ATM modulates the loading of recombination proteins onto a chromosomal translocation breakpoint hotspot

Chromosome translocations induced by DNA damaging agents, such as ionizing radiation and certain chemotherapies, alter genetic information resulting in malignant transformation. Abrogation or loss of the ataxia-telangiectasia mutated (ATM) protein, a DNA damage signaling regulator, increases the incidence of chromosome translocations. However, how ATM protects cells from chromosome translocations is still unclear. Chromosome translocations involving the MLL gene on 11q23 are the most frequent chromosome abnormalities in secondary leukemias associated with chemotherapy employing etoposide, a topoisomerase II poison. Here we show that ATM deficiency results in the excessive binding of the DNA recombination protein RAD51 at the translocation breakpoint hotspot of 11q23 chromosome translocation after etoposide exposure. Binding of Replication protein A (RPA) and the chromatin remodeler INO80, which facilitate RAD51 loading on damaged DNA, to the hotspot were also increased by ATM deficiency. Thus, in addition to activating DNA damage signaling, ATM may avert chromosome translocations by preventing excessive loading of recombinational repair proteins onto translocation breakpoint hotspots.     

Chromatin structural elements and chromosomal translocations in leukemia

Recurring chromosome abnormalities are strongly associated with certain subtypes of leukemia, lymphoma and sarcomas. More recently, their potential involvement in carcinomas, i.e. prostate cancer, has been recognized. They are among the most important factors in determining disease prognosis, and in many cases, identification of these chromosome abnormalities is crucial in selecting appropriate treatment protocols. Chromosome translocations are frequently observed in both de novo and therapy-related acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS). The mechanisms that result in such chromosome translocations in leukemia and other cancers are largely unknown. Genomic breakpoints in all the common chromosome translocations in leukemia, including t(4;11), t(9;11), t(8;21), inv(16), t(15;17), t(12;21), t(1;19) and t(9;22), have been cloned. Genomic breakpoints tend to cluster in certain intronic regions of the relevant genes including MLL, AF4, AF9, AML1, ETO, CBFB, MYHI1, PML, RARA, TEL, E2A, PBX1, BCR and ABL. However, whereas the genomic breakpoints in MLL tend to cluster in the 5' portion of the 8.3 kb breakpoint cluster region (BCR) in de novo and adult patients and in the 3' portion in infant leukemia patients and t-AML patients, those in both the AML1 and ETO genes occur in the same clustered regions in both de novo and t-AML patients. These differences may reflect differences in the mechanisms involved in the formation of the translocations. Specific chromatin structural elements, such as in vivo topoisomerase II (topo II) cleavage sites, DNase I hypersensitive sites and scaffold attachment regions (SARs) have been mapped in the breakpoint regions of the relevant genes. Strong in vivo topo II cleavage sites and DNase I hypersensitive sites often co-localize with each other and also with many of the BCRs in most of these genes, whereas SARs are associated with BCRs in MLL, AF4, AF9, AML1, ETO and ABL, but not in the BCR gene. In addition, the BCRs in MLL, AML1 and ETO have the lowest free energy level for unwinding double strand DNA. Virtually all chromosome translocations in leukemia that have been analyzed to date show no consistent homologous sequences at the breakpoints, whereas a strong non-homologous end joining (NHEJ) repair signature exists at all of these chromosome translocation breakpoint junctions; this includes small deletions and duplications in each breakpoint, and micro-homologies and non-template insertions at genomic junctions of each chromosome translocation. Surprisingly, the size of these deletions and duplications in the same translocation is much larger in de novo leukemia than in therapy-related leukemia. We propose a non-homologous chromosome recombination model as one of the mechanisms that results in chromosome translocations in leukemia. The topo II cleavage sites at open chromatin regions (DNase I hypersensitive sites), SARs or the regions with low energy level are vulnerable to certain genotoxic or other agents and become the initial breakage sites, which are followed by an excision end joining repair process.     

