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Molecular and Cellular Biology, July 2001, p. 4140-4148, Vol. 21, No. 13
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.13.4140-4148.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Early Cell Cycle Box-Mediated Transcription of
CLN3 and SWI4 Contributes to the Proper
Timing of the G1-to-S Transition in Budding Yeast
Vivian L.
MacKay,
Bernard
Mai,
Laurie
Waters, and
Linda L.
Breeden*
Fred Hutchinson Cancer Research Center, Basic
Sciences Division, Seattle, Washington 98109-1024
Received 18 December 2000/Returned for modification 27 February
2001/Accepted 9 April 2001
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ABSTRACT |
The Cln3-Cdc28 kinase is required to activate the
Swi4-Swi6 transcription complex which induces CLN1 and
CLN2 transcription in late G1 and drives the
transition to S. Cln3 and Swi4 are both rate limiting for
G1 progression, and they are coordinately transcribed to
peak at the M/G1 boundary. Early cell cycle box (ECB)
elements, which confer M/G1-specific transcription, have
been found in both promoters, and elimination of all ECB elements from
the CLN3 promoter causes both a loss of periodicity and
Cln3-deficient phenotypes, which include an extended G1
interval and increased cell volume. Mutants lacking the ECB elements in
both the CLN3 and SWI4 promoters have low
and deregulated levels of CLN transcripts, and the
G1-to-S transition for these mutants is delayed and highly
variable. These observations support the view that the coordinated rise
of Cln3 and Swi4 levels mediated by ECB-dependent transcription
controls the timing of the G1-to-S phase transition.
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INTRODUCTION |
Cell cycle-regulated transcription
is a motive force for the transitions into and out of
G1 in Saccharomyces cerevisiae.
The M-to-G1 transition involves elimination of
the mitotic cyclins (B-type cyclins [Clbs]). This occurs, in
part, through targeted proteolysis of these B-type cyclins by the
anaphase-promoting complex (APC) (45). However, even if
they are not degraded, the transition to G1 still
occurs (1, 35). This is due to the cessation of
CLB transcription in late M phase and the burst of
M/G1-specific transcription of SIC1
(16), a potent Clb-kinase inhibitor (36).
Sic1 and APC activity persist through G1
(1), resulting in the Clb-Cdk-deficient state that is
characteristic of G1 and required to set up
prereplication complexes on the DNA (29).
Exit from G1 also requires a wave of
transcription of CLN1 and CLN2, CLB5,
and CLB. Clb5 and Clb6 are initially inactive, but the
Cln-Cdk complexes are unaffected by Sic1 and the APC. The latter
serve to target Sic1 for degradation (34, 41) and may also
inactivate the APC (1), which in turn enables the Clb-Cdk
complexes to form and promote the transition into S phase. The third
G1 cyclin, Cln3, is required to activate this
late G1 wave of cyclin transcription (10,
40, 42). The direct target of Cln3-Cdk activation is unknown,
but the G1-specific promoter elements in
CLN1 (27) and CLN2 (39)
and the two transcription factors (Swi4 and Swi6) that activate these
elements have been identified (2, 3, 26). However, neither
the timing nor the mechanism by which Cln3-Cdk activity evokes this
abrupt transcriptional induction in late G1 has
been explained.
Cln3 is unique among the cyclins in that it does not undergo the same
radical oscillations in transcript levels as the others do
(43). This observation has led many investigators to view the Cln3-Cdk as constitutively active and has left open the question of
how a constant kinase activity can trigger the rapid induction of
CLN1 and CLN2 transcription in late
G1 that is associated with the transition to S
phase. Early studies suggested that a threshold level of Cln3-Cdk might
initiate CLN1 and CLN2 transcription and thus
more Cln-Cdk activity, which would then provide positive feedback and
induce more CLN transcription (9, 11). Later, it was shown that Cln1 and Cln2 play no discernible role in their own
activation (10, 19, 40), so that model cannot be correct. More recently, investigators have postulated that CLN3
expression is constitutive but that as G1 cells
grow their nucleocytoplasmic ratio decreases and their translational
capacity increases (5, 12). This could raise the level of
Cln3 in the late G1 nucleus above the threshold
required to start the cell cycle, but it is difficult to see how such a
gradual increase in Cln3-Cdk activity could give rise to the rapid
induction of late G1 transcription that is observed.
