Systematic yeast synthetic lethal and synthetic dosage lethal screens identify genes required for chromosome segregation

Abstract

Accurate chromosome segregation requires the execution and coordination of many processes during mitosis, including DNA replication, sister chromatid cohesion, and attachment of chromosomes to spindle microtubules via the kinetochore complex. Additional pathways are likely involved because faithful chromosome segregation also requires proteins that are not physically associated with the chromosome. Using kinetochore mutants as a starting point, we have identified genes with roles in chromosome stability by performing genome-wide screens employing synthetic genetic array methodology. Two genetic approaches (a series of synthetic lethal and synthetic dosage lethal screens) isolated 211 nonessential deletion mutants that were unable to tolerate defects in kinetochore function. Although synthetic lethality and synthetic dosage lethality are thought to be based upon similar genetic principles, we found that the majority of interactions associated with these two screens were nonoverlapping. To functionally characterize genes isolated in our screens, a secondary screen was performed to assess defects in chromosome segregation. Genes identified in the secondary screen were enriched for genes with known roles in chromosome segregation. We also uncovered genes with diverse functions, such as RCS1, which encodes an iron transcription factor. RCS1 was one of a small group of genes identified in all three screens, and we used genetic and cell biological assays to confirm that it is required for chromosome stability. Our study shows that systematic genetic screens are a powerful means to discover roles for uncharacterized genes and genes with alternative functions in chromosome maintenance that may not be discovered by using proteomics approaches.

Fig. 1.

2D hierarchical clustering of the synthetic genetic interactions determined by SL analysis. (A) Rows display 116 query genes; columns indicate 84 deletion mutant array genes. The central kinetochore query mutant cluster is indicated by a green line, the inner kinetochore query mutant cluster is indicated by a blue line, and the skp1-3 query mutant is indicated by an orange line. The cluster trees organize query and deletion mutant array genes that show similar patterns of genetic interactions. The yellow box outlines a cluster of array genes identified in the central and inner kinetochore query mutant SL screens. (B) The yellow outline of cluster in A is expanded to allow visualization of specific array genes. Clustered array genes are indicated by red lines (1, central kinetochore; 2, spindle checkpoint; 3, sister chromatid cohesion; 4, GimC/Prefoldin complex).

Fig. 2.

2D hierarchical clustering of the synthetic genetic interactions determined by SDL analysis. (A) Rows, nine SDL screens conducted at the indicated temperature; columns, 141 deletion mutant array genes. Blue indicates SDL interactions, and red indicates SDS interactions. (B) Yellow outline of cluster in A is expanded to allow visualization of specific array genes.

Fig. 3.

Rcs1p colocalizes with the Ndc10p kinetochore protein. Shown is a chromosome spread analysis of a bilobed chromatin mass carrying either Myc-tagged Cnn1p (YPH1733) or Rcs1p (YPH1731). Spreads were imaged for DNA (DAPI), Ndc10p (Ndc10p), and Myc-tagged protein (Myc). The preimmune panel is a control for the Ndc10p polyclonal antibodies. In the merged images (Overlay), DNA is colored blue, Ndc10p is colored green, and Myc-tagged proteins are colored red. Overlap of Ndc10p and Myc signal appears yellow. In 100 chromatin masses examined, 85% of single chromatin masses showed overlap between Rcs1p signal and Ndc10p signal, and 80% of bilobed chromatin masses showed overlap between Rcs1p and at least one Ndc10p signal.

Fig. 4.

Interaction of Rcs1p with the inner kinetochore protein Cbf1p. (A) Rcs1p interacts with Cbf1p in a yeast two-hybrid assay. Amino acids 1–413 of Rcs1p were fused to the Gal4 DNA-binding domain (Gal4BD), and full-length Cbf1p or amino acids 204–351 of Cbf1p were fused to the Gal4 activation domain (Gal4AD). Cbf1p (204–351) is the original clone isolated in the Rcs1p two-hybrid screen. Gal4BD and Gal4AD fusions were expressed either alone or together in PJ69-4A cells, and quantitative β-galactosidase assays were performed (see Materials and Methods). β-Galactosidase values represent the averages of at least two independent experiments with three independent colonies, and error bars indicate the standard deviations. (B) cbf1Δ rcs1Δ double mutants exhibit reduced fitness compared with either single mutant. An agar plate containing five dissected tetrads from a mating of an rcs1Δ mutant (YPH1735) to a cbf1Δ mutant (YPH792) is shown. The plate was grown at 25°C for 3 days. Arrows indicate cbf1Δ rcs1Δ double mutants.