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plo1+ regulates gene transcription at the M-G1 interval during the fission yeast mitotic cell cycle

2002; Springer Nature; Volume: 21; Issue: 21 Linguagem: Inglês

10.1093/emboj/cdf564

1460-2075

Mark Anderson, Szu Shien Ng, Vanessa Marchesi, Fiona H. MacIver, Frankie Stevens, Tracy Riddell, David M. Glover, Iain Hagan, Christopher McInerny,

Microtubule and mitosis dynamics

Article1 November 2002free access plo1+ regulates gene transcription at the M–G1 interval during the fission yeast mitotic cell cycle Mark Anderson Mark Anderson Division of Biochemistry and Molecular Biology, Institute of Biological and Life Sciences, University of Glasgow, Glasgow, G12 8QQ UK Search for more papers by this author Szu Shien Ng Szu Shien Ng Division of Biochemistry and Molecular Biology, Institute of Biological and Life Sciences, University of Glasgow, Glasgow, G12 8QQ UK Search for more papers by this author Vanessa Marchesi Vanessa Marchesi Division of Biochemistry and Molecular Biology, Institute of Biological and Life Sciences, University of Glasgow, Glasgow, G12 8QQ UK Search for more papers by this author Fiona H. MacIver Fiona H. MacIver Paterson Institute for Cancer Research, Christie Hospital, Wilmslow Road, Withington, Manchester, M20 4BX UK Search for more papers by this author Frances E. Stevens Frances E. Stevens Paterson Institute for Cancer Research, Christie Hospital, Wilmslow Road, Withington, Manchester, M20 4BX UK Search for more papers by this author Tracy Riddell Tracy Riddell Division of Biochemistry and Molecular Biology, Institute of Biological and Life Sciences, University of Glasgow, Glasgow, G12 8QQ UK Search for more papers by this author David M. Glover David M. Glover Department of Genetics, University of Cambridge, Downing Street, Cambridge, CB2 3EH UK Search for more papers by this author Iain M. Hagan Iain M. Hagan Paterson Institute for Cancer Research, Christie Hospital, Wilmslow Road, Withington, Manchester, M20 4BX UK Search for more papers by this author Christopher J. McInerny Corresponding Author Christopher J. McInerny Division of Biochemistry and Molecular Biology, Institute of Biological and Life Sciences, University of Glasgow, Glasgow, G12 8QQ UK Search for more papers by this author Mark Anderson Mark Anderson Division of Biochemistry and Molecular Biology, Institute of Biological and Life Sciences, University of Glasgow, Glasgow, G12 8QQ UK Search for more papers by this author Szu Shien Ng Szu Shien Ng Division of Biochemistry and Molecular Biology, Institute of Biological and Life Sciences, University of Glasgow, Glasgow, G12 8QQ UK Search for more papers by this author Vanessa Marchesi Vanessa Marchesi Division of Biochemistry and Molecular Biology, Institute of Biological and Life Sciences, University of Glasgow, Glasgow, G12 8QQ UK Search for more papers by this author Fiona H. MacIver Fiona H. MacIver Paterson Institute for Cancer Research, Christie Hospital, Wilmslow Road, Withington, Manchester, M20 4BX UK Search for more papers by this author Frances E. Stevens Frances E. Stevens Paterson Institute for Cancer Research, Christie Hospital, Wilmslow Road, Withington, Manchester, M20 4BX UK Search for more papers by this author Tracy Riddell Tracy Riddell Division of Biochemistry and Molecular Biology, Institute of Biological and Life Sciences, University of Glasgow, Glasgow, G12 8QQ UK Search for more papers by this author David M. Glover David M. Glover Department of Genetics, University of Cambridge, Downing Street, Cambridge, CB2 3EH UK Search for more papers by this author Iain M. Hagan Iain M. Hagan Paterson Institute for Cancer Research, Christie Hospital, Wilmslow Road, Withington, Manchester, M20 4BX UK Search for more papers by this author Christopher J. McInerny Corresponding Author Christopher J. McInerny Division of Biochemistry and Molecular Biology, Institute of Biological and Life Sciences, University of Glasgow, Glasgow, G12 8QQ UK Search for more papers by this author Author Information Mark Anderson1, Szu Shien Ng1, Vanessa Marchesi1, Fiona H. MacIver2, Frances E. Stevens2, Tracy Riddell1, David M. Glover3, Iain M. Hagan2 and Christopher J. McInerny 1 1Division of Biochemistry and Molecular