A new transcription factor for mitosis: in Schizosaccharomyces pombe, the RFX transcription factor Sak1 works with forkhead factors to regulate mitotic expression

Abstract

Mitotic genes are one of the most strongly oscillating groups of genes in the eukaryotic cell cycle. Understanding the regulation of mitotic gene expression is a key issue in cell cycle control but is poorly understood in most organisms. Here, we find a new mitotic transcription factor, Sak1, in the fission yeast Schizosaccharomyces pombe. Sak1 belongs to the RFX family of transcription factors, which have not previously been connected to cell cycle control. Sak1 binds upstream of mitotic genes in close proximity to Fkh2, a forkhead transcription factor previously implicated in regulation of mitotic genes. We show that Sak1 is the major activator of mitotic gene expression and also confirm the role of Fkh2 as the opposing repressor. Sep1, another forkhead transcription factor, is an activator for a small subset of mitotic genes involved in septation. From yeasts to humans, forkhead transcription factors are involved in mitotic gene expression and it will be interesting to see whether RFX transcription factors may also be involved in other organisms.

Figure 1.

Fkh2 ChIP enrichment and analysis of ChIP-seq targets. (A) ChIP analysis by qPCR of asynchronous cultures of No Tag cells (F108) and cells expressing Fkh2-TAP tag (JLP1586). qPCR was done for two positive controls, cdc15 and spo12, and two negative controls, adh1 and srp7. Data show the mean ± S.E.M, (N = 7). P-value < 0.021. (B) Sequencing reads aligned to the Schizosaccharomyces pombe genome comparing Fkh2-TAP ChIP to No Tag ChIP upstream of cdc15 transcript. Blue and red indicate sequencing reads aligning to opposite strands (Also see Supplementary Figure S1). (C) Cell cycle time of peak expression of Fkh2 cell cycle ChIP-seq targets. Each target is assigned a cell cycle phase at peak expression, in degrees (58). (D) Motif analysis from the sequence of Fkh2 and Sak1 peaks. Motif comparison of New Motif (Fkh2 ChIP binding region) to the known motifs (all yeast) was by TOMTOM. (E) Frequency distribution of the distances (bp) between the centers of Fkh2 and Sak1 ChIP Seq peaks.

Figure 2.

Organization of Sak1 and Fkh motifs. Organization of the Sak1 (black) and Fkh (gray) motifs of (A) Fkh2 and (B) Sep1 targets. Motifs are displayed above or below the line depending on strandedness. The height of a motif is proportional to its quality (i.e. -log(P-value)) (max height; P-value = 10⁻¹⁰). Eight regions are shown for nine Sep1 targets because one pair of genes (SPAPB1E7.04c and gde1) is divergently transcribed.

Figure 3.

Sep1 targets. (A) Western analysis of Sep1-TAP tagged cells treated with Bortezomib or DMSO (solvent). (B) Venn diagram of the overlap between Fkh2, Sak1 and Sep1 coding targets. (C) Motif analysis of Sep1 binding regions by MEME. (D) A list of all Sep1p targets identified by ChIP-seq analysis. (E) Fkh2, Sep1 and Sak1 ChIP-seq coverage graphs upstream of ace2 and cdc15. All graphs depict sequence tag counts per base (average: 25 bp window; normalization: count/million reads); scale: 60. They are visualized in the integrated genome viewer, IGV (Broad Institute).

Figure 4.

Selective spore germination assays for fkh2Δ, sak1Δ and sep1Δ cells. (A) Flow cytometry analysis of DNA content for the selective spore germination of WT, fkh2Δ, sak1Δ and sep1Δ mutants. (B) Flow cytometry analysis as in Figure 4A for sak1–891 at restrictive temperature. 0 time: permissive temperature. Figure 4A, B: Y axis, cell number; Z axis, hours. (C) Gene expression analysis from the selective spore germination (S.G.) of the fkh2Δ, sak1Δ and sep1Δ deletion mutants and the sak1–891-ts mutant. The control for the fkh2Δ S.G., sak1Δ S.G. and sep1Δ S.G. was the WT S.G. experiment at each of the time points respectively. The control for sak1–891-ts strain was a WT (F14) strain kept at that temperature for an equal duration of time. The cluster represents both direct and indirect roles of each transcription factor (whole-genome cluster analysis: Dataset S1). The clustering process is described in the methods section. Each “ChIP-Seq” column represents the results described above of the ChIP-Seq experiments with the relevant transcription factors (Fkh2, Sak1, or Sep1) for each gene in the cluster (e.g., cdc15 bound to Fkh2 and Sak1 in the ChIP-Seq experiments, but not to Sep1). Every row represents a gene and every column represents a microarray or ChIP-Seq experiment. Red signifies upregulation (Experiment/Control > 1), green signifies downregulation (Experiment/Control < 1). Black signifies no change. Dynamic range is 16-fold from reddest to greenest (key: fold change). HU: Hydroxyurea.

Figure 5.

Genetic phenotypes and interactions. (A) Sak1 overexpression. DIC (Differential Interference Contrast) microscopy and fluorescence images (DAPI/Calcofluor) of WT (JLP988) cells either containing an empty vector (pJR-3XL) or a plasmid overexpressing Sak1 from the nmt1 promoter (pJR-3XL-sak1) under derepressed (-thiamine) conditions at 20 h. Scale bar: 10 μm. (B) Percentage of cells from the experiment in Figure 5A showing 2 nuclei (bi-nucleate), 2 nuclei with septa in between (septate) or showing an aberrant phenotype (e.g. multiple septa). Hundred cells were counted for each experiment at the given times. (C) The lengths of septated control cells and septated Sak1-overexpressing cells from the experiment of Figure 5A (t-test, P-value < 3.51 × 10⁻⁷). (D) The lengths of septated control cells and septated fkh2Δ cells at 12 h into the selective spore germination experiment (t-test, P-value < 1.5 × 10⁻⁵). (E) cdc25–22 block release of WT (cdc25–22, JLP1679) and fkh2–3 cdc25–22 (JLP1770) cells. Septation is shown as a function of time. (F) Serial dilutions of cdc25–22 (JLP1635), fkh2Δ (JLP1501) and cdc25–22 fkh2Δ (JLP1741, JLP1745) were grown on YES plates at the indicated temperatures. (G) Growth assays of two biological duplicates each of WT, fkh2Δ, sep1Δ and fkh2Δ sep1Δ mutants obtained from a cross of fkh2::ura4⁺ (JLP1501) and sep1::LEU2 (JLP1823) on a YES plate. (H) Percentage of cells bearing 1 septum, or 2 or more septa, in WT, fkh2Δ, sep1Δ, or fkh2Δ sep1Δ mutants. Two hundred cells from each of three biological replicates grown in liquid YES medium were counted and are represented by the mean ± S.E.M.