QFA identifies gene deletions that interact with bir1-17

To identify enhancers and suppressors of bir1-17 that might indicate specific functional interactions, we used SGA technology to cross bir1-17 to the collection of ∼4200 viable systematic gene deletion strains, followed by QFA (Addinall et al. 2011) to identify suppressing or enhancing genetic interactions. bir1-17 was originally isolated in the W303 genetic background and confers a recessive, temperature-sensitive growth defect that is clearly evident at 37° (Makrantoni and Stark 2009). To investigate the best approach for identifying bir1-17 suppressors and enhancers, we first compared growth of the S288C background control and bir1-17 strains after arraying by either pinning or spotting. Figure S2 shows that when strains were arrayed by pinning, the fitness defect of bir1-17 was hard to detect even at higher incubation temperatures (Figure S2A). In contrast, when dilutions of the two strains were arrayed by spotting onto the screening plates then the fitness defect of bir1-17 was readily detectable at 37° (Figure S2B). We therefore carried out QFA analysis by spotting rather than pinning the arrays of control and bir1-17 double mutants generated by SGA.

Double bir1-17 yfg∆ mutants were generated at 23°. QFA was subsequently performed by spotting out and screening growth at 20, 27, and 37°, calculating the GIS and q-value (FDR-corrected p-value) for each double mutant to indicate the magnitude of the genetic interaction between bir1-17 and each yfg∆ gene knockout and its statistical significance, respectively (Tables S2–S4 in File S2; data ranked in ascending order of GIS, starting with the most negative interactions). Since bir1-17 cells spotted at 37° show a clear growth defect, we chose to focus primarily on this dataset. A q-value threshold of ≤0.05 was applied to identify a subset of statistically significant genetic interactions at each screening temperature. QFA data were summarized in the form of fitness plots, in which the fitness of each bir1-17 yfg∆ strain is plotted against the fitness of the corresponding control ura3∆ yfg∆ strain.

Figure 1 shows the fitness plot for bir1-17 yfg∆ mutants screened at 37°, indicating all statistically significant enhancers (negative GIS; blue triangles) and suppressors (positive GIS; red triangles). Table S5 and Table S8 in File S2 list the statistically significant bir1-17 enhancers and suppressors, respectively, identified by screening at 37°, while Table S6 and Table S7 in File S2 present the statistically significant enhancers at 20 and 27° for comparison. In the fitness plot, the dashed gray line indicates where points should lie when deletion mutants show identical fitness in combination with either bir1-17 or the control ura3∆ mutation (the line of equal growth). The regression line of the actual data points is indicated by the solid line. Downward displacement of this regression line away from the line of equal growth (Figure 1) is consistent with the temperature-sensitive phenotype of bir1-17 that was clearly evident following spotting out under QFA conditions (Figure S2), and is in contrast to the fitness plots from the 20 and 27° screens (Figure S3).

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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.

To look in an unbiased manner for relationships between genes identified as statistically significant enhancers and suppressors of bir1-17 at 37°, we searched for GO terms within the process, function, and component ontologies that showed significant enrichment in the enhancers and suppressors. We focused primarily on strong interactions (|GIS| ≥ 25; mapped onto the 37° fitness plot in Figure S4), but also searched using enhancers and suppressors with |GIS| ≥ 10 for comparison. These data are summarized in File S3 (enhancers) and File S4 (suppressors). We also manually examined the position on the 37° fitness plot of the members of each of the core protein complexes defined by Benschop et al. (2010), as a means of identifying consistent patterns of genetic interaction with bir1-17 that might reflect functional interactions between Bir1 and specific cellular processes. Table 2 summarizes the proportion of bir1-17 enhancers or suppressors identified at each screening temperature. Around 3% of yfg∆ knockouts strongly enhanced bir1-17 in the 37° screen (GIS ≤ −25), while 2.2% of knockouts strongly suppressed (GIS ≥ 25). This was in contrast to the 20 and 27° screens, where the proportion of strong interactions was far less.

