Schematic model for HU-induced expression responses. Kinases Rad3p, Cds1p, Chk1p, and Sty1p are shown in rectangles and transcription factors are in ovals. Kinases directly or indirectly regulate transcription factors. Arrows and blunted lines indicate positive and negative regulations, respectively. Expression profiles of the CESR-, MCB-, and FKH-clusters in presence (+HU) and absence (−HU) of HU are shown.

The FKH- and ACE2-clusters. (A) Magnified view of the FKH-cluster as in Figure 2B. Individual genes in the cluster are shown on the right. (B–E) Average expression profiles of the FKH-cluster. The x- and y-axis indicate expression ratios and time points after HU treatment, respectively. An arrow indicates the time of expression level changes. Asterisks indicate the defect in expression induction. Expression levels of the FKH-cluster in synchronized cells supplemented with HU (+HU) and without (−HU) are separated compared with a common reference (B). Expression levels in HU-treated samples as in B are directly compared with those in the control (C). Expression levels of the FKH-cluster in asynchronized cells supplemented with HU are compared with those without HU (D and E). (F) Magnified view of the ACE2-cluster as in Figure 2B. Individual genes in the cluster are shown on the right. (G) Average expression profiles of the ACE2-cluster. The x- and y-axis indicate expression ratios and time points after HU treatment, respectively. Expression levels of the histone-cluster in synchronized cells supplemented with HU (+HU) and without (−HU) are separated compared with a common reference.

Average expression profiles of highly coregulated clusters CESR(1), CESR(2), and RIBO(1). The x- and y-axis indicate expression ratios and time points after HU treatment, respectively. Arrowheads indicate expression levels in cells before HU addition. An arrow indicates the time of expression level changes. (A–C) Average expression profiles of the CESR(1)-cluster. Expression levels of the CESR(1)-cluster in synchronized cells supplemented with HU (+HU) and without (−HU) are separated compared with a common reference (A). Expression levels in HU-treated samples as in (A) are directly compared with those in the control (B). Expression levels of the CESR(1)-cluster in asynchronized cells supplemented with HU are compared with those without HU (C). (D and E) Average expression profiles of the CESR(2)-cluster. Comparisons of expression levels in D and E are the same as described in A and B, respectively. (F) Average expression profiles of the RIBO(1)-cluster. Comparisons of expression levels is the same as described in A.

The MCB- and histone-clusters. (A) Magnified view of the MCB-cluster as in Figure 2B. Individual genes in the cluster are shown on the right. (B–E) Average expression profiles of the MCB-cluster. The x- and y-axis indicate expression ratios and time points after HU treatment, respectively. An arrow indicates the time of expression level changes. Asterisks indicate the defect in expression induction. Expression levels of the MCB-cluster in synchronized cells supplemented with HU (+HU) and without (−HU) are separated compared with a common reference (B). Expression levels in HU treated samples as in B are directly compared with those in the control (C). Expression levels of the MCB-cluster in asynchronized cells supplemented with HU are compared with those without HU (D and E). (F) Magnified view of the histone-cluster as in Figure 2B. Individual genes in the cluster are shown on the right. (G) Average expression profiles of the histone-cluster. The x- and y-axis indicate expression ratios and time points after HU treatment, respectively. Expression levels of the histone-cluster in synchronized cells supplemented with HU (+HU) and without (−HU) are separated compared with a common reference.

Regulation of protein levels of forkhead transcription factors Sep1p and Fkh2p upon HU treatment. (A–C) Cells treated with 8 mM HU were sampled at various time points as indicated for Western blot analysis. Strains bearing a sole copy of fhk2⁺-3HA and sep1⁺-3HA were used for analysis of Fkh2p and Sep1p levels after HU treatment in A and B, respectively. Tubulin levels were used as loading control. Membranes in B were stripped and reblotted for determination of Cdc2-Y15-P levels. Levels of PSATIR were used as loading control. Individual bands are quantified using ImageJ software. Fold-changes of Sep1p and Cdc2-Y15-P levels are plotted after 0-h normalization. (D) Levels of Sep1p are oscillated along cell cycle. Cells bearing a sole copy of sep1⁺-3HA in the cdc25-22 background were synchronized by temperature shift. G₂-arrested cells were released to permissive temperature and sampled at various time points for Western blot analysis of Sep1p levels. Arrowheads indicate the peak levels of Sep1p coincident with peak of mitotic indexes. (E) Plating assays for sensitivity to HU. Approximately 5 μl of 10-fold series diluted samples were inoculated onto plates containing 2.5 mM HU. An arrow indicates that phenotype of rad3Δ strain is alleviated when sep1Δ is in the background. (F) Frequencies of HU-induced cut phenotype. The cut phenotype was examined after rad3Δ and rad3Δ sep1Δ cells were challenged with 8 mM HU for 3 h. Numbers of cells with cut phenotype are countered from >500 cells.

