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First published online 4 December 2007
doi: 10.1242/jcs.019489

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Regulation of gene expression during M-G1-phase in fission yeast through Plo1p and forkhead transcription factors

¹ Division of Biochemistry and Molecular Biology, Faculty of Biomedical and Life Sciences, University of Glasgow, Glasgow, G12 8QQ, UK
² Institute of Cell and Molecular Biology, University of Edinburgh, Edinburgh, EH9 3JR, UK
³ Stem Cell Biology and Developmental Genetics, National Institute for Medical Research, The Ridgeway, Mill Hill, London, NW7 1AA, UK

View larger version (56K): [in this window] [in a new window]   Fig. 1. Genetic interactions between plo1+, components of PBF and M-G1 transcription. (A) Synthetic defective cell separation phenotypes revealed by double mutants in plo1, fkh2, sep1 and mbx1. Cells containing combinatorial mutants of fkh2, sep1, mbx1 and plo1-ts35 were grown on solid YE medium at 25°C, and viewed with DIC optics. Scale bar, 5 µm. (B) Effect on transcription of genes expressed at M-G1 phase by overexpressing mutant versions of Plo1p. Cells containing either pREP1: plo1+ (wild-type), pREP1: plo1DHK625AAA [Polo-box domain (PBD) mutant], or pREP1: plo1K69R [kinase domain (KD) mutant] were grown to mid-exponential stage before the removal of thiamine to induce overexpression. After 16 hours cells were removed for microscopic examination and northern blot analysis of cdc15+, spo12+ and plo1+ mRNA levels. Quantification of each transcript against rRNA is shown. Scale bar, 5 µm.

View larger version (29K): [in this window] [in a new window]   Fig. 2. Plo1p binds to Mbx1p in vivo. (A) Two-hybrid interaction between Plo1p and Mbx1p. Wild-type plo1+ in the two-hybrid bait vector was transformed into budding yeast, together with empty prey vector, or prey vector containing either mbx1+, fkh2+, sep1+ or sck1+ (the latter encoding a known Plo1p-interacting protein) (Reynolds and Ohkura, 2003). Colour reactions were quantified in cells grown in liquid medium and are plotted; error bars indicate two standard errors. Table indicates combinations of two-hybrid plasmids used. (B) Two-hybrid interaction between Plo1p and Mbx1p requires both the kinase and the Polo-box domains. Wild-type plo1+ or either kinase (plo1K69R) or Polo-box domain (plo1.472-684 and plo1DHK625AAA) mutants of plo1 in the two-hybrid bait vector were transformed into budding yeast, together with the empty prey vector, or the prey vector containing either mbx1+ or sck1+. Colour reactions were quantified in cells grown in liquid medium and are plotted; error bars indicate two standard errors. Table indicates combinations of two-hybrid plasmids used. (C) Mbx1p is co-immunoprecipitated with Plo1p from fission yeast extract. Plo1p was immunoprecipitated with antibody against the native protein from soluble extracts of fission yeast expressing Mbx1p-13myc from its endogenous promoter. The soluble extracts and the immunoprecipitates were analysed by western blotting with antibodies against Plo1p and Myc (the latter to detect Mbx1p-13myc). As controls, extracts from mbx1 and precipitates without antibodies (beads only) from tagged strains are also included. Approximately ten times more of the precipitates were loaded than of the soluble input fractions. Arrow indicates co-immunoprecipitated Mbx1p-13myc with Plo1p.

View larger version (75K): [in this window] [in a new window]   Fig. 3. Plo1p binds directly to and phosphorylates Mbx1p in vitro. (A) In vitro kinase assay with purified 6His-Plo1p and recombinant components of PBF. Separate kinase reactions with 6His-Plo1p and either 6His-Fkh2p, GST-Sep1p or GST-Mbx1p were separated by SDS PAGE, stained with Coomassie Blue and autoradiographed. Arrow indicates phosphorylated GST-Mbx1p, near to autophosphorylated 6His-Plo1p. Table indicates combinations of tagged proteins used. (B) In vitro kinase assay with purified 6His-Plo1p and GST-Mbx1p bound to glutathione-Sephadex beads after washes with 1% NP-40 to remove 6His-Plo1p, separated by SDS PAGE, stained with Coomassie Blue and autoradiographed. Arrow indicates phosphorylated GST-Mbx1p. Table indicates combinations of tagged proteins used.

View larger version (35K): [in this window] [in a new window]   Fig. 4. Mbx1p binds to Plo1p throughout the cell cycle. cdc25-22 mbx1-13myc cells were synchronised by transient temperature arrest and samples taken every 20 minutes after returning the cells to the permissive temperature. Septation indices were counted microscopically and are plotted to indicate the synchrony of the culture. Soluble extracts from each time point were prepared, Plo1p was immunoprecipitated with antibody against the native protein, and the immunoprecipitates analysed by western blotting with antibodies against Plo1p and Myc (the latter to detect Mbx1p-13myc). asy, control sample from asynchronous cells prior to synchronisation. Quantification of co-immunoprecipitated Mbx1p against immunoprecipitated Plo1p is shown.

View larger version (49K): [in this window] [in a new window]   Fig. 5. Sep1p binds to Fkh2p both in vivo and in vitro. (A) Fkh2p is co-immunoprecipitated with Sep1p from fission yeast extract. Sep1p-3HA was immunoprecipitated with antibody against HA from soluble extracts of fission yeast expressing Fkh2p-13myc from its endogenous promoter. The soluble extracts and the immunoprecipitates were analysed by western blotting with antibodies against HA and Myc (the latter to detect Fkh2p-13myc). As controls, extracts from Sep1p-3HA-expressing cells alone and precipitates without antibodies (beads only) from the double-tagged strain are also included. Approximately ten times more of the precipitates were loaded than of the soluble input fractions. Arrow indicates Fkh2p-13myc co-immunoprecipitated with Sep1p-3HA. (B) 6His-Fkh2p is pulled down by GST-Sep1p in vitro. Bacterially expressed 6His-Fkh2p and GST-Sep1p were purified and mixed together. When GST-Sep1p was bound to GST beads, 6His-Fkh2p was present in the pull-down fraction, whereas GST alone was unable to pull down 6His-Fkh2p. Arrow indicates pulled-down 6His-Fkh2p with GST-Sep1p.

