Fkh2p and Sep1p regulate mitotic gene transcription in fission yeast

doi:10.1242/jcs.01473

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First published online October 27, 2004
doi: 10.1242/10.1242/jcs.01473

Journal of Cell Science 117, 5623-5632 (2004)
Published by The Company of Biologists 2004

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Fkh2p and Sep1p regulate mitotic gene transcription in fission yeast

Vicky Buck¹^,*, Szu Shien Ng²^,*, Ana Belen Ruiz-Garcia¹^,*^,, Kyriaki Papadopoulou², Saeeda Bhatti², Jane M. Samuel¹, Mark Anderson², Jonathan B. A. Millar¹^, and Christopher J. McInerny²^,

¹ Division of Yeast Genetics, National Institute for Medical Research, The Ridgeway, Mill Hill, London, NW7 1AA, UK
² Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow, G12 8QQ, UK

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Fig. 1. S. pombe fkh2⁺. (A) Schematic representation of the predicted 642 amino acid S. pombe Fkh2p, forkhead-like protein (SPBC16G5.15c) (Wood et al., 2002

). Positions of the forkhead domain (FHD; 232-342) and the forkhead associated domain (FHA; 78-158) are shown. Arrow indicates position of the C-terminal truncation clone (1-392) that suppresses mcs3-12 wee1-50 cdc25-22. Phylogenetic tree showing the relationship between S. cerevisiae and S. pombe forkhead polypeptides, based on multiple sequence alignment, generated using the DNA cluster method in the Megalign programme based on a Jotun Hein algorithm (DNASTAR). (B) fkh2⁺ is non-essential. Heterozygous diploids containing a fkh2::kan^R (fkh2 ${Delta}$ ) and a wild-type allele were sporulated on EMM. Asci were separated by tetrad dissection and spores germinated on YE for 4 days, before replica plating on YE plus G418. (C) fkh2 ${Delta}$ cells have a variable length at division. Micrographs of wild-type (GG 1) and fkh2 ${Delta}$ (JM 2286) cells, growing in liquid EMM at 28°C. The length of 100 septating cells was measured and the mean length and standard deviation measured. Bar, 10 µm. (D) fkh2 ${Delta}$ (JM 2286) cells show abnormal septation. Fluorescent micrographs of fkh2 ${Delta}$ cells growing in liquid EMM at 28°C. Cells were fixed, the DNA stained with DAPI and septa marked with calcofluour. Representative images illustrating the range and incidence of septation defects (estimated from 100 cells) are shown. Arrows indicate positions of septa. Bar, 5 µm.

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Fig. 2. Role of fkh2⁺ in M/G1-specific transcription. (A) fkh2⁺, ace2⁺ and slp1⁺ are periodically expressed during M-phase. cdc25-22 (GG 308) cells were synchronised by transient temperature arrest and samples taken every 15 minutes upon release to the permissive temperature. RNA was analysed by northern blot using fkh2⁺, slp1⁺, ace2⁺, cdc15⁺, spo12⁺, fin1⁺ and adh1⁺ as probes, the latter as a loading control. Quantification of each transcript against adh1⁺ is shown. Septation indices were counted microscopically and are plotted to indicate the synchrony of the culture. (B) Loss of M/G1 periodic transcription in fkh2 ${Delta}$ cells. cdc25-22 (GG 308) and fkh2 ${Delta}$ cdc25-22 (JM 2285) cells were synchronised by transient temperature arrest and samples taken every 15 minutes upon release to the permissive temperature. RNA was analysed by northern blot using slp1⁺, ace2⁺, cdc15⁺, spo12⁺, fin1⁺ and adh1⁺ as probes, the latter as a loading control. Quantification of each transcript against adh1⁺ is shown. Septation indices were counted microscopically and are shown in black (fkh2 ${Delta}$ cdc25-22) or grey (cdc25-22).

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Fig. 3. fkh2⁺ and sep1⁺ share an overlapping function. (A) sep1 ${Delta}$ rescues the lethal phenotype caused by overexpression of fkh2⁺. pREP3X:fkh2⁺ and pREP3X were transformed into wild-type and sep1 ${Delta}$ cells, and colonies streaked on EMM agar to induce overexpression of fkh2⁺. Micrographs of wild-type and sep1 ${Delta}$ cells overexpressing fkh2⁺, grown on EMM agar. Bar, 30 µm. (B) Loss of periodic transcription in sep1 ${Delta}$ cells. sep1 ${Delta}$ cdc25-22 (A 62) cells were synchronised by transient temperature arrest and samples taken every 15 minutes for northern blot analysis after release to the permissive temperature. The blot was probed consecutively with fkh2⁺, slp1⁺, ace2⁺, cdc15⁺, spo12⁺, fin1⁺ and adh1⁺ probes, the latter as a loading control. Quantification of each transcript against adh1⁺ is shown.

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Fig. 4. Requirement of fkh2⁺, sep1⁺ and the PCB sequence for M/G1-specific transcription. (A) A cdc15⁺ promoter fragment containing the PCB sequence confers M/G1-specific transcription. cdc25-22 (GG 308) cells containing pSP ${Delta}$ 178.15UAS (Anderson et al., 2002

) were synchronised by transient temperature arrest and samples taken every 15 minutes upon release to the permissive temperature. RNA was analysed by northern blot using lacZ, cdc15⁺ and adh1⁺ as probes, the latter as a loading control. Quantification of each transcript against adh1⁺ is shown. Septation indices could not be counted for this culture, because of sep1 ${Delta}$ septation defect. (D) PCB sequence is required for M/G1-specific transcription in fission yeast. cdc25-22 (GG 308) cells containing pSP ${Delta}$ 178.15UAS.MUT (GB 344) were synchronised by transient temperature arrest and samples taken every 15 minutes upon release to the permissive temperature. RNA was analysed by northern blot using lacZ, cdc15⁺ and adh1⁺ as probes, the latter as a loading control. Quantification of each transcript against adh1⁺ is shown. Septation indices were counted microscopically and are plotted to indicate the synchrony of the culture.