Biology, Institute of Biological and Life Sciences, University of Glasgow, Glasgow, G12 8QQ UK 2Paterson Institute for Cancer Research, Christie Hospital, Wilmslow Road, Withington, Manchester, M20 4BX UK 3Department of Genetics, University of Cambridge, Downing Street, Cambridge, CB2 3EH UK *Corresponding author. E-mail: [email protected] The EMBO Journal (2002)21:5745-5755https://doi.org/10.1093/emboj/cdf564 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The regulation of gene expression plays an important part in cell cycle controls. We describe the molecular machinery that co-ordinates gene transcription at the M–G1 interval during the fission yeast mitotic cell cycle. A sequence is identified in the cdc15+ promoter that we call a PCB (pombe cell cycle box), which confers M–G1-specific transcription. Sequences similar to the PCB are present in the promoters of seven other genes, spo12+, cdc19+, fin1+, sid2+, ppb1+, mid1+/dmf1+ and plo1+, which we find to be transcribed at M–G1. A transcription factor complex is identified that binds to the PCB sequence, which we name PBF, for PCB-binding factor. Finally, we show that PBF binding activity and consequent gene transcription are regulated by the Plo1p protein kinase, thus invoking a potential auto-feedback loop mechanism that regulates mitotic gene transcription and passage through septation and cytokinesis. Introduction Many forms of control have been shown to regulate the mitotic cell division cycle, such as protein kinase activity, specific proteolytic degradation and changes in intracellular location. Transcription has also been found to play an important role in controlling cell cycle progress, and the cell cycle-specific regulation of gene expression is widespread. Microarray analysis has revealed in the budding yeast, Saccharomyces cerevisiae, that the transcript profile of ∼700 genes, out of a total of ∼6000, varies throughout the mitotic cell cycle (Cho et al., 1998; Spellman et al., 1998). These genes fall into a number of groups whose transcript abundance peaks at different cell cycle times. Each group of genes is regulated co-ordinately by a common DNA sequence present in their promoters, which are bound by a transcription factor complex. Examples of such groupings are the MCB–MBF group of genes at G1–S, and Mcm1p/Fkhp genes during mitosis (Futcher, 2000). Cell cycle-regulated transcription has also been studied in the fission yeast, Schizosaccharomyces pombe. A group of genes, including cdc22+, cdc18+, cig2+, cdt1+ and mik1+, are transiently expressed at the beginning of S phase, and their products are required, either directly or indirectly, for DNA synthesis (Fernandez-Sarabia et al., 1993; Kelly et al., 1993; Connolly and Beach, 1994; Hofmann and Beach, 1994; Ng et al., 2001). The molecular components that control G1–S transcription in fission yeast comprise a transcription factor complex named DSC1 (DNA synthesis control; also called MBF), that contains the products of the cdc10+, res1+, res2+, rep1+ and rep2+ genes (Lowndes et al., 1992; Tanaka et al., 1992; Caligiuri and Beach, 1993; Miyamoto et al., 1994; Sugiyama et al., 1994; Zhu et al., 1994; Nakashima et al., 1995). DSC1 binds to MCB sequences (MluI cell cycle box; ACGCGT) that are present in the promoters of cdc22+, cdc18+, cdt1+ and cig2+, all of which are expressed maximally at the G1–S boundary during the mitotic cell cycle. We are interested in identifying other groups of genes that are regulated co-ordinately in fission yeast at different cell cycle times, with the aim of characterizing the molecular components that control their transcription. To this end, we have studied cdc15+, a gene that is transcribed specifically during the M–G1 phase of mitotic division (Fankhauser et al., 1995; Utzig et al., 2000). We report the identification of seven other genes that are expressed coincidentally with cdc15+, and describe a promoter sequence and a transcription factor complex that regulates their cell cycle transcription. Finally, we show that Plo1p regulates the expression of these genes. We propose that Plo1p controls M–G1 cell cycle transcription during mitosis in fission yeast, as part of an auto-activatory loop that results in cytokinesis and septation. Results Mitotic cell