The activities of the Cln3 protein and the Cln3-Cdc28 kinase have been
difficult to measure due to the instability of the Cln3 protein
(8, 44), but a modest oscillation of Cln3 protein through
the cell cycle is evident from the results of Western analysis of
epitope-tagged Cln3 (42). There is also a reproducible three- to fivefold oscillation in CLN3 mRNA in late M/early
G1, just before Cln3 is required to activate
transcription of its target genes (21). Swi4, a component
of the late G1 transcription complex activated by
Cln3-Cdk, is also periodically expressed and peaks during the
M/G1 interval, and both genes contain
M/G1-specific promoter elements called early cell
cycle boxes (ECBs). CLN3 and SWI4 heterozygotes
delay the G1-to-S transition, indicating that even twofold drops in their gene dosages disrupt normal
G1 progression (21). This
haploinsufficiency led us to infer that even the modest transcriptional
increase observed for CLN3 during late M and throughout
early G1 could have a significant impact upon the
timing of the transition to S phase.
To test this hypothesis, we eliminated ECB elements from the
CLN3 promoter and showed that they are responsible for most
of the periodicity of the CLN3 transcript. We then
characterized the impact of the loss of ECB-mediated activation of
CLN3 and/or SWI4 transcription on the start of
the cell cycle. Our data support the view that ECB elements mediate a
coordinated increase in the synthesis of these two rate-limiting gene
products to a level which triggers the start of the cell cycle.
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MATERIALS AND METHODS |
Yeast strains, plasmids, and growth conditions.
All strains
used were derived from W303 and are listed in Table
1; one-step gene replacements
(33), lithium acetate transformations (14),
or genetic crosses (37) were used to construct the
derivatives. DNA integrations and replacements were confirmed by PCR
(23), and site-directed mutations (18) were
sequenced before and after transplacement. Unless noted otherwise,
cultures were grown at 30°C in YEP (37) medium
supplemented with adenine (55 mg/liter) and containing 2% galactose
(YEPGal) or glucose (YEPGlu) as the sole carbon source. Synthetic
minimal media were as described previously (37).
Elutriations from late-log-phase cultures (<5 × 107 cells/ml) were done as described previously
(15) with isolated daughter-cell fractions subsequently
grown in fresh medium.
The
swi4ecb (here and throughout, the suffix -
ecb
occurring with a genotypic designation indicates the mutation of an ECB
element encoded by the gene [in this case,
swi4]; if the
suffix
also includes a number [e.g., -
ecb5 or
-
ecb6], the number indicates
how many ECB elements have
been mutated) mutant has been described
previously
(
21); the
cln3ecb mutants have either five or
six
putative ECB elements replaced with mutant forms, as shown in
Fig.
1. These mutations were introduced into
the
CLN3 promoter
by recombining them into a strain (BY2270)
in which the
CLN3 promoter
(positions
1028 to
414
[positions numbered from the translational
start]) was deleted and
replaced with the
URA3 gene. BY2270 was
constructed by
introducing an
EcoRI site at position
1028 into
the
CLN3 promoter in pBD1865 to produce pBD2175. This DNA was
cut with
EcoRI and
XhoI (position
414), and a
URA3 fragment with
like ends from pBD1918 was inserted. The
cln3::
URA3 DNA construct
was used to
replace the
CLN3 locus of BY2125 and generate BY2270.
BY2270
produces no detectable
CLN3 mRNA and was used as our
cln3 null strain. Replacement with the ECB mutant promoter
sequences
was carried out by a series of site-directed mutageneses of
pBD1865;
then these DNAs were used to replace the
cln3::
URA3 locus of BY2270.
Candidates
were sequenced. The
cln3ecb5 swi4ecb strain was generated
by
crossing the
cln3ecb5 and
swi4ecb strains and
sequenced to
verify that the mutations had been maintained. The plasmid
carrying
CLN2 under
MET3 promoter control
(pDS846) was a kind gift from
D. Stuart and C. Wittenberg. This
plasmid was integrated into
BY2679 and BY2680 at
leu2
to produce BY2786 and BY2788, respectively.