Phenotypic enhancers of bir1-17

It was quickly apparent from looking at the bir1-17 enhancers identified at 37° (Table S5 in File S2) that knockouts of almost any of the nonessential components of the Ctf19 kinetochore complex (CHL4, CTF3, CTF19, IML3, IRC15, MCM16, MCM21, MCM22, and NKP1; see Biggins 2013) strongly enhanced bir1-17; only NKP2 was not identified as an enhancer. IRC15 is included here because of the overlap of its full-length deletion with CTF19, as discussed below. All of these knockouts except nkp1∆ fell into the strong enhancer category (i.e., GIS ≤ −25), and five out of the six strongest enhancers were members of the Ctf19 complex (Table S5 in File S2). Only MCM21 and CTF19, encoding the two nonessential members of the COMA subcomplex (Ame1 and Okp1 are both essential; De Wulf et al. 2003; Ortiz et al. 1999), were among the strongest enhancer of bir1-17 at all three screening temperatures. The other members of the Ctf19 complex were either weaker enhancers at 20° and 27° than at 37°, or in some cases, not identified as significant enhancers at all. It is striking that in contrast to the two lower temperature screens, the 37° screen placed knockouts of genes encoding all the Ctf19 complex components except NKP1 and NKP2 in a tight cluster at the lower right of the plot, indicating that the knockouts had little or no defect in the control (ura3∆) background, but in contrast had a very strong defect when combined with bir1-17 (see Figure S5). Thus, screening at 37° when double mutants are close to the bir1-17 maximum permissive temperature greatly assisted in identifying genetic interactions between the core components of the Ctf19 complex and bir1-17.

GO analysis of the strong bir1-17 enhancers (GIS ≤ −25) identified at 37° (see Table S5 in File S2) highlighted a number of specific GO terms concerned with chromosome segregation, sister chromatid cohesion, and microtubule-based processes, as well as more general terms including nuclear division and chromosome organization that encompassed many of the strongest negative genetic interactions (File S3). In addition to the Ctf19 complex, this analysis highlighted genes encoding all the S. cerevisiae kinesin-like proteins except KIP1 (i.e., KIP2, KIP3, KAR3, and CIN8), VIK1 and CIK1 (encoding Kar3-associated proteins; Manning et al. 1999), BIK1 (a plus-end tracking protein related to CLIP-170; Berlin et al. 1990; Blake-Hodek et al. 2010), TUB3 (α-tubulin), CIN1 (encoding a β-tubulin folding factor; Hoyt et al. 1990, 1997) and KAR9 (encoding a spindle positioning factor; Beach et al. 2000; Miller et al. 2000), although kar9∆ was inviable at 20 and 37° but was synthetic lethal with bir1-17 at 27°. bim1∆, removing another plus-end tracking protein that binds to Kar9 and is also involved in spindle positioning (Beach et al. 2000; Miller et al. 2000), was also clearly a strong bir1-17 enhancer at the two lower temperatures, although it fell outside our statistical significance cut-off at 37°. Thus cells lacking proper Bir1 function appear to become particularly dependent on the normal functioning of kinesins and microtubules.

GO analysis, together with consideration of the core protein complexes defined by Benschop et al. (2010), also identified knockouts of genes encoding any of the three members of the Ctf8/Ctf18/Dcc1 complex that is related to replication factor C (RFC^Ctf18) as enhancers of bir1-17 (although ctf18∆ fell just outside our cut-offs of q ≤ 0.05 and GIS ≤ −25 for significant strong enhancers). All three members of the Tof1/Mrc1/Csm3 complex that acts at stalled replication forks to promote sister chromatid cohesion (Bando et al. 2009; Tourriere et al. 2005; Xu et al. 2004) were also highlighted (although tof1∆ fell just outside our q-value cut-off). Like the Tof1 complex, the Ctf8 complex is also involved in sister chromatid cohesion (Mayer et al. 2001, 2004), highlighting the link between this process and CPC-dependent error correction. Examination of the core protein complexes also revealed negative enhancement of bir1-17 by those viable knockouts in the systematic collection that affected RFC^Rad24 (RAD24), RFC^Elg1 (ELG1), a PCNA-like clamp (RAD17 and possibly DDC1) and DNA polymerase epsilon [DPB3, YBR277c (::DPB3), and DPB4 if the q-value cut-off is relaxed].