cycle transcription of cdc15+ cdc15+ was first identified by mutation in the original fission yeast cell cycle screens (Nurse et al., 1976), and ascribed a function late in the cell cycle between nuclear division and early cell plate formation. Subsequent characterization of the gene product (Fankhauser et al., 1995) showed that cdc15+ is a key element in the control of the re-organization of the F-actin ring at cytokinesis (Marks et al., 1986). This ring later constricts to effect cytokinesis, once genomic segregation has occurred (Demeter and Sazer, 1998). Fankhauser et al. (1995) also showed that cdc15+ transcript abundance varied during the cell cycle, with a peak at metaphase during vegetative growth, which suggested that control of cdc15+ transcription may play a role in promoting cytokinesis. We confirmed and extended the observation concerning cdc15+ mRNA abundance during the mitotic cell cycle, by two different synchronization methods. In synchronously dividing cells, which were either wild type (972h−) size selected by centrifugal elutriation (Figure 1A), or synchronized by transient arrest at the G2–M boundary by reversible temperature shifts of a cdc25-22 mutant (Figure 1B), cdc15+ mRNA varied in abundance in a cell cycle manner. By directly comparing the peak level of cdc15+ transcript with that of cdc22+, we confirmed that cdc15+ peaks slightly earlier in the cell cycle. This was especially apparent in the cdc25-22 mutant experiment (Figure 1B), where we took samples every 10 min after temperature release for northern analysis. As cdc22+ mRNA peaks at the G1–S boundary (White et al., 2001), these two experiments suggested that cdc15+ mRNA was present at M–G1. Figure 1.cdc15+ is expressed before MCB-regulated genes in the fission yeast cell cycle, and is not controlled by DSC1. (A) A population of wild-type cells (972h−), synchronous for division, were size selected by centrifugal elutriation at 32°C, and cell samples taken every 20 min for northern blot analysis of RNA. ‘as’ indicates RNA prepared from asynchronously dividing cells prior to elutriation. The blot was hybridized consecutively with cdc15+, cdc22+ and adh1+ probes, the latter as a loading control. Quantification of each transcript against adh1+ is shown. (B) cdc25-22 cells were synchronized for cell division by transient temperature shifts. Northern blot analysis was performed on RNA samples prepared from cell samples taken at 10 min intervals following release from restrictive temperature. The blot was hybridized consecutively with cdc15+, cdc22+ and adh1+ probes, the latter as a loading control. Quantification of each transcript against adh1+ is shown. The degree of synchrony is indicated by the septation index. (C) RNA was prepared from cultures of wild-type (972h−) and cdc10-C4 cells grown at 25°C, and subjected to northern blot analysis. The membrane was hybridized consecutively with cdc15+, cdc22+ and adh1+ probes, the latter as a loading control. Quantification of each transcript against adh1+ is shown. Download figure Download PowerPoint cdc22+ is a member of a group of genes whose transcription is regulated co-ordinately at the G1–S boundary by the combination of a common promoter sequence present in all the genes' promoters which is bound by a transcription factor complex. The promoter sequence is called an MCB, and the transcription factor complex, DSC1, contains the cdc10+ gene product (Lowndes et al., 1992). cdc15+ is unlikely to be under DSC1–MCB control, because it is not transcribed at the G1–S interval. Furthermore, cdc15+ does not contain MCB sequences in its promoter region. Finally, a mutant of a component of DSC1, cdc10-C4, which results in overexpression of all known MCB-regulated genes compared with wild type (McInerny et al., 1995; Ng et al., 2001), did not affect cdc15+ transcription (Figure 1C). These experiments showed that cdc15+ is representative of a new type of mitotic cell cycle-expressed gene in fission yeast, which is transcribed at the M–G1 boundary. Regulation of cdc15+ transcription To see whether variation in cdc15+ mRNA levels during the cell cycle was due to transcriptional regulation, we tested the ability of DNA fragments from the promoter region of cdc15+ to confer M–G1 transcription to