The Forkhead Transcription Factor Fkh2 Regulates the Cell Division Cycle of Schizosaccharomyces pombe

Abstract

In eukaryotes the regulation of gene expression plays a key role in controlling cell cycle progression. Here, we demonstrate that a forkhead transcription factor, Fkh2, regulates the periodic expression of cdc15 ؉ and spo12 ؉ in the M and G 1 phases of the cell division cycle in the fission yeast Schizosaccharomyces pombe. We also show that Fkh2 is important for several cell cycle processes, including cell morphology and cell separation, nuclear structure and migration, and mitotic spindle function. We find that the expression of fkh2 ؉ is itself regulated in a cell cycle-dependent manner in G 1 coincident with the expression of cdc18 ؉ , a Cdc10-regulated gene. However, fkh2؉ expression is independent of Cdc10 function. Fkh2 was found to be phosphorylated during the cell division cycle, with a timing that suggests that this posttranslational modification is important for cdc15؉ and spo12 ؉ expression. Related forkhead proteins regulate G 2 and M phase-specific gene expression in the evolutionarily distant Saccharomyces cerevisiae, suggesting that these proteins play conserved roles in regulating cell cycle processes in eukaryotes.Forkhead transcription factors in eukaryotes have been implicated in a number of cellular processes including embryogenesis, development, and the cell division cycle. Forkhead proteins contain a conserved region spanning a monomeric DNA binding (FKH) domain (for reviews, see references 26 and 31). Indeed, a core DNA consensus sequence has been identified to which many forkhead proteins bind (27). Mutations in genes encoding forkhead proteins have been linked with several pathological conditions including thyroid agenesis (11), cleft palate (11), glaucoma (38), speech development defects (30), and tumorigenesis (16,32,39).In eukaryotes the periodic expression of genes plays an important role in the regulation of the cell division cycle. Global approaches in the budding yeast Saccharomyces cerevisiae have provided insight into the groups of genes that are expressed in this manner and also the transcription factors responsible for this cell cycle-dependent expression (10,44,47). The MBF (MluI binding factor), consisting of Mbp1 and Swi6, and the related SBF (Swi4/Swi6 cell cycle box binding factor), consisting of Swi4 and Swi6, transcription factor complexes are important for regulation of genes that are expressed early in the cell division cycle (see reference 51 for a review). In contrast, the forkhead proteins Fkh1 and Fkh2 regulate cell cycle-dependent expression of a group of genes, named the CLB2 gene cluster, that includes CLB1 and CLB2, encoding mitotic cyclins (18, 48), in the G 2 and M phases (28,29,40,53). Regulation of the CLB2 gene cluster involves the formation of a ternary complex between Fkh2 and the MADS-box protein, Mcm1, and the coactivator Ndd1 (28, 29, 40, 53; see references 8, 17, and 25 for reviews). The altered expression of the CLB2 gene cluster in a fkh1⌬ fkh2⌬ mutant is associated with defects in the structure of the mitotic spindle, nuclear migration, cell morp...

Promoter-driven splicing regulation in fission yeast

“…It is involved in septum formation, cytokinesis, nuclear structure and mitotic spindle function (Bulmer et al, 2004). It was also suggested that Fkh2 was implicated in the periodic gene expression of M and G1 phases (Buck et al, 2004), (when it is phosphorylated (Buck et al, 2004)) by negatively regulating the transcription of a cluster of genes (Rustici et al, 2004).…”

“…It was also suggested that Fkh2 was implicated in the periodic gene expression of M and G1 phases (Buck et al, 2004), (when it is phosphorylated (Buck et al, 2004)) by negatively regulating the transcription of a cluster of genes (Rustici et al, 2004). The null mutant displays longer length, coldsensitive, heat-sensitive and slow-growing phenotypes (Buck et al, 2004), (Bulmer et al, 2004). Regarding meiosis, a null mutant presents low levels of ste11 mRNA, dephosphorylation at Fkh2 residues T314 and S462 is required for binding to ste11 promoter region and induction of ste11 transcription during nitrogen starvation (Shimada et al, 2007).…”

A homeodomain transcription factor regulates the DNA replication checkpoint in yeast

“…Protein extracts were prepared as described previously using lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.5% Nonidet P-40, 10 mM imidazole, 0.1% leupeptin, 0.1% pepstatin A, 1% aprotinin, 1% phenylmethylsulfonyl fluoride, 0.2% Na 3 VO 4 , 5% NaF). 33 Proteins to be in MBF-dependent gene expression occurs in yox1Δ cells treated with HU and, significantly, both Rad3 and Cds1 are essential for replication stress-induced phosphorylation of Yox1. Since the timing of Rad3/Cds1-dependent phosphorylation of Yox1 coincides with the upregulation of MBF-dependent gene expression, we suggest that this phosphorylation of Yox1 is essential for dissociation of Yox1 from MBF target promoters and activation of gene expression (Fig.…”

An Atg10-like E2 enzyme is essential for cell cycle progression but not autophagy inSchizosaccharomyces pombe

“…Cultures were grown and extracts prepared using lysis buffer (50 mM TRIS-HCl pH 7.5, 150 mM NaCl, 0.5% Nonidet P-40, 10 mM imidazole, 0.1% leupeptin, 0.1% pepstatin A, 1% aprotinin, 1% PMSF, 0.2% Na 3 VO 4 , 5% NaF). 31 Protein samples were then analyzed by SDS-PAGE and transferred onto a nitrocellulose transfer membrane (Protran ® ). To investigate autophagic activity, the levels of Atg8-GFP and free GFP were examined using a 1:2,000 dilution of mouse anti-GFP antibody (Invitrogen) and a 1:2,000 dilution of anti-mouse HRP-conjugated secondary antibody (Sigma-Aldrich).…”

“…To analyze microtubules in mitotic cells, cells were fixed and prepared for microscopy as described previously. 31 To visualize microtubules, a 1:1,000 dilution of anti-TAT1 antibody (Cancer Research UK) and a 1:200 dilution of secondary antibody [Alexa Fluor ® FITC-conjugated anti-mouse antibody (Invitrogen)] were used for indirect immunofluorescence. Cells were mounted onto poly-L-lysine-coated microscope slides with Vectashield mounting medium (containing DAPIVector Laboratories).…”