpoints at the fragile sites Xq27 or 3pl4.2 (FRA3B) or at points so close to the fragile sites as to be cytogenetically indistinguishable. Southern blot analysis of DNA from a panel of chromosome 3 deletion and translocation hybrids was then utilized to detect loss or retention of markers flanking FRA3B and to corroborate the cytogenetic evidence that the breakpoints were at this fragile site. One cell line with a reciprocal translocation between human chromosome 3 (with breakpoint at 3pl4.2) and a hamster chromosome showed cytogenetically that the fragile site was expressed on both derivative chromosomes, supporting the hypothesis that the fragile site represents a repeated sequence. The approach described provides a means of generating specific rearrangements in somatic cell hybrids with a breakpoint at a fragile site. This will allow generation of translocation hybrids which should be useful as part of gene mapping panels, for physical mapping of probes in the region of fragile sites, and in strategies to clone fragile sites based on isolation of the human-hamster DNA junction at the translocation breakpoint. Introduction
Chromosomal fragile sites are, by definition, specific points on chromosomes that exhibit "fragility" under appropriate conditions of induction (reviewed in Sutherland et al. 1985). Cytologically, this fragility is almost always seen as a nonstaining chromosome or chromatid gap, less frequently as a break, and only rarely as a deletion. Both rare and common fragile sites have been described. Aside from the fragile X which causes a common form of mental retardation, no pathologic role for any other fragile site has been shown. However, the coincident locations of some fragile sites and chromosome Received December 17, 1987; revision received March 15, 1988. Address for correspondence and reprints: Dr. Thomas W. Glover, Department of Pediatrics, Division of Pediatric Genetics, D1225 Medical Professional Building, Box 0718, Ann Arbor, MI 48109. 0 1988 by The American Society of Human Genetics. All rights reserved. 0002-9297/88/4303-0006$02.00
rearrangement breakpoints seen in certain forms of cancer have been noted (Hecht and Glover 1984; Hecht and Sutherland 1984; Lebeau and Rowley 1984; Yunis and Soreng 1984). The genetic or molecular basis for chromosome fragility at these sites is not known, nor are the biological consequences of fragile site expression fully understood. We and others have previously shown that fragile sites predispose to intrachromosomal recombination as measured by sister-chromatid exchanges (Tommerup 1986; Glover and Stein 1987). This finding suggested that fragile sites often, if not always, result in DNA strand breakage at some point during expression. We now report that fragile sites can also predispose to deletions and interchromosomal recombination (translocations) following induction in a somatic cell hybrid system. These findings demonstrate a further consequence of fragile site expression and support the view that fragile site expression is a consequence of, or can result from, DNA 265
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strand breakage. Furthermore, the resulting translocation chromosomes with one breakpoint at a fragile site in a human chromosome and the other in a rodent chromosome provide useful tools for gene mapping around fragile sites and for cloning fragile sites by isolation of translocation breakpoints containing heterologous human-rodent DNA junctions. Material and Methods Cell Culture Two somatic cell hybrids were utilized in these studies (fig. 1). UCTP-2A is a hybrid containing normal human chromosome 3 in a CHO uridine auxotroph cell line deficient for the last two enzymes of dUMP biosynthesis which map to the long arm of human chromosome 3 (Patterson et al. 1983). Human chromosome 3 is maintained by selection in uridine-deficient medium (DMEM) with 10% dialyzed fetal calf serum (FCS). This hybrid was utilized to test for breakage and recombination at the common fragile site at 3pl4.2 (FRA3B). FX-629 is a somatic cell hybrid constructed by polyethylene glycol (PEG) fusion of a human male fragile X lymphoblastoid cell line and CHO cell line RJK-629 which is deficient in HPRT and G6PD activities (HPRT -, G6PD I. The human fragile X chromosome is maintained by selection in HAS medium (DMEM + 10% FCS with 1 x 10-4 M hypoxanthine and 1 x 10-5 M azaserine). The majority of cells in this hy-
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Glover and Stein
brid also contained, by coincidence, human chromosome 3 as the only other human chromosome. This cell line was used to detect translocations at Xq27 as well as deletions and translocations in chromosome 3. Chemical Treatments and Chromosome Analysis
Hybrid cell lines were plated in appropriate selective medium at a low cell density (approximately 1 x 105 cells) in 25-cm2 tissue culture flasks and treated with aphidicolin (0.4-0.6 gM) or FUdR (0.1 jiM) for fragile site expression (Glover 1981; Tommerup et al. 1981; Glover et al. 1984). They were grown in the presence of drugs for 5 days with one subculturing. Medium was then changed with drugs omitted and cells were allowed to "recover" for 24 h. Cells were then trypsinized and a culture was established from about 1-2 x 105 cells for chromosome harvest 24 h later in order to determine the relative frequency of human chromosome rearrangements in the population. Cells were also plated at a density of 100-200 cells/100 mm tissue culture dish to generate cell colonies originating from a single cell. Resulting colonies were isolated with glass cloning cylinders and were replica plated in 24 well dishes. One replicate was harvested for chromosome analysis. Colonies showing deletions or translocations at fragile sites or at other areas of interest on chromosomes 3 or X were propagated as cell lines for future studies. Initial scoring of UCTP-2A hybrids was done on Qbanded slides using (CMA)2S fluorescent stain
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Figure I Parental cell lines. a and b, UCTP-2A containing chromosome human 3. c and d, FX-629 containing chromosomes 3 (left) and X (right). a, Fluorescent staining; b and c, G-11 staining; d, GTG-banding.