Broadening the GO analysis to consider enhancers with a GIS ≤ −10 did not change this overall picture but gave greater emphasis to some categories such as genes involved in chromatin modification (components of the Set3C, ISW, Compass, Ada, and Rpd3S complexes), histone exchange (SWC5, VPS71, and VPS72), tRNA wobble uridine modification (components of Elongator, ATS1, KTI12, URM1, UBA4, NCS2, NCS6, and SAP190), iron transport (FET3 and FTR1), and peroxisomal function. Multiple components in each of these categories were strong, significant enhancers of bir1-17 at 37° (Table S5 in File S2). While a potential functional connection between Bir1 and either iron transport or peroxisomes is not at all obvious, many of these other enhancers may function indirectly by altering the pattern of expression of proteins that have a direct functional relationship with Bir1. Other notable strong enhancers not falling into any of the above-mentioned categories included knockouts of the CENP-T-related kinetochore component CNN1 that interacts with the Ndc80 kinetochore subcomplex (Bock et al. 2012; Malvezzi et al. 2013) and the CPR6 peptidyl-prolyl cis-trans isomerase and its interacting protein encoded by STI1 (Mayr et al. 2000). Figure S6 shows these groups of genes mapped onto the 37° fitness plot as an indicator of how consistently they interacted with bir1-17 in the screen.

Phenotypic suppressors of bir1-17

QFA carried out at 37° identified many potential suppressors of bir1-17 that caused relief of the temperature-sensitive growth defect, including 94 strong suppressors (GIS ≥ 25; Table S8 in File S2). GO analysis of these genes (File S4) revealed highly significant enrichment for genes encoding components of the large ribosomal subunit (LRSU) or proteins involved in its rRNA processing and assembly, as well as for genes involved in mRNA catabolism, and in particular nonsense-mediated mRNA decay (NMD). This was further supported by our analysis of the core protein complexes of Benschop et al. (2010). Thus four out of the six strongest suppressors and almost one-third of all the strong (GIS ≥ 25) suppressors could be assigned roles either in ribosome biogenesis or as components of the ribosome. Strikingly, the vast majority of knockouts affecting the ribosome were specific for the LRSU: we isolated 27 RPL gene deletions (removing LRSU proteins) but only three RPS gene deletions (removing small ribosomal subunit proteins) as statistically significant bir1-17 strong suppressors. Taking the unbiased approach of mapping all genes annotated as RPL and RPS in SGD onto the 37° fitness plot, we confirmed that genes in these two groups in general behaved very differently when knocked out and combined with bir1-17 (Figure S7, compare A and B), while Figure S7C shows that the vast majority of all gene knockouts affecting either biogenesis of the LRSU or LRSU components caused phenotypic suppression of bir1-17.

Knockouts of the NMD genes EBS1, NMD2, NAM7, and UPF3 were all within the strong suppressor set (nam7∆ was the second strongest suppressor), while the remaining components of the NMD pathway present in the systematic deletion collection (DCN1 and NMD4) were also statistically significant suppressors but falling just below our GIS ≥ 25.0 cut-off (Table S8 in File S2). SKI2 and SKI3, encoding two components of the Ski complex that are involved in a variety of RNA decay processes, including NMD and processes mediated by the exosome, were also identified. The remaining functionally related genes (RRP6, SKI7, SKI8, and YKL023w) were also suppressors falling slightly below our GIS cut-off. Figure S8A summarizes how the SKI, NMD, and exosome gene knockouts mapped onto the 37° fitness plot.

Although GO terms directly related to cell division were not specifically highlighted by our GO analysis even when suppressors with GIS ≥ 10 were included, one gene encoding a kinetochore component (YBP2) was found within the top 30 suppressors (File S4). Mapping the core protein complexes defined by Benschop et al. (2010) onto the 37° fitness plot also identified mutations affecting the COP9 signalosome as suppressors of bir1-17. The COP9 signalosome removes the NEDD8 homolog Rub1 from the yeast cullin Cdc53 and is required for cell cycle regulation at the G1/S boundary (Wee et al. 2002). Thus, both csi1∆ and pci8∆ were strong significant suppressors, while csn9∆, rri1∆, and rri2∆ were suppressors that fell just outside our GIS and/or q-value cut-offs (Figure S8B).

The basis for suppression by each of these classes of genes is not yet clear, but suppression by the first two groups of gene knockout is most likely related to alterations in the expression of proteins caused by changes in mRNA stability or alterations in the ribosome population. Since bir1-17 is a temperature-sensitive loss of function mutant, it may be that these knockouts lead to elevated Bir1-17 protein levels at higher temperatures that can compensate for the effect of the mutations it contains. In a previous QFA analysis, both NMD gene deletions and RPL (but not RPS) gene deletions were also found to suppress the temperature-sensitivity of a cdc13-1 query mutation (Addinall et al. 2011). In this case, the NMD deletions were shown to operate through affecting levels of another protein (Stn1), which like Cdc13, is specifically involved in telomere function (Addinall et al. 2011). Conversely, both NMD and RPL gene deletions enhanced the phenotype of yku70∆ in a parallel QFA analysis (Addinall et al. 2011). It is therefore possible that loss of either the NMD pathway or specific RPL genes may lead to generalized effects on gene expression that can suppress or enhance specific mutations through effects on expression of functionally related genes.

