Yeast Screens Identify the RNA Polymerase II CTD and SPT5 as Relevant Targets of BRCA1 Interaction
Figure 1
Identification and characterization of genetic suppressors of BRCA1-induced lethality in yeast.
(A) Suppression of BRCA1-induced lethality following transformation of isogenic deletion strains with BRCA1 plasmid. BRCA1 suppressor strains were initially identified by sequencing rapid growing strains arising on GAL-URA plates following transformation of the diploid deletion strain pool with the GAL::BRCA1 expression plasmid. Putative BRCA1 suppressor strains identified from the pool were selected from the arrayed deletion strain library and transformed with the BRCA1 expression plasmid, grown to stationary phase in GLU and 2 ul aliquots were replica spotted onto GLU and GAL plates from serial 5-fold dilutions in water (arrows indicate direction of decreasing cell concentration). Typical results are depicted for seven strains. Five deletion strains (apt1Δ, nst1Δ, oca1Δ, ygr039WΔ and ylr412WΔ) failed to suppress BRCA1 lethality and were omitted from further study. Two deletion strains, bbc1Δ and ygr064WΔ (which deletes the N terminus of SPT4) were confirmed to suppress BRCA1-induced lethality. (B) Deletion strains defective in mRNA export suppress BRCA1-induced lethality. Deletions of the nuclear pore associated genes NUP2, POM34 and MLP2 (from deletion pool) and deletions of the IR resistance genes NUP120, NUP170, NUP188 as well as NUP133 (not shown, Table S1) suppress BRCA1 lethality. Relative plating efficiencies for colony formation on GAL vs GLU (GAL/GLU) are from Table S1. With the exception of nup84Δ, all IR sensitive nuclear pore defective strains significantly suppressed BRCA1-induced lethality. (C) Deletion of the transcriptional elongation genes SPT4 or CTK1 completely suppress BRCA1-induced lethality. Reduced colony forming ability on GAL (Galactose) vs GLU (Glucose) plates is completely suppressed by deletion of SPT4 or CTK1 when compared to WT strains containing the BRCA1 plasmid. Plates depicted are following 3 (WT and spt4Δ strains) and 4 days (ctk1Δ) incubation at 30°C. (D) Suppressor strains defective in transcription elongation express BRCA1. WT, spt4Δ, def1Δ and ctk1Δ strains containing the BRCA1 expression plasmid were induced in liquid GAL for the indicated times (hrs). Western blot has been probed with anti-BRCA1 antibody MS110. Full-length BRCA1 has an apparent MW of 220–250 kDa or larger (arrow). (E) Nuclear exclusion of BRCA1 is not responsible for suppression of BRCA1-induced lethality. In situ immunofluorescence of BRCA1 was determined in uninduced yeast cells (GLU) or induced for BRCA1 expression for 6 hours (GAL). Hoechst staining was used to position the nucleus. No partitioning defects were observed for BRCA1 in other nuclear pore mutants (nup2Δ, nup120Δ, nup133Δ, nup188Δ, pom34Δ or mlp2Δ; data not shown) that suppressed BRCA1 lethality. In all cells examined, BRCA1 was localized to both the nucleus and cytoplasm. Bar is 10 um. (F) Suppression of BRCA1-induced lethality is not due to second site suppressors. Diploid deletion strains (spt4Δ, ctk1Δ and def1Δ) containing the GAL::BRCA1 expression plasmid were “covered” with the corresponding gene expressed from a second selectable plasmid and assayed for the reinstatement of BRCA1 lethality by determining the relative plating efficiencies of colony forming ability on GAL vs GLU with selection for both plasmids. WT+BRCA1 (-HIS) is the relative survival of colony forming ability for the WT strain containing the GAL::BRCA1-HIS3 expression plasmid on GAL-HIS vs GLU-HIS medium. Error bars are +/− 1 SD about the mean and each bar represents 3-6 experiments.