a heterologous gene. A number of cdc15+ promoter fragments were cloned into the reporter plasmid pSPΔ178 (Lowndes et al., 1992), and the cell cycle-dependent transcription of lacZ analysed by northern blot analysis. One fragment, corresponding to bases −157 to −53 relative to the cdc15+ ATG, conferred M–G1 transcription to lacZ, coincident with endogenous cdc15+ cell cycle expression in synchronized wild-type cells size selected by elutriation (Figure 2A). Similar results were obtained in cdc25-22 cells synchronized by transient temperature arrest (data not shown). These experiments showed that the cyclic behaviour of cdc15+ mRNA abundance was due to transcriptional regulation and not mRNA stability, and that this fragment of DNA contained the cis-acting DNA element that controlled this expression. Figure 2.Characterization of a region of the cdc15+ promoter that confers M–G1 transcription, and identification of a transcription factor complex, PBF, that binds to it. (A) A fragment from the cdc15+ promoter was inserted into pSPΔ178 (Lowndes et al., 1992) to create pSPΔ178. 15UAS, transformed into wild-type cells, and cells synchronous for division were size selected by centrifugal elutriation at 32°C, with samples taken every 20 min for northern blot analysis of RNA. ‘as’ indicates RNA prepared from asynchronously dividing cells prior to elutriation. The blot was hybridized consecutively with lacZ, cdc15+ and adh1+ probes, the latter as a loading control. Quantification of each transcript against adh1+ is shown. (B) The same cdc15+ promoter fragment as in (A) was used as labelled probe in gel retardation analysis with total fission yeast protein extracts. Lane F, free probe; lane P, 20 μg of protein with probe. Competition reactions were performed with the same unlabelled cdc15+ promoter DNA with 1/100, 1/10 and 1 M excess. The large arrow indicates PBF, and the small arrow indicates free probe. Download figure Download PowerPoint This same cdc15+ promoter fragment was then labelled and used as the probe in gel retardation experiments with protein extracts from wild-type fission yeast cells to identify the protein complex that bound to this sequence (Figure 2B). Because this complex potentially regulates cdc15+ cell cycle transcription, we named it PBF for pombe cell cycle box (PCB)-binding factor (see later). We mapped the region of contact between PBF and the cdc15+ promoter by competition experiments using fragments of DNA from the cdc15+ promoter region (Figure 3). Significantly, whereas fragment F was able to compete, fragment G could not (compare lanes 14 and 15), with the difference between these two DNAs being only 3 bp. This observation implicated this region of DNA as being important for interaction with PBF. The sequence around this region, which corresponded to −141 to −134 relative to the ATG of cdc15+, is AGGCAACG. Figure 3.Mapping of the interaction site between PBF and the cdc15+ promoter. Gel retardation analysis was performed using the cdc15+ promoter fragment as labelled probe. Lane F, free probe; lane P, 20 μg of protein with probe. Competition reactions were performed with unlabelled DNAs corresponding to different fragments of the cdc15+ promoter, designated with the letters A–I, with 1/100, 1/10 and 1 M excess, as indicated. Download figure Download PowerPoint A family of M–G1-regulated genes Using the sequence defined by gel retardation analysis in the cdc15+ promoter, we searched the fission yeast genome database (Wood et al., 2002) for other genes that contained similar sequences in their promoter regions. We took two approaches to this, both examining gene promoters randomly and looking specifically in the promoter regions of genes that are known to be cell cycle regulated in budding yeast at M–G1. Having identified potential genes (Figure 4A), we analysed their transcription profile during a mitotic cell cycle. These data are shown in Figure 4B. Interestingly, spo12+, cdc19+, fin1+, mid1+/dmf1+, sid2+, ppb1+ and plo1+ all showed a similar transcription profile to that of cdc15+, suggesting that the same molecular processes may regulate transcription of all of these genes. spo12+ previously has been reported to be cell cycle expressed, coincident with cdc15+ (Samuel et al., 