A Functional Genomics Approach to Idenitfy Novel Breast Cancer Gene Targets in Yeast
Final rept. 15 Apr 2003-14 Apr 2006
DUKE UNIV DURHAM NC
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We selected Saccharomyces cerevisiae mutants that suppressed the G1 arrest and lethality observed following expression of BRCA1 in yeast. This genetic screen identified conserved interactive components of the CCR4 damage response network that participate in mRNA elongation, transport and decay. These genes confer resistance to IR and UV as well as transcription elongation inhibitors. Since transcription elongation is regulated by phosphorylation of the RNA polymeraseII RNAPII carboxy terminal domain CTD, we examined the status of RNAPII CTD phosphorylation following BRCA1 induction. BRCA1-induced cleavage of the phospho-CTD was observed in WT, but not in mutant suppressor strains. Both lethality and CTD cleavage was suppressed by cancer-related mutations in the BRCT domain of BRCA1 in WT yeast. Using co-immunoprecipitation, we determined that Spt4p and Dhh1p physically interact with BRCA1 in yeast, while the conserved human orthologs of Dhh1p Ddx6p and Spt5p interact with BRCA1 in human MCF7 cells following DNA damage. Immunofluorescent colocalization of BRCA1, Spt5p and Ddx6p at cytoplasmic P-bodies in MCF7 cells suggests that BRCA1 shuttling plays a key role in mRNA decay. We hypothesize that defects in BRCA1-mediated RNAPII CTD cleavage and cytoplasmic mRNA shuttling following DNA damage are critical early events in the onset of breast cancer.
- Genetic Engineering and Molecular Biology
- Medicine and Medical Research