Fitness profiling links topoisomeraseⅡregulation of centromeric integrity to doxorubicin resistance in fission yeast

摘要

OBJECTIVE To address the molecular implication of Top2 in the context of its interaction with doxorubicin resistance(DXR)genes.METHODS To perform epistasis analyses of top2 with 63genes representing doxorubicin resistance(DXR)genes in fission yeast.Fission yeast cells with single and double mutants were serial diluted and spotted to plates containing 15-75μg·mL-1 doxorubicin.Plates were scanned after 3and 7d.Cell growth was measured and compared between single mutants and double mutants.Nucleus morphology was performed by staining the cells with 4′,6-diamidino-2-phenylindole(DAPI)to observe chromosome segregation.Reverse transcriptase PCR(RT-PCR)was employed to visualize the changes in transcription level and evaluate the stability of chromatin structure.RESULTS Our findings revealed a subset that synergistically collaborate with Top2 to confer DXR and showed that the chromatin-regulating RSC and SAGA complexes act with Top2 in a cluster that is functionally distinct from the Ino80 complex.In various DXR mutants,doxorubicin hypersensitivity was unexpectedly suppressed by a concomitant top2 mutation.Several DXR proteins showed centromeric localization,and their disruption resulted in centromeric defects and chromosome missegregation.An additional top2 mutation could restore centromeric chromatin integrity,suggesting a counterbalance between Top2 and these DXR factors in conferring doxorubicin resistance.CONCLUSION The findings reported here show a functional interaction between Top2 and factors that confer genomic stability at centromeric chromatin under doxorubicin condition.Overall,this molecular basis for mitotic catastrophe associated with doxorubicin treatment will help to facilitate drug combinatorial usage in Doxorubicin-related chemotherapeutic regimens.