Journal of Cell Science, Supplement 18, 63-68 (1994)Printed in Great Britain © The Company of Biologists Limited 1994

63

Regulation of the cell cycle timing of Start in fission yeast by the rum1+ gene

Sergio Moreno1’*, Karim Labib1, Jaime Correa2 and Paul Nurse211nstituto de Microbiología Bioquímica, CSIC/Universidad de Salamanca, Edificio Departamental, Campus Miguel de Unamuno, 37007 Salamanca, Spain2ICRF Cell Cycle Group, Lincoln’s Inn Fields, London WC2A 3PX, UK'Author for correspondence

SUMMARY

We have identified the rum l+ gene as a new regulator of the Gi-phase of the fission yeast cell cycle. rum l+ deter­mines the cell cycle timing of Start, by maintaining cells in a pre-Start state until they have attained a minimal critical mass. Cells lacking rum l+ are unable to arrest in pre-Start Gi in response to nitrogen starvation and are subsequently

sterile. In addition, rum l+ prevents entry into mitosis from pre-Start Gi, as shown by the fact that cdclO mutants in the absence of rum l+ undergo lethal mitosis without entering S-phase.

Key words: ruml+, Start, cell cycle, Schizosaccharomyces pombe

INTRODUCTION

Progression from the Gi-phase of the cell cycle to the onset of chromosomal DNA replication requires a process of cell cycle commitment in a wide variety of eukaryotes from yeast to humans. In the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe this process of commitment, termed Start, defines a period in late Gi beyond which cells can no longer undergo other developmental fates, such as sexual differentiation (Hartwell, 1974; Nurse, 1975; Nurse and Bisset, 1981). At Start, yeast cells monitor the extracellular microenviroment (presence or absence of nutrients, sexual pheromones, etc.) and either make a commitment to progres­sion through the mitotic cell cycle, appropiate for cells growing in rich medium, or undergo cell cycle arrest in Gi as a prelude to conjugation and meiosis, appropiate for nutritionally starved cells (Hartwell, 1974; Nurse, 1975; Nurse and Bisset, 1981). Before cells can pass Start, they must grow sufficiently to attain a critical cell mass, and small cells in Gi cannot undergo S-phase until they reach this mass (Hartwell, 1974; Nurse, 1975; Nurse and Thuriaux, 1977; Nasmyth et al., 1979). In fission yeast the p34cdc2 protein kinase is known to be required for progression past Start (Nurse and Bisset. 1981), together with the transcription factors encoded by the genes cdcl0+ (Nurse, 1981) and resl+/sctl+ (Tanaka et al., 1992; Caligiuri and Beach, 1993). Candidate Gi cyclins are encoded by pu cl+ (Forsburg and Nurse, 1991) and cig2+ (Bueno and Russell, 1993; Connolly and Beach, 1994; Obara-Ishihara and Okayama, 1994) but a clear role for these cyclins in the mitotic cell cycle remains to be established.

Here we describe the identification and characterization of the gene rum l+, encoding a 25 kDa protein important for deter­mining the duration of the Gi-phase. Overexpression of ruml+ initially causes cell cycle delay in Gi, since the critical mass required for Start is increased relative to wild-type cells. These

cells undergo multiple rounds of S-phase in the absence of mitosis, leading to very long cells with increased ploidy. When rum l+ is deleted, the critical cell mass required at Start is reduced and the pre-Start Gi interval is eliminated, indicating that ruml+ is a major element determining the timing of Start. rum l+ is also important in restraining mitosis until Gi is finished: when ruml+ is deleted in a mutant that normally blocks at Start, cells proceed to undergo mitosis and cell division. Therefore ruml+ is important for defining the Gi status of a fission yeast cell.

MATERIALS AND METHODS

Strains and mediaS. pombe strains used in this study were 972 hr, leul-32 hr, ade6- M210/ade6-M216 ura4-D18/ura4-D18 leul-32/leul-32 h+/h~, ade6- 704 leul-32 h+, cdclO-129 h~, cdc2-L7 hr, cdc2-M26 hr and w eel- 50 hr. Escherichia coli strain DH5a was used for routine cloning. S. pombe and E. coli were manipulated following standard procedures (Moreno et al., 1991; Sambrook et al., 1989).