2000). It may be significant that most of these genes form a group that is implicated in the execution or regulation of cytokinesis and septation. Outside this group is cdc19+, a member of the MCM class of protein required for DNA replication that have been shown to be loaded onto chromosomes during metaphase (Kearsey et al., 2000). Figure 4.A group of seven genes are expressed during mitosis in fission yeast coincident with cdc15+. (A) Predicted consensus sequence for the PCB element in fission yeast. Sequences related to the PCB consensus present in M–G1-expressed gene promoters are listed, with numbers referring to their position relative to each gene‘s ATG. (B) cdc25-22 cells were synchronized for cell division by transient temperature shifts. Northern blot analysis was performed on RNA from cell samples taken at 20 min intervals following release from the restrictive temperature. ’as' indicates RNA prepared from asynchronously dividing cells prior to temperature shifts. The blot was hybridized consecutively with cdc15+, spo12+, cdc19+, mid1+/dmf1+, fin1+, sid2+, ppb1+, plo1+ and adh1+ probes, the latter as a loading control. Quantification of each transcript against adh1+ is shown. The degree of synchrony achieved is indicated by the septation index. Download figure Download PowerPoint Defining the PCB sequence To define more precisely the consensus sequence required for M–G1 transcription in fission yeast, we adopted two approaches. First, we carried out a series of competitive gel retardation experiments using a fragment of DNA from the spo12+ promoter that contained a sequence related to the sequence defined in the cdc15+ promoter. A 20 bp fragment from the spo12+ promoter containing the putative upstream sequence (position −344; Figure 4A) successfully competed with PBF (Figure 5A, lane 2), demonstrating that both promoters recognize the same transcription factor complex. Individual base pairs required for PBF to bind spo12+ DNA were determined by assaying the effect of mutating single base pairs on competitive binding activity. A series of DNAs were synthesized containing consecutive single base pair mutations (A/T to G, or C/G to T), and their ability to bind PBF assayed. As shown in Figure 5A, the central GT and ACA (lanes 9, 10 and 12–14) were all critical for binding, as their individual mutation resulted in loss of PBF binding activity. The fact that mutating the T in the central GT had such an effect was surprising, as this base pair is not conserved amongst other genes (Figure 4A); possibly this base pair is important in the spo12+ promoter in combination with some other base(s). Figure 5.Defining the PCB sequence. (A) Base pairs required in spo12+ PCB to bind PBF. Gel retardation analysis was performed using the cdc15+ promoter fragment as labelled probe. Lane P, 20 μg of protein with probe. Competition reactions were performed using cold DNA fragments corresponding to the spo12+ PCB, where single consecutive bases were mutated in separate DNAs, with A/T to G, or C/G to T, with 1 M excess. The central GT and ACA are required for successful competition with PBF. (B) PBF binds promoter fragments containing PCB sequences from other genes transcribed at M–G1 during mitosis. Gel retardation analysis was performed using the cdc15+ promoter fragment as labelled probe. Lane F, free probe; lane P, 20 μg of protein with probe. In alternate lanes, 1 and 1/10 M excess unlabelled competitor promoter DNA fragments from various fission yeast mitotic M–G1-expressed genes was added to the reaction mixture prior to electrophoresis. Download figure Download PowerPoint In the second approach, we tested whether PBF bound DNA from the promoters of other M–G1-transcribed genes containing sequences related to the PCB sequence (Figure 5B). We performed competitive gel retardation experiments using fragments from the cdc19+ and mid1+/dmf1+ promoters, and demonstrated that these DNAs also bound PBF. These experiments allowed us to predict GNAACg/a as a putative consensus sequence for the fission yeast M–G1 cis-acting promoter element (Figure 4A). We suggest naming this sequence PCB for pombe cell cycle box. Cell cycle-dependent behaviour of PBF binding to PCBs A possible mechanism for PBF and PCB regulation of cell