Functional Genomics Approach to Identify Novel Breast Cancer Gene Targets in Yeast

摘要

We selected Saccharomyces cerevisiae mutants from the diploid deletion collection that suppressed Gi arrest and lethality following heterologous expression of BRCAl. Expression of BRCAl was confirmed by immunofluorescence or Western blot analysis. We also screened the diploid deletion strains for altered sensitivity to the transcription inhibitor zymocin to identify those that also suppress BRCAl-induced lethality and are radiation sensitive. We identified conserved components of the CCR4 damage response network and many of these genes, including CCR4, DHHl, DEFi, HCMl, SPT4, SPT5, SUBi, YAF9, YAP3 and numerous components of the nuclear pore complex are required for transcription. These genes confer resistance to radiation and the transcription elongation inhibitor zymocin. We hypothesize that BRCAl and zymocin stall transcription elongation in Gl. When cells progress into S phase, the stalled transcription complexes serve as replication blocks that are processed into lethal DNA double-strand breaks. Consistent with this model is the observation that BRCAl enhanced plasmid degradation and loss in WT as compared to SPT4 deleted cells. Using co-immunoprecipitation (but not two hybrid analysis), we determined that Spt4p (and Dhhlp) physically interact with BRCAl in a complex while the highly conserved human orthologue of Dhhlp (DDX6) physically interacts with BRCAl in human cells.