rum1+ overexpression experiments, DAPI staining, DNA and protein content determinationTo induce rum l+ expression from the regulatable nm tl+ promoter, a S. pombe leul-32 hr strain transformed with pREP3X-rwm/+ or an integrant constructed by transforming the strain ade6-704 leul-32 h+ with the plasmid pREP3X(sup3-5)-ram/+ were grown to mid-expo­nential phase in minimal medium containing 5 |J.g/ml thiamine at 32°C. Cells were fixed in cold 70% ethanol, stained with DAPI and photographed using a Zeiss Axioscop photomicroscope (Moreno et al., 1991). DNA content was determined by a FACScan (fluorescence activated cell analyser; Becton-Dickinson; Sazer and Sherwood, 1990) and by the diphenylamine method of Bostock (1970) with the modification of Gendimenico et al. (1988). Protein content was deter­mined using the BCA kit assay (Pierce). Cell number was measured using a Coulter counter.

64 S. Moreno and others

Constructing a rum1+ deletionA 2.1 kb Clai fragment in rum l+ was replaced with the ura4+ gene in a 6.1 kb rum l+ genomic clone. This construction was used to delete the rum l+ gene using a diploid strain ade6-M210/ade6-M216 ura4- D18/ura4-D18 leul-32/leul-32 h+/h~. Stable ura+ diploids were obtained and tetrads were dissected after sporulation. Although there was a general reduction in the viability of the spores from these tetrads in comparison with a wild-type control, uracil prototrophic haploid colonies with the rum l+ gene deleted grew well in YE5S indicating that the rum l+ gene was not essential in a wild-type background.

Nitrogen starvationCells were grown in minimal medium to mid-exponential phase, washed 6 times with nitrogen-free minimal medium and resuspended in this medium at a concentration of 2 x l0 6 cells/ml.

A

RESULTS

rum1+ overexpression inhibits the cell cycle both in Gi and G2The rum l+ gene was isolated screening for genes that when overproduced induce extra rounds of DNA replication in the absence of mitosis (Moreno and Nurse, 1994). A 1.5 kb cDNA derived from a gene we called rum 1+ (for replication «ncoupled from mitosis) induced over-replication when over­expressed, generating very long cells with a giant nucleus (Fig. 1 A). Flow cytometry analysis during a time course revealed an increase in DNA content, showing discrete peaks of 1C, 2C, 4C, 8C, 16C, etc., indicating that the increase in DNA content was a consequence of complete rounds of DNA replication (Fig. IB). This is also supported by the fact that a short pulse of rum l+ overexpression converts more than 50% of cells into viable diploids and tetraploids. Overproduction of the ruml protein therefore produces a cell cycle consisting exclusively of complete rounds of DNA replication, without intervening mitosis.

Examination of very early time points shows that the first effect of ru m l+ overexpression is the accumulation of cells with a 1C DNA content (10-20% of the total), indicating a delay in Gi. We measured the DNA and protein content during ru m l+ induction and found that whilst both DNA and protein content per cell increased in parallel, the protein/DNA ratio was elevated in the over-replicating cells by 10% in relation to wild type and by 65% in relation to a weel mutant (see Table 1), indicating that the cell mass at which S-phase takes place is increased. This means that elevated rum 1+ expression results in a delay of S-phase until cells attain a larger mass, suggest­ing that the rum 1+ gene product acts as a transient inhibitor of progression through Gi into S-phase. This provides an expla­nation for the appearance of the 1C population early on in the induction.

B

3z

"3U

O FF

ON 14

U L _ON 16

ON 18

1C 2C 4C 8C

DNA Content

Table 1. Cell mass at S phase in wild type, rum l A, weel-50 and weel-50 rum l A and rum l overexpressor

Strain

W ild type rum lA w eel-50 w eel-50 rum lA rum l OVP

Cell mass at S phase

1.491.431.000.821.65

Fig. 1. rum l+ overexpression induces over-replication. (A) S. pombe leul-32 hr cells transformed with pREP3X-rM/w/+; promoter off (left), promoter on (right). (B) Time course of induction o f rum l+ up to 18 hours in the integrant strain. FACS analysis (1C, 2C, 4C, 8C peaks are indicated).

rum1+ cell cycle regulation 65

ru m l+ also acts as an inhibitor of mitotic entry. When rum l+ is overexpressed the percentage of mitotic cells falls to zero and the p34cdc2/p56cdc13 associated protein kinase activity is very low (Moreno and Nurse, 1994). Recent experiments

A wild type

indicate that the ruml protein expressed and purified from E. coli acts directly as a very potent inhibitor of the p 3 4 cdc2/p 5 6 c d c i 3 complex in vitro (J. Correa, S. Moreno and P. Nurse, unpublished results).

