Chapter 31

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The Detection and Analysis of Chromosome Fragile Sites ... Key words Common fragile site, Rare fragile site, Telomere fragility, Mitosis, DNA replication stress,.

Chapter 31 The Detection and Analysis of Chromosome Fragile Sites Victoria A. Bjerregaard, O¨zg€ un O¨zer, Ian D. Hickson, and Ying Liu Abstract A fragile site is a chromosomal locus that is prone to form a gap or constriction visible within a condensed metaphase chromosome, particularly following exposure of cells to DNA replication stress. Based on their frequency, fragile sites are classified as either common (CFSs; present in all individuals) or rare (RFSs; present in only a few individuals). Interest in fragile sites has remained high since their discovery in 1965, because of their association with human disease. CFSs are recognized as drivers of oncogene activation and genome instability in cancer cells, while some RFSs are associated with neurodegenerative diseases. This review summaries our current understanding of the nature and causes of fragile site “expression”, including the recently characterized phenomenon of telomere fragility. In particular, we focus on a description of the methodologies and technologies for detection and analysis of chromosome fragile sites. Key words Common fragile site, Rare fragile site, Telomere fragility, Mitosis, DNA replication stress, Karyotype, Fluorescence in situ hybridization (FISH), Ultrafine DNA bridge (UFB), Micronuclei, 53BP1 body


Introduction The faithful replication of DNA is fundamental to all organisms as it ensures that the genetic material is passed on to the next generation of cells. In human cells, the genetic information is stored in 23 pairs of chromosomes (22 pairs of autosomes and one pair of sex chromosomes). The replication and segregation of these 46 chromosomes is orchestrated by the cell division cycle. This cycle comprises interphase, during which the chromosomes are replicated, and mitosis when the chromosomes are segregated. During mitosis, the duplicated chromosomes are first condensed (compacted) and then evenly distributed to the two daughter cells. Mitosis is subdivided into six subphases: prophase, prometaphase, metaphase, anaphase, telophase, and cytokinesis. In metaphase, which is when chromosomes are fully condensed and adopt the iconic X-shaped structure, it is well documented that some chromosomal loci tend to form a gap, break or constriction that is not stained by the most commonly used DNA dye, DAPI. These gaps/breaks are generally

Marco Muzi-Falconi and Grant W. Brown (eds.), Genome Instability: Methods and Protocols, Methods in Molecular Biology, vol. 1672, DOI 10.1007/978-1-4939-7306-4_31, © Springer Science+Business Media LLC 2018



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referred to as chromosomal fragile sites, and are particularly prevalent in cells exposed to agents that partially inhibit DNA synthesis [1]. Based on their frequency in population, these loci are classified as either common fragile sites (CFSs), which are present in all individuals, or rare fragile sites (RFSs), which exist in less than 5% of individuals [2, 3]. To date, more than 200 CFSs [4], and up to 33 RFS [5] have been identified in human cells. Interest in chromosomal fragile sites has remained high since their discovery over 50 years ago [6]. This is because CFSs are recognized as a driver of genome instability in cancer cells [7], while some RFSs are associated with neurodegenerative diseases, such as the Fragile X syndrome [8–11]. One model for the appearance (usually termed “expression”) of fragile sites is that they result from a failure in chromatin condensation during early mitosis due to the incomplete replication of the locus [5, 12]. This failure to fully condense the locus, and hence to make it insufficiently compact to be visualized using DAPI staining, helps to explain why the apparently broken fragile site is never detached fully from the host chromosome. Instead, the site appears to be loosely connected, perhaps by undetectable DNA “tethers”; see below. It is also relevant that all of the known cell culture conditions that promote expression of fragile sites perturb DNA replication. Great progress has been made in recent years toward understanding why and how fragile sites arise. This progress can be summarized in the following areas: (1) cataloging of the genomic features common to different fragile sites (i.e., CG/AT content, repeats features, location on the chromosomes, or evolutionary conservation locus) [13, 14]; (2) defining the cell culture conditions that induce fragile site expression [15–18]; (3) examining the replication program within fragile sites [19]; (4) analyzing how gene transcription can influence the replication of fragile sites [20, 21]; (5) identifying the proteins that are recruited to fragile sites in either S phase or mitosis [22–25]; and, more recently, (6) investigating how fragile site replication is completed, which is now known to occur in early mitosis [26]. This review discusses our current understanding of the nature and causes of chromosome fragility, with a particular focus on the methodologies used for the detection and analysis of these unstable chromosomal loci.


