Biomedical subjects
Publications and source records attributed to Michelle Debatisse.
The origins of gene amplifications in mammalian cells have been difficult to analyze because of secondary genome rearrangements. Recent studies in budding yeast, including in this issue of Cell, have provided new insights into the role of palindromic sequences in gene amplification.
Common fragile sites (CFS) are evolutionary conserved loci where damage appears recurrently upon treatments perturbing DNA synthesis. Although long studied, the mechanisms underlying CFS fragility are still incompletely understood and CFS function is unknown. We have mapped most of them at the junction of chromosomal bands replicating at different times in S phase, indicating that specific replication programs take place at CFS. In good agreement with this finding, we obtained results suggesting that CFS remain incompletely replicated up to late G(2), even in cells that went unperturbed through S phase. The recent demonstration that the function of ATR and its downstream targets are crucial to CFS stability may thereby indicate that mitotic onset is delayed until completion of their replication. Altogether, available results now suggest that CFS constitute integral "cis" components of the G(2)-M checkpoint.
Various studies suggest a tight relationship between chromosome rearrangements driving tumor progression and breaks at loci called common fragile sites. Most of these sites are induced after perturbation of the replication dynamics, notably by aphidicolin treatment. We have mapped the majority of these sites to the interface of R and G bands, which calls into question the previous assignment of aphidicolin-sensitive sites to R bands. This observation suggests that most of them correspond to loci that ensure the transition between early and late replicating domains. We show that calyculin A, which triggers chromosome condensation at any phase of the cell cycle but does not markedly impair replication, induces damage in the chromosomes of human lymphocytes treated in G(2) but not in G(1) phase. We demonstrate that these lesions colocalize with those induced by aphidicolin treatment. Hence, common fragile site stability is compromised, whether aphidicolin delays replication or calyculin A advances condensation. We also show that, in cells that go through an unperturbed S phase, completion of their replication and/or replication-associated chromatin reorganization occur all along the G(2) phase, which may explain their inability to condense properly after calyculin A treatment during this phase of the cell cycle.
Fragile sites are classified as common or rare depending on their occurrence in the populations. While rare sites are mainly associated with inherited diseases, common sites have been involved in somatic rearrangements found in the chromosomes of cancer cells. Here we study a mouse locus containing the ionotropic glutamate receptor delta 2 (grid2) gene in which spontaneous chromosome rearrangements occur frequently, giving rise to mutant animals in inbred populations. We identify and clone common fragile sites overlapping the mouse grid2 gene and its human ortholog GRID2, lying respectively at bands 6C1 and 4q22 in a 7-Mb-long region of synteny. These results show a third example of orthologous common sites conserved at the molecular level, and reveal an unexpected link between an inherited disease and an aphidicolin-sensitive region. Recurrent deletions of subregions of band 4q22 have been previously described in human hepatocellular carcinomas. This 15-Mb-long region appears precisely centered on the site described here, which strongly suggests that it also plays a specific role in hepatic carcinogenesis.
Amplification of the epidermal growth factor receptor gene on double minutes is recurrently observed in cells of advanced gliomas, but the structure of these extrachromosomal circular DNA molecules and the mechanisms responsible for their formation are still poorly understood. By using quantitative PCR and chromosome walking, we investigated the genetic content and the organization of the repeats in the double minutes of seven gliomas. It was established that all of the amplicons of a given tumor derive from a single founding extrachromosomal DNA molecule. In each of these gliomas, the founding molecule was generated by a simple event that circularizes a chromosome fragment overlapping the epidermal growth factor receptor gene. In all cases, the fusion of the two ends of this initial amplicon resulted from microhomology-based nonhomologous end-joining. Furthermore, the corresponding chromosomal loci were not rearranged, which strongly suggests that a postreplicative event was responsible for the formation of each of these initial amplicons.
Accurate duplication of genetic material is central to cell proliferation. In eukaryotes, S phase is tightly controlled during development. For example, initiation events occur at random sites in early embryos but later appear restricted to preferred DNA regions. Epigenetic changes depending on chromatin organization and/or availability of specific factors likely control origin choice. By using the dynamic molecular combing technology, coupled with specific labeling of neo-synthesized DNA and FISH, we recently demonstrated that the efficiency and spacing of initiation sites are still flexible in mammalian somatic cells, and strongly rely on nucleotide availability. In all conditions, initiation events appear confined to short AT-rich sequences previously identified as matrix attachment regions, which suggests a direct involvement of these features in origin specification. Functional relationships between matrix anchorage and origin selection are discussed.
Selection of active origins and regulation of interorigin spacing are poorly understood in mammalian cells. Using tricolor analysis of combed DNA molecules, we studied an amplified locus containing the known origin, oriGNAI3. We visualized replication firing events at this and other discrete regions and established a strict correlation between AT richness and initiation sites. We found that oriGNAI3 is the prominent origin of the domain, the firing of which correlates with silencing of neighboring sites and establishes large interorigin distances. We demonstrate that cells reversibly respond to a reduction in nucleotide availability by slowing the rate of replication fork progression; in addition, the efficiency of initiation at oriGNAI3 is lowered while other normally dormant origins in the region are activated, which results in an overall increase in the density of initiation events. Thus, nucleotide pools are involved in the specification of active origins, which in turn defines their density along chromosomes.
Gene amplification plays a critical role in tumor progression. Hence, understanding the factors triggering this process in human cancers is an important concern. Unfortunately, the structures formed at early stages are usually unavailable for study, hampering the identification of the initiating events in tumors. Here, we show that the region containing the PIP gene, which is overexpressed in 80% of primary and metastatic breast cancers, is duplicated in the breast carcinoma cell line T47D. The two copies are organized as a large palindrome, lying 'in loco' on one chromosome 7. Such features constitute the landmark of the breakage-fusion-bridge (BFB) cycle mechanism. In hamster cells selected in vitro to resist cytotoxic drugs, common fragile site (CFS) activation has been shown to trigger this mechanism. Here, we characterize FRA7I at the molecular level and demonstrate that it lies 2 Mb telomeric to the PIP gene and sets the distal end of the repeated sequence. Moreover, our results suggest that the BFB process was frozen within the first cycle by healing of the broken chromosome. T47D cells thus offer a unique opportunity to observe the earliest products of the BFB cycle mechanism. Our findings constitute the first evidence that this amplification mechanism can be initiated in vivo by fragile site activation.
Gene amplification is frequently associated with tumor progression, hence, understanding the underlying mechanisms is important. The study of in vitro model systems indicated that different initial mechanisms accumulate amplified copies within the chromosomes (hsr) or on extra-chromosomal elements (dmin). It has long been suggested that formation of dmin could also occur following hsr breakdown. In order to check this hypothesis, we developed an approach based on the properties of the I-SceI meganuclease, which induces targeted DNA double-strand breaks. A clone containing an I-SceI site, integrated by chance close to an endogenous dhfr gene locus, was used to select for methotrexate resistant mutants. We recovered clones in which the I-SceI site was passively co-amplified with the dhfr gene within the same hsr. We show that I-SceI-induced hsr breakdown leads to the formation of dmin and creates different types of chromosomal rearrangements, including inversions. This demonstrates, for the first time, a direct relationship between double-strand breaks and inversions. Finally, we show that activation of fragile sites by aphidicolin or hypoxia in hsr-containing cells also generates dmin and a variety of chromosomal rearrangements. This may constitute a valuable model to study the consequences of breaks induced in hsr of cancer cells in vivo.
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