Leu
+
control transformants (BY2785 and BY2787) were made by integrating
pRS305 (
38) at the
leu2 locus.
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FIG. 1.
CLN3 promoter region showing the positions of the ECB
elements (solid boxes) and the glucose response elements (GREs)
(open boxes). Below and to the left the sequence alignment of the six
potential ECB elements of the CLN3 promoter is shown.
Bases identical to the consensus sequence are shaded. To the right the
mutations (in italics and lowercase) that have been introduced to
prevent Mcm1 binding and to inactivate the ECB elements are shown. The
boldface type indicates key residues for Mcm1 binding. uORF,
untranslated ORF.
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Synchronies and Northern blots
For analysis
of cell cycle-regulated transcripts, cells were synchronized either by
-factor treatment (4) or by elutriation (15) and, during outgrowth, samples were removed for
isolation of RNA. Aliquots (10 µg) of total RNA were run on 1%
agarose gels containing 0.6 M formaldehyde in 40 mM
morpholinepropanesulfonic acid (MOPS)-acetate buffer, pH 7.2, transferred to nylon membranes, and hybridized with probes specific for
CLN3, SWI4, CLN2, CLN1, HO, and ACT1.
Hybridization intensities were quantified with a PhosphorImager and
ImageQuant software (Molecular Dynamics, Sunnyvale, Calif.), and the
results were normalized to the ACT1 signal.
Cell sizing, fluorescence-activated cell sorter (FACS)
analysis, and budding index
Median cell volumes
(in femtoliters) were determined for log-phase cultures (5 × 106 to 2 × 107 cells/ml) with a particle
counter and size analyzer (model Z2) and AccuComp software (both
provided by Coulter Corp., Miami, Fla.). Culture aliquots were washed
and resuspended in 2 mM EDTA, sonicated lightly to disperse aggregates,
and diluted to 0.5 × 105 to 2.0 × 105 cells/ml into Isoton II diluent (Coulter Corp.) for analysis.
Cell samples for FACS analysis were fixed overnight in 67% ethanol at
room temperature, washed with 50 mM Tris-HCl (pH 7.8),
incubated in 50 mM Tris-HCl containing 0.2 mg of RNase A/ml at
37°C for 2 to 4 h, and resuspended in a solution containing 200
mM Tris-HCl (pH 7.5),
200 mM NaCl, and 78 mM MgCl
2. After addition
of
propidium iodide (final concentration, 30 µg/ml), the samples
were
sonicated lightly and analyzed on a FACS Calibur flow cytometer
(Becton-Dickinson, San Jose, Calif.). The percentage of
G
1 cells
in the population was determined using
MultiCycle software (Phoenix
Flow Systems, San Diego, Calif.)
To obtain the budding index, cell growth was arrested with 0.2% sodium
azide, the samples were held overnight and sonicated,
and then cells
were counted microscopically (at least 200 cells
were scored per
sample). Bud scars on mother cells were visualized
by Calcofluor
staining (
31).
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RESULTS |
ECB elements confer a coordinated burst of
M/G1-specific transcription of CLN3 and
SWI4.
ECB elements were first identified in the
SWI4 promoter and shown to be sufficient to confer
M/G1-specific transcription to a heterologous
transcript (21). There are six sequences in the promoter
region of the CLN3 gene (Fig. 1) that have various degrees of homology to the 16-bp ECB consensus sequence originally identified. The first four putative ECBs are clustered approximately 1 kb upstream
of the translational start. Two more potential ECBs are located about
0.5 kb downstream from these. Previous experiments (21)
showed that DNA fragments including sequences from positions 994 to 439 confer M/G1-specific transcription
to a heterologous transcript. Moreover, mutations that disrupt three of
the clustered sites (sites 1, 3, and 4) eliminate cell cycle regulation
of the same reporter construct. This result suggests that sites 2, 5, and 6 are not functional, at least in this heterologous context. However, the activities of individual ECBs have not been investigated in their native locations. Rather, we mutated all six possible ECB
elements (Fig. 1) in order to assess their role in the regulation of
CLN3 transcription. The CLN3 promoter also
contains five A2GA5 sequences (glucose response elements), which have been reported to induce CLN3 transcription in response to glucose
(28). CLN3 transcription starts at about
position 350, and its untranslated leader sequence contains an
upstream open reading frame. There is evidence that this upstream open
reading frame imparts an additional level of translational regulation
to the CLN3 message (30).