The Induction and Detection of Fragile Sites The first chromosomal fragile site to be defined in human cells was described by Dekaban et al. in 1965 through an analysis of the karyotypes of blood lymphocytes [6]. In the following years, cytogeneticists remained puzzled by the fact that these fragile sites were observed in some laboratories but not in others. It was not until 1977 that it was shown that the type of medium in which the blood

The Detection and Analysis of Chromosome Fragile Sites


lymphocytes were cultured played a role in the induction of fragile site expression [27]. By 1984, 17 heritable fragile sites had been identified, amongst which 14 were known to be induced by cell culture medium deficient in folic acid [28], or by the so-called “thymidylate stress” (e.g., by exposure to 5-fluorodeoxyuridine, FUdR, a potent inhibitor of thymidylate synthetase) [29]. At that time, it was noticed that thymidylate stress often produces fragile site “hot spots” that occur nonrandomly in the genome [30]. It was subsequently discovered that aphidicolin (APH), an inhibitor of replicative DNA polymerases (but not polymerases β or γ), could also induce fragility at several nonrandom sites [15]. The term “common fragile site” was then introduced to describe the sites that can be induced by both APH and thymidylate stress, while “rare fragile sites” were defined as those induced by thymidylate stress, but not APH [15]. However, soon after, it was discovered that some CFSs could also be induced by FUdR, although at a much lower frequency [16]. It was therefore suggested that the fragile site classification should be based on their frequency in the population, rather than on the form of induction [16]. To date, it is known that many agents can induce the expression of CFSs and/or RFSs. These include APH, BrdU, 5-azacytidine, caffeine, and FUdR for CFSs [1, 16], and FUdR, thymidine, distamycine A, BrdU, APH, and 5-azacytidine for RFSs [18]. Conventionally, fragile sites are detected in blood lymphocytes that are cultured for approximately 72 h in medium supplemented with 2% phytohemoagglutinin [4]. Various fragile-site “inducers” can then be added to the cell culture medium according to the type of fragile site of interest. For example, to induce the expression of CFSs in lymphocytes, the cells are generally incubated with APH (0.2 μM) for 24 h, and then arrested for 2 h with the mitotic spindle inhibitor Colcemid (0.04 μg/ml) to trap the cells in metaphase when the chromosomes are fully condensed [4]. To observe the chromosomes under a microscope, the cells are swollen with hypotonic buffer, fixed with methanol–acetic acid (3:1), dropped onto slides, and stained by a DNA dye (usually DAPI). For more detailed analysis of chromosome structure, such as gaps or constrictions, Gbanding (digestion of chromosomes with trypsin followed by Giemsa staining that yields a series of lightly and darkly stained bands) or Q-banding (a fluorescence based technique using quinacrine staining) are commonly performed [31]. With the increase in interest in fragile site research, a number of variations have been introduced to the above standard procedure. First, CFSs are now widely studied in human fibroblasts and various cancer cells [24–26, 32], as well as in mouse or rat cells [33–36]. As a result of this change, the conditions and procedures for cell culture, fragile site induction, and cell harvesting may vary from study to study. For example, to harvest mitotic cells from adherent cell lines, a method called “mitotic shake-off” is frequently used to


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enrich for cells in prometaphase [23]. More recently, because of the need to define which proteins bind to and process CFSs, immunofluorescence (IF) analysis, EdU incorporation, or Fluorescence in situ hybridization (FISH), or a combination of these, are widely performed on metaphase chromosomes [23–25]. Such developments require that the chromosome preparation step be modified such that the detection of proteins bound to a fragile site is still feasible. For example, in the conventional cytogenetic protocol, the mitotic cells are fixed with methanol–acetic acid (3:1). This can interfere with IF analysis, which requires that protein conformation be preserved as much as possible for antibody binding. To overcome this obstacle, a commonly used method is to centrifuge mitotic cells onto slides (instead of dropping them) using a “cytospin” apparatus, and then to fix the metaphases with paraformaldehyde for analysis by IF and then by FISH [37]. It is important to bear in mind also that the protocol for this type of analysis can vary according to the antibodies and FISH probes used [22–24]. Therefore, each step of this protocol needs to be optimized. Figures 1 and 2 show examples of metaphase chromosomes analyzed by IF and an EdU incorporation assay (to mark sites of active DNA synthesis), or by IF combined with FISH to define the location of the FRA16D CFS locus in human cells. The detection of fragile sites using FISH has enabled researchers to define the fragility of specific loci in greater detail, particularly in cases where fragility might be difficult to detect using simple DNA staining (Fig. 3). In addition, this method has confirmed the identity of a new class of fragile sites: telomere fragile sites. Telomeres are the sequences located at the ends of eukaryotic chromosomes. Telomeres in human cells contain a repeated TTAGGG sequence that ranges in length from 4 to 14 kb [38]. Interest in telomeres has surged since the discovery of the telomere-specific reverse transcriptase, telomerase [39], and, the fact that the maintenance of the telomeric repeat sequence, either by telomerase [40] or by an alternative mechanism (ALT) [41], is essential for the long-term proliferation of cells in culture and in vivo. In the past decade, emerging evidence has shown that telomeric repeats pose a challenge to the DNA replication process, which can generate the so-called “telomere fragile site” (TFS) instability that is similar to what is seen at APH-induced CFSs. Indeed, pronounced TFS expression is seen in cancer cells or immortalized cells, especially when the cells are treated with APH [42] (Fig. 4). The widespread usage of a peptide nucleic acid (PNA) FISH probe that binds specifically to the telomere repeat sequence has been instrumental in the analysis of this type of fragile site, as conventional chromosome staining methods cannot detect minor changes at the tip of chromosomes [43]. Equally importantly, the usage of telomeric PNA probes is compatible with IF analysis [42, 44–47].

