Identification of cell cycle-regulated genes in fission yeast

Abstract

Cell cycle progression is both regulated and accompanied by periodic changes in the expression levels of a large number of genes. To investigate cell cycle-regulated transcriptional programs in the fission yeast Schizosaccharomyces pombe, we developed a whole-genome oligonucleotide-based DNA microarray. Microarray analysis of both wild-type and cdc25 mutant cell cultures was performed to identify transcripts whose levels oscillated during the cell cycle. Using an unsupervised algorithm, we identified 747 genes that met the criteria for cell cycle-regulated expression. Peaks of gene expression were found to be distributed throughout the entire cell cycle. Furthermore, we found that four promoter motifs exhibited strong association with cell cycle phase-specific expression. Examination of the regulation of MCB motif-containing genes through the perturbation of DNA synthesis control/MCB-binding factor (DSC/MBF)-mediated transcription in arrested synchronous cdc10 mutant cell cultures revealed a subset of functional targets of the DSC/MBF transcription factor complex, as well as certain gene promoter requirements. Finally, we compared our data with those for the budding yeast Saccharomyces cerevisiae and found approximately 140 genes that are cell cycle regulated in both yeasts, suggesting that these genes may play an evolutionarily conserved role in regulation of cell cycle-specific processes. Our complete data sets are available at http://giscompute.gis.a-star.edu.sg/~gisljh/CDC.

Figure 2.

The expression profiles of 747 cell cycle-regulated genes in S. pombe. Genes correspond to the rows, and the time points (10-min intervals) in each experiment form the columns. The intensities of the colors indicate the magnitudes of induction (red) or repression (green) for each gene (see key at bottom right). Black, no change of expression levels; gray, data not available. The color bars on top indicate the cell cycle phases according to Figure 1. (A) Genes are ordered by their times of peak expression; phases of peak expression are indicated by the colored bars on the right. The graph on the left shows the distributions of promoter motifs indicated as densities relative to the background density for that motif (see Materials and Methods). The distribution of the PCB motif (asterisk) was not significantly enriched at any stage of the cell cycle. (B) Genes that share similar expression profiles are grouped by cluster analysis as described in the text. The dendrogram on the left shows the structures of the clusters; the bars on the right indicate sets of genes sharing the indicated promoter motifs.

Figure 1.

Characterization of the synchronous cultures. (A) FACS analysis of a synchronous wild-type culture. For each time point, 10,000 propidium iodine-stained cells were measured for fluorescence intensity. 2C indicates the DNA content of an interphase cell containing a G2 nucleus or a mitotic cell containing two G1 nuclei. 4C indicates the DNA content of a septated cell containing two G2 nuclei. (B) Mitotic (binucleate-cell) and septation indexes of synchronous cultures of wild-type (top) and cdc25–22 (bottom) cells. Black arrows, start times for the samples analyzed; red arrows, times of coincident peaks of 4C cells and septated cells in the wild-type culture; colored bars, cell cycle phases.

Figure 3.

The G1-phase clusters. The transcription profiles are displayed as in Figure 2B. The systematic names plus the gene symbols (where available) are listed on the right. Genes possessing the indicated promoter motifs are marked by a “+” on the right. (A) Expanded view of the MCB cluster (see Figure 2B). The red + indicates that the MCB motifs are not within 1 kb of the start codon (see text). The expression profiles from three independent asynchronous cultures of the cdc10-C4 mutant at permissive temperature (vs. asynchronous wild-type cells as reference) are also shown. (B) Perturbation of the DSC transcriptional activity by use of a cdc10 mutation (see Materials and Methods). The top two panels show mitotic index (binuc), septation index (septa), and DNA contents of the elutriation-synchronized wild-type and cdc10-V50 mutant cultures at 36°C. The bottom panel shows the expression profiles of the MCB cluster genes. The solid boxes on the right indicate genes whose G1 expression was reduced in the cdc10 mutant and thus are classified as DSC dependent. The numbers of MCB and ACE2 motifs associated with each gene are also indicated. (C) Expanded view of the ACE2 cluster (see Figure 2B). The expression profiles from three independent asynchronous cultures of the Δace2 mutant (vs. asynchronous wild-type cells as reference) also are shown.

Figure 4.

The S- and G2-phase clusters. The transcription profiles and gene symbols are displayed as in Figures 2B and 3. (A) The S-phase cluster. (B and C) The two RiP clusters containing ribosomal protein genes. (D) The ATF cluster. The presence or absence of ATF motifs is indicated on the right.

Figure 5.

The M-phase clusters. The transcription profiles and gene symbols are displayed as in Figures 2B and 3. The presence or absence of SFF motifs is indicated on the right. The expression profiles from three independent asynchronous Δsep1 mutant cultures (vs. asynchronous wild-type cells as reference) also are shown. (A) The SFF (1) cluster. Most genes have peak expression in M phase. (B) The SFF (2) cluster. Most genes have peak expression in either M or G1 phase.

Figure 6.

S. pombe and S. cerevisiae orthologues showing cell cycle-regulated expression. (A) Venn diagram summarizing orthologies and cell cycle-regulated expression in the two yeasts. The thin red (blue) circle and the sum of red (blue) numbers represent the 4970 (6000) ORFs in S. pombe (S. cerevisiae). The thick circles and enclosed numbers represent the cell cycle-regulated genes. Thus, 139 S. pombe genes with 142 S. cerevisiae orthologues (including multiple-gene pairs, or co-orthologs) were cell cycle regulated in both yeasts; 417 S. pombe genes with 550 S. cerevisiae orthologues were cell cycle regulated in S. pombe but not in S. cerevisiae; and so on. (B) Expression profiles of the ∼140 orthologues as separated into 194 one-to-one pairs (see text). Expression profiles of the 139 S. pombe genes were displayed based on the elutriation experiment and ordered based on the timing of their peak expression (cf. Figure 2A). Expression profiles of the 142 S. cerevisiae genes were based on the α-factor experiment (Spellman et al., 1998) and ordered according to their orthologues in S. pombe. (C) Distribution of the peak-expression phases for the 194 ortholog pairs. The numbers of gene pairs in each category are based on the assignment of peak-expression phases for the individual genes in S. pombe (this study) and S. cerevisiae (Spellman et al., 1998). Numbers in parentheses are the expected numbers of pairs based on the subtotals for each yeast (gray-shaded boxes) and the assumption of no correlation in peak-expression phases between the two yeasts. Pink-shaded and unshaded boxes indicate gene pairs with concordant and discordant expressions, respectively.

Figure 7.

Expression profiles of the 108 orthologue pairs with concordant phases of peak expression. The transcription profiles are displayed as in Figure 6B. The assigned phases of peak expression and functions in biological processes (where known) of the gene pairs are given. Genes encoding proteins involved in DNA replication or chromosome structure are shown in blue; genes encoding proteins involved in cell wall biogenesis or cytokinesis are shown in brown.

Figure 8.

Expression profiles of the 86 orthologues with discordant phases of peak expression. Information is displayed as described in Figure 7.