View larger version (49K): [in this window] [in a new window]   Fig. 6. Fkh2p, Sep1p and Plo1p bind to PCB sequences in vivo. (A) Promoter regions amplified during the ChIP procedure together with the positions of the PCB and FLEX1 motifs. (B-E) Chromatin immunoprecipitation (ChIP) experiments with tagged versions of (B) Fkh2p-13myc, (C) Fkh2p-3HA, (D) Sep1p-13myc and (E) Plo1p-3HA, on the cdc15+, fkh2+ and plo1+ promoters. WCE, whole-cell extracts (non-immunoprecipitated input sample); IP, immunoprecipitates. Approximately ten times more of the precipitates were loaded than of the WCE input DNA. As negative controls, beads alone and normal mouse IgG were used for precipitations, and DNA of the act1+-coding sequence was used as substrate. For all Myc- and HA-tagged strains, control ChIPs were completed with a wild-type untagged strain. As a positive control for all four strains, histone H3 was analysed by ChIP with appropriate antibody on the cdc15+, fkh2+ and plo1+ promoters. In each case the ChIP was shown to be quantitative, because a serial reduction of the WCE input DNA resulted in a corresponding reduction of the observed PCR signal.

View larger version (40K): [in this window] [in a new window]   Fig. 7. Requirement of Plo1p, Mbx1p and Sep1p for Fkh2p promoter binding in vivo. ChIP experiments with Fkh2p-13myc and Fkh2p-3HA from extracts of cells containing mutants of plo1, mbx1 and sep1. (A-C) Crosslinked DNA was prepared from (1) wild-type and (A) plo1-ts35 cells at (2) permissive and (3) restrictive temperatures, or from cells with chromosome deletions of (B) mbx1 or (C) sep1. In each case, Fkh2p-13myc and Fkh2p-3HA were analysed by ChIP using anti-Myc and anti-HA antibodies respectively, and binding to PCB promoter fragments from cdc15+, fkh2+ and plo1+ detected by PCR. WCE, whole-cell extracts (non-immunoprecipitated input sample); IP, immunoprecipitates. Approximately ten times more of the precipitates were loaded than of the WCE input DNA. As negative controls, beads alone and normal mouse IgG were used for precipitations, and DNA of the act1+-coding sequence was used as substrate. As a positive control, histone H3 was analysed by ChIP with appropriate antibody. Graph in A shows the quantification of Fkh2p binding against WCE input DNA. In each case the ChIP was shown to be quantitative, because a serial reduction of the WCE input DNA resulted in a corresponding reduction of the observed PCR signal (data not shown).

View larger version (54K): [in this window] [in a new window]   Fig. 8. Cell-cycle-specific binding of Fkh2p, Sep1p and Plo1p to PCB promoter DNAs. (A-C) Separate populations of (A) cdc25-22 fkh2-3HA, (B) cdc25-22 sep1-13myc and (C) cdc25-22 nmt41:plo1-3HA cells were synchronised by transient temperature arrest, and samples taken every 20 minutes after returning the cells to the permissive temperature for ChIP and northern blot analysis. Septation indices were counted microscopically and are plotted to indicate the synchrony of each culture. Crosslinked DNA was prepared from each sample and Fkh2p-3HA, Sep1p-13myc and Plo1p-3HA were analysed by ChIP using anti-HA and anti-Myc antibodies. Binding of Fkh2p-3HA, Sep1p-13myc and Plo1p-3HA to the cdc15+, fkh2+ and plo1+ promoters was detected by PCR. As a loading control, PCR was performed with 10% whole-cell extracts (WCE; non-immunoprecipitated sample) containing input DNA, and plo1+ (A, B) and cdc15+ (C) oligonucleotides. Graphs show the quantification of Fkh2p, Sep1p and Plo1p binding against WCE input DNA. RNA was prepared from duplicate samples to detect mRNA levels of cdc15+, fkh2+ and plo1+ by northern blot analysis. mRNA levels of cdc22+, a known G1-S phase expressed transcript independent of PBF-PCB controls were also detected to allow comparison between experiments. Quantification of each transcript against rRNA is shown under each northern blot panel. asy, control DNA and RNA samples from asynchronous cells prior to synchronisation.

View larger version (24K): [in this window] [in a new window]   Fig. 9. Plo1p, Fkh2p and Sep1p regulation of genes expressed during the M-G1 phase in fission yeast. Expression of genes during late M phase in fission yeast is controlled by a transcription factor complex containing at least three proteins, the MADS box protein Mbx1p, and the forkhead transcription factors Fkh2p and Sep1p. Fkh2p and Sep1p both control cell cycle expression through cell-cycle-specific binding to PCB sequences, with Fkh2p repressing and Sep1p activating gene expression. Additionally, Mbx1p changes in a cell-cycle-specific manner. It is directly bound to and phosphorylated by Plo1p, whereby Plo1p stimulates transcription by binding to PCB DNA after Fkh2p. As plo1+ and fkh2+ are also transcribed at M-G1 phase, and both contain PCB sequences in their promoters to which Plo1p and Fkh2p bind, these genes regulate their own expression.