The Detection and Analysis of Chromosome Fragile Sites


Fig. 1 A representative example of the colocalization of FANCD2 and EdU with a common fragile site. The U2OS cells were treated with APH (0.4 μM), synchronized using RO3306 and then pulse-labeled with EdU as described previously [26]. Metaphase cells were collected by mitotic shake-off, and then spun onto coverslips for metaphase chromosome preparation. The metaphase chromosomes were stained with FANCD2 antibody (green), EdU (click IT reaction) (red), and observed using DAPI (blue). In the upper panel, the blue arrow indicates a chromosome fragile site. In the lower panel, the yellow arrow indicates the colocalization of FANCD2 and EdU at the fragile site, as revealed by the yellow signal in this merged image


The Characterization of Fragile Sites CFSs cover megabase-pair-long regions and tend to contain or overlap with large genes. In addition, they are located in evolutionally conserved chromosomal regions, such as in syntenic regions of mouse and human chromosomes [1]. Several hypotheses have been put forward to explain how completion of CFS replication is delayed compared to that in other regions of the genome. For example, evidence has been provided that CFS expression can be caused by either a lack of replication origins within the locus [19], or by collisions between replication forks and the long gene transcripts located there [48]. Furthermore, the molecular events leading to the fragile site expression are beginning to be revealed, following the findings that key components of DNA repair and


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Fig. 2 A representative example of the analysis of colocalization of FANCD2 with a specific common fragile site: FRA16D. GM06865 lymphocytes were cultured and treated with APH as described previously [4]. Metaphase spreads were prepared by the “Cytospin” method. The metaphase chromosomes were subjected to IF analysis with FANCD2 antibody (red) followed by FISH analysis using a BAC probe that binds specifically to the FRA16D locus (green). DNA was observed with DAPI (blue). The yellow arrow indicates FANCD2 “twin foci” colocalized with FRA16D FISH probes where the merged signal is yellow

Fig. 3 A representative example of the analysis of a rare fragile site, FRAXA, using FISH. GM09237 lymphocytes, which contain over 900 CGG repeats in the promoter region of FMR1 gene, were cultured and treated with FUdR as described previously [58]. Metaphase spreads were prepared by the conventional “dropping” method. The metaphase chromosomes were subjected to FISH analysis using a BAC probe that binds specifically to FRAXA (green). DNA was stained with DAPI (blue). The yellow arrow indicates the FRAXA FISH probe signal that is apparently detached from the X chromosome

The Detection and Analysis of Chromosome Fragile Sites


Fig. 4 A representative example of the analysis of telomere fragility. U2OS cells were treated with APH (0.4 μM) and RO3306 as described previously [26]. Metaphase cells were collected by mitotic shake-off and dropped onto slides for metaphase FISH analysis with a PNA telomere probe following the manufacturer’s instructions (Panagene, Korea). The chromosomes were stained with DAPI (blue) and the telomeres were detected with FAM labeled PNA telomere probe (green). The blown-up images below show examples of atypical telomere signals

DNA damage response pathways, including γH2AX [22], phospho-DNA-PKcs [22], and SMC1 [49], are recruited to CFSs. We showed recently that the Fanconi anemia proteins, FANCD2 and FANCI, bind specifically to CFS loci in cells treated with a low dose of APH irrespective of whether the chromosome is broken or not in metaphase [23]. These proteins form a focus on each sister chromatid at the location of the CFS. Most interestingly, when cells harboring these foci are analyzed in anaphase, the foci are often linked by a DNA bridge called an ultrafine DNA bridge (UFB). It is possible that at CFS loci where DNA replication is disrupted or otherwise delayed, the sister chromatids are still interlinked by regions of underreplicated DNA when cells enter anaphase. This would then lead to the formation of a UFB. Most of the time, these replication intermediates can be processed or resolved by proteins including PICH, BLM, and RPA [50]. However, some of the bridges/UFBs may lead to detrimental consequences, such


