Genetic interactions derived from high-throughput phenotyping of 6589 yeast cell cycle mutants

Over the last 30 years, computational biologists have developed increasingly realistic mathematical models of the regulatory networks controlling the division of eukaryotic cells. These models capture data resulting from two complementary experimental approaches: low-throughput experiments aimed at extensively characterizing the functions of small numbers of genes, and large-scale genetic interaction screens that provide a systems-level perspective on the cell division process. The former is insufficient to capture the interconnectivity of the genetic control network, while the latter is fraught with irreproducibility issues. Here, we describe a hybrid approach in which the 630 genetic interactions between 36 cell-cycle genes are quantitatively estimated by high-throughput phenotyping with an unprecedented number of biological replicates. Using this approach, we identify a subset of high-confidence genetic interactions, which we use to refine a previously published mathematical model of the cell cycle. We also present a quantitative dataset of the growth rate of these mutants under six different media conditions in order to inform future cell cycle models.


Parent Strain Construction
We used several strategies to generate the eight sets of parent strains used in this study (Table S1). We obtained most of the Set 1 and Set 3 strains by sporulation and tetrad analysis of the heterozygous diploid commercial collection of kanMX strains 1,2 , but we made some by de novo kanMX PCR-mediated gene deletions in BY4741 or BY4742 (using pFA6a-kanMX as the template and listed primers 3-6 , Table S2). We obtained most of the Set 2 and Set 4 strains by transformation of the heterozygous diploid strains with a natMX PCR product (using MX.for and MX.rev primers in Table S2 with pAG25 template. pAG25 and its sequences are available from Addgene) to switch markers, followed by selection of nourseothricinresistant/G418-sensitive transformants, sporulation, and tetrad analysis. We made the rest of sets 2 and 4 by de novo natMX PCR-mediated gene deletions in BY4741 or BY4742 (using pAG25 as the template and listed primers). We made most of Set 7 and Set 8 by de novo PCR-mediated gene deletions in the SGA strain Y8205 7 , but we made some of these strains by crossing one of the BY4741-derived gene deletion strain with Y8205, followed by tetrad dissection. We then used the SGA method to cross these strains to BY4741 and obtain MATa versions of these strains for Set 5 and Set 6. All strains were confirmed by PCR of genomic DNA using one set of test primers for the gene deletion and another for the wild-type gene 8 (Table S2). All strains (parents and progeny) are available upon request.

Double Mutant Progeny Construction
All crosses followed a standard format in which the MATa strains (Sets 3, 4, 7, and 8) which we will call the "hit" strains, were arrayed alphabetically by gene name so that each strain was a single well in a 36-well block, with two replicate MATa blocks per plate, leaving the first and fifth rows empty for the addition of the wildtype parents during phenotyping. If a deletion strain was missing in a MATa set, we left the position empty. We arrayed the MATa strains, which we will call the query or "bait" strains, so that each MATa strain in the set fills a block of 36 wells at the same positions as one of the two blocks of MATa strains (i.e., in rows 2-4 or 6-8).
For the crosses, we used a Rotor HDA (Singer Instruments, Somerset, UK) to replica-pin each MATa plate to 12-18 YPD plates using 96 long repads with 6 wet mix cycles and 4 dry mix cycles to ensure robust inoculation of each plate. Visual inspection of each plate ensured proper transfer of cells. We then pinned each MATa plate on top of one MATa plate using the same conditions to ensure good mixing of the two parent strains on the YPD plate. Matings were performed on YPD at 30 °C for two days.

Halo Assays
Halo assays were used to confirm the mating type of the parents and progeny and identify potential cell signaling and chromosome segregation defects. We performed halo assays as described 9 using Y955 lawns to test for a-halos, and Y991 to test for a-halos. We prepared halo assay plates by growing Y995 and Y991 in YPD broth at 30 °C overnight in a shaking incubator. The next morning, we diluted each strain 1/5 in YPD broth, vortexed the tubes, and used sterile glass beads to spread 500 µl of the dilution per plate onto YPD PlusPlates. We allowed these plates to dry before pinning each plate of parent strains or haploid progeny (at 96 colony densities) onto both a Y995 lawn (to test for a-factor secretion) and a Y991 lawn (to test for a-factor secretion). We imaged halo assay plates after 48 h of growth (see Additional Data). Individual lines that did not behave as expected (most likely due to isolated genetic mishaps) were not excluded, as the consensus between multiple biological replicates was used to draw the conclusions presented.

