Regulation of a transcription factor network by Cdk1 coordinates late cell cycle gene expression

Abstract

To maintain genome stability, regulators of chromosome segregation must be expressed in coordination with mitotic events. Expression of these late cell cycle genes is regulated by cyclin-dependent kinase (Cdk1), which phosphorylates a network of conserved transcription factors (TFs). However, the effects of Cdk1 phosphorylation on many key TFs are not known. We find that elimination of Cdk1-mediated phosphorylation of four S-phase TFs decreases expression of many late cell cycle genes, delays mitotic progression, and reduces fitness in budding yeast. Blocking phosphorylation impairs degradation of all four TFs. Consequently, phosphorylation-deficient mutants of the repressors Yox1 and Yhp1 exhibit increased promoter occupancy and decreased expression of their target genes. Interestingly, although phosphorylation of the transcriptional activator Hcm1 on its N-terminus promotes its degradation, phosphorylation on its C-terminus is required for its activity, indicating that Cdk1 both activates and inhibits a single TF. We conclude that Cdk1 promotes gene expression by both activating transcriptional activators and inactivating transcriptional repressors. Furthermore, our data suggest that coordinated regulation of the TF network by Cdk1 is necessary for faithful cell division.

Figure 1. Characterization of cyclin-dependent kinase (Cdk)-transcription factors (TF) alleles

A Wild-type or cdc28-as1 strains carrying plasmids to express the indicated 3HA-tagged WT or Cdk-TF alleles from the GAL1 promoter were induced with galactose and levels of HA-tagged proteins compared by Western blot. Where indicated, strains were treated with 1NM-PP1 to inhibit cdc28-as1 for 10 min prior to collecting cells. Light exposure of the HA blot is shown to highlight changes in levels of Cdk-TFs, and dark exposure highlights the mobility shifts. B Cells from (A) were arrested in G1 and TF expression induced by galactose addition. TFs were then immunoprecipitated and incubated with or without Clb2/Cdk1 kinase, and phosphorylation analyzed by Western blotting. Note that unphosphorylated Yhp1-3HA co-migrates with IgG. C Cells expressing Tos4-3FLAG, Tos4-9A-3FLAG, Hcm1-3HA, Hcm1-15A-3HA, Yox1-3V5, Yox1-9A-3V5, Yhp1-13MYC, or Yhp1-13A-13MYC were arrested in G1, released into the cell cycle, and samples taken for Western blot and flow cytometry at 15-min time points. Western blots against epitope tags on WT and Cdk-TFs are shown. Quantification of protein levels and flow cytometry are shown in Supplementary Fig S2. D–G Cells from (C) expressing tagged WT and Cdk-alleles of Tos4 (D), Hcm1 (E), Yox1 (F), or Yhp1 (G) were treated with cycloheximide and samples collected for Western blot after the indicated number of minutes. For all blots, molecular weight markers are indicated to the left and Cdk1 levels are shown as a loading control. Flow cytometry showing cell cycle positions and quantification of half-life data are shown in Supplementary Fig S3. Source data are available online for this figure.

Figure 2. Regulation of transcription factors (TF) degradation

A–D Regulation of TF degradation by cyclin-dependent kinase 1 (Cdk1). Cells carrying the cdc28-as1 allele (or wild-type controls) and expressing Tos4-3FLAG (A), Hcm1-3HA (B), Yox1-3V5 (C), or Yhp1-13MYC (D) were treated with 1NM-PP1 for 2 h and half-lives compared by cycloheximide-chase assay. Flow cytometry controls showing cell cycle positions are shown in Supplementary Fig S4A. E–H Cells carrying the cdc53-1 temperature-sensitive allele (or wild-type controls), and expressing Tos4-3HA (E), Hcm1-3HA (F), Yox1-3HA (G), or Yhp1-3HA (H) from the inducible GAL1 promoter on plasmids, were treated with galactose for 30 min, shifted to 37°C for 2 h, and cycloheximide-chase assays was performed. Note that for Tos4, Yox1, and Yhp1, phosphorylated forms of each are stabilized in cdc53-1 cells. Flow cytometry controls showing cell cycle positions are shown in Supplementary Fig S4B. I Cycloheximide-chase assay comparing the half-lives of HA-tagged WT and Cdk-alleles (integrated at the endogenous locus) in wild-type cells to cells deleted for the anaphase-promoting complex (APC) activator Cdh1, or arrested with nocodazole. Flow cytometry profiles for each strain are shown in Supplementary Fig S4C. J Wild-type and apc1Δ strains were treated with galactose to induce expression of constitutively active Cdh1 (Cdh1-m11) and levels of Tos4-3FLAG, Cdh1-m11, Clb2 (an established APC target), and Cdk1 were analyzed by Western blotting. Source data are available online for this figure.