The genetic enhancement profiles of bir1-17 and ipl1-321 show both common and distinct features, many of which are shared with mcd1-1

Since Ipl1 functions together with Sli15, Bir1, and Nbl1 within the yeast CPC complex, it might be expected that strains with loss-of-function mutations in each of these genes would show synthetic lethality or strong negative genetic interactions with a common set of nonessential gene knockouts that reflect their shared functional roles. Comprehensive analysis has yet to be performed with conditional alleles of either sli15 or nbl1, but our study now enables comparison of the strong genetic enhancers of both ipl1-321 and bir1-17 to be made. Figure 2 shows that 13 of the strong genetic interactions of bir1-17 established in our work are indeed shared with the known synthetic lethal interactions of ipl1-321 (colored yellow and red). Overlap between strong negative genetic interactors of ipl1-321 and those of the cohesin mutant mcd1-1 (also called scc1-73) was noted previously (Ng et al. 2009) and is also the case here for bir1-17, with nine strong negative genetic interactions shared by all three mutant alleles (colored red in Figure 2). These data underline the strong functional links between sister chromatid cohesion, chromosome biorientation, and faithful sister chromatid segregation that operate during cell division.

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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.

Remarkably, the ipl1-321 and bir1-17 alleles each individually show at least as many strong negative genetic interactions with mcd1-1 that they do not share with each other. For example, strains lacking several nonessential components of the Ctf19 complex that were identified in this study as the strongest negative enhancers of bir1-17 are also synthetic lethal with, or strong enhancers of mcd1-1, but not ipl1-321. Conversely, both ipl1-312 and mcd1-1 show synthetic lethality with the spindle assembly checkpoint gene knockouts (BUB1, BUB3), whereas these mutations are only moderate negative genetic interactors of bir1-17 at 27°, consistent with our earlier finding that bir1-17 does not require a functional checkpoint for viability (Makrantoni and Stark 2009). The substantial differences between the profiles of ipl1-321 and bir1-17, and in particular between those subsets of interactions that are shared with mcd1-1, support the notion that the ipl1-321 and bir1-17 mutations confer distinct effects on CPC function, although some of these differences could reflect either false positives or false negatives in one or another SGA screen. However, the apparent overlap between the strong enhancers of bir1-17 and mcd1-1 is nonetheless striking. We therefore next individually assessed a selection of the bir1-17 genetic interactors identified in our screen both to verify the interactions and to address more systematically whether some interactions were really specific to bir1-17 and not shared with ipl1-321. As a more robust approach to verification, we chose to do this in the W303 genetic background in which ipl1-321 and bir1-17 were both isolated (Biggins et al. 1999; Makrantoni and Stark 2009), and in which much of the work on CPC-mediated error correction in yeast has been carried out.

Confirmation of the negative genetic interactions between bir1-17 and the Ctf19, Ctf8-Ctf18-Dcc1, and Csm3-Mrc1-Tof1 complexes in W303

To verify a selection of the interactions identified in the bir1-17 QFA screen, we deleted a range of hits in the W303 background and carried out tetrad analysis following crosses with bir1-17. Table S9 summarizes those crosses where bir1-17 yfg∆ strains were found to be unconditionally lethal. In instances where such double mutants were viable, their relative fitness was examined by spotting out equivalent serial dilutions of strains on agar plates and assessing growth at different temperatures (Figure S9). Table S10 summarizes both sets of data and shows that, although some interactions could be readily verified in W303, other interactions could not.

