Frequent chromosomal translocations induced by DNA double-strand breaks

The faithful repair of DNA damage such as chromosomal double-strand breaks (DSBs) is crucial for genomic integrity. Aberrant repair of these lesions can result in chromosomal rearrangements, including translocations, which are associated with numerous tumours. Models predict that some translocations arise from DSB-induced recombination in differentiating lymphoid cell types or from aberrant repair of DNA damage induced by irradiation or other agents; however, a genetic system to study the aetiology of these events has been lacking. Here we use a mouse embryonic stem cell system to examine the role of DNA damage on the formation of translocations. We find that two DSBs, each on different chromosomes, are sufficient to promote frequent reciprocal translocations. The results are in striking contrast with interchromosomal repair of a single DSB in an analogous system in which translocations are not recovered. Thus, while interchromosomal DNA repair does not result in genome instability per se, the presence of two DSBs in a single cell can alter the spectrum of repair products that are recovered.     

Aberrant rearrangement of the kappa light-chain locus involving the heavy-chain locus and chromosome 15 in a mouse plasmacytoma

The creation of a functional antibody gene requires the precise recombination of gene segments initially separated on the chromosome. Frequently errors occur in the process, resulting in the formation of a non-functional gene. The non-functional genes can be generated by incomplete rearrangements, frameshifts, or the use of pseudo V or J joining segments. It is likely that these aberrant rearrangements arise by the same mechanism as is used in generating functional genes, a process which we have suggested may involve unequal sister chromatid exchange. Aberrant rearrangements of immunoglobulin genes occur in normal lymphocytes and play a major part in allelic exclusion. However, it has recently been suggested that aberrant rearrangements involving immunoglobulin and non-immunoglobulin genes may be involved in tumorigenesis. This suggestion has been stimulated by the frequent occurrence of translocations involving chromosomes known to carry immunoglobulin genes in B-cell malignancies. The rearrangement of non-immunoglobulin DNA to the heavy-chain locus has recently been reported. Some aberrant rearrangements of the kappa locus appear to be due to rearrangements to sites that do not include the conventional sequence for V gene segment joining. Here we describe an aberrant kappa rearrangement that has led to the joining of DNA from chromosomes 15, 6 and 12, and so appears to be the result of chromosomal translocations or transpositions. As 15/6 or 15/12 translocations have frequently been found in mouse plasmacytomas (as have analogous translocations in human lymphocyte tumours) this aberrant kappa rearrangement may be unique to the plasmacytoma from which it was isolated.     

Chromosomal evolution in Saccharomyces

The chromosomal speciation model invokes chromosomal rearrangements as the primary cause of reproductive isolation. In a heterozygous carrier, chromosomes bearing reciprocal translocations mis-segregate at meiosis, resulting in reduced fertility or complete sterility. Thus, chromosomal rearrangements act as a post-zygotic isolating mechanism. Reproductive isolation in yeast is due to post-zygotic barriers, as many species mate successfully but the hybrids are sterile. Reciprocal translocations are thought to be the main form of large-scale rearrangement since the hypothesized duplication of the whole yeast genome 10(8) years ago. To test the chromosomal speciation model in yeast, we have characterized chromosomal translocations among the genomes of six closely related species in the Saccharomyces 'sensu stricto' complex. Here we show that rearrangements have occurred between closely related species, whereas more distant ones have colinear genomes. Thus, chromosomal rearrangements are not a prerequisite for speciation in yeast and the rate of formation of translocations is not constant. These rearrangements appear to result from ectopic recombination between Ty elements or other repeated sequences.     

ATM stabilizes DNA double-strand-break complexes during V(D)J recombination

The ATM (ataxia-telangiectasia mutated) protein kinase mediates early cellular responses to DNA double-strand breaks (DSBs) generated during metabolic processes or by DNA-damaging agents. ATM deficiency leads to ataxia-telangiectasia, a disease marked by lymphopenia, genomic instability and an increased predisposition to lymphoid malignancies with chromosomal translocations involving lymphocyte antigen receptor loci. ATM activates cell-cycle checkpoints and can induce apoptosis in response to DNA DSBs. However, defects in these pathways of the DNA damage response cannot fully account for the phenotypes of ATM deficiency. Here, we show that ATM also functions directly in the repair of chromosomal DNA DSBs by maintaining DNA ends in repair complexes generated during lymphocyte antigen receptor gene assembly. When coupled with the cell-cycle checkpoint and pro-apoptotic activities of ATM, these findings provide a molecular explanation for the increase in lymphoid tumours with translocations involving antigen receptor loci associated with ataxia-telangiectasia.     