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Fragile Site Breakage and Recombination (Deagau et al. 1978). Human chromosome 3 and its derivatives could readily be scored owing to the presence of marker-centromeric heterochromatin. The cloned cells were further characterized with G-11 staining (modified from Wyandt et al. 1976). FX-629 hybrids were scored following G-11 staining and subsequently characterized by conventional GTG-banding. DNA Analysis of Translocation and Deletion Hybrids
DNA Isolation and Southern blots. High-molecular-
weight DNA was isolated from cell lines originating from single cell colonies cytogenetically characterized to contain a deletion or translocation at or near FRA3B. DNA was isolated by means of phenol/chloroform extraction of N-lauroylsarcosine-proteinase K-treated cell lysates. DNA was recovered by ethanol precipitation and dissolved in TE buffer (10 mM Tris-HCI pH8.0, 1.0 mM EDTA). Restriction endonuclease digestions were accomplished by digesting aliquots of DNA to completion with MspI (BRL, Bethesda, MD) under conditions recommended by the manufacturer. DNA fragments were resolved by electrophoresis in 1% agarose gels. Denatured DNA was transferred (Southern 1975) onto HybondO nylon membranes (Amersham, Arlington Heights, IL) which were rinsed in 2 x SSC and air-dried prior to hybridization. Prehybridization and hybridization were done in a solution containing 1 M NaCl, 10% dextran sulfate, 1 x Denhardt's solution, and 1% SDS. Filters were prehybridized overnight and hybridized for 18 h at 65 C. DNA probes were labeled with 32P by the oligolabeling technique (Feinberg and Vogelstein 1983) to specific activities >109 cpm/ pg. Filters were then washed with several changes of SSC plus SDS and exposed to X-ray film for 24-48 h.
Probes. -Two probes recognizing random human
genomic sequences on human chromosome 3 were utilized. pl2-32 (D3S2) detects a polymorphic locus at band p21 in MspI-digested human DNA (Naylor et al. 1984, 1985, 1987; Harris et al. 1986). pMS1-37 (D3S3) detects a polymorphic locus reported to be at band p14.2 in MspI-digested human DNA (Barker et al. 1984; Gerber et al. 1986). More recent data obtained from a chromosome 3 somatic cell hybrid panel defining the region 3pl4.1 to 3p2l.1 by means of a series of translocations confirm the order 3pl4.1, D3S3, 3pl4.2, D3S2, 3p2l.1 (H. Drabkin, personal communication). Thus, the best available data placed D3S3 at 3pl4.2 and D3S2 at 3pl4.2 or 3p2l.l. These two probes then detect the nearest two currently known loci that flank FRA3B. Results Induction of Rearrangements
UCTP-2A hybrids were treated with aphidicolin and scored for breaks, gaps, deletions, and rearrangements of chromosome 3 in three separate experiments. The effects of this treatment on human chromosome 3 are shown in table 1. In total, 113 deletions or translocations were observed with a breakpoint in human chromosome 3, and, of these, 52 (46%) had the breakpoint at FRA3B or at a site so near FRA3B as to be cytologically indistinguishable. Therefore, in 409 cells, 52 deletions or translocations were observed with a breakpoint at or near FRA3B, for a total relative population frequency of 13%. Separating data for the two aphidicolin treatments shows 12 (6.5%) of 85 cells so affected at 0.4 gM and 40/224 cells (18%) at 0.6 gM. Breakpoints were clearly clustered at 3pl4. An examination of the distribution of breakpoints along chromosome
Table I Aphidicolin-induced Deletions or Translocations of Chromosome 3
Aphidicolin
(AM) .4
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.6
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Total
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Cells Scoreda 185
Total Deletions or Translocations, Chromosome 3 40
224
73
409
113
Deletions or Translocations,
3p14 12 6.5% of cells (5%-7.5%)b 30% of breaks (25%-32%)C 40 18% of cells (15%-20%)b 55% of breaks (44%_60%)C 52 13% of cells, 46% of breaks
Pooled data from three experiments. Percentage of cells with translocation at 3pl4. Average and range from three experiments. c Percentage of deletion or translocation breakpoints on chromosome 3 that occurred at 3pl4. Average and range from three experiments. a
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Figure 2 Deletion and translocation hybrids shown with G-11 staining (a, b, and e-g) and fluorescent staining (c and d). a and c, Deletions at 3pl4; b and d, translocations at 3pl4; e-g, translocations at Xq27.