To see if ECB elements are responsible for the
M/G
1-specific transcription of
CLN3,
we synchronized wild-type and ECB mutant
cells and measured RNA levels
through the cell cycle. To avoid
glucose-mediated effects on
CLN3 transcription, we grew the cells
in rich medium
containing galactose as the carbon source. Wild-type
cells clearly
exhibited the same oscillating pattern of
CLN3 mRNA
levels
observed with glucose-grown cells (Fig.
2A), and
CLN3 mRNA
levels
peaked 10 min before the
CLN2 mRNA levels did (reference
21 and data not shown). In contrast, strains with
mutations
in either the first five or all six of the potential ECB
elements
displayed dramatically reduced transcript levels throughout
the
cell cycle. These cells retained a modest but reproducible peak
of
CLN3 mRNA coincident with the ECB-induced peak, which
suggests
that there may be another unrecognized promoter element or a
component
of posttranscriptional control which makes a minor
contribution
to
CLN3 expression. However, this
non-ECB-mediated induction never
exceeded the trough level of
CLN3 transcript from a wild-type
cell, so its significance
is unclear.
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FIG. 2.
ECB elements are important for cell cycle regulation of
CLN3. (A and B) Cycling of CLN3 mRNA in
wild type (BY2125) and cln3ecb6 mutant (BY2690),
respectively. Cells were grown in YEPGal and synchronized with
-factor. Cells were released into fresh medium at time point 0 and
samples were taken every 10 min, as indicated by the numbers above the
lanes. The expression of CLN3 and ACT1
was analyzed by Northern blot hybridizations. The blots were hybridized
at the same time to the same probes, and exposure times were identical.
(C) CLN3 transcript levels in the wild-type strain
(BY2125) (closed squares) and cln3ecb5 (BY2278) (open
squares) and cln3ecb6 (BY2690) (open circles) mutant
strains grown in parallel were measured throughout the cell cycle, and
the results of Northern blotting were quantitated with a
PhosphorImager.
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We also noted that mutation of the sixth potential ECB had no impact on
the transcription pattern through the cell cycle.
This provides further
evidence that this poor match to the consensus
is not an active ECB.
Thus, all subsequent experiments were carried
out with a strain
carrying mutations in the first five putative
ECBs, and this multiple
mutant is referred to as
cln3 ecb.
ECB-regulated transcription of SWI4 and
CLN3 affects the length of G1 and size
control.
Since Cln3 and Swi4 are rate-limiting activators of the
transition to S phase (7, 21, 24) and their transcription
is coordinately regulated by ECB elements, we wished to know how ECB
elements contribute to the regulation of G1
progression. To address this issue, we measured the length of
G1 and the sizes of cells carrying mutations in
the CLN3 ECB elements and/or the SWI4 ECB. Table
2 shows that cells lacking ECB-activated
transcription of either SWI4 or CLN3 or both
spend a larger proportion of their cell cycle in
G1 than wild-type cells. Perhaps due to the
glucose induction of CLN3 transcription, the effects of
these mutations were reduced when YEPGlu was used, but they are
qualitatively similar to those observed when YEPGal was used. Loss of
the ECB elements from both SWI4 and CLN3
promoters caused a G1 delay roughly equivalent to
that of a cln3 null strain, which we found to maintain the
level of cells in G1 at 43% in YEPGal medium.
The swi4 deletion strain is lethal in this background so
that comparison cannot be made.
In addition to prolonging G
1, elimination of
ECB-mediated activation of
CLN3 and
SWI4
expression also caused a concomitant
increase in cell volume (Fig.
3A and B). The data presented in
Fig.
3
indicate that the M/G
1-specific transcription of
these
two genes makes a substantial and additive contribution to the
control of the G
1-to S-transition, especially for
cells grown
in the absence of glucose. Figure
3C and D show a direct
comparison
of the size distribution of
cln3ecb cells with
that of cells with
no Cln3 at all. Consistent with the FACS analysis
data, the absence
of Cln3 results in the most severe phenotype and loss
of the ECB-dependent
transcription of
CLN3 has an
intermediate phenotype. Thus, both
basal and cell cycle-regulated
transcription of
CLN3 contributed
to size control under the
conditions tested.