DNA repair protein Ku80 suppresses chromosomal aberrations and malignant transformation

Cancer susceptibility genes have been classified into two groups: gatekeepers and caretakers. Gatekeepers are genes that control cell proliferation and death, whereas caretakers are DNA repair genes whose inactivation leads to genetic instability. Abrogation of both caretaker and gatekeeper function markedly increases cancer susceptibility. Although the importance of Ku80 in DNA double-strand break repair is well established, neither Ku80 nor other components of the non-homologous end-joining pathway are known to have a caretaker role in maintaining genomic stability. Here we show that mouse cells deficient for Ku80 display a marked increase in chromosomal aberrations, including breakage, translocations and aneuploidy. Despite the observed chromosome instabilities, Ku80-/- mice have only a slightly earlier onset of cancer. Loss of p53 synergizes with Ku80 to promote tumorigenesis such that all Ku80-/- p53-/- mice succumb to disseminated pro-B-cell lymphoma before three months of age. Tumours result from a specific set of chromosomal translocations and gene amplifications involving IgH and c-Myc, reminiscent of Burkitt's lymphoma. We conclude that Ku80 is a caretaker gene that maintains the integrity of the genome by a mechanism involving the suppression of chromosomal rearrangements.     

Role of genomic instability and p53 in AID-induced c-myc-Igh translocations

Chromosomal translocations involving the immunoglobulin switch region are a hallmark feature of B-cell malignancies. However, little is known about the molecular mechanism by which primary B cells acquire or guard against these lesions. Here we find that translocations between c-myc and the IgH locus (Igh) are induced in primary B cells within hours of expression of the catalytically active form of activation-induced cytidine deaminase (AID), an enzyme that deaminates cytosine to produce uracil in DNA. Translocation also requires uracil DNA glycosylase (UNG), which removes uracil from DNA to create abasic sites that are then processed to double-strand breaks. The pathway that mediates aberrant joining of c-myc and Igh differs from intrachromosomal repair during immunoglobulin class switch recombination in that it does not require histone H2AX, p53 binding protein 1 (53BP1) or the non-homologous end-joining protein Ku80. In addition, translocations are inhibited by the tumour suppressors ATM, Nbs1, p19 (Arf) and p53, which is consistent with activation of DNA damage- and oncogenic stress-induced checkpoints during physiological class switching. Finally, we demonstrate that accumulation of AID-dependent, IgH-associated chromosomal lesions is not sufficient to enhance c-myc-Igh translocations. Our findings reveal a pathway for surveillance and protection against AID-dependent DNA damage, leading to chromosomal translocations.     

Hypermutation of multiple proto-oncogenes in B-cell diffuse large-cell lymphomas.

Genomic instability promotes tumorigenesis and can occur through various mechanisms, including defective segregation of chromosomes or inactivation of DNA mismatch repair. Although B-cell lymphomas are associated with chromosomal translocations that deregulate oncogene expression, a mechanism for genome-wide instability during lymphomagenesis has not been described. During B-cell development, the immunoglobulin variable (V) region genes are subject to somatic hypermutation in germinal-centre B cells. Here we report that an aberrant hypermutation activity targets multiple loci, including the proto-oncogenes PIM1, MYC, RhoH/TTF (ARHH) and PAX5, in more than 50% of diffuse large-cell lymphomas (DLCLs), which are tumours derived from germinal centres. Mutations are distributed in the 5' untranslated or coding sequences, are independent of chromosomal translocations, and share features typical of V-region-associated somatic hypermutation. In contrast to mutations in V regions, however, these mutations are not detectable in normal germinal-centre B cells or in other germinal-centre-derived lymphomas, suggesting a DLCL-associated malfunction of somatic hypermutation. Intriguingly, the four hypermutable genes are susceptible to chromosomal translocations in the same region, consistent with a role for hypermutation in generating translocations by DNA double-strand breaks. By mutating multiple genes, and possibly by favouring chromosomal translocations, aberrant hypermutation may represent the major contributor to lymphomagenesis.     