3 demonstrated that 30% and 55% of all breaks in chromosome 3 produced by 0.4 and 0.6 iM aphidicolin, respectively, occurred at 3pl4, for a combined average of 46%. Examples of deletion and translocation chromosomes are shown in figure 2a-2d. FX-629 cells treated with FUdR or aphidicolin in three separate experiments were scored only for translocations, since deletions would be too small to be accurately detected cytologically, expecially with G-11 banding. Data for the frequency of translocations occurring in the X chromosome and, in particular, at or near the Xq27 fragile site, are shown in table 2. An average of 13% and 6% of cells showed a translocation at or very near the fragile X site following treatment with either FUdR or aphidicolin, respectively. The X
chromosome breaks were clearly clustered at Xq27-28, with as many as 95% occurring there in one experiment with FUdR. Almost all other breaks were seen at or near Xp22 and Xq22, the location of common fragile sites (data not shown). Examples of translocations are shown in figure 2e-2g. Within a given treated culture and between cultures, some of the translocation chromosomes appeared cytologically identical. Thus, it is possible that some of the translocation chromosome cells could have arisen from a common parent or that a small number of sites in the hamster genome are preferentially broken. Analysis of Clones
A series of deletion and translocation clonal cell lines
Fragile Site Breakage and Recombination
269
Table 2 FUdR- and Aphidicolin-induced Translocations of the X Chromosome
Inducing Agent 0.1
pM FUdR
0.6
piM Aphidicolin
Cells Scoreda
Total Trans X Chromosome
486
83
175
26
............... ...........
Trans Xq27-28
62 13% of cells (2%_30%)b 75% of breaks (25%-95%)c 11 6% of cells (4%-8%)b 42% of breaks (20%-67%)C
Pooled data from three experiments. Percentage of cells with translocation at Xq27-Xq28. Average and range from three experiments. c Percentage of translocation breakpoints on the human X chromosome that occurred at Xq27-28. Average and range from three experiments. a
b
were isolated from aphidicolin- and FUdR-treated popu-
lations. One such cell line, B-15 (fig. 3a), had an apparently balanced reciprocal translocation between human and hamster chromosomes with a breakpoint at 3pl4.2. As a further test that the breakpoint was at FRA3B, B-15 cells were treated with 0.4 gM aphidicolin for 24
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h to induce fragile site expression. Breaks and gaps at the translocation breakpoint were seen in both derivative chromosomes (fig. 3b-3f). In 150 cells scored, the derivative 3 chromosomes showed 19% gaps or breaks while the derivative hamster chromosomes showed 8% gaps or breaks at the translocation breakpoint. The pa-
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e Figure 3 Hybrid cell line B-15 showing reciprocal translocation (a) and aphidicolin-induced breakage at 3pl4 (b-J). b, Breakage on both translocation chromosomes; c and e, breakage on derivative 3 at 3pl4; d and f, breakage on the derivative hamster chromosome.