Figure 3. Simultaneous mutation of S-phase transcription factors (TFs) delays mitotic progression

A, B Cells expressing differentially tagged WT TFs (TOS4-3FLAG HCM1-3HA YOX1-3V5 YHP1-13MYC), or phosphomutant TFs (4P, tos4-9A-3FLAG hcm1-15A-3HA yox1-9A-3V5 yhp1-13A-13MYC), were arrested in G1, released, and collected at 15-min intervals for Western blot (A) and flow cytometry (B). Levels of TFs and cyclin-dependent kinase 1 (Cdk1) are shown in (A). Quantification of proteins levels are shown in Supplementary Fig S5A. DNA content at each time point is shown (B, top). Overlay of WT (blue) and 4P (red) at 75 and 90 min highlight the mitotic delay in 4P cells (B, bottom). A representative of three replicate experiments is shown. C–G mRNA from WT and 4P cells collected as in (A) were compared to mRNA from asynchronous WT cells. Data from one of two biological replicates are shown. Average expression of all 6,237 genes at each time point in WT (blue) and 4P (red) cells (C). Average expression of 930 cell cycle-regulated genes (D). For further explanation and breakdown of individual groups, see Supplementary Fig S5C. Average expression of 97 genes that have Hcm1-binding motifs in their promoters and whose expression peaks during S-phase (E) (Pramila et al, 2006). Average expression of 39 MCM cluster genes (F) (Spellman et al, 1998). Average expression of 31 CLB2 cluster genes (G) (Spellman et al, 1998). Data from biological replicates of all cell cycle-regulated genes, and lists of genes in each cluster, are included in Supplementary Dataset S1. H RT-qPCR of representative Hcm1 targets (CIN8, HTZ1) and MCM cluster genes (DBF2, KIN3) at the indicated time points after release from G1. For each gene, mean expression from three biological replicates, with standard deviations, is plotted. Asterisks indicate that all four comparisons are statistically significant with a P-value of ≤ 0.01 (CIN8, P = 0.01; HTZ1, P = 0.005; DBF2, P = 0.007; KIN3, P = 0.003). Data information: In all parts, wild-type cells are graphed in blue, 4P cells are red. Source data are available online for this figure.

Figure 4. Phosphorylation inactivates Yox1 and Yhp1

A–C Wild-type cells carrying plasmids expressing YOX1 or yox1-13A from the GAL1 promoter, or an empty vector control (EV), were grown in raffinose and arrested in G1. Cells were then released into medium containing raffinose and galactose, to induce overexpression of YOX1 or yox1-13A. DNA content 5 h after release from G1 is shown in (A). Western blots for Yox1-3HA, Clb2 (a marker of mitosis), and cyclin-dependent kinase 1 (Cdk1; loading control) as cells progressed through mitosis (90, 120, 150, and 180 min after release from G1) are shown in (B). Expression of the Yox1 target genes DBF2, HST4, MCM3, and CDC20 at the indicated time points is shown in (C). All values were normalized to ACT1. Mean and standard deviations from technical replicates of a representative experiment are shown. For each gene, values are shown relative to empty vector control cells at 90 min after release. Flow cytometry profiles for all time points are shown in Supplementary Fig S7A. D Expression of Yox1/Yhp1 targets genes in asynchronous yhp1-13A, yox1-9A, or yhp1-13A yox1-9A cells compared to wild-type. All values were normalized to ACT1. Mean and standard deviations from technical replicates of a representative experiment are shown. See Supplementary Fig S7B and C for corresponding Western blots and cell cycle positions. E ChIP-qPCR of 3V5-tagged Yhp1, Yhp1-13A, Yox1, and Yox1-9A, compared to an untagged control. Mean and standard deviations for three biological replicates are shown. For each primer set, binding is shown relative to Yhp1. See Supplementary Fig S7C and D for corresponding Western blots and cell cycle positions from a representative experiment. Source data are available online for this figure.