Regulation of protein levels of individual MBF factors Cdc10, Res1p, Res2p, and Rep2p upon HU treatment. (A–C) Cells treated with 8 mM HU were sampled at various time points as indicated for Western blot analysis. Strains bearing a sole copy of cdc10⁺-3HA, res1⁺-3HA, and res2⁺-3HA were used for analysis of protein levels after HU treatment in A, and strains bearing a sole copy of rep2⁺-3HA were used as indicated in B. The tubulin levels were used as loading control. Membranes in B were stripped and reblotted for determination of Cig2p levels. Individual bands were quantified using ImageJ software (National Institutes of Health, Bethesda, MD). Fold changes of Rep2p and Cig2p levels are plotted after 0-h normalization. (D) Average expression profiles of the MCB-cluster in cig2Δ cells. The x- and y-axis indicate expression ratios and time points after HU treatment, respectively. (E) Plating assays for HU susceptibility. Approximately 5 μl of 10-fold series diluted samples were inoculated onto plates supplemented with 5 mM HU and grown at 30°C for ∼2–3 d. Arrows and arrowhead indicate strains with and without noticeable sensitivities to HU, respectively. (F) Plating assays for sensitivity to HU. ∼5 μl of 10-fold series diluted samples were inoculated onto plates containing 2.5 mM HU and supplemented with thiamine (repressed for rep2⁺) or without thiamine (induced for rep2⁺). Arrows indicate that phenotypes of rad3Δ and cds1Δ strains are alleviated when rep2⁺ is over produced.

Phenotypic assessment and expression profiling of wild-type and kinase mutant cells upon HU treatment. (A) Plating assay for kinase mutant sensitivities to prolonged or transient exposure to HU. For prolonged HU treatment, ∼5 μl of 10-fold series diluted wild-type, rad3Δ, cds1Δ, and chk1Δ strains were spotted and grown on YES plates supplemented with 8 mM HU at 30°C. For transient HU treatment, cells were treated with 8 mM HU for 3 h and recovery on plate without supplement with HU. (B) Images of wild-type and rad3Δ cells after HU treatment for 3 h. Nuclei and division septa were stained using DAPI and Aniline blue, respectively. Arrowheads indicate cells with a cut phenotype. (C) Mitotic indexes of synchronized G₂ cell cultures. Mitotic indexes or percentage of binucleate cells was determined in synchronized cultures at various time points as indicated after addition of HU (+HU) or without as control (−HU). (D) DNA content profiles in synchronized cultures. DNA contents in samples (C) were determined by FACS. Red arrows indicate a 4C-peak and a 1C-peak in cultures without and with HU supplement, respectively. (E) Hierarchical cluster analysis of expression profiles in various strains supplemented with or without HU. Expression profile clustering of the 263 differentially expressed genes from wild-type, rad3Δ, cds1Δ, and chk1Δ strains supplemented with or without HU. The dendrogram on the left depicts the structure of the clusters, and three groups—CESR, RIBO, and CDCE—are indicated. The phasogram on the right show the levels of gene expression at various time points as in C. Genes correspond to rows and time points form the columns. Color intensities reflect the magnitudes of induction (red) or repression (green) for each gene (see key at bottom right). Black indicates no change of expression levels; and gray refers to data not available. (F) Characterization of genes in CESR, RIBO, and CDCE groups. Numbers of genes belong to induced (up) CESR, repressed (down) CESR, and cell cycle–regulated (CDC), Rad3p-dependent, and Cds1p-dpenedent genes in three groups are indicated. Enrichment of particular gene types are indicated as *** and ** for p < 0.001 and p < 0.01, respectively.