A number of conclusions can be drawn from these data. First, all five knockouts of core Ctf19 complex components that we tested from within the group of strong bir1-17 enhancers were synthetic lethal with bir1-17 in the W303 background, as was the knockout of the one example of the kinesin group that we tested (cin8∆). nkp1∆ (identified as a weaker enhancer in our screen) showed no clear enhancement of bir1-17 in W303, although deletion of its paralog NKP2 caused strong enhancement of bir1-17. This verifies in W303 all five core Ctf19 complex components we identified by QFA as strong negative genetic interactors of bir1-17. Remarkably, the genetic interactions seen in W303 were actually stronger (synthetic lethality) compared with the QFA screen (strong enhancement). The unconditional lethality of either iml3∆ or chl4∆ with bir1-17 in W303 (Figure 3A) is particularly striking in this regard, since the QFA screen only identified these knockouts as enhancers at 37° (see Tables S5–S7 in File S2). Although we identified cnn1∆ (deleting the yeast homolog of the kinetochore protein CENP-T) as a strong enhancer in the screen, this could not be reproduced in the W303 background. Second, negative genetic interactions between both the Ctf8-Ctf18-Dcc1 complex (synthetic lethality with dcc1∆) and the Csm3-Mrc1-Tof1 complex (negative genetic interaction with mrc1∆) could be confirmed in the W303 background, although the strong negative enhancement by loss of Csm3 seen by QFA could not be recapitulated. Finally, of the bir1-17 enhancers identified that fell into the other functional groups discussed above, three could be verified in W303 as bir1-17 enhancers (iki3∆, vps71∆, and yku70∆) but several others affecting the COMPASS (bre2∆, spp1∆), ISW (chd1∆) and Rpd3S (sin3∆) complexes showed little or no enhancement of bir1-17 in W303.

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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°.

Although we reproduced the negative genetic interaction between bir1-17 and irc15∆ in W303 using a precise ORF knockout identical to that in the systematic collection used for the QFA screen (data not shown), this knockout also deletes the last eight sense codons of CTF19, with which IRC15 overlaps on the opposite strand. Using a construct that removed the first 332 codons of the 499-codon IRC15 ORF and thus left the CTF19 ORF intact, we found no genetic interaction with bir1-17 (Figure S9A), and so we conclude that irc15∆ was identified in our QFA screen because the systematic knockout interferes with Ctf19 function. It is also therefore likely that other mitotic phenotypes reported for irc15∆ mutants may result from effects on the overlapping CTF19 gene rather than indicating functional consequences of Irc15 loss.

bir1-17, but not ipl1-321, is dependent on the Iml3-Chl4 subcomplex for viability

Given that we identified several verifiable strong negative genetic interactions in our QFA analysis between bir1-17 and Ctf19 complex gene knockouts that were not seen in a similar ipl1-321 screen (Ng et al. 2009), we looked in more detail at iml3∆ and chl4∆. When iml3∆ and chl4∆ strains were crossed with ipl1-321, ipl1-2, and sli15-3 mutants (all in the W303 background), viable double mutants were readily isolated in each case and grew essentially normally at 26°, which is permissive for all three Ts^– alleles (Figure 3B). Thus bir1-17 strains show complete dependence on functional Iml3 and Chl4 in W303, whereas ipl1-321, ipl1-2, and sli15-3 strains are virtually independent of Iml3 and Chl4 for normal proliferation. This confirms that at least some of the differences between the genetic interaction profiles of ipl1-321 and bir1-17 are genuine and implies that there is a fundamental difference in the way that the ipl1 and sli15-3 mutations affect CPC function in comparison with bir1-17.

The sli15(∆NT) allele removes the first 228 residues of Sli15 and thereby prevents its interaction with Bir1. Surprisingly, this is not lethal as anticipated, despite the delocalization of Ipl1 from kinetochores as a result of the truncation (Campbell and Desai 2013), leading to the idea that while targeting of the CPC to the kinetochore may be important for efficient chromosome biorientation, it is not essential for it to occur. However, the combination of sli15(∆NT) with either ctf19∆ or mcm21∆ was almost lethal (Campbell and Desai 2013). We therefore crossed sli15(∆NT) with iml3∆ and chl4∆ and, although the double mutants could be obtained, in contrast to sli15-3 (Figure 3B), there was a clear negative genetic interaction in both cases and the chl4∆ sli15(∆NT) double mutant was inviable at 35°, a temperature at which each single mutant grew normally (Figure 3C). Thus bir1-17 and sli15(∆NT) share a clear negative genetic interaction with loss of Iml3 or Chl4 that is not seen in ipl1 mutants or sli15-3. Double nbl1-6 chl4∆ and nbl1-6 iml3∆ mutants could also be obtained, but again showed a strong negative genetic interaction at 35° or above (Figure 3C). In summary, mutations that affect the targeting of the CPC [nbl1-6, sli15(∆NT) and bir1-17] show strong negative genetic interactions with loss of either IML3 or CHL4, whereas mutations primarily affecting CPC’s protein kinase activity (ipl1-2, ipl1-321, and sli15-3) do not.