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as the formation of micronuclei or 53BP1 bodies in the next G1 phase [51]. We showed recently that underreplicated CFSs could be detected and processed by structure-specific endonucleases (e.g., the MUS81-EME1 complex) in early mitosis, and that this promotes CFS expression [25]. This discovery is consistent with the notion that the expression of CFSs is not an accidental event, but instead is a programmed and regulated process. Moreover, we have proposed that CFS expression is beneficial for the maintenance of genome stability because it prevents much more hazardous events, such as irreversible chromosome missegregation. Most recently, we have demonstrated that, following treatment of cells with APH, entry into the mitotic prophase triggers the recruitment of MUS81-EME1 to CFSs, and the nuclease activity of MUS81 then promotes POLD3-dependent DNA synthesis at CFSs. When analyzed on metaphase chromosomes, the sites of new DNA synthesis in mitosis correspond to the expressed CFSs, indicating that the gap/break in the chromosome is the location of a repair event that delays condensation of the locus. This DNA repair-based replication “salvage” pathway serves as a mechanism to minimize chromosome missegregation and nondisjunction, particularly in cancer cells with elevated levels of chromosome instability (CIN þve) [26]. In the case of RFSs, most of the studies have been focused on the FRAXA (fragile X) locus that is associated with Fragile X syndrome. This disorder is characterized by a spectrum of intellectual disabilities, as well as atypical physical characteristics (including an elongated face, large or protruding ears, and large testicles), and behavioral characteristics such as stereotypic movements (e.g., hand-flapping), and social anxiety. It is well established that Fragile X syndrome is caused by the expansion of the CGG trinucleotide repeat that affects the expression of the Fragile X mental retardation 1 gene (FMR1) [52, 53]. In the past three decades, great progress has been made in deciphering the mechanism underlying the expansion of the repeats that denote such folate-sensitive RFSs [54–56]. However, the mechanism underlying the fragility of these loci and other CGG trinucleotide repeat regions remains unknown. Undoubtedly, the methodologies that have been developed to define the fragility of CFSs can be applied to the analysis of this type of fragile site.


Concluding Remarks In the past five decades, a considerable amount has been learned about chromosome fragile sites in the human genome. The methods involved in the detection and analysis of chromosome fragile sites have progressed from the original cytogenetic-based techniques to fluorescence based ones (i.e., IF and FISH) (summarized in Fig. 5). This has allowed us to understand why fragile sites are

The Detection and Analysis of Chromosome Fragile Sites


Fig. 5 A flowchart of the methodology involved in the induction, detection, and analysis of human chromosome fragile sites. References for the methods applied are indicated where appropriate


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unusually “fragile”, and the strategies utilized by cells to cope with this fragility to ensure the faithful replication and segregation of DNA during the cell division cycle. In addition to the methods mentioned above, great strides have been made in imaging technologies that allow for more sensitive, faster, and reliable detection of genomic loci. Moreover, high content array CGH technology has enabled researchers to detect DNA copy number changes in an interval of 1 marker per 1737 bases cross the whole human genome with a input of DNA as little as 250 ng (e.g., via the CytoScan® HD platform developed by Affymetrix). It seems likely that chromosome fragile sites will remain an intriguing feature of human cells and a fascinating topic for research for the following reasons: (1) CFSs exist in every individual and many are in conserved chromosome regions in mammals. This begs the question as to why that is the case, and whether there is any advantage for the cells to maintain those apparently fragile loci during evolution? (2) Several different types of DNA synthesis inhibitors can induce the expression of either CFS or RFS, and hence it would be interesting to find out why that is the case. Is this because of the DNA sequence differences at each fragile site, or because different inhibitors initiate a different DNA damage response? (3) Errors arising during the repair or replication at fragile sites following replication stress (that can be either endogenous, such as oncogene associated, or exogenous, such as folate deficiency) lead to chromosome translocations or an abnormal number of chromosomes. This can, in turn, drive the development of several debilitating disorders (e.g. cancer, cognitive dysfunction, and infertility). It will be a high priority to identify the causes of those errors. (4) The recently identified fragility at telomeric sites raises questions regarding which factors increase or decrease the expression of TFSs in cancer cells, considering that there is evidence to show that APH can increase the expression of TFSs in ALT cells [57]. The answer to this question would potentially allow us to develop novel anticancer drug treatments given that cancer cells have shown unusual telomere length maintenance either via an active telomerase or the ALT mechanism.

Acknowledgment We thank Theresa Wass, Malgorzata Clausen, and Wei Wu for experimental help. Work in the authors’ laboratory is supported by a Faculty Ph.D. fellowship from the University of Copenhagen (V.A.B.), the European Union (O.O., I.D.H., and Y.L.), The Danish National Research Foundation (DNRF115; I.D.H. and Y. L.), and The Nordea Foundation (I.D.H.).

The Detection and Analysis of Chromosome Fragile Sites


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