A Functional Link Between Bir1 and the Saccharomyces cerevisiae Ctf19 Kinetochore Complex Revealed Through Quantitative Fitness Analysis

Abstract

The chromosomal passenger complex (CPC) is a key regulator of eukaryotic cell division, consisting of the protein kinase Aurora B/Ipl1 in association with its activator (INCENP/Sli15) and two additional proteins (Survivin/Bir1 and Borealin/Nbl1). Here, we report a genome-wide genetic interaction screen in Saccharomyces cerevisiae using the bir1-17 mutant, identifying through quantitative fitness analysis deletion mutations that act as enhancers and suppressors. Gene knockouts affecting the Ctf19 kinetochore complex were identified as the strongest enhancers of bir1-17, while mutations affecting the large ribosomal subunit or the mRNA nonsense-mediated decay pathway caused strong phenotypic suppression. Thus, cells lacking a functional Ctf19 complex become highly dependent on Bir1 function and vice versa. The negative genetic interaction profiles of bir1-17 and the cohesin mutant mcd1-1 showed considerable overlap, underlining the strong functional connection between sister chromatid cohesion and chromosome biorientation. Loss of some Ctf19 components, such as Iml3 or Chl4, impacted differentially on bir1-17 compared with mutations affecting other CPC components: despite the synthetic lethality shown by either iml3∆ or chl4∆ in combination with bir1-17, neither gene knockout showed any genetic interaction with either ipl1-321 or sli15-3 Our data therefore imply a specific functional connection between the Ctf19 complex and Bir1 that is not shared with Ipl1.

Keywords: Bir1; Chromosome biorientation; Iml3-Chl4 complex; Kinetochore; yeast.

Figure 1

Fitness plot of bir1-17 double mutants at 37°. Following four replicate crosses of bir1-17 with the yeast genome knockout collection, quantitative fitness analysis of each bir1-17 yfg∆ (“your favorite gene deletion”) strain was carried out at 37° and mean fitness plotted against the mean fitness observed from eight replicates of a control cross between a ura3∆ strain and the knockout collection. Gene deletions that significantly enhanced (blue triangles) or suppressed (red triangles) the growth defect of a bir1-17 strain are indicated, with all other nonsignificant deletions indicated as gray circles. A significant interaction was defined as one with a q-value (FDR-corrected p-value; see Addinall et al. 2011) ≤ 0.05, with enhancers having a negative genetic interaction strength (GIS) and suppressors having a positive GIS. The line of equal growth (gray dashed) and a population model of expected fitness under the assumption of genetic independence (solid gray; a regression line based on all the data points) are also indicated. The blue lines show the average position of his3∆ strains as a proxy for wild-type growth.

Figure 2

Comparison of the strong negative genetic interactors of ipl1-321, bir1-17, and mcd1-1 revealed by SGA analysis. Genes showing synthetic lethality or strong negative genetic interaction with two or more of ipl1-321, bir1-17, or mcd1-1 are connected to the relevant query mutations by lines that are color-coded according to the number of shared interactions as follows: yellow, negative genetic interactors shared by ipl1-321 and bir1-17; dark blue, negative genetic interactors shared by ipl1-321 and mcd1-1; light blue, negative genetic interactors shared by bir1-17 and mcd1-1; and red, negative genetic interactors shared by all three query genes. The gray box with dashed outline encloses genes encoding components of the Ctf19 complex. Strong negative genetic interactors shown in the diagram were defined as follows: ipl1-321, all genes identified by Ng et al. (2009) as ipl1-321 negative genetic interactors together with additional genes listed in SGD (Cherry et al. 2012, queried December 2016); mcd1-1, all genes identified by Ng et al. (2009) or present in the DRYGIN database (Koh et al. 2010, queried December 2016) as mcd1-1 negative genetic interactors, together with additional genes listed in SGD (Cherry et al. 2012, queried December 2016); bir1-17, all strong negative enhancers identified in this work at any of the three screening temperatures that were shared with at least one of the other two mutations (ipl1-321 or mcd1-1). Note that we find that deletion of CTF19 is synthetic lethal with ipl1-321 as shown (100% inviability of 25 ctf19::KanMX ipl1-321 spores in 31 tetrads from a W303 background ctf19::KanMX × ipl1-321 cross where single ctf19::KanMX and ipl1-321 segregants showed 100% and >97% viability, respectively). IRC15 is included within the Ctf19 complex because its knockout also affects CTF19: see Confirmation of the negative genetic interactions between bir1-17 and the Ctf19, Ctf8-Ctf18-Dcc1 and Csm3-Mrc1-Tof1 complexes in W303 for details.

Figure 3

Genetic interactions of iml3∆ and chl4∆ with bir1-17, ipl1, and sli15 mutations in the W303 genetic background. (A) iml3∆ and chl4∆ each show synthetic lethality with bir1-17. Progeny from five tetrads are shown, indicating the relevant genotypes of viable progeny and the deduced genotypes of inviable progeny. (B) iml3∆ and chl4∆ are viable when combined with ipl1-2, ipl1-321, sli15-3, and sli15∆2-228. Equivalent 10-fold dilutions of representative single and double mutants were grown at 26 or 35° for 2 d. Although the strong temperature-sensitive phenotype of ipl1-2, ipl1-321, and sli15-3 is clearly evident at 35°, all double mutant combinations involving these alleles grew normally at 26°. (C) iml3∆ and chl4∆ are viable when combined with either sli15∆2-228 or nbl1-6 but show synthetic negative genetic interaction with both. While iml3∆ sli15∆2-228 double mutants grew normally at 26°, chl4∆ sli15∆2-228 grew poorly, and both iml3∆ sli15∆2-228 and chl4∆ sli15∆2-228 strains showed temperature sensitivity at 35° in comparison to the corresponding single mutant strains. iml3∆ nbl1-6 and chl4∆ nbl1-6 strains were also viable, but unlike the three individual mutant strains, were unable to grow at 35°.

Figure 4

Accumulation of cohesin at the centromere and in the pericentromeric region is not defective in bir1-17. Analysis of Mcd1-6HA in wild-type, bir1-17, and chl4∆ cells, first synchronized in G1 with α-factor at 25° and then released for 3 h at temperatures either permissive (25°; P) or restrictive (37°; R) for bir1-17 in the presence of nocodazole and benomyl to induce a metaphase arrest. (A) Mcd1 association with the centromeric (CEN), pericentromeric (PERICEN), and arm (ARM) regions of chromosome IV in the two mutant strains relative to the wild-type strain was examined by chromatin immunoprecipitation (ChIP) using an anti-HA antibody. The mean of three independent experiments is shown with error bars indicating the SE. (B) FACS analysis of DNA content confirming synchronization in mitotic metaphase at either temperature.

Figure 5

Boosting Sgo1 expression does not overcome the requirement for Chl4 and Iml3 in bir1-17 strains. Strains with the indicated genotypes (in a TOR1-1 background) were grown on YPAD medium containing 2% raffinose and 2% galactose to induce expression of GAL-SGO1 where present, either in the absence (left panels) or presence (right panels) of 10 µg/ml rapamycin to induce nuclear exclusion of Iml3 or Chl4. Plates were photographed after 2 d of growth at 25°. Several other independent isolates of each ura3::GAL-SGO1 strain showed the same properties.