weel mutant

r~ \12J fSl

B

Ur<u»oB=3

z

u

uoX!E3

z13U

wild type-1 **x*s S1 *12 HOURS 4 HOURS 6 HOURS

rumlA

' e MX« " 2 HOURS 4 HOUiS * t HORS ,

D l- Iâ - I j L .2*0 4M 6M MM l«M *** 6M 8*« 1606 I ^ 4^ M IM l«M 4M 4M 8M 1(88

DNA Content

weel-50

«> - 1 KM? - g HOURS

I . .. —j —se 2®e 4<« tee eee ieee 2M 4 M fcèe eee îeee ^ «ie (ie eee i m

¡Rt4 hours 1

. iA ................. ..eée 4 M w e eee i«

weel-50 ruml A

"1 e hours - 1 1 MOuB S t® HOUBS 8 HOURS 4 HOURS

A L iu . D * -j I X - ,a n n n r v s . T m : ar^a. t ¿¿¿'ïci. »j— 5^*1 U.

DNA Content

Fig. 2. ru m l A m utants do not arrest in G i in response to nitrogen starvation or in com bination with a w eel-5 0 mutant. (A) Cell cycle diagram s o f w ild-type and w eel m utant strains. (B) FACS profile o f the wild-type (upper) and rum l A (low er) strains after nitrogen starvation at 32°C. (C) FACS profile o f a tim e course after shifting to 36°C a w eel-50 (upper) and w eel-50 rum l A (lower) strain.

66 S. Moreno and others

To demonstrate that cells can undergo DNA replication without intervening mitosis, we overexpressed rum l+ in a cdc25-22 temperature-sensitive mutant. At the restrictive tem­perature these cells, arrested in G2 , were able to undergo multiple rounds of S-phase (Moreno and Nurse, 1994). However, the over-replication phenotype was blocked in tem­perature-sensitive mutants of cdc2 or cdclO at 36°C (Moreno and Nurse, 1994). Since these two mutants block cell cycle progression through Gi at Start, overreplicating cells with elevated ru m l+ expression must pass Start before they can initiate each S-phase.

rum 1+ inhibits cell cycle progression in G1If the rum \+ gene product acts as a transient inhibitor of Gi progression, reducing the normal level of the rum 1+ gene product should shorten the Gi interval and reduce the cell mass at which S-phase takes place. Rapidly growing wild-type cells have no pre-Start Gi interval because cells completing mitosis already have the cell mass required to pass Start (Fig. 2A). The pre-Start Gi interval is extended in small cells, such as nitrogen starved cells or in wee\ mutants, which divide at reduced cell mass (Fig. 2A). Reducing the normal level of the rum l+ gene product should therefore decrease the Gi interval in these small cells, but have little effect in rapidly growing wild-type cells.

As expected, deleting the rum ]+ gene has no effect on rapidly growing wild-type cells, but a dramatic effect is seen when these cells are nitrogen starved. When wild-type cells are nitrogen starved there is an accumulation of cells blocked in Gi (Fig. 2B). In contrast, cells deleted for rum 1+ show no extension of Gi (Fig. 2B). This is not due to cells failing to divide after shifting into medium lacking nitrogen, as the cell number increase in the two cultures is identical (data not shown). The failure to extend Gi is because the small cells initiate S-phase immediately after cell division. These cells were also found to be sterile, probably because conjugation requires Gi arrest at Start after nitrogen starvation.

A dramatic effect is also seen when rum 1+ is deleted in the temperature-sensitive wee 1-50 mutant. Two hours after shift to 36°C, w eel-50 cells begin to accumulate in Gi but no such accumulation was seen in weel-50 cells deleted for ru m l+ (Fig. 2C). The Gi interval observed in a wee 1-50 strain is elim­inated when ru m l+ is deleted. Therefore rum l+ must encode a major element that determines the length of Gi in these small cells. In the absence of the ru m l+ gene product the require­ment to attain a critical cell mass before passing Start is reduced. This was verified by measuring the protein/DNA ratio, which was reduced by 18% when rum l+ was deleted and increased by 65% when ruml + was over-produced (Table 1, values are calculated with respect to the values obtained for a w eel mutant after two hours at 36°C). In the absence of rum l+, weel-50 cells undergo Start and initiate S phase as soon as they complete mitosis.