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FIG. 3.
Loss of ECB-mediated transcription of key cell cycle
regulators leads to increased cell volume. Exponential-phase cultures
of strains BY2679, BY2680, BY2681, and BY2682 grown in YEPGlu
(A) or YEPGal (B) and of strains BY2125, BY2270, and BY2278 grown in
YEPGlu (C) or YEPGal (D) were analyzed for cell size profile as
described in Materials and Methods. The relevant genotypes are
indicated.
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To further characterize G
1 progression and size
heterogeneity in cells lacking ECB function in their
SWI4
and/or
CLN3 promoters,
we purified small
G
1 daughters of each genotype and compared their
behavior to that of wild-type cells. Several size fractions of
G
1 daughter cells of each genotype were
inoculated into fresh
medium, and their growth, budding kinetics, and
DNA profiles were
monitored. As has been previously shown
(
20), wild-type cells
show remarkable uniformity in that
they shift from 0 to 50% budded
as the cells enlarge from a volume of
26 to 31 fl (Fig.
4A). This
concerted
transition reflects a uniform response to the signal
to start the cell
cycle which is tightly correlated with cell
size. Cells that lack the
ECB-mediated burst of either
CLN3 or
SWI4
expression (Fig.
4A and B, respectively) bud at larger cell
sizes and
those lacking ECB activation of both
CLN3 and
SWI4 are
even larger and more heterogeneous at budding (Fig.
4C). In contrast
to the uniform behavior observed with wild-type cells,
the
cln3ecb swi4ecb cultures nearly doubled in volume as
they went from 0%
to 50% budding. Figure
4D shows wild-type and
cln3ecb swi4ecb mutant cells at the time point when 50% of
the cells had budded.
It is clear that the wild-type cells were of
uniform size, as
were their buds, indicating that bud initiation
occurred at about
the same time in the growth of this population.
However, budding
occurred more slowly and heterogeneously in the double
mutant,
leading to cells with buds of various sizes. This effect was
not
due to the size heterogeneity of the starting population, because
when we started with cells with precisely the same size distribution,
buds accumulated in the double-mutant population much more gradually
than in the wild type population (Fig.
5.) This heterogeneity
of response also
was not due to contamination of the double-mutant
population with
petite mutants, as measured by growth of the starting
population on
glycerol, or to the presence of mother cells, as
measured by Calcofluor
staining for bud scars (data not shown.)
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FIG. 4.
ECB elements confer rapid, synchronous progression
through G1 in daughter-cell fractions isolated by
elutriation. Late-exponential-phase cultures (ca. 5 × 107 cells/ml) grown in YEPGal were fractionated by
elutriation. Fractions containing <5% budded cells were diluted into
fresh YEPGal at densities between 2.4 × 106 and
2 × 107 cells/ml, grown at 30°C, and sampled at
15-min intervals for determinations of size and budding indexes. The
median cell volumes (in femtoliters) were determined by sizing on a
Coulter model Z2 analyzer), as described in Materials and Methods. (A)
Budding profiles of cells lacking the ECB-mediated burst of
CLN3 expression. Open symbols, wild-type
[BY2679(CLN3 SWI)] (starting fractions had median
volumes of from 13.0 to 15.8 fl); closed circles,
cln3ecb mutant (BY2681) (starting fractions had median
volumes of from 22.7 to 25.9 fl). (B) Budding profiles of cells lacking
the ECB-mediated burst of SWI4 expression. Circles,
swi4ecb (BY2682) (starting fractions had median volumes
of from 14.8 to 17.2 fl); open squares, wild type (profile shown for
comparison). (C) Budding profiles of cells lacking ECB activation of
both CLN3 and SWI4. Circles,
cln3ecb swi4ecb (BY2680) (starting fractions had median
volumes of from 15.0 and 28.4 fl); open squares, wild type (profile
shown for comparison). (D) Phase-contrast photomicrographs of fractions
of wild-type (initial median volume = 16.9 fl) and cln3ecb
swi4ecb (initial median volume = 18.8 fl) elutriated cells
after outgrowth to ca. 50% budded cells. The images
shown are at the same magnification.