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Fig. 5. S. pombe mbx1⁺. (A) Schematic representation of the predicted 436 amino acid S. pombe Mbx1p, MADS box-like protein (SPBC19G7.06). Position of the MADS box motif (M; 20-80) is shown. Phylogenetic tree showing the relationship between S. cerevisiae and S. pombe MADS box-like polypeptides, based on multiple sequence alignment, generated using the DNA cluster method in the Megalign programme based on a Jotun Hein algorithm (DNASTAR). (B) Fluorescent micrographs of mbx1 ${Delta}$ (JM 2331) cells, growing exponentially in EMM at 28°C. DNA stained with DAPI and septa marked with calcofluor. Representative images illustrating cytokinetic defects are shown. Bar, 10 µm. (C) Genetic interaction between mbx1⁺ and fkh2⁺. fkh2 ${Delta}$ sep1 ${Delta}$ (GG 631), fkh2 ${Delta}$ mbx1 ${Delta}$ (GG 551), sep1 ${Delta}$ mbx1 ${Delta}$ (GG 543) and sep1 ${Delta}$ fkh2 ${Delta}$ mbx1 ${Delta}$ (GG 754) cells were streaked on YE agar and grown at 28°C for 3 days. (D) sep1 ${Delta}$ , fkh2 ${Delta}$ and mbx1 ${Delta}$ show synthetic cytokinetic defects. Micrographs of cells growing in liquid EMM at 28°C: wild-type (GG 1), fkh2 ${Delta}$ (JM 2286), mbx1 ${Delta}$ (JM 2331), sep1 ${Delta}$ (GG 515), fkh2 ${Delta}$ mbx1 ${Delta}$ (GG 551), fkh2 ${Delta}$ sep1 ${Delta}$ (GG 631), sep1 ${Delta}$ mbx1 ${Delta}$ (GG 543) and fkh2 ${Delta}$ mbx1 ${Delta}$ sep1 ${Delta}$ (GG 754) were shown. Bar, 10 µm.

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Fig. 6. Mbx1p is a component of PBF and interacts with Fkh2p. (A) Effect of gene deletions on PBF binding activity. Gel retardation analysis was performed using a cdc15⁺ promoter fragment as labelled probe with 20 µg of protein extracts from wild-type (GG 1), sep1 ${Delta}$ (GG 515), fkh2 ${Delta}$ (JM 2286), mbx1 ${Delta}$ (JM 2331), fkh2 ${Delta}$ sep1 ${Delta}$ (GG 631), fkh2 ${Delta}$ mbx1 ${Delta}$ (GG 551) and sep1 ${Delta}$ mbx1 ${Delta}$ (GG 543) cells are shown. Lane `f' indicates free probe. (B) Effect of gene tags on PBF binding activity. Gel retardation analysis was performed with protein extracts from wild-type (GG 1) cells or protein extracts from mbx1-13myc (JM 2349), fkh2-3HA (JM 2257) and fkh2-13myc (JM 2252) cells, either without plasmid (-) or transformed with a plasmid expressing a genomic copy of the equivalent wild-type gene (g), or transformed with a control plasmid (p). Lane `f' indicates free probe. (C) mbx1⁺ is not required for periodic transcription in M phase. cdc25-22 (GG 308) and mbx1 ${Delta}$ cdc25-22 (JM 2332) cells were synchronised by transient temperature arrest, and upon release to the permissive temperature samples taken every 15 minutes for northern blot analysis. The blot was probed consecutively with fkh2⁺, slp1⁺, ace2⁺, cdc15⁺, spo12⁺, fin1⁺ and adh1⁺ probes, the latter as a loading control. Quantification of each transcript against adh1⁺ is shown. Septation indices were counted microscopically and are shown in black (mbx1 ${Delta}$ cdc25-22) or grey (cdc25-22).

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Fig. 7. Fkh2p and Mbx1p are phosphorylated during M-phase. (A) Cell cycle-specific changes in Fkh2p and Mbx1p mobility. Cultures of fkh2-3HA cdc25-22 (JM 2253) and mbx1-13myc cdc25-22 (JM 2252) cells were synchronised by transient temperature arrest and upon release to the permissive temperature, samples taken every 5 minutes for western blot analysis. Blots were probed with anti-HA antibodies to detect Fkh2p, anti-myc antibodies to detect Mbx1p, and anti-PSTAIRE antibodies to detect Cdc2p as an invariant loading control. The synchrony of the culture was confirmed by measurement of the septation indices over 300 minutes. (B) Fkh2p is a phosphoprotein. Fkh2-3HAp was immuno-precipitated from lysates prepared from an exponentially growing culture of fkh2-3HA cells (JM 2257) using the anti-HA antibody. Sample A was untreated, and samples B and C were incubated with calf intestinal phosphatase, either with or without phosphatase inhibitors. Samples were separated by SDS-PAGE and analysed by western blot with anti-HA antibodies.

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