rum 1+ defines a cell as being in Gi pre-StartThe above results suggested that the rum l+ gene product may be required to define a cell as being in the pre-Start Gi-phase of the cell cycle. If this is so, cells lacking rum l+ should be unable to block in this interval of the cell cycle. This was inves­tigated by deleting rum l+ in the cdclO-129 mutant and in a cdc2-M26 mutant. The cdcl0+ gene encodes a transcription factor that is required for cells to pass Start and activate the

transcription of a number of S-phase genes (Lowndes et al., 1992). When a cdclO-129 culture is shifted to 36°C, most cells are initially post-Start and so proceed to divide once before arresting, resulting in a cell number doubling. But cdclO-129 cells deleted for ru m l+ continue to divide further (Moreno and Nurse, 1994). Analysis of a synchronous culture of the cdclO- 129 rumlA mutant showed that cells were unable to remain arrested at the pre-Start interval, and underwent mitosis and cell division even though S-phase had not take place, resulting in a ‘cut’ phenotype (Fig. 3A). A similar experiment was performed with a cdc2-M26 strain deleted for rum l+. Most cdc2ts mutant alleles cause arrest in Gi and G2 upon shift to the restrictive temperature, though the Gi block is transient and leaks through after several hours. cdc2-M26 is unusual in that the block in Gi is very tight and cells can be arrested before Start for at least 12 hours. A cdc2-M26 rum lA double mutant does not remain arrested in pre-Start Gi, however, it blocks

B cdclO-129 cdclO-129 rum lA

"3U

660 800 1000

DNA Content

Fig. 3. cdclO-129 at the restrictive tem perature blocks Gi progression at START. (A) M icroscopic appearance o f cdclO-129 (right) and cdclO-129 rum lA (left) 5 and 7 hours, respectively, after the shift from 25 to 36°C. Cells w ere doubled-stained with DAPI and calcofuor. (B) FACS analysis o f cdclO -129 (left) and cdclO-129 ru m lA (right) after shift from 25°C to 36°C.

rum1+ cell cycle regulation 67

very transiently in Gi, and all cells have passed Start and completed S-phase by 4 hours (K. Labib, S. Moreno and P. Nurse, in preparation). This confirms that cells lacking ruml+ are unable to remain in a pre-Start Gi state, and also suggests a close interaction between cdc2 and rami in Gi.

Since rum l+ is important to prevent entry into mitosis from pre-Start Gi we tested whether it was required during S-phase to prevent entry into mitosis. We found that addition of hy­droxyurea to wild-type and rum l +-deleted cells was identical in effect (Moreno and Nurse, 1994). Cell division stopped and cells became elongated with no mitotic features.

These experiments indicate that the ruml+ gene product defines a cell as being in the pre-Start Gi interval. When rum\ + is deleted then a cell blocked in this interval cannot recognise this situation and proceeds with the program of cell division. But if the same cells are allowed to proceed into the post-Start Gi interval where progression is blocked using hydroxyurea, then the cell now recognises this situation and prevents mitosis and cell division, suggesting that different checkpoint controls must be operative in the Gi interval, one prior to Start involving ruml+ and a second post-Start, which does not involve rum\+ (Moreno and Nurse, 1994).

The rum1 proteinThe ruml+ gene potentially encodes a protein of 25 kDa (Moreno and Nurse, 1994). This gene product contains several interesting motifs: there is a putative bipartite nuclear locali­sation signal suggesting the protein may be targeted to the nucleus, as well as potential phosphorylation sites for both the cdc2 and MAP protein kinases, located in the amino terminus of the protein, that might be important for cell cycle regula­tion.

DISCUSSION

Three aspects of ruml+ gene function are involved in regulat­ing the pre-Start Gi interval. Firstly, it acts as a major element determining the length of Gi. It functions as a transient inhibitor of S-phase onset, delaying Start until the cell has attained a critical minimal mass. When rum\+ is deleted this mass is reduced and when ruml+ is overexpressed this mass is increased. The only other class of genes known to affect the length of Gi are the Gi-cyclins of S. cerevisiae and vertebrate cells. Dominant mutants in CLN genes shorten the G[ interval in budding yeast (Sudbery et al., 1980; Nash et al., 1988; Cross, 1988; Hadwiger et al., 1989). This is analogous to the effects of ruml+, except that Gi cyclins act positively whilst rum\+ acts negatively. In mammalian cells ectopic expression of cyclins D or E also shortens the Gi interval (Ohtsubo and Roberts, 1993; Quelle et al., 1993).