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FIG. 5.
Wild-type (BY2679) and cln3ecb swi4ecb
(BY2680) daughter cells exhibit very different budding kinetics in
spite of their identical initial size distributions. Elutriated
fractions of wild-type and cln3ecb swi4ecb daughter
cells with identical size distributions (A) and starting median volumes
of 15.1 and 15.0 fl, respectively, were sampled during outgrowth and
monitored for budding and median cell size (B).
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As expected, the double-mutant cells were more sensitive to
-factor
than wild-type cells are. In addition, we found that
their transit to S
phase after release from the arrest was slower
and more heterogeneous
than that of wild-type cells. Figure
6 shows the FACS profiles for wild-type and
cln3ecb swi4ecb
mutant
cells obtained as they were released from
-factor arrest. Not
only was initiation of the first S phase at least 15 min slower
for the
cln3ecb swi4ecb cells but the synchrony of the culture
was
lost within the first cell cycle. The delay of S phase, as
well as the
size heterogeneity and FACS profiles observed in unperturbed,
growing
populations of
cln3ecb swi4ecb cells, leads us to conclude
that the defects in G
1 progression manifested by
the ECB mutants
are not artifacts of recovery from elutriation, nor are
they specific
to daughter cells. Rather, they reflect an inefficient
and deregulated
G
1-to-S transition. However, the
heterogeneity between different
elutriated fractions that was
especially evident with the double
mutant (Fig.
4C) may have resulted
in part from the elutriation
treatment. We see a general tendency for
the larger daughter-cell
fractions to bud at larger cell volumes,
suggesting that the double
mutant is particularly sensitive to
disruptions in the growth
cycle in late G
1.
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FIG. 6.
Coupling between the G1-to-S phase
transition and initiation of budding is maintained in cln3ecb
swi4ecb mutants. Unbudded daughter-cell fractions from
elutriation were sampled at 15-min intervals during outgrowth for
budding (squares) and initiation of DNA synthesis (circles). The
percentage of cells in each sample that had exited G1 was
determined by subtracting the percentage of cells in G1
(determined by analysis of the FACS profiles with MultiCycle software)
from 100. Open symbols, wild-type (BY2679); closed symbols,
cln3ecb swi4ecb (BY2680).
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Budding is typically coordinated with the transition to S phase, so we
also monitored exit from G
1 in wild-type and
cln3ecb swi4ecb cells by FACS analysis. As indicated in Fig.
7, the percentage
of cells that exited
G
1 increased smoothly and in parallel to
the
budding profile, which lagged by about the same amount in
both strains.
Thus, the coupling between budding and the transition
to S phase was
not altered in the double mutant.
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FIG. 7.
ECB mutants are more sensitive to -factor and recover
from -factor arrest more slowly than wild-type cells. The upper
portion of the figure shows 3,000, 1,000 and 300 cells of the genotypes
indicated (strains BY2125, BY2680, BY2681, and BY2684) that were
spotted on YEPglu plates containing 0 or 0.6 µM -factor and
allowed to grow for 2 days at 30°C. The lower portion of the figure
shows FACS profiles of wild-type (BY2125) and cln3ecb
swi4ecb (BY2680) cells that were grown in YEPGal. Growth was
arrested by treatment with 5 µg of -factor/ml for 135 min, and
then the cells were released into the same medium and sampled at 15-min
intervals.
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CLN1 and CLN2 are critical targets of
CLN3 and SWI4.
The long
G1 phase and heterogeneous cell size of
cln3ecb swi4ecb cells suggest that their ability to induce
CLN1 and CLN2 transcription is highly impaired.
To see if this is the case, we carried out Northern blot analysis of
mRNAs from elutriated daughter cells harvested at intervals as they
progressed through the cell cycle with the wild-type and double mutant
cells. The starting population of wild-type G1
cells had high levels of CLN3 and SWI4 mRNAs
which continued to rise throughout G1 and then fell as cells began to bud. The pattern of CLN1 and
CLN2 transcription paralleled the pattern of ECB-mediated
transcription but showed a slight lag. However, in the double-mutant
population, both CLN3 and SWI4 transcripts
remained at a low constitutive level (Fig. 8). CLN1 and CLN2
mRNA levels also remained low but rose gradually, as they did in a
cln3 mutant population (10, 40). HO,
another target gene which is not expressed in daughter cells
(25), peaked only in the second cycle.