Translocation breakpoint in two unrelated Tourette syndrome cases,within a region previously linked to the disorder

Tourette syndrome (TS) is a complex neuropsychiatric disorder characterized by both motor and vocal tics. The etiology of TS is poorly understood; however, evidence of genetic transmission arises from family and twin studies. A complex mode of inheritance has been suggested, likely involving contributions of several genes with different effect size. We describe here two unrelated families wherein balanced t(6;8) chromosomal translocations occur in individuals diagnosed with TS. In one of these families, the transmission of the translocation is associated with learning and behavioral difficulties; in the other family, one parent is unaffected and the other cannot be traced, thus transmission cannot be demonstrated and it is possible that the translocation may have occurred de novo. The breakpoint on chromosome 8 occurs within the q13 band in both families, suggesting that a gene or genes in this region might contribute to the TS phenotype. Existing linkage and cytogenetic data, suggesting involvement of chromosome 8 in TS families and individuals, further support this hypothesis. We have identified two YAC clones mapping distal and proximal to the chromosome 8 translocation site, as determined by fluorescent in situ hybridization (FISH). PCR amplification of genetic markers in this region, using isolated chromosomes from one of the patients, followed by BAC screening with the closest flanking genetic markers, has identified a 200-kb BAC, which, by FISH, we have demonstrated encompasses the chromosome 8 breakpoint in both families. The fact that the chromosomal breaks in the TS cases from both families occur within such a small region of chromosome 8 further supports the hypothesis that disruption of a gene or genes in this part of chromosome 8 contributes to the clinical phenotype.     

Chromosome landing at the tomato Bs4 locus

The tomato (Lycopersicon esculentum) Bs4 gene confers resistance to strains of Xanthomonas campestris pathovar vesicatoria that express the avirulence protein AvrBs4. As part of a map-based cloning strategy for the isolation of Bs4, we converted Bs4-linked amplified fragment length polymorphism (AFLP) and restriction fragment length polymorphism (RFLP) markers into locus-specific sequence-tagged-site (STS) markers. The use of these markers for the analysis of 1972 meiotic events allowed high-resolution genetic mapping within a 1.2-cM interval containing the target gene. Two tomato yeast artificial chromosome (YAC) clones, each harboring inserts of approximately 250 kb, were identified using the marker most closely linked to Bs4. YAC end-specific markers were established and employed to construct a local YAC contig. The ratio of physical to genetic distance at Bs4 was calculated to be 280 kb/cM, revealing that recombination rates in this region are about three times higher than the genome-wide average. Mapping of YAC end-derived markers demonstrated that the Bs4 locus maps within a region of 250 kb, corresponding to a genetic interval of 0.9 cM.     

Characterization of a complex philadelphia translocation (1p-;9q+;22q-) by gene mapping

Summary Human-Chinese hamster somatic cell hybrids were obtained using circulating leucocytes from a chronic myeloid leukaemia (CML) patient carrying a complex Philadelphia (Ph¹) translocation (1p-; 9q+; 22q-). Hybrid clones which showed segregation of the translocation chromosomes were studied. The chromosome 22 markers ACO2, ARSA, and NAGA segregated with the 1p- derivative; and the chromosome 1 markers UMPK, PGD, and ENO1 segregated with the 9q+ derivative. Hence, molecular evidence has been obtained for the translocation of the distal part of 22q to chromosome 1 and for the translocation of the distal part of 1p to chromosome 9. No conclusions could be drawn either about translocation of chromosome 9 material or about a possible difference in breakpoint in chromosome 22 when compared with six cases of 9;22 translocations similarly studied and previously reported. In addition, a more precise mapping of PGM1 was obtained, the gene being proximal to UMPK and the breakpoint in 1p32.     

The MLLT3 Gene Maps between D9S156 and D9S171 and Contains an Unstable Polymorphic Trinucleotide Repeat

MLLT3, one of the genes shown to be a translocation breakpoint partner for the acute lymphocytic leukemia (MLL) gene, has been mapped to 9p22. We have identified a polymorphic trinucleotide repeat within this gene that shows somatic instability. The inheritance pattern of this polymorphism in recombinant individuals from families previously typed for other chromosome 9 markers indicates that the gene lies in the interval bounded by D9S156 and D9S171.     

A common breakpoint on 11q23 in carriers of the constitutional t(11;22) translocation

Structural chromosomal rearrangements occur commonly in the general population. Individuals that carry a balanced translocation are at risk of having unbalanced offspring; therefore, the frequency of translocations in couples with recurrent spontaneous abortions is higher than that in the general population. The constitutional t(11;22) translocation is the most common recurrent non-Robertsonian translocation in humans and may serve as a model to determine the mechanism that causes recurrent meiotic translocations. We previously localized the t(11;22) translocation breakpoint to a region on 22q11 within a low-copy repeat, termed "LCR22." To define the breakpoint on 11q23 and to ascertain whether this region shares homology with LCR22 sequences, we performed haplotype analysis on patients with der(22) syndrome. We found that the breakpoint on 11q23 occurred between two genetic markers, D11S1340 and APOC3-tetra, both being present within a single bacterial-artificial-chromosome clone. To determine whether the breakpoint occurred within the same region among a larger set of carriers, we performed FISH mapping studies. The breakpoints were all within the same clone, suggesting that this region may harbor sequences that are prone to breakage. We narrowed the breakpoint interval, in both derivative chromosomes from two unrelated carriers, to a 190-bp, AT-rich repeat, which indicates that this repeat may mediate recombination events on chromosome 11. Interestingly, the LCR22s harbor AT-rich repeats, suggesting that this sequence motif may mediate recombination events in nonhomologous chromosomes during meiosis.     