cycle transcription of cdc15+ is by periodic binding of the transcription factor to the promoter sequence during the cell cycle. We tested this hypothesis in three different experiments. In the first experiment, we size selected small wild-type cells (972h−) by centrifugal elutriation, and followed a synchronous population during two divisions. Gel retardation analysis of PBF in such synchronized cells revealed constitutive binding of PBF to PCBs during the cell cycle (Figure 6). Similar results were obtained with synchronized cells made by transient temperature arrest using the cdc10-M17 mutation (data not shown; Kim and Huberman, 2001). Figure 6.Cell cycle binding of PBF to PCBs. A population of wild-type cells (972h−) synchronous for division were size selected by centrifugal elutriation at 25°C, and cell samples taken every 20 min for gel retardation analysis. PBF was detected using the cdc15+ promoter fragment as labelled probe with 20 μg of protein in each sample. Lane P, 20 μg of protein from asynchronous cells with probe. The large arrow indicates PBF, and the small arrow indicates free probe. The ratio of PBF to free probe is plotted. Download figure Download PowerPoint In the third experiment, we examined PBF binding to PCBs in protein extracts from fission yeast cells arrested at discrete stages of the cell cycle (G1, S, G2 and M), using a variety of cdc− mutants. In agreement with the previous experiments, PBF bound to PCBs in cells arrested at all cell arrest points (data not shown). These three experiments suggested that PBF binds to PCBs throughout the cell cycle. plo1-ts35 affects PBF binding activity in vitro and PCB-regulated gene transcription in vivo We were interested in identifying genes that regulated cell cycle expression of the cdc15+ group of genes through the PBF transcription factor complex. One potential candidate is the plo1+ polo-like kinase, which is known to have a central role in regulating late mitotic events in fission yeast (Ohkura et al., 1995; Bahler et al., 1998; Tanaka et al., 2001). If Plo1p controls PCB-regulated genes, then compromising Plo1p function might affect both PBF activity and the periodic transcription of these genes. We therefore initially determined PBF binding activity in vitro by gel retardation analysis in five different temperature-sensitive plo1− alleles. Interestingly, the mutant with the strongest phenotype, plo1-ts35, resulted in the loss of PBF binding at both permissive and restrictive temperatures (Figure 7A, lanes 5–7). plo1-ts35 cells block mitotic progression as cells are unable to form a mitotic spindle. We therefore asked whether the lack of PBF binding in plo1-ts35 at 36°C was an indirect consequence of the early mitotic arrest, by assessing PBF activity in cut7-24. cut7-24 mutant cells arrest at the same stage of mitosis as plo1-ts35, because of defects in a mitotic motor protein that is required to form the spindle (Hagan and Yanagida, 1990). cut7-24 cells contained PBF at 36°C, indicating that loss of PBF in plo1-ts35 was not a consequence of the cell cycle arrest in this strain (Figure 7A, lanes 11–13). Furthermore, the loss of PBF activity in plo1-ts35 was not due to dominant inhibition of binding in plo1-ts35 cells, as mixing extracts from wild-type and mutant cells had no effect on PBF (Figure 7A, lanes 8–10). plo1-ts35 is a recessive, loss-of-function, allele: haploid plo1-ts35 cells containing a single copy of plo1+ are phenotypically wild type (data not shown). Therefore, loss of PBF in plo1-ts35 was likely to be due to loss of a Plo1p function, rather than dominant interference with activation by another effector. Figure 7.plo1-ts35 affects PBF in vitro and PCB-regulated gene transcription in vivo. (A) Gel retardation analysis was performed using the cdc15+ promoter fragment as labelled probe with 20 μg of protein extracts from wild-type (972h−), plo1-ts35 and cut7-24 cells grown at permissive (25°C) and restrictive temperatures (36°C), for the indicated times. In the mixing experiment, protein extracts from wild-type and plo1-ts35 cells were mixed before electrophoresis. Lane F indicates free probe. (B) Cell cycle transcription of cdc15+, spo12+ and plo1+ in plo1-ts35 cells. A population of plo1-ts35 cells, synchronous for