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FIG. 8.
ECB elements are required for transcriptional regulation
of CLN3 and SWI4 and their target genes
(CLN1, CLN2, and HO) through the cell
cycle. Unbudded daughter-cell fractions from elutriation were grown in
YEPGal, and samples were taken at 15-min intervals for isolation of
total RNA, as well as for size determination, FACS analysis, and
budding index. Data shown are from a single Northern blot iteratively
hybridized with probes for the indicated transcripts; multiple blotting
experiments (data not shown) were carried out to confirm the validity
of the cycling pattern shown. Initial cell volumes of these preparative
elutriation fractions were 18.4 fl (wild-type strain [BY2679]) (open
squares) and 23.4 fl (cln3ecb swi4ecb strain [BY2680])
(filled squares). The graph at the upper left shows budding kinetics,
and the other five graphs show quantitated hybridization signals for
the transcripts indicated (data normalized to the ACT1
signal and plotted with the lowest point in the wild-type samples set
at 1.0). Below, hybridization patterns for each transcript scanned by a
PhosphorImager are shown.
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From these results one would predict that enhanced expression of
CLN2 in these cells would effectively suppress these
phenotypes.
As shown in Fig.
9, ectopic
expression of
CLN2 from the
MET3 promoter
in
cln3ecb swi4ecb cells shifted their size distribution to a
uniform and smaller cell volume comparable to that observed for
wild-type cells. This decrease in cell size was accompanied by
a
concomitant decrease in the percentage of G
1
cells in the population
(data not shown).
View larger version (18K):
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|
FIG. 9.
Ectopic expression of CLN2 suppresses the
increased cell size and heterogeneity of cln3ecb swi4ecb
cells. A DNA construct with CLN2 under the control of
the MET3 promoter or a control vector was integrated at
leu2 in the cln3ecb swi4ecb strain and
the comparable wild-type strain. For analysis, cultures were grown in
synthetic minimal glucose medium lacking methionine to derepress the
promoter, and size profiles were determined on a Coulter model Z2
analyzer.
|
|
| |
DISCUSSION |
ECB elements, which are sufficient to promote
M/G1-specific transcription, have been identified
in both the SWI4 and CLN3 promoters. In a
previous study, it was shown that loss of the ECB element from
SWI4 caused a 10-min delay of its transcription and a change
of start site, but SWI4 expression was still periodic owing
to the MluI cell cycle box elements that also reside
in the SWI4 promoter. This 10-min delay led to a lengthening
of G1 and an enlarged cell size
(21). By introducing inactivating mutations in all six of
the potential ECB elements in the CLN3 promoter, we have
shown that these elements are responsible for most of the cell cycle
regulation of the CLN3 transcript. The resulting strain also
suffers a lengthening of G1 and an increase in
cell size, indicating that ECB-mediated transcription of
CLN3 is required for efficient transit through
G1 and into S phase. Thus, elimination of the
modest oscillation of CLN3 transcription or decoupling the
peak of CLN3 transcription from that of SWI4 by
10 min has a negative effect upon G1 progression.
Loss of the ECB elements from both promoters has a more dramatic
effect, consistent with our previous findings that Swi4 and Cln3 make
independent, rate-limiting contributions to G1
progression (21). The tight correlation between budding
and the transition to S phase is not perturbed in the double mutant.
This is not surprising because CLN1 and CLN2, the
critical targets of Swi4 and Cln3, initiate both the budding cycle and
the transition to S, so both should be equivalently delayed, as they
are in the absence of Cln3 (10). However, the mechanism
which triggers the late G1 transcription
responsible for these events at a specific time and at a specific cell
size is absent from these cells.