Tetrad Analysis
To identify synthetic lethality by tetrad analysis, several tetrads (usually 12) were dissected for one or more biological replicates of each of the 58 gene combinations listed in Figure 4. The surviving spores were patched onto YPD plates, and then replica plated onto YPD+G418 (600ug/ml), YPD+nat (150ug/ml), and Y995 and Y991 lawn plates (as described above but using 300ul diluted culture).
Dissections from which we could recover very few live spores of any genotype were identified as having a likely meiotic defect (MD). Recovery of a live spore that was resistant to both antibiotics was considered evidence for viability (V). Cases where a dead spore in a tetrad could be inferred to have the double mutant phenotype based on allele segregation were considered evidence for synthetic lethality (SL). If the ratio of spores supporting synthetic lethality to spores supporting viability (SL:V) was 4:1 or greater, we considered the gene combination SL. If the SL:V ratio was between 4:1 and 1:1, we considered the gene combination to have reduced viability (RV). If the SL:V ratio was less than 1:1, we considered the gene combination to be viable (V). Cases where we were able to identify fewer than two spores as potentially SL or V (due to low viability overall) were also designated MD.  Normalizations based on the mean growth rate of the wildtype controls on each plate were used to account for edge effects. Heat maps show a visual representation of growth rates across each plate. The X and Y axis are the coordinates for the 384 positions where a colony may appear. In every case, wild-type controls are in rows A, B, I, and J, columns 1-4, 11-14, and 21-24. Histograms compare the growth rate of colonies that are on the edge of the plate or adjacent to an empty position (distance level 0) with those that are one or more positions away from an edge (non-zero distance levels). The p-value reported above the histogram marks the significance of the difference between the growth rate of edge-adjacent colonies and internal colonies. In each case, raw, unnormalized heat maps, histograms, and p-values are shown just above their normalized counterparts.    We have simulated all the mutants in this figure. We compared simulations and published phenotypes when possible. Mutants highlighted in red indicate inconsistencies between simulation and phenotypes. Mutants we have simulated for which no observed phenotype has been published are indicated with *. Mutants highlighted in bold have different phenotypes in the 2015 and 2020 models. New mutant simulations reported in the "New Data" column on the left. bck2D cdh1D  bck2D cln3D  bck2D swi4D  bck2D swi6D  bfa1D lte1D  bub2D cdc55D  cdc55D cdh1D  cdc55D clb5D  cdc55D lte1D  cdc55D swi4D  cdh1D clb5D  cdh1D cln3D  cdh1D

New Data
Wild-type In glucose In galactose

Bck2 mutants bck2D
Multi-copy BCK2 bck2Δ cln3Δ cdh1Δ * bck2D cln3D sic1D bck2D cln3D GAL-CLB5 * bck2D cln3D multi-copy CLN2 bck2D cln3D GAL-CLN3 bck2D cln3D cdc6D sic1D * bck2D cln3D GAL-CLN2 Cln mutants cln3D GAL-CLN2 GAL-CLN3 cln1D cln2D cln1Dcln2D bck2D cln1D cln2D cdh1D cln1D cln2D cln3D cln1D cln2D sic1D cln1D cln2D GAL-SIC1 cln1D cln2D cdc6D sic1D cln1D cln2D bck2D cdh1D * cln1D cln2D cdh1D GAL-CLN2 cln1D cln2D cdh1D GAL-SIC1 cln1D cln2D cln3D apc ts cln1D cln2D cln3D cdh1D cln1D cln2D cln3D sic1D cln1D cln2D cln3D multi-copy BCK2 cln1D cln2D cln3D GAL-CLB2 cln1D cln2D cln3D GAL-CLB5 cln1D cln2D cln3D multi-copy CLB5 cln1D cln2D cln3D GAL-CLN2 cln1D cln2D cln3D GAL-CLN3 cln1D cln2D GAL-SIC1 GAL-CLN2 cln1D cln2D cln3D sic1D cdc6D * cln1D cln2D GAL-SIC1 GAL-CLN2 cdh1D cln1D cln2D cln3D bck2D GAL-CLN2 Figure S6. Comparison of fitness scores for double mutants in all four sets of crosses on YPR media. White cells indicate zero growth and grey cells indicate missing or excluded data. Royal blue is used to designate fitness scores that differ from WT by fewer than 2 standard deviations. Cyan and green indicate fitness scores that are greater than WT by up to or more than 6 standard deviations respectively. Magenta and red indicate fitness scores that are less than WT by up to or more than 6 standard deviations respectively.  Figure S7. Comparison of fitness scores for double mutants in all four sets of crosses on YPG media. White cells indicate zero growth and grey cells indicate missing or excluded data. Royal blue is used to designate fitness scores that differ from WT by fewer than 2 standard deviations. Cyan and green indicate fitness scores that are greater than WT by up to or more than 6 standard deviations respectively. Magenta and red indicate fitness scores that are less than WT by up to or more than 6 standard deviations respectively.  White cells indicate zero growth and grey cells indicate missing or excluded data. Royal blue is used to designate fitness scores that differ from WT by fewer than 2 standard deviations. Cyan and green indicate fitness scores that are greater than WT by up to or more than 6 standard deviations respectively. Magenta and red indicate fitness scores that are less than WT by up to or more than 6 standard deviations respectively.  White cells indicate zero growth and grey cells indicate missing or excluded data. Royal blue is used to designate fitness scores that differ from WT by fewer than 2 standard deviations. Cyan and green indicate fitness scores that are greater than WT by up to or more than 6 standard deviations respectively. Magenta and red indicate fitness scores that are less than WT by up to or more than 6 standard deviations respectively.  White cells indicate zero growth and grey cells indicate missing or excluded data. Royal blue is used to designate fitness scores that differ from WT by fewer than 2 standard deviations. Cyan and green indicate fitness scores that are greater than WT by up to or more than 6 standard deviations respectively. Magenta and red indicate fitness scores that are less than WT by up to or more than 6 standard deviations respectively.

Supplementary Tables
Supplementary tables are provided as Excel files in the Supplementary Data file.