270
rental UCTP-2A hybrid exhibited 35% FRA3B expression under these conditions. The results of Southern analysis of a series of isolated deletion and translocation hybrid cell lines with 3pl4.2 breakpoints are shown in figure 4. DNA from normal human control cells, parental UCTP-2A hybrids, and all the deletion and translocation hybrids were positive for presence of D3S3 alleles which map to 3pl4.2 (fig. 4a), whereas the hamster parent cell line was negative. Thus, all deletion and translocation breakpoints in the panel tested are consistent in mapping distal to D3S3, as is FRA3B. Figure 4b shows results for analysis of the distally mapped locus D3S2 which is located in band 3p21. Normal human and UCTP-2A cells were positive, whereas the parental hamster cells were negative for D3S2 alleles. All deletion and translocation hybrids were negative except the reciprocal translocation B-15 (fig. 4, lane 9), as expected, and del B-6 (fig. 4, lane 7). A followup cytogenetic analysis of del B-6 showed the presence of a minority of cells with a normal chromosome 3, thus rendering the line mosaic for cells with a del 3pl4.2 chromosome and for those with a normal 3. The presence of a subline containing a normal 3 was not observed during initial cytogenetic characterization of 15 cells from this line but presumably was present since initial colony isolation and may have had a selective growth advantage in mass culture. Thus, with this taken in account, all deletion and translocation hybrids produced results consistent with breakage proximal to D3S2 and in the region of FRA3B. All breakpoints, and presumably FRA3B, therefore lay between the D3S2 and D3S3 loci. Discussion
As shown elsewhere (Tommerup 1986; Glover and Stein 1987), expressed fragile sites can lead to intrachromosomal recombination. The present data show that fragile sites can also predispose to deletions and interchromosomal recombination and do so at rather high frequencies under the appropriate conditions. These findings support our earlier suggestions (Glover and Stein 1987) that fragile sites result from, or lead to, DNA strand breakage during expression following failure of DNA replication over the fragile site "region." Such breakage can lead to SCEs, deletions, and translocations under appropriate conditions. Warren et al. (1987) have recently reported the induction, isolation, and characterization of breakage and translocation at the fragile X site in a somatic cell hy-
Glover and Stein brid system similar to that described here. In the present experiments, we have confirmed this finding and have further shown that this approach can be extended to a common fragile site at 3pl4. The somatic cell hybrid systems utilized in the present experiments were designed such that breakage at FRA3B or the fragile X site could occur with subsequent deletion of chromosomal material distal to the fragile sites occurring without selective disadvantage to the cells. We speculate that should deletion at fragile sites occur in human cells in vivo or in vitro, such cells would have greatly reduced fitness and probably die, being seen only rarely in cytogenetic preparation. Without the availability of DNA probes to the fragile sites studied here, the evidence that breakage has occurred exactly at the fragile sites is circumstantial. However, four lines of evidence support the belief that breakage and recombination have occurred directly at the fragile X and FRA3B in a high proportion of treated cells. First, cytologically, breaks were observed at or so near the fragile sites as to be cytogenetically indistinguishable. Second, the location of all breaks on human chromosomes were determined and an obvious clustering of breaks was observed at 3p14 and Xq27, the location of the fragile sites under study. Third, treatment of reciprocal translocation B-15 cells with aphidicolin resulted in fragile site expression on both sides of the translocation. In addition to supporting the view that B-15 is broken precisely at FRA3B, this observation supports the hypothesis that fragile sites are regions of repeated sequences or amplified DNA (Sutherland et al. 1985; Ledbetter et al. 1986; Nussbaum et al. 1986). Furthermore, treatment of cell populations containing chromosome 3 or X nonreciprocal translocations with aphidicolin or FUdR, respectively, resulted in some cells with a gap at the translocation breakpoint and no instances of apparent fragile X or FRA3B elsewhere on translocation chromosomes (data not shown). Last, DNA markers flanking FRA3B are lost or retained in a panel of deletion and translocation hybrids, as expected if the breakpoints are all at FRA3B. A similar observation was previously made for closely linked probes flanking the fragile X site by Warren et al. (1987) in two X-translocation cell lines generated in a fashion much like those described here. Translocations at or near the fragile X site were induced by both FUdR and aphidicolin. Although previous work (Glover et al. 1984) had failed to show induction of the fragile X with 0.2 gM aphidicolin, we were able to demonstrate breakage at Xq27 by using a longer exposure with a higher concentration of aphidicolin
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272 (0.6 pM). Ledbetter et al. (1986) have also recently shown that fragile sites at or near the fragile X site can be induced in all individuals by aphidicolin. It is not presently known whether both FUdR and aphidicolin cause breakage at the same location. The aphidicolin locus may coincide with the fragile X site or represent a nearby common fragile site. Molecular characterization and comparison of isolated translocation cell lines induced by aphidicolin and FUdR will provide one approach to this question. These findings have several theoretical and practical implications. They are relevant to suggestions that fragile sites may predispose to specific translocations and deletions in vivo. Chromosome translocations with breakpoints at fragile sites may be useful in gene mapping and cloning studies utilizing somatic cell hybrids and could conceivably be created at any fragile site occurring on a human chromosome in a rodent cell background. Such hybrids are also potentially useful for physical mapping of probes in the region of fragile sites by use of pulsed-field gel electrophoresis. Finally, as also suggested by Warren et al. (1987), hybrid cells with translocations at fragile sites can be utilized in strategies to clone fragile sites. The translocation breakpoints mark a junction between human and hamster DNA that should be amenable to isolation and cloning.
Acknowledgments We thank Dr. C. Scoggin for hybrid cell line UCTP-2A, Dr. T. Caskey for cell line RJK629, and Drs. M. Leppert and R. White for probes D3S3 and D3S2. We also thank Dr. H. Drabkin, Eleanor Roosevelt Institute, for sharing unpublished data with us. Dr. R. Gemmill provided helpful discussion. We are grateful to Joanne Owens for assistance in preparation of the manuscript. This work was supported by grant CA 43222 from the National Institutes of Health.
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