Interplay of p53 and DNA-repair protein XRCC4 in tumorigenesis, genomic stability and development

XRCC4 is a non-homologous end-joining protein employed in DNA double strand break repair and in V(D)J recombination. In mice, XRCC4-deficiency causes a pleiotropic phenotype, which includes embryonic lethality and massive neuronal apoptosis. When DNA damage is not repaired, activation of the cell cycle checkpoint protein p53 can lead to apoptosis. Here we show that p53-deficiency rescues several aspects of the XRCC4-deficient phenotype, including embryonic lethality, neuronal apoptosis, and impaired cellular proliferation. However, there was no significant rescue of impaired V(D)J recombination or lymphocyte development. Although p53-deficiency allowed postnatal survival of XRCC4-deficient mice, they routinely succumbed to pro-B-cell lymphomas which had chromosomal translocations linking amplified c-myc oncogene and IgH locus sequences. Moreover, even XRCC4-deficient embryonic fibroblasts exhibited marked genomic instability including chromosomal translocations. Our findings support a crucial role for the non-homologous end-joining pathway as a caretaker of the mammalian genome, a role required both for normal development and for suppression of tumours.     

Human subtelomeres are hot spots of interchromosomal recombination and segmental duplication

Human subtelomeres are polymorphic patchworks of interchromosomal segmental duplications at the ends of chromosomes. Here we provide evidence that these patchworks arose recently through repeated translocations between chromosome ends. We assess the relative contribution of the principal mechanisms of ectopic DNA repair to the formation of subtelomeric duplications and find that non-homologous end-joining predominates. Once subtelomeric duplications arise, they are prone to homology-based sequence transfers as shown by the incongruent phylogenetic relationships of neighbouring sections. Interchromosomal recombination of subtelomeres is a potent force for recent change. Cytogenetic and sequence analyses reveal that pieces of the subtelomeric patchwork have changed location and copy number with unprecedented frequency during primate evolution. Half of the known subtelomeric sequence has formed recently, through human-specific sequence transfers and duplications. Subtelomeric dynamics result in a gene duplication rate significantly higher than the genome average and could have both advantageous and pathological consequences in human biology. More generally, our analyses suggest an evolutionary cycle between segmental polymorphisms and genome rearrangements.     

Spi-1 is a putative oncogene in virally induced murine erythroleukaemias

Retroviral insertional mutagenesis has been proposed as an efficient mechanism to turn on or to increase the expression of oncogenes in several avian or mammal models. Integration site studies of avian leukosis virus, murine leukaemia and murine mammary tumour viruses led to the coleutification of highly conserved genes whose expression is induced or increased during leukaemogenesis, probably through enhancer elements present in the retroviral long terminal repeats. This is reminiscent of the activation of cellular proto-oncogenes or putative oncogenes in numerous human tumours and leukaemias as a result of chromosomal translocations or DNA rearrangements. Here we report the characterization of a new putative oncogene isolated from a murine erythroleukaemia induced by the acute leukaemogenic retrovirus spleen focus forming virus (SFFV). An important and unusual feature of this genomic locus Spi-1 (for SFFV proviral integration) is that rearrangements due to SFFV integration were found in 95% of the erythroid tumours studied. A 4.0-kilobase messenger RNA was detected in rearranged tumours. No Spi-1 rearrangement was detected in other virally induced myeloid, lymphoid or erythroid tumours tested.     

A non-B-DNA structure at the Bcl-2 major breakpoint region is cleaved by the RAG complex

The causes of spontaneous chromosomal translocations in somatic cells of biological organisms are largely unknown, although double-strand DNA breaks are required in all proposed mechanisms. The most common chromosomal abnormality in human cancer is the reciprocal translocation between chromosomes 14 and 18 (t(14;18)), which occurs in follicular lymphomas. The break at the immunoglobulin heavy-chain locus on chromosome 14 is an interruption of the normal V(D)J recombination process. But the breakage on chromosome 18, at the Bcl-2 gene, occurs within a confined 150-base-pair region (the major breakpoint region or Mbr) for reasons that have remained enigmatic. We have reproduced key features of the translocation process on an episome that propagates in human cells. The RAG complex--which is the normal enzyme for DNA cleavage at V, D or J segments--nicks the Bcl-2 Mbr in vitro and in vivo in a manner that reflects the pattern of the chromosomal translocations; however, the Mbr is not a V(D)J recombination signal. Rather the Bcl-2 Mbr assumes a non-B-form DNA structure within the chromosomes of human cells at 20-30% of alleles. Purified DNA assuming this structure contains stable regions of single-strandedness, which correspond well to the translocation regions in patients. Hence, a stable non-B-DNA structure in the human genome appears to be the basis for the fragility of the Bcl-2 Mbr, and the RAG complex is able to cleave this structure.     