Fine mapping and cloning of the breakpoint associated with Menkes syndrome in a female patient.

The gene responsible for Menkes syndrome has been assigned to Xq13 by a combination of comparative mapping and linkage analysis. A previous report has mapped the translocation breakpoint associated with the disease in a female patient to an interval delimited by PGK1 and a group of six more proximal Xq13 markers, including DXS56. We have characterized a number of PGK1- or DXS56-positive YACs, from which we have generated six new markers. One of them identifies a small overlap region between a PGK1-positive YAC and three DXS56-positive YACs, distal to the Menkes breakpoint. A 560-kb region covered by a DXS56-positive YAC has been restriction-mapped and subcloned, disclosing a 187-kb MluI fragment astride the breakpoint. A probe mapping distal to the rearrangement in the same interval reveals altered PGFE fragments in a hybrid constructed from the translocation patient's DNA. We describe the development of a cosmid contig extending 150 kb from a nearby CpG island across the breakpoint. This contig includes four adjacent clones displaying cross-specific hybridization.     

Closing in on the Rieger syndrome gene on 4q25: mapping translocation breakpoints within a 50-kb region.

Rieger syndrome (RGS) is an autosomal dominant disorder of morphogenesis affecting mainly the formation of the anterior eye chamber and of the teeth. RGS has been localized to human chromosome 4q25 by linkage to epidermal growth factor (EGF). We have constructed a detailed physical map and a YAC contig of the genomic region encompassing the EGF locus. Using FISH, several YACs could be shown to cross the breakpoint in two independent RGS patients with balanced 4q translocations. Alu- and LINE-fragmentation of a 2.4-Mb YAC generated a panel of shorter YACs ranging in size from 2.4 Mb to 75 kb. Several fragmentation YACs were subcloned in cosmids, which were mapped to specific subregions of the original YAC by hybridization to the fragmentation panel to further refine the localization of the translocation breakpoints, allowing mapping of the breakpoints to within the most-telomeric 200 kb of the original 2.4-Mb YAC. FiberFISH of cosmids located in this 200-kb region mapped the two translocation breakpoints within a 50-kb region approximately 100-150 kb centromeric to D4S193, significantly narrowing down the candidate region for RGS. The mapping data and resources reported here should facilitate the identification of a gene implicated in Rieger syndrome.     

Molecular characterisation of the t(1;15)(p22;q22) translocation in the prostate cancer cell line LNCaP

Although chromosome translocations are well-documented recurrent events in hematological malignancies and soft tissue sarcomas, their significance in carcinomas is less clear. We report here the molecular characterization of the reciprocal translocation t(1;15)(p22;q22) in the prostate carcinoma cell line, LNCaP. The chromosome 1 breakpoint was localized to a single BAC clone, RP11-290M5, by sequential FISH analysis of clones selected from the NCBI chromosome 1 map. This was further refined to a 580-bp region by Southern blot analysis. A 2.85-kb fragment spanning the der(1) breakpoint was amplified by long-range inverse PCR. The breakpoint on chromosome 1 was shown to lie between the CYR61 and the DDAH1 genes with the der(1) junctional sequence linking the CYR61 gene to the TSPAN3 (TM4SF8) gene on chromosome 15. Confirmatory PCR and FISH mapping of the der(15) showed loss of chromosome material proximal to the breakpoint on chromosome 15, containing the PSTPIP1 and RCN2 genes. On the available evidence we conclude that this translocation does not result in an in-frame gene fusion. Comparative expressed sequence hybridization (CESH) and comparative genomic hybridization (CGH) analysis, showed relative down-regulation of gene expression surrounding the breakpoint, but no gross change in genomic copy number. Real-time quantitative RT-PCR for genes around the breakpoint supported the CESH data. Therefore, here we may have revealed a gene down-regulation mechanism associated with a chromosome translocation, either through small deletion at the breakpoint or through another means of chromosome domain related gene regulation.     

Breakpoints of t(4;11) translocations in the human MLL and AF4 genes in ALL patients are preferentially clustered outside of high-affinity matrix attachment regions.