With a clonal population of wild-type daughter cells under constant
environmental conditions, the balance between biosynthetic capacity and
the activity of key regulators causes cells to progress through
G1 at a particular pace and achieve a
characteristic and uniform cell size before budding and the transition
to S phase is triggered. In contrast, cells lacking ECB-mediated
transcription of CLN3 and SWI4 exit
G1 in a delayed and highly asynchronous manner,
which is reflected in the diverse sizes these cells attain. Thus,
instead of the highly regulated and sharp transition observed with
wild-type cells, the cln3ecb swi4ecb cells behave as though the triggering mechanism has been lost and commitment to start the cell
cycle has become a stochastic process, subject to random fluctuations
in the concentration of key regulators from one cell to another. These
observations show that the ECB elements, which coordinate the rise of
both SWI4 and CLN3 mRNA levels, are important components of the mechanism which controls the timing of the
G1-to-S phase transition in daughter cells.
Due to the asymmetric growth pattern of budding yeast, daughters are
typically smaller than their mothers and spend more time in their first
G1 before achieving a characteristic cell size and committing to another round of division (for a review, see reference 32). This homeostatic mechanism has long been
thought to involve accumulation of an unstable activator
(22), and experimental evidence has shown that Cln3 could
be that activator (7, 24). Our data demonstrate a clear
role for ECB-mediated transcription of CLN3 and
SWI4 in determining the timing of the
G1-to-S transition in daughter cells. There is an
ECB-mediated, early G1 peak of both
CLN3 and SWI4 transcription in elutriated
daughters which is necessary for the normal induction of late
G1 transcripts. In the absence of this
transcriptional control, the transition to S phase is delayed and
highly heterogeneous. We also observed heterogeneous cell sizes and
G1 delays in exponentially growing mixtures of
cln3ecb swi4ecb mothers and daughters, so we expect that
G1 progression is also ECB-mediated in mother
cells, but the specific pattern of ECB-mediated transcription could
differ in mother and daughter cells.
Our initial observations showed that CLN3 mRNA levels peak
in very late M phase and remain high throughout
G1 in mixed populations of mothers and daughters
(21). In this paper, we show that elutriated G1 daughters start with high levels of
CLN3 and SWI4 mRNA which continue to rise as
cells transit through G1 and peak just minutes before the peak of their target genes. This is consistent with the
possibility that ECB activity peaks later in daughter cells than in
mothers. This is an interesting possibility, as it could explain how
G1 is prolonged in daughter cells. However, the
transcript pattern we observed in these daughters could also be an
artifact of elutriation.
The discovery of the importance of transcriptional regulation of
CLN3 and SWI4 by ECB elements provides a simple
model for how the timing of the G1-to-S
transition is controlled. Cln3 and Swi4 are unstable proteins
(44; L. L. Breeden, unpublished data). This
does not favor models in which either protein must accumulate to some
threshold level to trigger start. However, if their stability were
constant through the cell cycle, the transcriptional oscillation mediated by the ECB elements would result in a parallel oscillation in
the protein levels, enabling the concentration of both proteins to rise
coordinately and peak in early G1. The
coordinated increase in the levels of these two rate-limiting
regulators would promote the efficient formation and activation of the
Swi4-Swi6-DNA complex and result in a burst of transcription of
the next wave of cyclins that are responsible for an efficient
transition to S phase. In vivo binding studies indicate that Swi4-Swi6
complexes are detectable on the DNA throughout
G1, during the interval of maximum
SWI4 and CLN3 transcription, but activation of
the complex occurs in late G1 (6, 13,
17). This lag between the formation of the complex and its
activation suggests that there may be other steps involved in the
activation process.
| |
ACKNOWLEDGMENTS |
We gratefully acknowledge helpful discussions with and technical
assistance from other members of the lab, particularly J. Sidorova, S. Plante, C. McInerny, and J. Partridge.
This work was supported by a grant from the National Institutes of
Health (GM41073) to L.L.B.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Fred Hutchinson
Cancer Research Center, Basic Sciences Division, Seattle, WA
98109-1024. Phone: (206) 667-4484. Fax: (206) 667-6526. E-mail:
lbreeden{at}fhcrc.org.
Present address: Department of Biochemistry, University of
Washington, Seattle, WA 98195.
Present address: Aventis Pharma GmbH, Martinsried, Germany.
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Molecular and Cellular Biology, July 2001, p. 4140-4148, Vol. 21, No. 13
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.13.4140-4148.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
