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. 2017 Jun 7;7(6):1753-1766.
doi: 10.1534/g3.117.042317.

Mapping the Synthetic Dosage Lethality Network of CDK1/CDC28

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Free PMC article

Mapping the Synthetic Dosage Lethality Network of CDK1/CDC28

Christine Zimmermann et al. G3 (Bethesda). .
Free PMC article

Erratum in

  • Corrigendum.
    G3 (Bethesda). 2019 Aug 8;9(8):2787. doi: 10.1534/g3.119.400472. G3 (Bethesda). 2019. PMID: 31395747 Free PMC article. No abstract available.

Abstract

Cdk1 (Cdc28 in yeast) is a cyclin-dependent kinase (CDK) essential for cell cycle progression and cell division in normal cells. However, CDK activity also underpins proliferation of tumor cells, making it a relevant study subject. While numerous targets and processes regulated by Cdc28 have been identified, the exact functions of Cdc28 are only partially understood. To further explore the functions of Cdc28, we systematically overexpressed ∼4800 genes in wild-type (WT) cells and in cells with artificially reduced Cdc28 activity. This screen identified 366 genes that, when overexpressed, specifically compromised cell viability under conditions of reduced Cdc28 activity. Consistent with the crucial functions of Cdc28 in cell cycle regulation and chromosome metabolism, most of these genes have functions in the cell cycle, DNA replication, and transcription. However, a substantial number of genes control processes not directly associated with the cell cycle, indicating that Cdc28 may also regulate these processes. Finally, because the dataset was enriched for direct Cdc28 targets, the results from this screen will aid in identifying novel targets and process regulated by Cdc28.

Keywords: Cdc28; Cdk1; Mutant Screen Report; cell cycle; synthetic lethality.