Secondly, rum\+ also influences the need to complete mitosis before a cell can undergo Start and initiate the next S- phase. When rum\+ is overexpressed in G2, cells can proceed to Start and initiate a new round of DNA replication in the absence of mitosis. The checkpoint control that monitors whether mitosis has been completed is disturbed in these cells. This phenomenon may occur naturally during endoreduplica- tion in certain tissues in multicellular organisms, such as salivary glands of Drosophila (Smith and Orr-Weaver, 1991).

Thirdly, the rum l+ gene product defines the cell as being in

pre-Start

G l

post-Start

G2

© ©

rum i

Fig. 4. Passage through START and entry into mitosis are brought about by the Gi and G2 forms of p34cdc2, respectively. The rum l+ gene product inhibits p34cdc2 in Gi until the cell mass required for START is attained. On attainment of the required minimal cell mass the inhibitory effect of the rum l+ gene product is lost allowing START to take place and p34cdc2 to be converted to the G2 form. In the absence of the rum l+ gene product START and S-phase occur prematurely at a reduced cell mass and the G2 form of p34cdc2 can be generated even if cells are not allowed to complete START, resulting in premature mitosis.

the pre-Start Gi interval, and restrains such a cell from under­going mitosis. When ruml+ is overexpressed, onset of mitosis is inhibited, and when ruml+ is deleted cells that normally block at Start can proceed with the cell division program and undergo mitosis even though S-phase has not taken place. Fission yeast cells, arrested in late Gi post-Start or in S-phase, send a signal that blocks p34cdc2 function at mitosis (Enoch and Nurse, 1991). This signal may be generated by the assembly of replication complexes post-Start, and is mediated via inhibitory phosphorylation of a residue of tyrosine 15 in the p34cdc2 protein kinase (Enoch et al., 1992). Cells prior to Start are unlikely to have assembled replication complexes, and so a separate checkpoint mechanism restraining mitosis in pre-Start cells is required; we propose a role for rum\+ in such a control. Separate mechanisms restraining mitosis before and after Start are further suggested by the fact that mutants such as cdc2-3w, lacking the post-Start checkpoint, are still able to arrest cell cycle progression pre-Start without entering mitosis (Enoch and Nurse, 1991).

Based on the above discussion and recent biochemical experiments, we propose that ruml might be an inhibitor of the cell cycle machinary acting in a similar way to FAR1 (Chang and Herskowitz, 1990) and p40 (Mendenhall et al., 1987; Mendenhall, 1993) in Saccharomyces cerevisiae, or p21CIP1 (Wade Harper et al., 1993; Xiong et al., 1993) and p27KIP1 (Polyak et al., 1994) in animal cells (Fig. 4). In Gi, when cells are too small to undergo Start, p34cdc2 is inacti­vated by the rum l+ gene product until the critical mass is attained. This inhibition of the Gi form of p34cdc2 automati­cally prevents conversion into the G2 form and thus blocks mitotic onset. Once the critical mass is reached the rum l+ inhibitory effect is lost and the cell executes Start and proceeds to S-phase. At the same time p34cdc2 is converted to the G2 form but is restrained from bringing about mitosis by a new checkpoint control, activated by the assembly of

68 S. Moreno and others

replication complexes, working through p34cdc2 tyrosine 15 phosphorylation.

Cells lacking the rum l+ inhibitor cannot delay Start until the correct critical mass is attained, explaining why Gi is not extended in small cells deleted for ruml+. A cell arrested prior to Start in the absence of the rum l+ gene product cannot block the conversion of p34cdc2 to the G2 form, and as a consequence the cell undergoes premature mitosis. If the rum\+ gene product is present at high levels in a G2 cell, this results in a potent inhibition of p34cdc2/p56cdc13 and cells never enter mitosis. However these cells can undergo S-phase when cells attain the critical cell mass.

The presence of potential MAP kinase phosphorylation sites suggests an interaction with MAP kinase homologues in fission yeast, perhaps analogous to the regulation of FAR1 in response to pheromone action in budding yeast. Such interaction could delay Gi progression in the presence of S. pombe pheromones.

We dedicate this paper to Professor Murdoch Mitchison. He helped to initiate the study of the cell cycle problems that are the subject of this article, and has continued to influence our thinking. We thank Dr Bruce Edgar for his kind gift of the cDNA library and his help at the initial stages of this work and all the members of our groups for many discussions and ideas during the course of this work. This project was supported by ICRF, SERC, the MRC, the EC, EMBO, the Worship­ful Company of Bakers, and the DGICYT.

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