A systematic screen for transcriptional regulators of the yeast cell cycle

Abstract

Transcription factors play a key role in the regulation of cell cycle progression, yet many of the specific regulatory interactions that control cell cycle transcription are still unknown. To systematically identify new yeast cell cycle transcription factors, we used a quantitative flow cytometry assay to screen 268 transcription factor deletion strains for defects in cell cycle progression. Our results reveal that 20% of nonessential transcription factors have an impact on cell cycle progression, including several recently identified cyclin-dependent kinase (Cdk) targets, which have not previously been linked to cell cycle transcription. This expanded catalog of cell-cycle-associated transcription factors will be a valuable resource for decoding the transcriptional regulatory interactions that govern progression through the cell cycle. We conducted follow-up studies on Sfg1, a transcription factor with no previously known role in cell cycle progression. Deletion of Sfg1 retards cells in G(1), and overexpression of Sfg1 delays cells in the G(2)/M phase. We find that Sfg1 represses early G(1), Swi5/Ace2-regulated genes involved in mother-daughter cell separation. We also show that Sfg1, a known in vitro cyclin-dependent kinase target, is phosphorylated in vivo on conserved Cdk phosphorylation sites and that phosphorylation of Sfg1 is necessary for its role in promoting cell cycle progression. Overall, our work increases the number of transcription factors associated with cell cycle progression, strongly indicates that there are many more unexplored connections between the Cdk-cyclin oscillator and cell cycle transcription, and suggests a new mechanism for the regulation of cell separation during the M/G(1) phase transition.

Figure 1.—

Screen for cell cycle phenotypes among transcription factor deletions. (A) Scoring procedure for the initial high-throughput screen: strains were tested in 96-well plates, each of which contained eight replicates of the wild-type strain and a single copy of each deletion strain. Cell cycle profiles of the wild-type strains were averaged to produce a baseline cell cycle distribution. Cell cycle distributions of the deletion strains were compared against the baseline by calculating a z-score, as described in materials and methods. (B) Outline of the procedure for identifying transcription factor deletions with altered cell cycle profiles.

Figure 2.—

Distribution of cell cycle phenotypes and association with cell-size control. (A) Distribution of cell cycle mutants by phase. Most hits exhibited an accumulation of cells in G₁, although all phases of the cell cycle were represented among the hits. (B) Cell cycle transcription factor mutants are enriched in cell-size mutants categorized as the largest or the smallest 5% of the strains in Jorgensen et al. (2002).

Figure 3.—

Impact of Sfg1 on the cell cycle. (A) Flow cytometry results demonstrate that a Δsfg1 strain exhibits an accumulation of cells in the G₁ phase of the cell cycle relative to the wild-type (WT) strain. (B) Overexpression of SFG1 results in the accumulation of cells in G₂/M relative to a strain harboring an empty plasmid. The difference in wild-type %G₁ is due to differences in growth media–rich media with 2% dextrose in A and synthetic uracil dropout media with 2% galactose in B. Error bars indicate standard error.

Figure 4.—

Sfg1 is an in vivo cyclin-dependent kinase target. (A) Sfg1 contains three conserved Cdc28 consensus sequences (S/T-P-X-X-K/R) with partial consensus sequences (S/T-P) conserved in Saccharomyces kluyveri. (B) Phosphatase-treated wt-Sfg1 from cell extracts migrates faster on SDS–PAGE, indicating that it is phosphorylated in vivo; untreated 3xA-sfg1 migrates with phosphatase-treated Sfg1, indicating that abolition of the three Cdc28 consensus sites inhibits in vivo phosphorylation. (C) Abolition of Sfg1 phosphorylation sites recapitulates the cell cycle defect of Δsfg1, assayed by flow cytometry in synthetic uracil dropout media. The plasmids listed correspond to the following plasmids described in materials and methods: pEmpty, pMW100; pSFG1, pMW104; p3xA-sfg1, pMW113. Error bars indicate standard error.