Figures

Figure 1
Figure 1
Set-up of the screen. (A) Schematic overview of the screen. (B) Example of the data. Red boxes indicate genes that aggravate the cdc28-as1 phenotype, whereas green boxes indicate genes that ameliorate the slow-growth defect of cdc28-as1 mutants on 1-NM-PP1. Yellow boxes indicate genes that induce the SDL phenotype even under noninducing conditions, whereas blue boxes indicate genes that induce the SDL phenotype even in the absence of 1-NM-PP1. DMSO, dimethyl sulfoxide; ORF, open reading frame; SDL, synthetic dosage lethality; WT, wild-type.
Figure 2
Figure 2
Comparison of the SDL dataset with other genome-wide screens. (A) Overlap between the “hub” SDL networks of CDC28 and the kinases SLT2, BCK1, ELM1, and PHO85. Data were obtained from Sharifpoor et al. (2012). (B) Overlap between the SDL screen and previously identified genes with a negative genetic relationship with CDC28. Data were obtained from BioGRID. (C and D) Overlap between the SDL screen and the SGA screen described in Zimmermann et al. (2011). (E) Overlap between the SDL screen and previously identified genes that cause dosage lethality of cdc28 mutants (data from BioGRID). SDL, synthetic dosage lethality; SGA, synthetic genetic array.
Figure 3
Figure 3
Analysis of the data reveals overrepresentation of genes involved in the cell cycle, DNA metabolism, and transcription. (A) GO Biological Process analysis of the genes that aggravate the cdc28-as1 phenotype (negative SDL interactions). GO analysis was performed using Metascape. (B) Network plot of the relationships among GO terms. Nodes represent enriched terms colored by its cluster ID. (C) The same network as presented in (B) but showing P values for the nodes. (D) Analysis of the negative SDL interactors using the GO Slim mapper tool of the SGD. (E) Analysis of the macromolecular complex components of the top 154 genes in the SDL network. Only those GO terms are shown for which at least five terms genes were identified. GO, gene ontology; ID, identifier; SDL, synthetic dosage lethality; SGD, Saccharomyces Genome Database.
Figure 4
Figure 4
The effect of overexpression of cyclins. (A) Overexpression of CLN2 and CLN3 causes SDL in cdc28-as1 mutants even under noninducing conditions. Cultures of cells transformed with plasmids containing the ORFs YEL074W (“Mock”), CLN2, or CLN3 were spotted on glucose-containing SC-URA plates supplemented with either DMSO or 1-NM-PP1, followed by incubation at 30° until colonies appeared. (B) Overexpression of CLN2 and CLN3 suppresses the growth defect of cdc28-4 mutants. Cultures of cells transformed with plasmids containing the control ORF YEL074W (“Mock”), CLN2, CLN3, or MSA1 were spotted on YPD or on YP-galactose and incubated at either 30° or 34° until colonies appeared. (C) cdc28-as1 strains transformed with plasmids harboring either the control ORF YEL074W (Mock) or CLN3 were grown to log phase and treated with 500 nM 1-NM-PP1, after which transcription was induced with galactose as described in the Materials and Methods. Subsequently, mRNA levels of CLN2, CLB6, ACT1, and CLN3 were determined by RT-qPCR. DMSO, dimethyl sulfoxide; ORF, open reading frame; RT-qPCR, quantitative reverse transcription polymerase chain reaction; SC-URA, synthetic complete-uracil; SDL, synthetic dosage lethality; WT, wild-type; YP, yeast extract peptone; YPD, YP and dextrose.
Figure 5
Figure 5
Overexpression of FIN1 in cdc28-as1 mutants induces mitotic spindle aberrancies. (A) No obvious differences in spindle misalignment between WT cells and cdc28-as1 mutants after overexpression of KAR9, SPC97, FIN1, or CIN8. TUB1-GFP-expressing WT cells and cdc28-as1 mutants were transformed with plasmids containing the indicated ORFs (Mock: YEL074W), after which cells were treated with 1-NM-PP1 and overexpression was induced by galactose as described in Materials and Methods. Spindle alignment in at least 100 M phase cells was imaged and quantified using fluorescence microscopy. (B) Overexpression of FIN1 in the cdc28-as1 mutant background results in aberrant spindle assembly. Cells were treated and imaged as in (A). At least 300 cells were analyzed per treatment and genotype. (C) Quantification of the data shown in (B). Error bars indicate SD. *P < 0.05. NS, not significant; WT, wild-type.
Figure 6
Figure 6
Genes that may promote dosage rescue of cdc28-as1 mutant cells. (A) Comparison of previously identified genes that mediate dosage suppression of cdc28 alleles with the ORFs that mediate dosage suppression identified by the synthetic dosage lethality (SDL) screen. The only ORF in common between these datasets was YBR160W, which encodes Cdc28. (B) Putative dosage suppressors identified by the SDL screen organized by their cellular functions.
Figure 7
Figure 7
The SDL dataset is enriched for proteins that are phosphorylated in a Cdc28-dependent manner. (A) Venn diagram depicting overlap between the SDL dataset and proteins that are phosphorylated in a Cdc28-dependent manner in vivo and in vitro, and which have previously been confirmed to be Cdc28 substrates. (B) Most proteins that have been found to be phosphorylated in a Cdc28-dependent manner in vivo cause dosage lethality when overexpressed in a cdc28-as1 mutant. The Venn diagram shows overlap between the SDL dataset and proteins that are phosphorylated in a Cdc28-dependent manner in vitro and in vivo. (C) The SDL dataset is enriched for known Cdc28 targets. The Venn diagram shows overlap between the SDL screen, proteins that have been found to be phosphorylated in a Cdc28-dependent manner in vivo, and previously confirmed Cdc28 substrates. (D and E) Overexpression of proteins phosphorylated on potential Cdc28 sites cause an SDL phenotype. The Venn diagrams show overlap between the SDL dataset, confirmed Cdc28 targets, and proteins that are known to be phosphorylated in vivo on either minimal Cdc28 sites (D) or on full Cdc28 consensus sites (E) in a Cdc28-dependent manner. (F–H) Domain structures of Nbp1 (F), Sak1 (G), and Epo1 (H), and location of sites known to be phosphorylated in vivo in a Cdc28-dependent manner. CDK, cyclin-dependent kinase; SDL, synthetic dosage lethality.
Figure 8
Figure 8
Aberrant nuclear localization of Whi5 in M phase upon overexpression of several SDL genes. Overexpression of CIN8, FIR1, and RFA1 results in a significant increase in nuclear localization of Whi5-GFP in M phase cells. Whi5-GFP-expressing cells containing YEL074W (“Mock”), CDC6, SLY41, CIN8, CLN2, IML1, FIR1, and RFA1 expression plasmids were grown to log phase in the presence of glucose and arrested in M phase with nocodazole. Cells were then washed and incubated in nocodazole-containing medium supplemented with galactose to induce expression for 3 hr, after which nuclear localization of Whi5-GFP was assessed by fluorescence microscopy. Error bars indicate SD. ns, not significant; **P < 0.001, ***P < 0.0001. GFP, green fluorescent protein; SDL, synthetic dosage lethality; WT, wild-type.

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