Chromosomal translocations t(4;11) are based on illegitimate recombinations between the human MLL and AF4 genes, and are associated with high-risk acute leukemias of infants and young children. Here, the question was asked, whether a correlation exists between the location of translocation breakpoints within both genes and the location of S/MARs. In "halo mapping experiments" (to define SARs), about 20 kb of MLL DNA was found to be attached to the nuclear matrix. Similar experiments performed for the translocation partner gene AF4 revealed that SARs are spanning nearly the complete breakpoint cluster region of the AF4 gene. By using short DNA fragments in "scaffold reassociation experiments" (to define MARs), similar results were obtained for both genes. However, Distamycin A competition experiments in combination with "scaffold reassociation experiments" revealed specific differences in the affinity of each tested DNA fragment to bind the isolated nuclear matrix proteins. When the latter data were compared with the known location of chromosomal breakpoints for both genes, an unexpected correlation was observed. DNA areas with strong MAR affinity contained fewer translocation breakpoints, while areas with weak or absent MAR affinity showed a higher density of chromosomal breakpoints.     

Physical mapping and cloning of a translocation in sugar beet (Beta vulgaris L) carrying a gene for nematode (Heterodera schachtii) resistance from B. procumbens

Two diploid (2n=18) sugar beet (Beta vulgaris L.) lines which carry monogenic traits for nematode (Heterodera schachtii Schm.) resistance located on translocations from the wild beet species Beta procumbens were investigated. Short interspersed repetitive DNA elements exclusively hybridizing with wild beet DNA were found to be dispersed around the translocations. The banding pattern as revealed by genomic Southern hybridization was highly conserved among translocation lines of different origins indicating that the translocations are not affected by recombination events with sugar beet chromosomes. Physical mapping revealed that the entire translocation is represented by a single Sal I fragment 300 kb in size. A representative YAC (yeast artifical chromosome) library consisting of approximately 13,000 recombinant clones (2.2 genome equivalents) with insert sizes ranging between 50 and 450 kb and an average of 130kb has been constructed from the resistant line A906001. Three recombinant YACs were isolated from this library using the wild beet-specific repetitive elements as probes for screening. Colinearity between YAC inserts and donor DNA was confirmed by DNA fingerprinting utilizing these repetitive probes. The YACs were arranged into two contigs with a total size of 215 kb; these represent a minimum of 72% of the translocation.     

Genomic DNA of leukemic patients: target for clinical diagnosis of MLL rearrangements

Genomic DNA is the optimal resource to analyze questions concerning genetic changes that are related to oncogenesis. This article tries to summarize recent efforts to analyze chromosomal changes that trigger the development of human acute myeloid and lymphoblastic leukemias. The aim of this study was to establish an universal method that enables the identification and characterization of chromosomal translocations of the human MLL gene at the genomic nucleotide level. Chromosomal translocations of the MLL gene are the result of illegitimate recombination events in hematopoietic stem or precursor cells, strictly associated with the onset of highly malignant leukemic diseases. The applied technology was able to identify specific fusion alleles that were generated by chromosomal translocations, chromosomal deletions, chromosomal inversions and partial tandem duplications. Moreover, it allowed us to investigate even highly complex genetic changes by applying systematic breakpoint analyses. On the basis of these analyses, patient-specific molecular markers were established that turned out to be a very good source for monitoring minimal residual disease (MRD). MRD analyses control the efficiency and efficacy of current treatment protocols and have become a very sensitive molecular tool to monitor therapeutic success or failure in individual leukemia patients.     

Etoposide metabolites enhance DNA topoisomerase II cleavage near leukemia-associated MLL translocation breakpoints

Chromosomal breakage resulting from stabilization of DNA topoisomerase II covalent complexes by epipodophyllotoxins may play a role in the genesis of leukemia-associated MLL gene translocations. We investigated whether etoposide catechol and quinone metabolites can damage the MLL breakpoint cluster region in a DNA topoisomerase II-dependent manner like the parent drug and the nature of the damage. Cleavage of two DNA substrates containing the normal homologues of five MLL intron 6 translocation breakpoints was examined in vitro upon incubation with human DNA topoisomerase IIalpha, ATP, and either etoposide, etoposide catechol, or etoposide quinone. Many of the same cleavage sites were induced by etoposide and by its metabolites, but several unique sites were induced by the metabolites. There was a preference for G(-1) among the unique sites, which differs from the parent drug. Cleavage at most sites was greater and more heat-stable in the presence of the metabolites compared to etoposide. The MLL translocation breakpoints contained within the substrates were near strong and/or stable cleavage sites. The metabolites induced more cleavage than etoposide at the same sites within a 40 bp double-stranded oligonucleotide containing two of the translocation breakpoints, confirming the results at a subset of the sites. Cleavage assays using the same oligonucleotide substrate in which guanines at several positions were replaced with N7-deaza guanines indicated that the N7 position of guanine is important in metabolite-induced cleavage, possibly suggesting N7-guanine alkylation by etoposide quinone. Not only etoposide, but also its metabolites, enhance DNA topoisomerase II cleavage near MLL translocation breakpoints in in vitro assays. It is possible that etoposide metabolites may be relevant to translocations.     