division, was size selected by centrifugal elutriation at 25°C. Cell samples were taken before elutriation (as) and at 30 min intervals after elutriation for northern blot analysis of RNA. Control RNA samples from asynchronous wild-type (972h−) cells (wt), and peak (P) and trough (T) cdc15+ mRNA cell cycle samples from the experiment shown in Figure 1A were included. The blot was hybridized consecutively with cdc15+, spo12+, cdc22+ and adh1+ probes, the latter as a loading control. Quantification of each transcript against adh1+ is shown. (C) Transcription of cdc15+, spo12+ and plo1+ in plo1-ts35 cdc2-33 arrested cells. plo1-ts35 cdc2-33 cells were cell cycle arrested by temperature shift after enriching for early G2 cells by elutriation (Tanaka et al., 2001). Cell samples were taken before elutriation (as), and at the arrest (A) after elutriation for northern blot analysis of RNA. Control RNA samples from asynchronous wild-type (972h−) cells (wt), asynchronous cdc2-33 cells (cdc2), and peak (P) and trough (T) cdc15+ mRNA cell cycle samples from the experiment shown in Figure 1B were included. The blot was hybridized consecutively with cdc15+, cdc22+ and adh1+ probes, the latter as a loading control. Quantification of each transcript against adh1+ is shown. Download figure Download PowerPoint To determine whether the loss of PBF activity affected the periodic transcription of its target genes, we examined the cell cycle profile of cdc15+ and spo12+ mRNA in plo1-ts35 cells synchronized by centrifugal elutriation at the permissive temperature of 25°C. The profiles of both transcripts were altered, and they were no longer expressed at the M–G1 boundary but instead were delayed and transcribed coincident with cdc22+ at G1–S (Figure 7B). Furthermore, plo1+ itself was no longer transcribed in a cell cycle-dependent manner in plo1-ts35 cells, but mRNA levels were constant at low levels throughout the cell cycle. The level of plo1+ mRNA in plo1-ts35 cells was similar to the lowest trough level of the profile seen in a wild-type cell cycle (Figure 7B; compare lane 3 with 5–14). This observation implies that plo1+ regulates its own expression, consistent with the presence of a PCB sequence in its promoter (Figure 4A). A reduction in plo1+ transcript levels at 25°C is consistent with the minor (5%) misshapen septum phenotype seen at this temperature in these cells (data not shown). The alteration in transcript periodicity in plo1-ts35 was specific to cdc15+, spo12+ and plo1+, as the cdc22+ transcript, which is under different cell cycle regulation, peaked in G1 phase (Figure 7B). We also tested the effect of a plo1-ts35 temperature arrest on PCB-regulated gene transcription. A plo1-ts35 cdc2-33 culture was incubated at 36°C for 150 min after enrichment for early G2 cells by elutriation. Cells taken for northern blot analysis contained very low levels of cdc15+, spo12+ and plo1+ transcript (Figure 7C, lane 6), consistent with the suggestion that plo1+ is regulating the expression of these genes. These transcripts were present at higher levels in similarly arrested cdc2-33 cells (data not shown), and in cdc25-22 cells arrested at the same cell cycle stage (Figure 1B). To summarize these three experiments, plo1-ts35 affected PBF binding in vitro, and cdc15+, spo12+ and plo1+ transcription in vivo, suggesting that plo1+ controls M–G1 PCB-regulated gene expression through PBF. Overexpression of plo1+ causes overexpression of PCB-regulated genes Previous experiments have demonstrated the critical role of plo1+ in controlling late cell mitotic events, as its overexpression in interphase cells resulted in premature septation (Ohkura et al., 1995). This control of septation by plo1+ is thought to be, in part, through it activating the regulatory septation initiation network (SIN; Tanaka et al., 2001). However, the active SIN needs to have the relevant target molecules in the F-actin ring in order to stimulate contraction of this ring and concomitant septation. Given that Plo1p is required for PBF activity and cell cycle transcription of PCB-regulated genes, we next asked whether overexpression of plo1+ would also provide the target molecules for the SIN by inducing transcription of PCB-regulated genes in interphase cells. plo