bir1-17 does not reduce accumulation of pericentromeric cohesin

The Ctf19 kinetochore complex is important for establishment of pericentromeric cohesion, while Csm3 is needed for ensuring that pericentromeric cohesin functions to hold sister centromeres together and shows an additive phenotype with mutations affecting the Ctf19 complex (Fernius and Marston 2009). We therefore examined whether the bir1-17 mutant might also have a defect in the accumulation of pericentromeric cohesin that could explain its strong negative genetic interactions with knockouts affecting the Ctf19 and Csm3-Mrc1-Tof1 complexes. After synchronizing cells in G1 and then releasing them at 37° in the presence of benomyl and nocodazole, association of cohesin at the centromere, pericentromere, and arm regions of chromosome IV was quantified in metaphase-arrested cells by chromatin immune precipitation using HA-tagged Mcd1. Relative to a wild-type control, the bir1-17 strain showed no obvious defect in the association of cohesin with the centromeric and pericentromeric regions (Figure 4). In contrast, a chl4∆ strain showed a clear deficiency in the level of cohesin at the centromere and pericentromere but not in accumulation of cohesin on the chromosome arm, as found previously (Fernius and Marston 2009). Thus defective accumulation of cohesin around the centromere in bir1-17 is unlikely to provide an explanation for its strong negative interaction with other mutations affecting pericentromeric cohesion. However, we cannot exclude the possibility that, as in csm3 mutants (Fernius and Marston 2009), the pericentromeric cohesin that accumulates is not fully functional.

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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.

Sgo1 overexpression does not relieve the requirement for Chl4 and Iml3 in bir1-17 strains

Since Iml3 and Chl4 are also needed for pericentromeric accumulation of Sgo1, which is involved both in CPC recruitment and the bias of sister kinetochores to form bioriented attachments (Verzijlbergen et al. 2014), we tested whether boosting Sgo1 expression could suppress the synthetic lethality between iml3∆ or chl4∆ and bir1-17. To do this and to provide a platform for further analysis of the defect in the double mutants, we utilized the “anchor away” system, in which proteins that function within the nucleus can be excluded by the addition of rapamycin, thereby triggering conditional loss of function (Haruki et al. 2008). This was carried out in the context of a TOR1-1 background so that cells were resistant to growth inhibition by rapamycin, and rapamycin-induced effects can therefore be ascribed solely to nuclear exclusion of the protein of interest. To verify the use of this approach with the Ctf19 kinetochore complex, we tagged each of the two essential Ctf19 complex members (Ame1 and Okp1) with FRB-GFP and demonstrated that cells could no longer grow when rapamycin was added, while strains in which the nonessential Iml3 and Chl4 were FRB-tagged allowed robust growth on rapamycin (Figure S10A). This analysis also confirmed that nuclear exclusion of Iml3 and Chl4 did not interfere with other essential components of the kinetochore, for example through them “piggy-backing” out of the nucleus with the tagged protein. Microscopy of both the Iml3-FRB-GFP and Chl4-FRB-GFP strains confirmed that kinetochore localization of either tagged protein was lost within 50 min of rapamycin treatment (Figure S10B).

To test whether boosting Sgo1 levels could reverse the synthetic lethality between bir1-17 and loss of Iml3 or Chl4, bir1-17 was introduced into the Iml3-FRB-GFP and Chl4-FRB-GFP strains, and then a galactose-inducible GAL-SGO1 construct introduced. As shown in Figure 5, addition of rapamycin to the growth medium recapitulated the synthetic lethal phenotype of bir1-17 with iml3∆ or chl4∆ gene knockouts. The rapamycin-induced lethality was not overcome by induction of the SGO1 overexpression construct. Furthermore, inducing SGO1 expression appeared, if anything, somewhat detrimental to the growth of strains lacking nuclear Iml3 or Chl4. Iml3 and Chl4 both have a specific role in ensuring correct sister chromatid separation in meiosis II that is not necessarily shared with other Ctf19 components (Marston et al. 2004), but Figure 5 confirms that the synthetic lethality seen in genetic crosses (Table S9) is also seen in mitotically proliferating cells.

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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.