Refinement of a Translocation Breakpoint Associated with Blepharophimosis–Ptosis–Epicanthus Inversus Syndrome to a 280-kb Interval at Chromosome 3q23

Blepharophimosis syndrome (BPES) is an autosomal dominant disorder of craniofacial development, the features of which include blepharophimosis, ptosis, and epicanthus inversus. Although it has been suggested that BPES is genetically heterogeneous, a major locus for this condition resides at chromosome 3q23. We have previously mapped a translocation breakpoint associated with BPES to the D3S1316–D3S1615 interval. The markers in this region have subsequently been shown to lie in a different order, with the BPES locus mapping to the 1-cM D3S1576 and D3S1316 interval. In the current investigation, a physical map, consisting of 60 yeast artificial chromosome (YAC) clones and 1 bacterial artificial chromosome, that spans this region has been constructed. Ten expressed sequence tags and the cellular retinol-binding protein I locus have been mapped to the contig. YAC end isolation has led to the creation of novel STSs that have been used to reduce the size of the BPES critical region to a 280-kb interval, which has been cloned in two nonchimeric YACs.     

Local gene density predicts the spatial position of genetic loci in the interphase nucleus

Specific chromosomal translocations are hallmarks of many human leukemias. The basis for these translocation events is poorly understood, but it has been assumed that spatial positioning of genes in the nucleus of hematopoietic cells is a contributing factor. Analysis of the nuclear 3D position of the gene MLL, frequently involved in chromosomal translocations and five of its translocation partners (AF4, AF6, AF9, ENL and ELL), and two control loci revealed a characteristic radial distribution pattern in all hematopoietic cells studied. Genes in areas of high local gene density were found positioned towards the nuclear center, whereas genes in regions of low gene density were detected closer to the nuclear periphery. The gene density within a 2 Mbp window was found to be a better predictor for the relative positioning of a genomic locus within the cell nucleus than the gene density of entire chromosomes. Analysis of the position of MLL, AF4, AF6 and AF9 in cell lines carrying chromosomal translocations involving these genes revealed that the position of the normal genes was different from that of the fusion genes, and this was again consistent with the changes in local gene density within a 2 Mbp window. Thus, alterations in gene density directly at translocation junctions could explain the change in the position of affected genes in leukemia cells.     

High-resolution, human–bovine comparative mapping based on a closed YAC contig spanning the bovine mh locus

A closed YAC contig spanning the mh locus was assembled by STS content mapping with seven microsatellite markers, eight genes or EST, and nine STS corresponding to YAC ends. The contig comprises 27 YACs, has an average depth of 4.3 YACs, and spans an estimated 1.2 Mb. A linkage map was constructed based on five of the microsatellite markers anchored to the contig and shown to span 7 cM, yielding a ratio of 160 kb/1 cM for the corresponding chromosome region. Comparative mapping data indicate that the constructed contig spans an evolutionary breakpoint connecting two chromosome segments that are syntenic but not adjacent in the human. Consolidation of human gene order by means of whole genome radiation hybrids and its comparison with the bovine order as inferred from the contig confirm conservation of gene order within segments. Received: 6 August 1998 / Accepted: 28 October 1998     

Topoisomerase II and the etiology of chromosomal translocations

Acute leukemias with balanced chromosomal translocations, protean morphologic and immunophenotypic presentations but generally shorter latency and absence of myelodysplasia are recognized as a complication of anti-cancer drugs that behave as topoisomerase II poisons. Translocations affecting the breakpoint cluster region of the MLL gene at chromosome band 11q23 are the most common molecular genetic aberrations in leukemias associated with the topoisomerase II poisons. These agents perturb the cleavage-religation equilibrium of topoisomerase II and increase cleavage complexes. One model suggests that this damages the DNA directly and leads to chromosomal breakage, which may result in untoward DNA recombination in the form of translocations. This review will summarize the evidence for topoisomerase II involvement in the genesis of translocations and extension of the model to acute leukemia in infants characterized by similar MLL translocations.