Discrete DNA integration events during Bridge-Induced Translocation generate partial chromosomal deletions and differential aneuploidy in Saccharomyces cerevisiae

Abstract

Chromosome aberrations and genome instability have a long history of being associated with human genetic diseases, including cancer. The feasibility to drastically reshape the genome with a single chromosomal translocation also gives this molecular event a powerful capacity to drive evolution. To date, numerous assays have been developed to study gross chromosomal rearrangements (GCRs), although the molecular mechanisms underlying GCRs remain unclear. Here, we have used the Bridge Induced Translocation (BIT) method to induced non-reciprocal translocations in Saccharomyces cerevisiae and to provide new insights into DNA integration mediated generation of chromosomal abberations and aneuploidy. We have devised a genetic system to monitor DNA integration events ensuing BIT in real time. Upon BIT induction using engineered linear DNA molecules, this system exploits the cellular homologous recombination machinery to allow reconstruction of individual fluorescent markers GFP and DsRed placed on chromosomes V and III, respectively. Our initial results confirm that targeted DNA integration into sequences placed on yeast chromosomes to allow BIT occurs infrequently in a two step process, resulting mostly in one-sided events scored as a single fluorescent signal. We have carried out BIT between chromosomes XV and IX in diploid wild type and mutant S. cerevisiae strains in an attempt to elucidate the molecular regulation of chromosomal bridging in yeast. In wild type cells, the induction of BIT events was intrinsically biased for preferential integration on chromosome IX. This integration bias was significantly altered in sgs1, mre11 and esc2 knockout backgrounds. We also noticed that a terminal 36Kbp fragment of chromosome IXL arising after BIT induction was lost not only from the translocation participant chromosome, but also from native chromosome IX. This loss was noticed to be more prevalent in the sgs1 and esc2 mutants in whom integration bias was also altered. A similar scenario held true for our observations in ku70 mutants. Our results suggest that DNA integration during BIT events is under redundant regulation, possibly during DNA replication, and nonreciprocal translocations arising as such may further lead to secondary aberrations when this regulation is compromised. We also describe at the molecular level, ten morphologically and physiologically different translocants ensuing from the induction of the same BIT event between chromosomes XVI and IX. We have demonstrated that despite their common origin from the integration of the same linear DNA construct, all ten translocation-carrying strains have different phenotypes, ability to sporulate, regulation of gene expression and morphology. Our observations provide insights on how heterogeneous phenotypic variations originate from the same initial genomic event. We show eight different ways in which yeast cells have dealt with a single initial event inducing translocation leading to variable aneuploidies in these cells. Thus despite harboring common translocation breakpoints, we noticed a distinct and separable pattern of chromosomal rearrangements among these translocant strains. Our results are in agreement with the formation of complex rearrangements and abnormal karyotypes described in many leukemia patients, thus confirming the modelistic value of the yeast BIT system for mammalian cells.