A chromosome 14 inversion in a T-cell lymphoma is caused by site-specific recombination between immunoglobulin and T-cell receptor loci

Specific chromosomal aberrations are associated with specific types of cancer (for review see ref. 1). The distinctiveness of each association has led to the belief that these chromosomal aberrations are clues to oncogenic events or to the state of differentiation in the malignant cell type. Malignancies of T lymphocytes demonstrate such an association characterized most frequently by structural translocations or inversions of chromosomes 7 and 14 (refs 7-9). Analyses of these chromosomally marked tumours at the molecular level may therefore provide insight into the aetiology of the cancers as well as the mechanisms by which chromosomes break and rejoin. Here we report such an analysis of the tumour cell line SUP-T1 derived from a patient with childhood T-cell lymphoma carrying an inversion of one chromosome 14 between bands q11.2 and q32.3, that is, inv(14) (q11.2; q32.2). These are the same chromosomal bands to which the T-cell receptor alpha-chain (14q11.2) and the immunoglobulin heavy-chain locus (14q32.3) have been assigned. Our analysis reveals that this morphological inversion of chromosome 14 was mediated by a site-specific recombination event between an immunoglobulin heavy-chain variable region (Ig VH) and a T-cell receptor (TCR) alpha-chain joining segment (TCR J alpha). S1 nuclease analysis shows that this hybrid gene is transcribed into poly(A)+ RNA.     

Carcinogen-specific mutation and amplification of Ha-ras during mouse skin carcinogenesis

Cellular proto-oncogenes can be activated by both point mutations and chromosomal translocations, suggesting that there may be a direct link between exposure to agents which damage DNA and genetic change leading to malignancy. Several groups have therefore analysed mutations found in cellular oncogenes of tumours induced by particular physical or chemical carcinogens. Here, we have analysed the molecular changes at different stages of carcinogenesis in mouse skin tumours induced by initiating and promoting agents. Over 90% of tumours, including premalignant papillomas, initiated with dimethylbenzanthracene (DMBA) have a specific A----T transversion at the second nucleotide of codon 61 of the Harvey-ras (Ha-ras) gene. The frequency of this mutation was dependent on the initiating agent used, but not on the promoter, suggesting that the mutation occurs at the time of initiation. The mutation was heterozygous in most papillomas tested, but was homozygous or amplified in some carcinomas. The development of further chromosomal changes at the c-Ha-ras gene locus is therefore a common feature of tumour progression.     

The human T-cell receptor alpha-chain gene maps to chromosome 14

The T-cell receptor for antigen has been identified as a disulphide-linked heterodimeric glycoprotein of relative molecular mass (Mr) 90,000 comprising an alpha- and a beta-chain. The availability of complementary DNA clones encoding mouse and human beta-chains has allowed a detailed characterization of the genomic organization of the beta-chain gene family and has revealed that functional beta-chain genes in T cells are generated from recombination events involving variable (V), diversity (D), joining (J) and constant (C) gene segments. Recently, cDNA clones encoding mouse and human alpha-chains have been described; the sequences of these clones have indicated that functional alpha-chain genes are also generated from multiple gene segments. It is possible that chromosomal translocations involving T-cell receptor alpha- and beta-chain genes have a role in T-cell neoplasms in much the same way as translocations involving immunoglobulin genes are associated with oncogenic transformation in B cells. In the latter case, the chromosomal localization of the immunoglobulin genes provided one of the first indications of the involvement of such translocations in oncogenic transformation. The chromosomal assignment of the alpha- and beta-chain genes may, therefore, provide equally important clues for T-cell neoplastic transformation. The chromosomal location of the mouse and human beta-chain gene family has been determined: the murine gene lies on chromosome 6 (refs 12, 13) whereas the human gene is located on chromosome 7 (refs 13, 14). Here we use a cDNA clone encoding the human alph-chain to map the corresponding gene to chromosome 14.     

Multiple pathways cooperate in the suppression of genome instability in Saccharomyces cerevisiae.