Figure 5. Phosphorylation of the Hcm1 N-terminus promotes SCF-dependent degradation

1. Expression of Hcm1-3N-3HA and Hcm1-8C-3HA over the cell cycle. Cells were arrested in G1, released into the cell cycle, and samples taken for Western blot and flow cytometry (Supplementary Fig S8A) at 15-min time points.

2. Cells expressing Hcm1-GFP or Hcm1-3N-GFP from the TEF1 promoter were arrested in G1 with alpha-factor, S-phase with HU, or mitosis with nocodazole and half-lives compared by cycloheximide-chase assay. Levels of Hcm1, Clb2, and cyclin-dependent kinase 1 (Cdk1) are shown. Cell cycle arrest was confirmed by flow cytometry (Supplementary Fig S8B).

3. Cells expressing the indicated Hcm1 mutants from the TEF1 promoter were arrested in mitosis (Supplementary Fig S8C) and half-lives compared by cycloheximide-chase assay. Two exposures of Hcm1 blots are shown to highlight differences in stability between the double phosphomutants.

4. Cycloheximide-chase assay of Hcm1(1-107)-GFP and Hcm1(1-107)3N-GFP fusion proteins in asynchronous cells.

5. CDC53, cdc53-1, sic1Δ CDC53, and sic1Δ cdc53-1 cells expressing Hcm1(1-107)-GFP were arrested in mitosis (Supplementary Fig S8D) at the permissive temperature, shifted to 37°C for 15 min, and half-lives compared by cycloheximide-chase assay.

6. Fivefold dilutions of cells with the indicated genotypes were spotted onto rich medium plates (YPD), or plates containing the indicated concentrations of benomyl.

Source data are available online for this figure.

Figure 6. Phosphorylation of the C-terminus of Hcm1 is required for activity

1. Cells with the indicated genotypes were synchronized in late S-phase by arresting in G1 and collecting 45 min after release (Supplementary Fig S10A). Expression of target genes was compared by RT-qPCR. All values are normalized to ACT1 and shown relative to Hcm1 WT cells. Mean and standard deviations from technical replicates of a representative experiment are shown.

2. Fivefold dilutions of cells with the indicated genotypes were spotted onto rich medium plates (YPD), or plates containing 15 μg/ml benomyl.

3. ChIP-qPCR of V5-tagged Hcm1, Hcm1-8C, Hcm1-16E, and an untagged control from cells that were arrested in G1 and collected 37 min after release (Supplementary Fig S10B). Mean and standard deviations from three biological replicates are shown. For each primer set, binding is shown relative to Hcm1 wild-type.

Figure 7. Coordinated phosphorylation of S-phase transcription factors (TFs) is important for fitness

1. Median cell volume of asynchronous cultures carrying cyclin-dependent kinase (Cdk)- mutations in the indicated TFs. The mean and standard deviations from three independent experiments are graphed.

2. Fivefold dilution of cells from (A) were spotted onto rich medium plates (YPD), or plates containing 15 μg/ml benomyl (top). Growth on benomyl plates (at a sub-saturating dilution) was quantified with ImageJ and normalized to growth on YPD. Mean and standard deviations of relative growth from three independent experiments are graphed (bottom). Benomyl sensitive (red bars), wild-type sensitivity (blue bars), intermediate sensitivity (purple bars).

3. The percentage of cells in co-cultures of strains carrying PGK1-URA3 (blue lines) and PGK1-GFP-URA3 (red lines) were determined at the indicated time points. 4P, hcm1-15A tos4-9A yox1-9A yhp1-13A; 3P, tos4-9A yox1-9A yhp1-13A; 4del, hcm1Δ tos4Δ yox1Δ yhp1Δ. Mean and standard deviations of 4–6 experiments are shown.

Figure 8. Model of regulation of the late cell cycle gene regulatory network by Cdk1

See text for details.