Gross chromosome rearrangements (GCRs), such as translocations, deletion of a chromosome arm, interstitial deletions and inversions, are often observed in cancer cells. Spontaneous GCRs are rare in Saccharomyces cerevisiae; however, the existence of mutator mutants with increased genome instability suggests that GCRs are actively suppressed. Here we show by genetic analysis that these genome rearrangements probably result from DNA replication errors and are suppressed by at least three interacting pathways or groups of proteins: S-phase checkpoint functions, recombination proteins and proteins that prevent de novo addition of telomeres at double-strand breaks (DSBs). Mutations that inactivate these pathways cause high rates of GCRs and show synergistic interactions, indicating that the pathways that suppress GCRs all compete for the same DNA substrates.     

Engineering evolution to study speciation in yeasts

The Saccharomyces 'sensu stricto' yeasts are a group of species that will mate with one another, but interspecific pairings produce sterile hybrids. A retrospective analysis of their genomes revealed that translocations between the chromosomes of these species do not correlate with the group's sequence-based phylogeny (that is, translocations do not drive the process of speciation). However, that analysis was unable to infer what contribution such rearrangements make to reproductive isolation between these organisms. Here, we report experiments that take an interventionist, rather than a retrospective approach to studying speciation, by reconfiguring the Saccharomyces cerevisiae genome so that it is collinear with that of Saccharomyces mikatae. We demonstrate that this imposed genomic collinearity allows the generation of interspecific hybrids that produce a large proportion of spores that are viable, but extensively aneuploid. We obtained similar results in crosses between wild-type S. cerevisiae and the naturally collinear species Saccharomyces paradoxus, but not with non-collinear crosses. This controlled comparison of the effect of chromosomal translocation on species barriers suggests a mechanism for the generation of redundancy in the S. cerevisiae genome.     

Cytokinesis failure generating tetraploids promotes tumorigenesis in p53-null cells

A long-standing hypothesis on tumorigenesis is that cell division failure, generating genetically unstable tetraploid cells, facilitates the development of aneuploid malignancies. Here we test this idea by transiently blocking cytokinesis in p53-null (p53-/-) mouse mammary epithelial cells (MMECs), enabling the isolation of diploid and tetraploid cultures. The tetraploid cells had an increase in the frequency of whole-chromosome mis-segregation and chromosomal rearrangements. Only the tetraploid cells were transformed in vitro after exposure to a carcinogen. Furthermore, in the absence of carcinogen, only the tetraploid cells gave rise to malignant mammary epithelial cancers when transplanted subcutaneously into nude mice. These tumours all contained numerous non-reciprocal translocations and an 8-30-fold amplification of a chromosomal region containing a cluster of matrix metalloproteinase (MMP) genes. MMP overexpression is linked to mammary tumours in humans and animal models. Thus, tetraploidy enhances the frequency of chromosomal alterations and promotes tumour development in p53-/- MMECs.     

Cell-type-specific replication initiation programs set fragility of the FRA3B fragile site

Common fragile sites have long been identified by cytogeneticists as chromosomal regions prone to breakage upon replication stress. They are increasingly recognized to be preferential targets for oncogene-induced DNA damage in pre-neoplastic lesions and hotspots for chromosomal rearrangements in various cancers. Common fragile site instability was attributed to the fact that they contain sequences prone to form secondary structures that may impair replication fork movement, possibly leading to fork collapse resulting in DNA breaks. Here we show, in contrast to this view, that the fragility of FRA3B--the most active common fragile site in human lymphocytes--does not rely on fork slowing or stalling but on a paucity of initiation events. Indeed, in lymphoblastoid cells, but not in fibroblasts, initiation events are excluded from a FRA3B core extending approximately 700 kilobases, which forces forks coming from flanking regions to cover long distances in order to complete replication. We also show that origins of the flanking regions fire in mid-S phase, leaving the site incompletely replicated upon fork slowing. Notably, FRA3B instability is specific to cells showing this particular initiation pattern. The fact that both origin setting and replication timing are highly plastic in mammalian cells explains the tissue specificity of common fragile site instability we observed. Thus, we propose that common fragile sites correspond to the latest initiation-poor regions to complete replication in a given cell type. For historical reasons, common fragile sites have been essentially mapped in lymphocytes. Therefore, common fragile site contribution to chromosomal rearrangements in tumours should be reassessed after mapping fragile sites in the cell type from which each tumour originates.